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CN111944056A - Apoptosis protein fusion type anti-HER-2 single chain antibody and preparation method and application thereof - Google Patents

Apoptosis protein fusion type anti-HER-2 single chain antibody and preparation method and application thereof Download PDF

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CN111944056A
CN111944056A CN202010678663.4A CN202010678663A CN111944056A CN 111944056 A CN111944056 A CN 111944056A CN 202010678663 A CN202010678663 A CN 202010678663A CN 111944056 A CN111944056 A CN 111944056A
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郭军
陆丹丹
郭艺辰
郑子慧
陈佳慧
王婷婷
王敏
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Nanjing University of Chinese Medicine
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Abstract

本发明公开了凋亡蛋白融合型抗HER‑2单链抗体及其制备方法和应用，凋亡蛋白融合型抗HER‑2单链抗体，由抗HER‑2单链抗体以不同串联方式与凋亡蛋白偶联而成，凋亡蛋白为细胞色素C或DNA片段化因子40，具体可与抗HER‑2单链抗体偶联成DFF40‑scFv融合型单链抗体、nCytc(串联)‑scFv融合型单链抗体。凋亡蛋白融合型抗HER‑2单链抗体通过凋亡蛋白的cDNA序列插入scFv上游并克隆入表达载体中构建重组质粒，重组质粒转入细胞，诱导表达凋亡蛋白融合型抗HER‑2单链抗体，制备方法易于构建和表达，构建的凋亡蛋白融合型抗HER‑2单链抗体能够特异性靶向HER‑2高表达的恶性肿瘤并介导癌细胞凋亡，可用于制备靶向治疗HER‑2高表达癌症的药物。

Figure 202010678663

The invention discloses apoptotic protein fusion type anti-HER-2 single-chain antibody and a preparation method and application thereof. The apoptotic protein is cytochrome C or DNA fragmentation factor 40. Specifically, it can be coupled with anti-HER-2 single-chain antibody to form DFF40-scFv fusion single-chain antibody, nCytc (tandem)-scFv fusion type single chain antibody. The apoptotic protein fusion anti-HER-2 single-chain antibody is inserted upstream of the scFv through the cDNA sequence of the apoptotic protein and cloned into the expression vector to construct a recombinant plasmid. Chain antibody, the preparation method is easy to construct and express, the constructed apoptotic protein fusion type anti-HER-2 single-chain antibody can specifically target malignant tumors with high HER-2 expression and mediate apoptosis of cancer cells, and can be used to prepare targeted Drugs for the treatment of HER-2-expressing cancers.

Figure 202010678663

Description

凋亡蛋白融合型抗HER-2单链抗体及其制备方法和应用Apoptosis protein fusion type anti-HER-2 single chain antibody and preparation method and application thereof

技术领域technical field

本发明属于生物医药领域，具体涉及凋亡蛋白融合型抗HER-2单链抗体及其制备方法和应用。The invention belongs to the field of biomedicine, and particularly relates to apoptotic protein fusion type anti-HER-2 single-chain antibody and a preparation method and application thereof.

背景技术Background technique

乳腺癌是全世界女性最常见的恶性肿瘤，全球乳腺癌发病率自20世纪70年代末开始一直呈上升趋势。2018年3月23日，国家癌症中心发布的最新数据显示，全国女性乳腺癌新发病例占女性恶性肿瘤发病16.51％，位居女性恶性肿瘤发病第1位。HER-2(HumanEpidermal Growth Factor receptor 2，又称为neu，ErBb-2，HER-2)为表皮生长因子受体超家族成员，是人类肿瘤中发生改变频率最高的癌基因之一，其基因表达水平和基因拷贝数目在几种人类肿瘤细胞中都有显著升高，特别是在乳腺癌、卵巢癌和胃癌中升高更为显著。研究表明30％以上的人类肿瘤中存在HER-2基因的扩增/过度表达(如乳腺癌、卵巢癌、子宫内膜癌、输卵管癌、胃癌和前列腺癌等)，HER-2过度表达的乳腺癌患者病情进展迅速，化疗缓解期短，内分泌治疗效果差，无病生存和总生存率低。HER-2基因的过表达不仅与肿瘤的发生发展相关，还是肿瘤靶向治疗药物选择的一个重要靶点。Breast cancer is the most common malignant tumor in women worldwide, and the global incidence of breast cancer has been increasing since the late 1970s. On March 23, 2018, the latest data released by the National Cancer Center showed that the new cases of female breast cancer in the country accounted for 16.51% of the incidence of female malignant tumors, ranking first in the incidence of female malignant tumors. HER-2 (Human Epidermal Growth Factor receptor 2, also known as neu, ErBb-2, HER-2) is a member of the epidermal growth factor receptor superfamily and is one of the most frequently altered oncogenes in human tumors. Levels and gene copy numbers were significantly elevated in several human tumor cells, especially in breast, ovarian, and gastric cancers. Studies have shown that HER-2 gene amplification/overexpression exists in more than 30% of human tumors (such as breast cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer and prostate cancer, etc.), and HER-2 overexpressed breast cancer Cancer patients have rapid disease progression, short chemotherapy remission, poor endocrine therapy, and low disease-free and overall survival rates. The overexpression of HER-2 gene is not only related to the occurrence and development of tumors, but also an important target for the selection of tumor-targeted therapy drugs.

目前临床批准的绝大部分HER-2为靶点的药物都集中在乳腺癌，比如拉帕替尼(泰立沙)、曲妥珠单抗(赫赛汀)、T-DM1、帕妥珠单抗。FDA在1998年批准曲妥珠单抗(Trastuzumab)用于有HER-2过量表达的乳腺癌的治疗。曲妥珠单抗中文商品名叫“赫赛汀”，是抗HER-2的单克隆抗体，它通过将自己附着在HER-2上来阻止人体表皮生长因子在HER-2上的附着，从而阻断癌细胞的生长，赫赛汀还可以刺激身体自身的免疫细胞去摧毁癌细胞。基因工程抗体是继多克隆抗体和单克隆抗体之后的第三代抗体，主要包括嵌合抗体、人源化抗体、完全人源抗体、单链抗体(single chain antibody fragment，scFv)、双特异性抗体等，其中单链抗体(single chain antibody fragment，scFv)是由抗体重链和轻链可变区片段用一条弹性短肽连接而成的小分子抗体片段。与完整抗体分子相比，单链抗体具有以下特点：(1)不含有抗体分子的恒定区片段，因而免疫原性弱，用于人体几乎不会产生抗鼠抗体；(2)对相应抗原具有较高的亲和力和特异性；(3)相对分子质量小，穿透力较强，在体内停留的时间较短，适用于疾病的免疫显像诊断和导向治疗；(4)由于相对分子质量较小，不需要进行糖基化修饰即可形成有功能的抗体分子，因而可以在原核表达系统中进行表达并容易获得。已有研究成功制备抗HER-2单链抗体用于肿瘤的靶向，然而，单链抗体稳定性差、易聚集，半衰期短效果不显著。Most of the currently approved HER-2-targeted drugs are concentrated in breast cancer, such as lapatinib (Talisa), trastuzumab (Herceptin), T-DM1, Pertuzumab monoclonal antibody. Trastuzumab was approved by the FDA in 1998 for the treatment of breast cancer with overexpression of HER-2. The Chinese trade name of trastuzumab is "Herceptin", which is an anti-HER-2 monoclonal antibody. It prevents the attachment of human epidermal growth factor to HER-2 by attaching itself to HER-2, thereby preventing the By stopping the growth of cancer cells, Herceptin can also stimulate the body's own immune cells to destroy cancer cells. Genetically engineered antibodies are the third generation of antibodies after polyclonal antibodies and monoclonal antibodies, mainly including chimeric antibodies, humanized antibodies, fully human antibodies, single chain antibody fragments (scFv), bispecific antibodies Antibodies, etc., wherein a single chain antibody (single chain antibody fragment, scFv) is a small molecule antibody fragment formed by linking the antibody heavy chain and light chain variable region fragments with an elastic short peptide. Compared with intact antibody molecules, single-chain antibodies have the following characteristics: (1) they do not contain constant region fragments of antibody molecules, so they are weak in immunogenicity and hardly produce anti-mouse antibodies when used in humans; High affinity and specificity; (3) Small relative molecular mass, strong penetrating power, and short stay in the body, suitable for immunoimaging diagnosis and guided treatment of diseases; (4) Due to the relatively high relative molecular mass It is small and does not require glycosylation to form functional antibody molecules, so it can be expressed in prokaryotic expression systems and easily obtained. Previous studies have successfully prepared anti-HER-2 single-chain antibodies for tumor targeting. However, single-chain antibodies have poor stability, easy aggregation, and short half-life effects.

单链抗体scFv也被用作药物递送平台递送效应分子杀伤靶细胞，但很多现有免疫毒素的杀伤功能域主要来源于植物或细菌毒素，分子量太大，组织渗透性低，且具有很高的免疫原性和强烈的副作用，限制了其临床应用。为了解决这些问题，据报道，一系列内源性细胞凋亡相关分子可以替代外源性毒素，经过大量文献研究发现，细胞色素C(Cytc)和DNA片段化因子40(DFF40)在细胞凋亡途径中发挥重要作用，Cytc释放到细胞浆的细胞色素C在dATP存在的条件下能与凋亡相关因子1(Apaf-1)结合，使其形成多聚体，并促使caspase-9与其结合形成凋亡小体，caspase-9被激活，被激活的caspase-9能激活其它的caspase如caspase-3等，从而诱导细胞凋亡；DFF40在凋亡细胞中，经caspase-3激活后，这种蛋白质可裂解核小体间DNA，导致特征性的DNA梯状，由于DFF40是凋亡级联中的下游组分，其表达和活化将导致不可逆的DNA损伤，导致明确的细胞死亡。Single-chain antibody scFv has also been used as a drug delivery platform to deliver effector molecules to kill target cells, but the killing domains of many existing immunotoxins are mainly derived from plant or bacterial toxins, which are too large in molecular weight, low in tissue permeability, and have high molecular weight. Immunogenicity and strong side effects limit its clinical application. In order to solve these problems, it has been reported that a series of endogenous apoptosis-related molecules can replace exogenous toxins. After a large number of literature studies, it was found that cytochrome c (Cytc) and DNA fragmentation factor 40 (DFF40) play a role in apoptosis. In the presence of dATP, cytochrome C released by Cytc into the cytoplasm can combine with apoptosis-related factor 1 (Apaf-1) to form a multimer, and promote the combination of caspase-9 with it to form a multimer. In apoptotic bodies, caspase-9 is activated, and the activated caspase-9 can activate other caspases such as caspase-3, etc., thereby inducing apoptosis; DFF40 is activated by caspase-3 in apoptotic cells. The protein cleaves internucleosomal DNA, resulting in a characteristic DNA ladder, and since DFF40 is a downstream component in the apoptotic cascade, its expression and activation will lead to irreversible DNA damage leading to definitive cell death.

基于上述研究，本发明将抗HER-2单链抗体scFv与凋亡蛋白DFF40或nCytc(n≥1)偶联，通过scFv将DFF40或nCytc递送至靶细胞，经细胞内化后通过激活内源性凋亡途径抑制肿瘤细胞活性，大大降低免疫原性和毒副作用。通过比较不同蛋白构建体的活性差异，以期寻求抗肿瘤活性最好且副作用最小的单链抗体免疫凋亡制剂，有望在乳腺癌的诊断和治疗方面发挥重要作用。Based on the above research, the present invention couples anti-HER-2 single-chain antibody scFv to apoptotic protein DFF40 or nCytc (n≥1), and delivers DFF40 or nCytc to target cells through scFv. The apoptosis pathway inhibits tumor cell activity and greatly reduces immunogenicity and toxic side effects. By comparing the activity differences of different protein constructs, we hope to find the single-chain antibody immunoapoptotic preparation with the best anti-tumor activity and the least side effects, which is expected to play an important role in the diagnosis and treatment of breast cancer.

发明内容SUMMARY OF THE INVENTION

本发明所要解决的技术问题是针对上述现有技术的不足，提供凋亡蛋白融合型抗HER-2单链抗体及其制备方法和应用。The technical problem to be solved by the present invention is to provide the apoptotic protein fusion type anti-HER-2 single-chain antibody and its preparation method and application in view of the above-mentioned deficiencies of the prior art.

为实现上述技术目的，本发明采取的技术方案为：一种凋亡蛋白融合型抗HER-2单链抗体，由抗HER-2单链抗体以不同串联方式与凋亡蛋白偶联而成，抗HER-2单链抗体包括序列如SEQ ID NO.1所示的重链可变区VH和序列如SEQ ID NO.2所示的轻链可变区VL，重链可变区和轻链可变区通过序列如SEQ ID NO.3所示的柔性肽(G4S)3连接，重链可变区VH上游含有6×his标签，抗HER-2单链抗体的氨基酸序列如SEQ ID NO.4所示，编码抗HER-2单链抗体的cDNA序列如SEQ ID NO.5所示，凋亡蛋白为细胞色素C串联集合体(nCytc，n≥1)或DNA片段化因子40(DFF40)。In order to achieve the above technical purpose, the technical solution adopted in the present invention is as follows: an apoptotic protein fusion type anti-HER-2 single-chain antibody, which is formed by coupling the anti-HER-2 single-chain antibody to apoptotic protein in different tandem manners, The anti-HER-2 single-chain antibody includes a heavy chain variable region VH whose sequence is shown in SEQ ID NO.1 and a light chain variable region VL whose sequence is shown in SEQ ID NO.2, a heavy chain variable region and a light chain The variable regions are connected by a flexible peptide (G 4 S) 3 whose sequence is shown in SEQ ID NO. 3, the VH upstream of the heavy chain variable region contains 6×his tags, and the amino acid sequence of the anti-HER-2 single-chain antibody is shown in SEQ ID As shown in NO.4, the cDNA sequence encoding anti-HER-2 single-chain antibody is shown in SEQ ID NO.5, and the apoptotic protein is cytochrome C tandem aggregate (nCytc, n≥1) or DNA fragmentation factor 40 ( DFF40).

进一步地，所述细胞色素C的氨基酸序列如SEQ ID NO.6所示，编码所述细胞色素C的cDNA序列如SEQ ID NO.7所示。Further, the amino acid sequence of the cytochrome C is shown in SEQ ID NO.6, and the cDNA sequence encoding the cytochrome C is shown in SEQ ID NO.7.

进一步地，所述DNA片段化因子40的氨基酸序列如SEQ ID NO.8所示，编码所述DNA片段化因子40的cDNA序列如SEQ ID NO.9所示。Further, the amino acid sequence of the DNA fragmentation factor 40 is shown in SEQ ID NO.8, and the cDNA sequence encoding the DNA fragmentation factor 40 is shown in SEQ ID NO.9.

进一步地，所述凋亡蛋白融合型抗HER-2单链抗体为Cytc-scFv融合型单链抗体，由所述的抗HER-2单链抗体与一个权利要求3所述的细胞色素C偶联而成，所述细胞色素C连接在6×his标签与抗HER-2单链抗体之间，所述Cytc-scFv融合型单链抗体的氨基酸序列如SEQ ID NO.10所示。Further, the apoptotic protein fusion-type anti-HER-2 single-chain antibody is a Cytc-scFv fusion-type single-chain antibody, and the anti-HER-2 single-chain antibody is coupled with a cytochrome C according to claim 3. The cytochrome C is linked between the 6×his tag and the anti-HER-2 single-chain antibody, and the amino acid sequence of the Cytc-scFv fusion single-chain antibody is shown in SEQ ID NO.10.

进一步地，所述凋亡蛋白融合型抗HER-2单链抗体为nCytc-scFv融合型单链抗体，由所述的抗HER-2单链抗体与细胞色素C串联集合体偶联而成，且细胞色素C串联集合体串联在6×his标签与抗HER-2单链抗体之间，细胞色素C串联集合体由若干个细胞色素C通过连接肽连接，所述连接肽为Caspase-3酶切位点DEVD，所述DEVD的cDNA序列如SEQ ID NO.11所示。Further, the apoptotic protein fusion type anti-HER-2 single-chain antibody is an nCytc-scFv fusion type single-chain antibody, which is formed by coupling the anti-HER-2 single-chain antibody and the cytochrome c tandem aggregate, And the cytochrome C tandem aggregate is connected in series between the 6×his tag and the anti-HER-2 single-chain antibody, and the cytochrome C tandem aggregate is connected by several cytochrome C through a connecting peptide, and the connecting peptide is Caspase-3 enzyme. Cut site DEVD, the cDNA sequence of DEVD is shown in SEQ ID NO.11.

进一步地，所述凋亡蛋白融合型抗HER-2单链抗体为DFF40-scFv融合型单链抗体，由所述的抗HER-2单链抗体与权利要求4所述的DNA片段化因子40连接而成，所述DNA片段化因子40连接在6×his标签与抗HER-2单链抗体之间，所述DFF40-scFv融合型单链抗体的氨基酸序列如SEQ ID NO.12所示。Further, the apoptotic protein fusion-type anti-HER-2 single-chain antibody is a DFF40-scFv fusion-type single-chain antibody, which is composed of the anti-HER-2 single-chain antibody and the DNA fragmentation factor 40 according to claim 4. The DNA fragmentation factor 40 is connected between the 6×his tag and the anti-HER-2 single-chain antibody, and the amino acid sequence of the DFF40-scFv fusion single-chain antibody is shown in SEQ ID NO.12.

本发明还提供了上述凋亡蛋白融合型抗HER-2单链抗体的制备方法，包括如下步骤：The present invention also provides a method for preparing the apoptotic protein fusion type anti-HER-2 single-chain antibody, comprising the following steps:

(1)合成如SEQ ID NO.5所示的编码抗HER-2单链抗体的cDNA序列和编码凋亡蛋白的cDNA序列；(1) Synthesize the cDNA sequence encoding anti-HER-2 single-chain antibody and the cDNA sequence encoding apoptotic protein as shown in SEQ ID NO.5;

(2)构建重组质粒：将步骤(1)所得的编码抗HER-2单链抗体的cDNA序列和编码凋亡蛋白的cDNA序列分别连入pET-32a(+)表达载体中，构建成重组质粒；(2) Construction of recombinant plasmid: The cDNA sequence encoding anti-HER-2 single-chain antibody and the cDNA sequence encoding apoptotic protein obtained in step (1) were respectively linked into the pET-32a(+) expression vector to construct a recombinant plasmid ;

(3)将步骤(2)所得重组质粒转化DH5α感受态细胞，筛选测序正确的重组质粒转化BL21(DE3)感受态细胞中，诱导表达凋亡蛋白融合型抗HER-2单链抗体。(3) Transform the recombinant plasmid obtained in step (2) into DH5α competent cells, screen and transform the recombinant plasmid with correct sequencing into BL21 (DE3) competent cells, and induce the expression of apoptotic protein fusion anti-HER-2 single chain antibody.

进一步地，步骤(2)中，采用限制性内切酶NcoⅠ和BamHⅠ酶切如SEQ ID NO.5所示的抗HER-2单链抗体的cDNA片段与pET-32a(+)表达载体的cDNA片段，然后将抗HER-2单链抗体的cDNA片段连接在pET-32a(+)表达载体的NcoⅠ和BamHⅠ酶切位点之间；凋亡蛋白为细胞色素C或DNA片段化因子40，采用限制性内切酶BglⅡ和NcoⅠ酶切pET-32a(+)表达载体的cDNA片段与如SEQ ID NO.7所示的Cytc的cDNA序列或如SEQ ID NO.9所示的DFF40的cDNA序列，然后将Cytc的cDNA序列或DFF40的cDNA序列连接在pET-32a(+)表达载体的BglⅡ和NcoⅠ酶切位点之间，其中，插入编码抗HER-2单链抗体的cDNA片段与插入编码凋亡蛋白的cDNA片段时在pET-32a(+)表达载体上采用的NcoⅠ酶切位点为同一酶切位点。Further, in step (2), the cDNA fragment of the anti-HER-2 single-chain antibody shown in SEQ ID NO.5 and the cDNA of the pET-32a(+) expression vector were digested with restriction enzymes NcoI and BamHI. The cDNA fragment of anti-HER-2 single-chain antibody was then ligated between the NcoI and BamHI restriction sites of the pET-32a(+) expression vector; the apoptotic protein was cytochrome C or DNA fragmentation factor 40, using Restriction endonucleases BglII and NcoI digested the cDNA fragment of the pET-32a(+) expression vector and the cDNA sequence of Cytc as shown in SEQ ID NO.7 or the cDNA sequence of DFF40 as shown in SEQ ID NO.9, Then the cDNA sequence of Cytc or the cDNA sequence of DFF40 was ligated between the BglII and NcoI restriction sites of the pET-32a(+) expression vector. The NcoI restriction site used in the pET-32a(+) expression vector was the same restriction site for the cDNA fragment of apoptotic protein.

进一步地，当凋亡蛋白为细胞色素C且细胞色素C的个数大于一个时，各个细胞色素C之间通过如SEQ ID NO.11所示的Caspase-3酶切位点DEVD串联连接。Further, when the apoptotic protein is cytochrome C and the number of cytochrome C is more than one, each cytochrome C is connected in series through the Caspase-3 enzyme cleavage site DEVD shown in SEQ ID NO.11.

进一步地，还包括步骤(4)采用Ni-NTA柱纯化步骤(4)所得的凋亡蛋白融合型抗HER-2单链抗体。Further, it also includes step (4) using Ni-NTA column to purify the apoptotic protein fusion type anti-HER-2 single-chain antibody obtained in step (4).

本发明还提供了上述抗HER-2单链抗体或凋亡蛋白融合型抗HER-2单链抗体在制备靶向治疗HER-2高表达癌症的药物中的应用。The present invention also provides the application of the above anti-HER-2 single-chain antibody or apoptotic protein fusion anti-HER-2 single-chain antibody in the preparation of a drug for targeted treatment of cancers with high HER-2 expression.

与现有技术相比，本发明的有益效果在于：在临床前研究中，许多重组免疫毒素与肿瘤细胞相关的不同细胞表面抗原相结合，通过毒素介导的翻译抑制作用导致细胞死亡，是有效的抗癌药物。但是，限制这些分子在临床上治疗实体癌的有效性的原因主要包括低特异性，高毒性。免疫毒素中的毒素分子主要是植物或细菌毒素，例如假单胞菌外毒素、金黄色葡萄球菌肠毒素、白喉毒素、海葵溶细胞素、白树素等等，具有可能导致胃肠道毒性和肝肾毒性等副作用，此外它们分子量大、渗透效率低，免疫原性高，半衰期短等。本发明以HER-2高表达型乳腺癌表面HER-2抗原为分子靶标，利用单链抗体的高特异性和低免疫原性等优点，以抗HER-2单链抗体scFv为药物递送平台，使用内源性细胞凋亡相关分子来替代外源性毒素，通过激活内源性凋亡途径来避免毒副作用的产生；将单链抗体的靶向性与凋亡蛋白的促凋亡活性相结合，设计构建三种凋亡蛋白融合型单链抗体：DFF40-scFv、Cytc-scFv以及nCytc-scFv(n>1，以实施例n＝3为例)，体内外实验均证明凋亡蛋白融合型单链抗体能够特异性靶向HER-2过表达乳腺癌并介导细胞凋亡，而对HER-2阴性乳腺癌细胞没有毒性，且三种融合型单链抗体的活性差异为DFF40-scFv>3Cytc-scFv>Cytc-scFv。据我们所知，这是将凋亡蛋白DFF40与单链抗体结合用于治疗HER-2阳性乳腺癌的第一项研究，更重要的是，引入凋亡执行蛋白Caspase3的特异性切割位点DEVD的凋亡蛋白融合型单链抗体蛋白构建体(nCytc-scFv)，通过酶切反应释放更多的效应分子，提高了抗肿瘤活性，是对通过内源性凋亡途径治疗肿瘤的策略的重要补充。nCytc-scFv作为本研究首次发现的较理想的一种免疫凋亡分子，其包含一个巧妙的良性循环，在扩大抗肿瘤活性的同时将肿瘤杀伤限制于细胞凋亡，符合高效低毒的治疗理念，是更新颖和有效的治疗途径。总之，本发明将促凋亡蛋白与单链抗体融合表达用于癌症的治疗可实现靶向性与抗肿瘤活性的良好结合，为癌症的靶向免疫治疗及抗体药物的研发提供新的思路。Compared with the prior art, the beneficial effect of the present invention is: in preclinical research, many recombinant immunotoxins are combined with different cell surface antigens related to tumor cells, and lead to cell death through toxin-mediated translation inhibition, which is effective. of anticancer drugs. However, the reasons that limit the effectiveness of these molecules in the clinical treatment of solid cancer mainly include low specificity and high toxicity. The toxin molecules in immunotoxins are mainly plant or bacterial toxins, such as Pseudomonas exotoxin, Staphylococcus aureus enterotoxin, diphtheria toxin, sea anemone cytolysin, leucanthin, etc., which may cause gastrointestinal toxicity and liver toxicity. Nephrotoxicity and other side effects, in addition they have large molecular weight, low penetration efficiency, high immunogenicity, short half-life, etc. The invention uses the HER-2 antigen on the surface of breast cancer with high HER-2 expression as the molecular target, utilizes the advantages of high specificity and low immunogenicity of the single-chain antibody, and uses the anti-HER-2 single-chain antibody scFv as the drug delivery platform. Use endogenous apoptosis-related molecules to replace exogenous toxins and avoid toxic side effects by activating endogenous apoptotic pathways; combine the targeting of single-chain antibodies with the pro-apoptotic activity of apoptotic proteins , designed and constructed three apoptotic protein fusion-type single-chain antibodies: DFF40-scFv, Cytc-scFv and nCytc-scFv (n>1, taking Example n=3 as an example). Single-chain antibodies can specifically target HER-2-overexpressing breast cancer and mediate apoptosis without being toxic to HER-2-negative breast cancer cells, and the difference in activity of the three fusion single-chain antibodies is DFF40-scFv> 3Cytc-scFv>Cytc-scFv. To the best of our knowledge, this is the first study to combine the apoptosis protein DFF40 with a single-chain antibody for the treatment of HER-2-positive breast cancer and, more importantly, to introduce the specific cleavage site DEVD of the apoptosis-executing protein Caspase3 The apoptotic protein fusion-type single-chain antibody protein construct (nCytc-scFv) releases more effector molecules through enzymatic cleavage reaction and improves the anti-tumor activity, which is an important strategy for the treatment of tumors through the endogenous apoptosis pathway. Replenish. As an ideal immune apoptosis molecule discovered for the first time in this study, nCytc-scFv contains an ingenious virtuous circle that expands anti-tumor activity while limiting tumor killing to apoptosis, which is in line with the therapeutic concept of high efficiency and low toxicity , is a more novel and effective treatment approach. In conclusion, the fusion expression of pro-apoptotic protein and single-chain antibody in the present invention for cancer treatment can achieve a good combination of targeting and anti-tumor activity, and provide new ideas for cancer-targeted immunotherapy and antibody drug development.

附图说明Description of drawings

图1为单链抗体蛋白二维结构示意图；Figure 1 is a schematic diagram of the two-dimensional structure of a single-chain antibody protein;

图2为实施例1和实施例2构建的重组质粒pET-32a(+)-scFv、pET-32a(+)-DFF40-scFv、pET-32a(+)-Cytc-scFv和pET-32a(+)-3Cytc-scFv的质粒图谱图；Figure 2 shows the recombinant plasmids pET-32a(+)-scFv, pET-32a(+)-DFF40-scFv, pET-32a(+)-Cytc-scFv and pET-32a(+) constructed in Example 1 and Example 2 )-3Cytc-scFv plasmid map;

图3是实施例3的scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv蛋白表达纯化鉴定结果，其中：A为纯化的scFv、DFF40-scFv、Cytc–scFv和3Cytc–scFv蛋白的SDS-PAGE凝胶电泳结果，M：蛋白Marker；B为纯化的scFv、DFF40-scFv、Cytc–scFv和3Cytc–scFv蛋白的WesternBlot鉴定结果图，一抗使用anti-6His tag，且B的Marker及泳道设置与A所用相同；Figure 3 shows the results of expression and purification of scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv proteins of Example 3, wherein: A is the SDS-PAGE of purified scFv, DFF40-scFv, Cytc-scFv and 3Cytc-scFv proteins Gel electrophoresis results, M: Protein Marker; B is the WesternBlot identification result of purified scFv, DFF40-scFv, Cytc–scFv and 3Cytc–scFv proteins, the primary antibody uses anti-6His tag, and the Marker and lane settings of B are the same as A used the same;

图4为实施例4的四种单链抗体蛋白(scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv)分别与SK-BR-3、MDA-MB-231、MCF-7共孵育后的免疫荧光图像，其中，绿色为FITC荧光二抗标记的单链抗体蛋白(图中亮色外边)，蓝色为DAPI染核，比例尺＝25μm；Figure 4 shows the immunity of the four single-chain antibody proteins (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) of Example 4 after co-incubating with SK-BR-3, MDA-MB-231 and MCF-7, respectively Fluorescence images, in which the green is the single-chain antibody protein labeled with FITC fluorescent secondary antibody (outside the bright color in the figure), and the blue is DAPI-stained nuclei, scale bar = 25 μm;

图5为实施例5的四种单链抗体蛋白(scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv)对HER-2过表达型乳腺癌细胞活力检测结果，其中：A为不同浓度的单链抗体蛋白对SK-BR-3的细胞毒性数据，B为不同浓度的单链抗体蛋白对MDA-MB-231的细胞毒性数据，C为不同浓度的单链抗体蛋白对MCF-7的细胞毒性数据,n＝3，与对照组相比，*P<0.05,**P<0.01,***p<0.001,ns：不显著；D为四种单链抗体蛋白(scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv)分别与MDA-MB-231及DFF40-scFv分别与MDA-MB-231和MCF-7共孵育48h后通过结晶紫染色结果；Figure 5 shows the results of the four single-chain antibody proteins (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) of Example 5 for the detection of the viability of HER-2 overexpressing breast cancer cells, wherein: A is the single-chain antibody of different concentrations Cytotoxicity data of single-chain antibody protein to SK-BR-3, B is the cytotoxicity data of single-chain antibody protein of different concentrations to MDA-MB-231, C is the cytotoxicity of single-chain antibody protein of different concentration to MCF-7 Data, n=3, compared with control group, *P<0.05, **P<0.01, ***p<0.001, ns: not significant; D is four single chain antibody proteins (scFv, Cytc–scFv, 3Cytc–scFv and DFF40-scFv) and MDA-MB-231 and DFF40-scFv were incubated with MDA-MB-231 and MCF-7 for 48h, respectively, and the results were obtained by crystal violet staining;

图6为实施例6的scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv融合型单链抗体对HER-2过表达型乳腺癌细胞诱导后Caspase3蛋白活性检测结果，其中：A为SK-BR-3分别与四种抗体蛋白共同作用后Caspase3活性检测结果，B和C分别为MDA-MB-231和MCF-7分别与四种抗体蛋白共同作用后Caspase3活性检测结果，n＝5，*P<0.05,**P<0.01,***p<0.001,ns：不显著；Fig. 6 is the detection result of Caspase3 protein activity after induction of scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv fusion single-chain antibody of Example 6 to HER-2 overexpressing breast cancer cells, wherein: A is SK-BR The detection results of Caspase3 activity after the interaction of -3 with the four antibody proteins respectively, B and C are the detection results of the Caspase3 activity after the interaction of MDA-MB-231 and MCF-7 with the four antibody proteins respectively, n=5, *P <0.05, **P<0.01, ***p<0.001, ns: not significant;

图7为实施例7的免疫印迹法分析凋亡相关蛋白表达量变化结果图，其中：A为四种单链抗体蛋白(scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv)作用于SK-BR-3细胞后，提取总蛋白，Bcl-2和Bax的含量变化检测结果，α-tubulin作为内参，B为A中蛋白条带的ImageLab定量结果，各柱形图中下段代表Bcl-2，上段代表Bax，两者总量为1；Figure 7 is a graph showing the changes in the expression of apoptosis-related proteins analyzed by immunoblotting in Example 7, wherein: A is the effect of four single-chain antibody proteins (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) on SK- After BR-3 cells, the total protein was extracted, and the content of Bcl-2 and Bax was detected. α-tubulin was used as an internal reference, and B was the ImageLab quantitative result of the protein band in A. The lower part of each bar represents Bcl-2, The upper segment represents Bax, and the total of the two is 1;

图8为实施例8的Hoechst核染色及细胞形态变化结果图，其中：A为四种单链抗体蛋白(scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv)分别与SK-BR-3细胞共孵育48h后使用倒置荧光显微镜观察细胞核固缩情况结果图，蓝色：Hoechst；白色：固缩的核(图中亮白色部分)；B为活细胞长时间动态影像成像分析仪观测的四种单链抗体蛋白(scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv)分别与SK-BR-3细胞共孵育0h、24h、48h后的细胞图像；Figure 8 is the result of Hoechst nuclear staining and cell morphological changes in Example 8, wherein: A is the four single-chain antibody proteins (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) and SK-BR-3 cells respectively After co-incubating for 48 hours, the results of nuclear pyknosis were observed using an inverted fluorescence microscope, blue: Hoechst; white: pyknotic nuclei (the bright white part in the figure); B is the four kinds of pyknotic cells observed by the long-term dynamic image analyzer of living cells Cell images of single-chain antibody proteins (scFv, Cytc–scFv, 3Cytc–scFv and DFF40-scFv) incubated with SK-BR-3 cells for 0h, 24h, and 48h, respectively;

图9为实施例8的四种单链抗体蛋白(scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv)与三种乳腺癌细胞(SK-BR-3、MDA-MB-231、MCF-7)共孵育48h后的细胞凋亡率统计结果，其中，n＝3，与对照组(MDA-MB-231、MCF-7)相比，*P<0.05,**P<0.01,***p<0.001,ns：不显著；Figure 9 shows the four single-chain antibody proteins of Example 8 (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) and three breast cancer cells (SK-BR-3, MDA-MB-231, MCF-7 ) Statistical results of apoptosis rate after co-incubation for 48h, where n=3, compared with the control group (MDA-MB-231, MCF-7), *P<0.05, **P<0.01, *** p<0.001, ns: not significant;

图10为实施例9的流式细胞术检测融合蛋白诱导的细胞凋亡实验图谱，其中：A为四种单链抗体蛋白(scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv)分别与SK-BR-3细胞共孵育48h，Annexin V-FITC/PI双染后避光孵育10min后立即用流式细胞仪检测的细胞凋亡结果，B～D：对于A中的凋亡数据的量化分析，n＝3，以scFv作用组为对照，*P<0.05,**P<0.01,***p<0.001，ns：不显著；Figure 10 is the experimental map of the apoptosis induced by the fusion protein detected by flow cytometry in Example 9, wherein: A is the four single-chain antibody proteins (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) and SK respectively - BR-3 cells were co-incubated for 48h, double-stained with Annexin V-FITC/PI, and incubated in the dark for 10min. The results of apoptosis were detected by flow cytometry immediately. B～D: Quantitative analysis of the apoptosis data in A , n=3, taking the scFv group as the control, *P<0.05, **P<0.01, ***p<0.001, ns: not significant;

图11为实施例10的凋亡蛋白融合型抗HER-2单链抗体的体内活性验证结果，其中：A为四种单链抗体蛋白(scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv)对裸鼠异种移植模型进行蛋白药物治疗后30天内肿瘤体积变化图，每3天测量一次并计算肿瘤体积，B为抗体蛋白药物治疗结束后，从scFv治疗组、Cytc–scFv治疗组、3Cytc–scFv治疗组及DFF40-scFv治疗组的裸鼠异种移植模型中分离出的肿瘤组织拍摄图像；C为抗体蛋白药物治疗结束后，从scFv治疗组、Cytc–scFv治疗组、3Cytc–scFv治疗组及DFF40-scFv治疗组的裸鼠异种移植模型中分离出的肿瘤组织质量结果，以scFv治疗组为对照，*P<0.05,**P<0.01,***p<0.001，ns：不显著。Figure 11 is the in vivo activity verification result of the apoptosis protein fusion anti-HER-2 single-chain antibody of Example 10, wherein: A is four single-chain antibody proteins (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) Tumor volume changes in nude mice xenograft model within 30 days after protein drug treatment, measured every 3 days and calculated tumor volume, B is after antibody protein drug treatment, from scFv treatment group, Cytc–scFv treatment group, 3Cytc– The images of tumor tissues isolated from the nude mouse xenograft models of scFv treatment group and DFF40-scFv treatment group; C is after antibody protein drug treatment, from scFv treatment group, Cytc-scFv treatment group, 3Cytc-scFv treatment group and Results of tumor tissue quality isolated from nude mouse xenograft model in DFF40-scFv treatment group, taking scFv treatment group as control, *P<0.05, **P<0.01, ***p<0.001, ns: not significant.

图12为实施例10的肿瘤组织石蜡切片Tunel染色结果，其中，凋亡细胞显示为深棕色(图中深色点)。Figure 12 shows the results of Tunel staining of the paraffin section of the tumor tissue in Example 10, wherein the apoptotic cells are shown as dark brown (dark dots in the figure).

具体实施方式Detailed ways

为了使本领域技术领域人员更好地理解本发明的技术方案，下面通过具体实施例对本发明作进一步说明。In order to make those skilled in the art better understand the technical solutions of the present invention, the present invention will be further described below through specific embodiments.

下述实施例中所使用的实验方法，如无特殊说明，均为常规方法，所用的试剂、方法和设备，如无特殊说明，均为本技术领域常规试剂、方法和设备。The experimental methods used in the following examples, unless otherwise specified, are conventional methods, and the used reagents, methods and equipment, unless otherwise specified, are conventional reagents, methods and equipment in the technical field.

实施例1：pET-32a(+)-scFv重组质粒的构建Example 1: Construction of pET-32a(+)-scFv recombinant plasmid

一、抗HER-2单链抗体scFv同源重组基因片段的获得First, the acquisition of anti-HER-2 single chain antibody scFv homologous recombination gene fragment

1.1重链可变区VH、轻链可变区VL和柔性肽(G4S)3序列的获取1.1 Acquisition of heavy chain variable region VH, light chain variable region VL and flexible peptide (G 4 S) 3 sequences

通过NCBI和Uniprot数据库查找及文献检索比对曲妥珠单抗单链抗体序列以及linker序列，确定如SEQ ID NO.1所示的重链可变区VH的氨基酸序列、如SEQ ID NO.2所示的轻链可变区VL的氨基酸序列以及如SEQ ID NO.3所示的柔性肽(G4S)3的氨基酸序列；The trastuzumab single-chain antibody sequence and linker sequence were compared through NCBI and Uniprot database search and literature search to determine the amino acid sequence of the heavy chain variable region VH shown in SEQ ID NO.1, as shown in SEQ ID NO.2 The amino acid sequence of the light chain variable region VL shown and the amino acid sequence of the flexible peptide (G 4 S) 3 shown in SEQ ID NO.3;

重链可变区VH序列(SEQ ID NO.1)：EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS；Heavy chain variable region VH sequence (SEQ ID NO. 1): EVQLVESGGGLVQPGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS;

轻链可变区VL序列(SEQ ID NO.2)：DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR；Light chain variable region VL sequence (SEQ ID NO. 2): DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR;

柔性肽(G4S)3序列(SEQ ID NO.3)：GGGGSGGGGSGGGGS；Flexible peptide (G 4 S) 3 sequence (SEQ ID NO. 3): GGGGSGGGGSGGGGS;

抗HER-2单链抗体的氨基酸序列(VH-linker-VL)如SEQ ID NO.4所示(抗HER-2单链抗体scFv的二维结构如图1所示，在上述重链可变区VH上游含有6×his标签用于镍柱纯化)，编码所述抗HER-2单链抗体scFv的cDNA序列如SEQ ID NO.5所示；The amino acid sequence (VH-linker-VL) of anti-HER-2 single-chain antibody is shown in SEQ ID NO. The upstream VH region contains 6×his tags for nickel column purification), and the cDNA sequence encoding the anti-HER-2 single-chain antibody scFv is shown in SEQ ID NO.5;

SEQ ID NO.4具体为：EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSG GGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR，下划线部分为柔性肽(G4S)3序列；SEQ ID NO.4具体为：EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS GGGGSG GGGSGGGGS DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR，下划线部分为柔性肽(G 4 S) 3序列；

SEQ ID NO.5具体为：5'-GAAGTTCAGCTGGTTGAATCTGGTGGTGGTCTGGTTCAGCCGGGTGGTTCTCTGCGTCTGTCTTGCGCTGCTTCTGGTTTCAACATCAAAGACACCTACATCCACTGGGTTCGTCAGGCTCCGGGTAAAGGTCTGGAATGGGTTGCTCGTATCTACCCGACCAACGGTTACACCCGTTACGCTGACTCTGTTAAAGGTCGTTTCACCATCTCTGCTGACACCTCTAAAAACACCGCTTACCTGCAGATGAACTCTCTGCGTGCTGAAGACACCGCTGTTTACTACTGCTCTCGTTGGGGTGGTGACGGTTTCTACGCTATGGACTACTGGGGTCAGGGTACCCTGGTTACCGTTTCTTCTGGGGGCGGGGGCTCTGGGGGCGGGGGCTCTGGGGGCGGGGGCTCTGATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGAGCGTGGGCGATCGCGTGACCATTACCTGCCGCGCGAGCCAGGATGTGAACACCGCGGTGGCGTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTATAGCGCGAGCTTTCTGTATAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCCGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCGACCTATTATTGCCAGCAGCATTATACCACCCCGCCGACCTTTGGCCAGGGCACCAAAGTGGAAATTAAACGC-3'；SEQ ID NO.5具体为：5'-GAAGTTCAGCTGGTTGAATCTGGTGGTGGTCTGGTTCAGCCGGGTGGTTCTCTGCGTCTGTCTTGCGCTGCTTCTGGTTTCAACATCAAAGACACCTACATCCACTGGGTTCGTCAGGCTCCGGGTAAAGGTCTGGAATGGGTTGCTCGTATCTACCCGACCAACGGTTACACCCGTTACGCTGACTCTGTTAAAGGTCGTTTCACCATCTCTGCTGACACCTCTAAAAACACCGCTTACCTGCAGATGAACTCTCTGCGTGCTGAAGACACCGCTGTTTACTACTGCTCTCGTTGGGGTGGTGACGGTTTCTACGCTATGGACTACTGGGGTCAGGGTACCCTGGTTACCGTTTCTTCTGGGGGCGGGGGCTCTGGGGGCGGGGGCTCTGGGGGCGGGGGCTCTGATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGAGCGTGGGCGATCGCGTGACCATTACCTGCCGCGCGAGCCAGGATGTGAACACCGCGGTGGCGTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTATAGCGCGAGCTTTCTGTATAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCCGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCGACCTATTATTGCCAGCAGCATTATACCACCCCGCCGACCTTTGGCCAGGGCACCAAAGTGGAAATTAAACGC-3'；

1.2聚合酶链式反应(PCR)获取用于同源重组的scFv基因1.2 Polymerase chain reaction (PCR) to obtain scFv gene for homologous recombination

根据scFv的cDNA序列以及pET-32a(+)载体上的序列，设计并合成与pET-32a(+)载体同源重组的scFv基因序列的PCR引物scFv-F(如SEQ ID NO.14所示)和scFv-R(如SEQ IDNO.15所示)，通过化学合成方法合成含有酶切位点NcoⅠ和BamHⅠ及补充碱基的核苷酸片段，随后通过引物scFv-F和scFv-R扩增scFv同源重组序列According to the cDNA sequence of scFv and the sequence on the pET-32a(+) vector, the PCR primer scFv-F (as shown in SEQ ID NO. 14) of the scFv gene sequence homologous recombination with the pET-32a(+) vector was designed and synthesized ) and scFv-R (as shown in SEQ ID NO. 15), nucleotide fragments containing enzyme cleavage sites NcoI and BamHI and supplementary bases were synthesized by chemical synthesis, and then amplified by primers scFv-F and scFv-R scFv homologous recombination sequence

scFv-F(SEQ ID NO.14)：5’-CGACGACGACGACAAGGCCATGGCTGAAGTTCAGCTGGTTGAATCTGG-3’，其中，下划线处表示pET-32a(+)载体同源序列，斜体处表示酶切位点NcoⅠ；scFv-F (SEQ ID NO. 14): 5'- CGACGACGACGACAAGG CCATGGCTGAAGTTCAGCTGGTTGAATCTGG-3', wherein the underline represents the homologous sequence of the pET-32a(+) vector, and the italics represents the enzyme cleavage site Nco I;

scFv-R(SEQ ID NO.15)：5’-CGACGGAGCTCGAATTCGGATCCTTAGCGTTTAATTTCCACTTTGGTGC-3’，其中，下划线处表示pET-32a(+)载体同源序列，斜体处表示酶切位点BamHⅠ；scFv-R (SEQ ID NO. 15): 5'- CGACGGAGCTCGAATTC GGATCCTTAGCGTTTAATTTCCACTTTGGTGC -3', wherein the underline represents the homologous sequence of the pET-32a(+) vector, and the italics represents the enzyme cleavage site BamHI;

Figure BDA0002585061130000081
Figure BDA0002585061130000081

PCR产物即为两端含有载体同源序列及酶切位点的单链抗体scFv基因片断，用于与酶切后的pET-32a(+)载体进行同源重组。The PCR product is a single-chain antibody scFv gene fragment containing vector homologous sequences and restriction sites at both ends, and is used for homologous recombination with the digested pET-32a(+) vector.

二、pET-32a(+)-scFv重组质粒的构建2. Construction of pET-32a(+)-scFv recombinant plasmid

将胶回收的scFv同源重组基因片段和双酶切(NcoⅠ和BamHⅠ)后的pET-32a(+)质粒通过同源重组进行连接，将连接产物转化到E.coli DH5α，并在菌液扩大培养后提取重组质粒pET-32a(+)-scFv送上海生工生物工程有限公司进行测序，结果显示，阳性重组质粒pET-32a(+)-scFv中插入的序列与实验设计一致，从而成功构建得到重组质粒pET-32a(+)-scFv(如图2A所示)。The scFv homologous recombination gene fragment recovered from the gel and the pET-32a(+) plasmid after double digestion (NcoI and BamHI) were connected by homologous recombination, and the ligation product was transformed into E.coli DH5α and expanded in bacterial liquid After culturing, the recombinant plasmid pET-32a(+)-scFv was extracted and sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing. The results showed that the sequence inserted in the positive recombinant plasmid pET-32a(+)-scFv was consistent with the experimental design, so the construction was successful. The recombinant plasmid pET-32a(+)-scFv was obtained (as shown in Fig. 2A).

实施例2：pET-32a(+)-Cytc-scFv、pET-32a(+)-3Cytc-scFv和pET-32a(+)-DFF40-scFv重组质粒的构建Example 2: Construction of pET-32a(+)-Cytc-scFv, pET-32a(+)-3Cytc-scFv and pET-32a(+)-DFF40-scFv recombinant plasmids

一、实验材料1. Experimental materials

1、PCR所需引物1. Primers required for PCR

通过NCBI和Uniprot数据库查找确定如SEQ ID NO.6所示的细胞色素C(Cytc)氨基酸序列和如SEQ ID NO.7所示的编码所述细胞色素C的cDNA序列；通过NCBI和Uniprot数据库查找确定如SEQ ID NO.8所示的DNA片段化因子40(DFF40)氨基酸序列和如SEQ ID NO.9所示的编码所述DFF40的cDNA序列；根据Cytc和DFF40的cDNA序列以及pET-32a(+)载体上的序列，设计并合成扩增上述Cytc的用于与pET-32a(+)载体同源重组的基因序列的PCR引物Cytc-F和Cytc-R；以及扩增DFF40的用于与载体同源重组的基因序列的PCR引物DFF40-F和DFF40-R，具体序列信息为：The amino acid sequence of cytochrome C (Cytc) shown in SEQ ID NO. 6 and the cDNA sequence encoding the cytochrome C shown in SEQ ID NO. 7 were determined by searching through NCBI and Uniprot databases; searched through NCBI and Uniprot databases Determine the DNA fragmentation factor 40 (DFF40) amino acid sequence shown in SEQ ID NO.8 and the cDNA sequence encoding the DFF40 shown in SEQ ID NO.9; according to the cDNA sequences of Cytc and DFF40 and pET-32a ( +) sequence on the vector, design and synthesize PCR primers Cytc-F and Cytc-R for amplifying the gene sequence of the above Cytc for homologous recombination with the pET-32a(+) vector; The PCR primers DFF40-F and DFF40-R of the gene sequence of the vector homologous recombination, the specific sequence information is:

细胞色素C的氨基酸序列(SEQ ID NO.6)：MGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNE；Amino acid sequence of cytochrome C (SEQ ID NO. 6): MGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNE;

细胞色素C的cDNA序列(SEQ ID NO.7)：5'-AGATCTCATGGGTGATGTTGAGAAAGGCAAGAAGATTTTTATTATGAAGTGTTCCCAGTGCCACACCGTTGAAAAGGGAGGCAAGCACAAGACTGGGCCAAATCTCCACGGTCTCTTTGGGCGGAAGACAGGTCAGGCCCCTGGATACTCTTACACAGCCGCCAATAAGAACAAAGGCATCATCTGGGGAGAGGATACACTGATGGAGTATTTGGAGAATCCCAAGAAGTACATCCCTGGAACAAAAATGATCTTTGTCGGCATTAAGAAGAAGGAAGAAAGGGCAGACTTAATAGCTTATCTCAAAAAAGCTACTAATGAGACCATGG-3'；细胞色素C的cDNA序列(SEQ ID NO.7)：5'-AGATCTCATGGGTGATGTTGAGAAAGGCAAGAAGATTTTTATTATGAAGTGTTCCCAGTGCCACACCGTTGAAAAGGGAGGCAAGCACAAGACTGGGCCAAATCTCCACGGTCTCTTTGGGCGGAAGACAGGTCAGGCCCCTGGATACTCTTACACAGCCGCCAATAAGAACAAAGGCATCATCTGGGGAGAGGATACACTGATGGAGTATTTGGAGAATCCCAAGAAGTACATCCCTGGAACAAAAATGATCTTTGTCGGCATTAAGAAGAAGGAAGAAAGGGCAGACTTAATAGCTTATCTCAAAAAAGCTACTAATGAGACCATGG-3'；

扩增Cytc同源重组序列的正向引物Cytc-F序列(SEQ ID NO.16)：5'-CCAGCACATG GACAGCCCAGATCTCATGGGTGATGTTGAGAAAGGC-3'，其中，下划线处表示pET-32a(+)载体同源序列，斜体处表示酶切位点BglⅡ；The forward primer Cytc-F sequence (SEQ ID NO. 16) for amplifying the Cytc homologous recombination sequence: 5'- CCAGCACATG GACAGCCC AGATCTCATGGGTGATGTTGAGAAAGGC-3', wherein the underline represents the pET-32a(+) vector homologous sequence, italics where represents the restriction enzyme cleavage site BglII;

扩增Cytc同源重组序列的反向引物Cytc-R序列(SEQ ID NO.17)：5'-CAACCAGCTG AACTTCAGCCATGGTCTCATTAGTAGCTTTTTTGAGATAAGC-3'，其中，下划线处表示pET-32a(+)载体同源序列，斜体处表示酶切位点NcoⅠ；The reverse primer Cytc-R sequence (SEQ ID NO. 17) for amplifying the Cytc homologous recombination sequence: 5'- CAACCAGCTG AACTTCAG CCATGGTCTCATTAGTAGCTTTTTTGAGATAAGC-3', wherein, the underline represents the pET-32a(+) vector homologous sequence, italics where represents the enzyme cleavage site Nco I;

DFF40的氨基酸序列(SEQ ID NO.8)：MLQKPKSVKLRALRSPRKFGVAGRSCQEVLRKGCLRFQLPERGSRLCLYEDGTELTEDYFPSVPDNAELVLLTLGQAWQGYVSDIRRFLSAFHEPQVGLIQAAQQLLCDEQAPQRQRLLADLLHNVSQNIAAETRAEDPPWFEGLESRFQSKSGYLRYSCESRIRSYLREVSSYPSTVGAEAQEEFLRVLGSMCQRLRSMQYNGSYFDRGAKGGSRLCTPEGWFSCQGPFDMDSCLSRHSINPYSNRESRILFSTWNLDHIIEKKRTIIPTLVEAIKEQDGREVDWEYFYGLLFTSENLKLVHIVCHKKT THKLNCDPSRIYKPQTRLKRKQPVRKRQ；DFF40的氨基酸序列(SEQ ID NO.8)：MLQKPKSVKLRALRSPRKFGVAGRSCQEVLRKGCLRFQLPERGSRLCLYEDGTELTEDYFPSVPDNAELVLLTLGQAWQGYVSDIRRFLSAFHEPQVGLIQAAQQLLCDEQAPQRQRLLADLLHNVSQNIAAETRAEDPPWFEGLESRFQSKSGYLRYSCESRIRSYLREVSSYPSTVGAEAQEEFLRVLGSMCQRLRSMQYNGSYFDRGAKGGSRLCTPEGWFSCQGPFDMDSCLSRHSINPYSNRESRILFSTWNLDHIIEKKRTIIPTLVEAIKEQDGREVDWEYFYGLLFTSENLKLVHIVCHKKT THKLNCDPSRIYKPQTRLKRKQPVRKRQ；

DFF40的cDNA序列(SEQ ID NO.9)：5'-ATGCTCCAGAAGCCCAAGAGCGTGAAGCTGCGGGCCCTGCGCAGCCCGAGGAAGTTCGGCGTGGCTGGCCGGAGCTGCCAGGAGGTGCTGCGCAAGGGCTGTCTCCGCTTCCAGCTCCCTGAGCGCGGTTCCCGGCTGTGCCTGTACGAGGATGGCACGGAGCTGACGGAAGATTACTTCCCCAGTGTTCCCGACAACGCCGAGCTGGTGCTGCTCACCTTGGGCCAGGCCTGGCAGGGCTATGTGAGCGACATCAGGCGCTTCCTCAGTGCATTTCACGAGCCACAGGTGGGGCTCATCCAGGCCGCCCAGCAGCTGCTGTGTGATGAGCAGGCCCCACAGAGGCAGAGGCTGCTGGCTGACCTCCTGCACAACGTCAGCCAGAACATCGCGGCCGAGACCCGGGCTGAGGACCCGCCGTGGTTTGAAGGCTTGGAGTCCCGATTTCAGAGCAAGTCTGGCTATCTGAGATACAGCTGTGAGAGCCGGATCCGGAGTTACCTGAGGGAGGTGAGCTCCTACCCCTCCACGGTGGGTGCGGAGGCTCAGGAGGAATTCCTGCGGGTCCTCGGCTCCATGTGCCAGAGGCTCCGGTCCATGCAGTACAATGGCAGCTACTTCGACAGAGGAGCCAAGGGCGGCAGCCGCCTCTGCACACCGGAAGGCTGGTTCTCCTGCCAGGGTCCCTTTGACATGGACAGCTGCTTATCAAGACACTCCATCAACCCCTACAGTAACAGGGAGAGCAGGATCCTCTTCAGCACCTGGAACCTGGATCACATAATAGAAAAGAAACGCACCATCATTCCTACACTGGTGGAAGCAATTAAGGAACAAGATGGAAGAGAAGTGGACTGGGAGTATTTTTATGGCCTGCTTTTTACCTCAGAGAACCTAAAACTAGTGCACATTGTCTGCCATAAGAAAACCACCCACAAGCTCAACTGTGACCCAAGCAGAATCTACAAACCCCAGACAAGGTTGAAGCGGAAGCAGCCTGTGCGGAAACGCCAG-3'；DFF40的cDNA序列(SEQ ID NO.9)：5'-ATGCTCCAGAAGCCCAAGAGCGTGAAGCTGCGGGCCCTGCGCAGCCCGAGGAAGTTCGGCGTGGCTGGCCGGAGCTGCCAGGAGGTGCTGCGCAAGGGCTGTCTCCGCTTCCAGCTCCCTGAGCGCGGTTCCCGGCTGTGCCTGTACGAGGATGGCACGGAGCTGACGGAAGATTACTTCCCCAGTGTTCCCGACAACGCCGAGCTGGTGCTGCTCACCTTGGGCCAGGCCTGGCAGGGCTATGTGAGCGACATCAGGCGCTTCCTCAGTGCATTTCACGAGCCACAGGTGGGGCTCATCCAGGCCGCCCAGCAGCTGCTGTGTGATGAGCAGGCCCCACAGAGGCAGAGGCTGCTGGCTGACCTCCTGCACAACGTCAGCCAGAACATCGCGGCCGAGACCCGGGCTGAGGACCCGCCGTGGTTTGAAGGCTTGGAGTCCCGATTTCAGAGCAAGTCTGGCTATCTGAGATACAGCTGTGAGAGCCGGATCCGGAGTTACCTGAGGGAGGTGAGCTCCTACCCCTCCACGGTGGGTGCGGAGGCTCAGGAGGAATTCCTGCGGGTCCTCGGCTCCATGTGCCAGAGGCTCCGGTCCATGCAGTACAATGGCAGCTACTTCGACAGAGGAGCCAAGGGCGGCAGCCGCCTCTGCACACCGGAAGGCTGGTTCTCCTGCCAGGGTCCCTTTGACATGGACAGCTGCTTATCAAGACACTCCATCAACCCCTACAGTAACAGGGAGAGCAGGATCCTCTTCAGCACCTGGAACCTGGATCACATAATAGAAAAGAAACGCACCATCATTCCTACACTGGTGGAAGCAATTAAGGAACAAGATGGAAGAGAAGTGGACTGGGAGTATTTTTATGGCCTGCTTTTTACCTCAGAGAACCTAAAACTAGTGCACATTGTCTGCCATAAGAAAACCACCCACAAGCTCAACTGTGACCCAAGCAGAATCTACAAACC CCAGACAAGGTTGAAGCGGAAGCAGCCTGTGCGGAAACGCCAG-3';

扩增DFF40的正向引物DFF40-F序列(SEQ ID NO.18)：5'-CCAGCACATGGACAGCCCAGATCTCATGCTCCAGAAGCCCAAGAGC-3'，其中，下划线处表示pET-32a(+)载体同源序列，斜体处表示酶切位点BglⅡ；The sequence of the forward primer DFF40-F (SEQ ID NO. 18) for amplifying DFF40: 5'- CCAGCACATGGACAGCC AGATCTCATGCTCCAGAAGCCCAAGAGC-3', wherein the underline represents the homologous sequence of the pET-32a(+) vector, and the italics represents the restriction site point BglII;

扩增DFF40的反向引物DFF40-R序列(SEQ ID NO.19)：5'-CAACCAGCTGAACTTCAGCCATGGT CTGGCGTTTCCGCACAGGCTG-3'，其中，下划线处表示pET-32a(+)载体同源序列，斜体处表示酶切位点NcoⅠ；The reverse primer DFF40-R sequence (SEQ ID NO. 19) for amplifying DFF40: 5'- CAACCAGCTGAACTTCAG CCATGGT CTGGCGTTTTCCGCACAGGCTG-3', where the underline represents the homologous sequence of the pET-32a(+) vector, and the italics represents the restriction enzyme cleavage site NcoI;

2、质粒：pET-32a(+)大肠杆菌表达载体购自Addgene质粒库；2. Plasmid: pET-32a(+) Escherichia coli expression vector was purchased from Addgene plasmid library;

二、实验方法2. Experimental method

1、细胞色素C同源重组基因片段的获得：通过化学合成方法合成在SEQ ID NO.7所示Cytc的cDNA序列两端添加有酶切位点BglⅡ和NcoⅠ及补充碱基的核苷酸片段，随后通过引物Cytc-F和Cytc-R扩增Cytc同源重组序列；1. Obtainment of cytochrome C homologous recombination gene fragment: a nucleotide fragment with enzyme cleavage sites BglII and NcoI and complementary bases added to both ends of the Cytc cDNA sequence shown in SEQ ID NO.7 was synthesized by chemical synthesis method. , and then amplified the Cytc homologous recombination sequence by primers Cytc-F and Cytc-R;

2、DFF40同源重组基因片段的获得：通过化学合成方法合成SEQ ID NO.9所示DFF40的cDNA序列两端添加有酶切位点BglⅡ和NcoⅠ及补充碱基的核苷酸片段，随后通过引物DFF40-F和DFF40-R扩增DFF40同源重组序列；2. Obtainment of DFF40 homologous recombination gene fragment: The cDNA sequence of DFF40 shown in SEQ ID NO. Primers DFF40-F and DFF40-R amplify the DFF40 homologous recombination sequence;

3、pET-32a(+)-Cytc-scFv重组质粒的构建3. Construction of pET-32a(+)-Cytc-scFv recombinant plasmid

将Cytc同源重组基因和双酶切(BglⅡ和NcoⅠ)后的pET-32a(+)-scFv重组质粒通过同源重组进行连接，将连接产物转化到E.coli DH5α；重组菌扩培后送上海生工生物工程有限公司进行测序，结果显示阳性重组质粒与预期cDNA序列完全一致，插入的Cytc-scFv融合型单链抗体的氨基酸序列如SEQ ID NO.10所示，构建得到重组质粒pET-32a(+)-Cytc-scFv(图2)。The Cytc homologous recombination gene and the pET-32a(+)-scFv recombinant plasmid after double digestion (BglII and NcoI) were connected by homologous recombination, and the ligated product was transformed into E.coli DH5α; the recombinant bacteria were expanded and sent to Shanghai Sangon Bioengineering Co., Ltd. performed sequencing, and the results showed that the positive recombinant plasmid was completely consistent with the expected cDNA sequence. The amino acid sequence of the inserted Cytc-scFv fusion single-chain antibody was shown in SEQ ID NO. 10, and the recombinant plasmid pET- 32a(+)-Cytc-scFv (Figure 2).

SEQ ID NO.10具体为：MGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNETMAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKRSEQ ID NO.10具体为：MGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNETMAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKR

4、pET-32a(+)-DFF40-scFv重组质粒的构建4. Construction of pET-32a(+)-DFF40-scFv recombinant plasmid

将DFF40同源重组基因和双酶切(BglⅡ和NcoⅠ)后的pET-32a(+)-scFv重组质粒通过同源重组进行连接，将连接产物转化到E.coli DH5α；测序，结果显示阳性重组质粒与预期cDNA序列完全一致，插入的DFF40-scFv融合型单链抗体的氨基酸序列如SEQ ID NO.12所示，构建得到重组质粒pET-32a(+)-DFF40-scFv(图2)；The DFF40 homologous recombination gene and the pET-32a(+)-scFv recombinant plasmid after double digestion (BglII and NcoI) were connected by homologous recombination, and the ligation product was transformed into E.coli DH5α; sequenced, the result showed positive recombination The plasmid is completely consistent with the expected cDNA sequence, and the amino acid sequence of the inserted DFF40-scFv fusion single-chain antibody is shown in SEQ ID NO.

SEQ ID NO.12具体为：MLQKPKSVKLRALRSPRKFGVAGRSCQEVLRKGCLRFQLPERGSRLCLYEDGTELTEDYFPSVPDNAELVLLTLGQAWQGYVSDIRRFLSAFHEPQVGLIQAAQQLLCDEQAPQRQRLLADLLHNVSQNIAAETRAEDPPWFEGLESRFQSKSGYLRYSCESRIRSYLREVSSYPSTVGAEAQEEFLRVLGSMCQRLRSMQYNGSYFDRGAKGGSRLCTPEGWFSCQGPFDMDSCLSRHSINPYSNRESRILFSTWNLDHIIEKKRTIIPTLVEAIKEQDGREVDWEYFYGLLFTSENLKLVHIVCHKKTTHKLNCDPSRIYKPQTRLKRKQPVRKRQTMAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRSEQ ID NO.12具体为：MLQKPKSVKLRALRSPRKFGVAGRSCQEVLRKGCLRFQLPERGSRLCLYEDGTELTEDYFPSVPDNAELVLLTLGQAWQGYVSDIRRFLSAFHEPQVGLIQAAQQLLCDEQAPQRQRLLADLLHNVSQNIAAETRAEDPPWFEGLESRFQSKSGYLRYSCESRIRSYLREVSSYPSTVGAEAQEEFLRVLGSMCQRLRSMQYNGSYFDRGAKGGSRLCTPEGWFSCQGPFDMDSCLSRHSINPYSNRESRILFSTWNLDHIIEKKRTIIPTLVEAIKEQDGREVDWEYFYGLLFTSENLKLVHIVCHKKTTHKLNCDPSRIYKPQTRLKRKQPVRKRQTMAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR

5、pET-32a(+)-3Cytc-scFv重组质粒的构建5. Construction of pET-32a(+)-3Cytc-scFv recombinant plasmid

采用如SEQ ID NO.11所示的Caspase-3酶切位点DEVD序列连接三段Cytc基因片段，组成3Cytc基因片段，按照上述与Cytc类似的方法获得3Cytc同源重组基因，随后和双酶切(BglⅡ和NcoⅠ)后的pET-32a(+)-scFv重组质粒通过同源重组进行连接，将连接产物转化到E.coli DH5α；测序，结果显示阳性重组质粒与预期cDNA序列完全一致，插入的3Cytc-scFv融合型单链抗体的氨基酸序列如SEQ ID NO.13所示，构建得到重组质粒pET-32a(+)-3Cytc-scFv(图2)；Use the Caspase-3 restriction site DEVD sequence shown in SEQ ID NO.11 to connect three Cytc gene fragments to form 3Cytc gene fragments, obtain the 3Cytc homologous recombination gene according to the above-mentioned method similar to Cytc, and then double-enzyme digestion (BglII and NcoI) pET-32a(+)-scFv recombinant plasmids were ligated by homologous recombination, and the ligated product was transformed into E.coli DH5α; sequencing results showed that the positive recombinant plasmid was completely consistent with the expected cDNA sequence, and the inserted The amino acid sequence of the 3Cytc-scFv fusion single-chain antibody is shown in SEQ ID NO.13, and the recombinant plasmid pET-32a(+)-3Cytc-scFv was constructed to obtain (Figure 2);

DEVD的cDNA序列(SEQ ID NO.11)：5'-GATGAAGTTGAT-3'；cDNA sequence of DEVD (SEQ ID NO. 11): 5'-GATGAAGTTGAT-3';

SEQ ID NO.13具体为：MGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNEDEVDMGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNEDEVDMGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNETMAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR，其中，下划线处为DEVD；SEQ ID NO.13具体为：MGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNE DEVD MGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNE DEVD MGDVEKGKKIFIMKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKATNETMAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR，其中，下划线处为DEVD；

进一步地，凋亡蛋白Cytc的个数不限于1个或3个，还可为2个或大于3个，各个Cytc之间均是通过Caspase-3酶切位点DEVD序列连接，DEVD的cDNA序列如SEQ ID NO.11所示。Further, the number of apoptotic protein Cytc is not limited to 1 or 3, but can also be 2 or more than 3, and each Cytc is connected by the Caspase-3 restriction site DEVD sequence, the cDNA sequence of DEVD. As shown in SEQ ID NO.11.

实施例3：scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv蛋白的诱导表达与鉴定Example 3: Induced expression and identification of scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv proteins

一、实验材料1. Experimental materials

大肠杆菌表达菌株感受态细胞BL21(DE3)购自北京天根生化科技；异丙基-L-硫代-β-D-吡喃半乳糖苷(IPTG)购自中国Aladdin；NI-NTA Resin购自北京全式金生物科技；E. coli expression strain competent cells BL21 (DE3) were purchased from Beijing Tiangen Biochemical Technology; isopropyl-L-thio-β-D-galactopyranoside (IPTG) was purchased from Aladdin, China; NI-NTA Resin was purchased from From Beijing Quanshijin Biotechnology;

二、实验方法2. Experimental method

1、scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv蛋白的诱导表达1. Induction and expression of scFv, Cytc–scFv, 3Cytc–scFv and DFF40-scFv proteins

将上述实施例1得到的阳性质粒pET28a-scFv和实施例2中得到的阳性质粒pET-32a(+)-Cytc-scFv、pET-32a(+)-3Cytc-scFv和pET-32a(+)-DFF40-scFv分别转化至大肠杆菌表达菌株感受态细胞BL21(DE3)，经IPTG诱导表达和相应条件摸索，诱导scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv蛋白表达，并通过NI-NTA Resin亲和层析纯化蛋白，scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv蛋白的二维结构如图1所示。The positive plasmid pET28a-scFv obtained in the above Example 1 and the positive plasmids pET-32a(+)-Cytc-scFv, pET-32a(+)-3Cytc-scFv and pET-32a(+)- DFF40-scFv was transformed into E. coli expression strain competent cells BL21(DE3), and the expression of scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv was induced by IPTG induction and corresponding condition exploration. The proteins were purified by affinity chromatography. The two-dimensional structures of scFv, Cytc–scFv, 3Cytc–scFv and DFF40-scFv proteins are shown in Figure 1.

2、scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv蛋白的鉴定2. Identification of scFv, Cytc–scFv, 3Cytc–scFv and DFF40-scFv proteins

将纯化的scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv蛋白进行SDS-PAGE电泳检测：蛋白液分别与SDS-PAGE上样缓冲液按比例混匀，金属浴加热变性10min，冷却至室温后上样，100V电泳70min；结束后，将凝胶切下来浸入考马斯亮蓝染色液染色25min，随后用脱色液室温摇床脱色3h，利用marker指示的分子量位置及甬道的背景是否有较多杂带以鉴定纯化效果；The purified scFv, Cytc–scFv, 3Cytc–scFv and DFF40-scFv proteins were detected by SDS-PAGE electrophoresis: the protein solution was mixed with SDS-PAGE loading buffer in proportion, heated in a metal bath for denaturation for 10 min, and cooled to room temperature. Load the sample, electrophoresis at 100V for 70min; after the end, cut off the gel and immerse it in Coomassie brilliant blue staining solution for 25min, then use the decolorizing solution to decolorize at room temperature for 3h, and use the molecular weight position indicated by the marker and the background of the channel to see if there are many impurity bands To identify the purification effect;

将纯化后的scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv蛋白进行Western Blot分析：如上所述通过煮沸变性制备蛋白样品，冷却后的样品上样到凝胶孔中，100V电泳70min，电泳结束后对照marker的指示带在相应位置切下凝胶，使用bio-rad半干转仪将蛋白转印到PVDF膜上，5％脱脂奶粉封闭1h，接着以鼠源6His Tag抗体为一抗，4摄氏度孵育过夜；次日，使用pH7.6的PBST洗膜后以山羊抗鼠IgG-FITC为二抗，室温孵育2h，随后使用增强型化学发光试剂盒进行曝光。The purified scFv, Cytc–scFv, 3Cytc–scFv and DFF40-scFv proteins were subjected to Western Blot analysis: protein samples were prepared by boiling denaturation as described above, and the cooled samples were loaded into gel wells, electrophoresed at 100V for 70 min, and electrophoresed After the end, the gel was cut at the corresponding position according to the indicator band of the marker, and the protein was transferred to the PVDF membrane using a bio-rad semi-dry spinner, 5% nonfat milk powder was blocked for 1 h, and then the mouse-derived 6His Tag antibody was used as the primary antibody. Incubate overnight at 4 degrees Celsius; the next day, the membrane was washed with PBST pH 7.6, then goat anti-mouse IgG-FITC was used as the secondary antibody, incubated at room temperature for 2 h, and then exposed using an enhanced chemiluminescence kit.

三、结果与分析3. Results and Analysis

将测序正确的重组质粒pET-32a(+)-scFv、pET-32a(+)-Cytc–scFv、pET-32a(+)-3Cytc-scFv和pET-32a(+)-DFF40-scFv分别转化BL21(DE3)感受态细胞中，通过氨苄青霉素在LB固体培养板上筛选出整合有质粒的重组子，融合基因的表达由pET32a(+)上的噬菌体T7启动子控制，并使用乳糖操纵子进行诱导，利用药瓶在37℃培养出菌体后加入IPTG，通过控制变量法确定最佳诱导条件为0.5mM IPTG，20℃反应12h。The correctly sequenced recombinant plasmids pET-32a(+)-scFv, pET-32a(+)-Cytc–scFv, pET-32a(+)-3Cytc-scFv and pET-32a(+)-DFF40-scFv were transformed into BL21, respectively In (DE3) competent cells, recombinants with integrated plasmids were screened on LB solid plates by ampicillin. The expression of the fusion gene was controlled by the phage T7 promoter on pET32a(+) and induced by the lactose operon. , using the bottle to cultivate the bacteria at 37 ℃ and then adding IPTG, the optimal induction condition was determined by the control variable method to be 0.5 mM IPTG, and the reaction was carried out at 20 ℃ for 12 h.

为了获得scFv蛋白及Cytc–scFv、3Cytc–scFv、DFF40-scFv重组蛋白，以进行蛋白鉴定，对0.5mM IPTG，20℃诱导反应12h条件下的pET-32a(+)-scFv/BL21(DE3)、pET-32a(+)-Cytc–scFv/BL21(DE3)、pET-32a(+)-3Cytc–scFv/BL21(DE3)和pET-32a(+)-DFF40–scFv/BL21(DE3)菌体沉淀进行超声破碎，取破碎悬液上清通过NI-NTA Resin亲和层析纯化蛋白，而后通过不同咪唑浓度的洗脱液洗脱下来，目的蛋白主要在100mM咪唑的洗脱液中。In order to obtain scFv protein and Cytc–scFv, 3Cytc–scFv, DFF40-scFv recombinant proteins for protein identification, pET-32a(+)-scFv/BL21(DE3) was induced to react with 0.5mM IPTG at 20℃ for 12h. , pET-32a(+)-Cytc–scFv/BL21(DE3), pET-32a(+)-3Cytc–scFv/BL21(DE3) and pET-32a(+)-DFF40–scFv/BL21(DE3) cells The precipitate was sonicated, and the supernatant of the crushed suspension was taken to purify the protein by NI-NTA Resin affinity chromatography, and then eluted with different imidazole concentrations. The target protein was mainly in the eluate of 100 mM imidazole.

使用SDS-PAGE验证蛋白的纯化效果，如图3A所示，scFv、DFF40-scFv、Cytc-scFv和3Cytc-scFv蛋白分子量分别为scFv：46kDa、DFF40-scFv：84kDa、Cytc-scFv：58kDa、3Cytc-scFv：83kDa，而后考马斯亮蓝染色后，每种蛋白根据其各自分子量的不同在相应位置都出现了条带，与预期结果一致，且背景干净无杂蛋白，说明纯化效果较好。SDS-PAGE was used to verify the purification effect of the protein. As shown in Figure 3A, the molecular weights of scFv, DFF40-scFv, Cytc-scFv and 3Cytc-scFv were scFv: 46kDa, DFF40-scFv: 84kDa, Cytc-scFv: 58kDa, 3Cytc -scFv: 83kDa, and after Coomassie brilliant blue staining, each protein appeared a band at the corresponding position according to its respective molecular weight, which was consistent with the expected result, and the background was clean and free of impurities, indicating that the purification effect was good.

进一步通过Western Blot实验进行scFv、DFF40-scFv、Cytc-scFv及3Cytc-scFv蛋白表达验证，如图3B所示，抗原抗体结合后进行生物发光成像，结果与凝胶染色结果一致，都在相应位置检测到条带。The protein expression of scFv, DFF40-scFv, Cytc-scFv and 3Cytc-scFv was further verified by Western Blot experiments. As shown in Figure 3B, bioluminescence imaging was performed after antigen-antibody binding. The results were consistent with the results of gel staining, all in the corresponding positions. Bands detected.

以上结果表明，重组蛋白scFv、DFF40-scFv、Cytc-scFv及3Cytc-scFv已成功诱导表达和纯化。The above results show that the recombinant proteins scFv, DFF40-scFv, Cytc-scFv and 3Cytc-scFv have been successfully induced to express and purified.

实施例4：scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv融合型单链抗体在体外对乳腺癌细胞的活性测定Example 4: In vitro activity assay of scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv fusion single chain antibodies on breast cancer cells

一、实验材料1. Experimental materials

人乳腺癌细胞系SK-BR-3、MDA-MB-231以及MCF-7购自美国模式培养物集存库(American type culture collection)ATCC；6His Tag抗体购自上海翊圣生物科技；山羊抗鼠IgG-FITC购自北京中杉金桥生物技术有限公司。Human breast cancer cell lines SK-BR-3, MDA-MB-231 and MCF-7 were purchased from American type culture collection ATCC; 6His Tag antibody was purchased from Shanghai Yisheng Biotechnology; goat antibody Mouse IgG-FITC was purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.

二、免疫荧光实验2. Immunofluorescence experiment

1、实验分组：HER-2高表达型细胞SK-BR-3作为实验组，HER-2阴性细胞MDA-MB-231和MCF-7，用作对照，分别将四种抗体构建体(scFv、Cytc-scFv、3Cytc-scFv及DFF40-scFv)与三种细胞于37℃共孵育30min。1. Experimental grouping: HER-2 high-expressing cells SK-BR-3 were used as the experimental group, HER-2 negative cells MDA-MB-231 and MCF-7 were used as controls, and four antibody constructs (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) were incubated with the three cells at 37°C for 30 min.

2、实验操作：2. Experimental operation:

(1)细胞准备：分别将三种乳腺癌细胞接种到预先放置有无菌爬片的12孔板培养皿中，待细胞贴壁后分别加入5ug/ml的不同抗体，于5％CO2培养箱中37℃培养30min；(1) Cell preparation: Three kinds of breast cancer cells were inoculated into 12-well culture dishes pre-placed with sterile slides. After the cells adhered, 5ug/ml of different antibodies were added, and cultured in 5% CO 2 Incubate at 37°C for 30min in an incubator;

(2)固定：吸弃培养基，PBS缓冲液洗涤细胞三次，使用新鲜的4％多聚甲醛固定细胞，于室温摇床30min，结束后可移至显微镜下观察细胞是否固定完全；(2) Fixation: Aspirate the culture medium, wash the cells three times with PBS buffer, fix the cells with fresh 4% paraformaldehyde, shake at room temperature for 30 minutes, and then move to a microscope to observe whether the cells are completely fixed;

(3)通透：吸弃固定液，将固定好的细胞用PBS洗涤3次，每次5min。使用PBST溶液(PBS+0.1％Triton X-100)于4℃冰箱通透细胞10min，结束后PBS洗涤3次，每次5min；(3) Permeabilization: Aspirate and discard the fixative, and wash the fixed cells three times with PBS for 5 min each time. Permeabilize cells with PBST solution (PBS+0.1% Triton X-100) at 4°C for 10 min, and wash with PBS for 3 times, 5 min each time;

(4)封闭：加入5％BSA封闭液室温摇床30min，封闭非特异性结合位点；(4) Blocking: Add 5% BSA blocking solution on a shaker at room temperature for 30 minutes to block non-specific binding sites;

(5)一抗结合：一抗使用鼠源6His Tag抗体，按照抗体说明书规定的if实验稀释比例稀释一抗，加入一抗稀释液，室温孵育2h或者4℃过夜。PBS洗涤3次，每次5min；(5) Primary antibody binding: Use mouse 6His Tag antibody as the primary antibody, dilute the primary antibody according to the if experiment dilution ratio specified in the antibody manual, add the primary antibody diluent, and incubate at room temperature for 2 hours or 4°C overnight. PBS washed 3 times, 5min each time;

(6)二抗结合：二抗使用山羊抗鼠IgG-FITC，室温避光孵育2h，PBS洗涤3次，每次5min；(6) Secondary antibody binding: Goat anti-mouse IgG-FITC was used as secondary antibody, incubated at room temperature for 2 hours in the dark, and washed with PBS for 3 times, 5 minutes each time;

(7)染核：加入DAPI染色液，避光结合10min，PBS洗涤3次，每次5min；(7) Nuclei staining: add DAPI staining solution, combine in the dark for 10 minutes, and wash with PBS for 3 times, 5 minutes each time;

(8)封片及检测：于载玻片上滴加封片剂一滴，取出爬片，细胞面接触封片剂进行封片，待爬片干燥固定后于共聚焦荧光显微镜观察并摄取图像。(8) Mounting and detection: drop a drop of mounting medium on the glass slide, take out the slide, contact the cell surface with the mounting medium for mounting, and observe and capture the image under a confocal fluorescence microscope after the slide is dried and fixed.

三、结果与分析3. Results and Analysis

使用630倍共聚焦显微镜观察，如图4所示，无论是独立的scFv还是凋亡蛋白融合型单链抗体DFF40-scFv、Cytc-scFv以及3Cytc-scFv均结合到SK-BR-3细胞表面，相比之下，HER-2阴性细胞MDA-MB-231和MCF-7表面没有观察到任何绿色目的蛋白，即scFv以及凋亡蛋白融合型scFv均能与HER-2过表达型乳腺癌细胞SK-BR-3表面HER-2抗原特异性结合；由此可见，由原核表达系统制备的抗HER-2单链抗体scFv特异性结合乳腺癌细胞表面抗原HER-2，与单克隆抗体具有等效靶向活性，且与凋亡蛋白DFF40或Cytc偶联后结合活性没有受到损伤。Using a 630x confocal microscope, as shown in Figure 4, whether it is an independent scFv or apoptotic protein fusion single-chain antibodies DFF40-scFv, Cytc-scFv and 3Cytc-scFv are bound to the surface of SK-BR-3 cells, In contrast, no green target protein was observed on the surface of HER-2 negative cells MDA-MB-231 and MCF-7, that is, scFv and apoptotic protein fusion scFv could interact with HER-2 overexpressing breast cancer cells SK. -Specific binding to HER-2 antigen on the surface of BR-3; it can be seen that the anti-HER-2 single-chain antibody scFv prepared by the prokaryotic expression system specifically binds to the breast cancer cell surface antigen HER-2, which is equivalent to the monoclonal antibody Targeting activity, and binding activity was not impaired after conjugation to apoptotic proteins DFF40 or Cytc.

实施例5：scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv融合型单链抗体对HER-2过表达型乳腺癌细胞活力影响验证Example 5: Verification of the effect of scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv fusion single-chain antibodies on the viability of HER-2 overexpressing breast cancer cells

一、实验材料1. Experimental materials

CCK-8细胞活力检测试剂盒购自美国Sigma；人乳腺癌细胞系SK-BR-3、MDA-MB-231以及MCF-7来源同实施例4；结晶紫染色液购自北京索莱宝；4％多聚甲醛购自美国Sigma；The CCK-8 cell viability detection kit was purchased from Sigma in the United States; the sources of human breast cancer cell lines SK-BR-3, MDA-MB-231 and MCF-7 were the same as those in Example 4; the crystal violet staining solution was purchased from Beijing Soleibo; 4% paraformaldehyde was purchased from Sigma in the United States;

二、实验方法2. Experimental method

CCK-8细胞活力检测试剂盒含有WST-8，在电子载体1-Methoxy PMS存在的情况下，细胞内脱氢酶将WST-8还原成水溶性的橙黄色甲臢染料，其能够溶解在培养基中并可通过酶标仪于450nm处检测吸光度，其生成量与活细胞数量成正比。结晶紫染色液，是一种碱性染色液，可以把细胞核染成深紫色，常用于细胞计数以及细胞增殖分析。本发明通过在SK-BR-3，MDA-MB-231和MCF7细胞系上进行CCK-8分析以及细胞增殖分析，确定免疫细胞凋亡蛋白的体外细胞毒性作用。The CCK-8 Cell Viability Assay Kit contains WST-8. In the presence of the electron carrier 1-Methoxy PMS, intracellular dehydrogenase reduces WST-8 to a water-soluble orange-yellow formazan dye, which can be dissolved in cultured In the base, the absorbance can be detected at 450nm by a microplate reader, and the amount generated is proportional to the number of living cells. Crystal violet staining solution is an alkaline staining solution that can stain cell nuclei into deep purple, and is often used for cell counting and cell proliferation analysis. The present invention determines the in vitro cytotoxicity of immune cell apoptosis proteins by performing CCK-8 analysis and cell proliferation analysis on SK-BR-3, MDA-MB-231 and MCF7 cell lines.

1、细胞活力检测实验1. Cell viability assay

(一)实验分组：HER-2高表达型细胞SK-BR-3作为实验组，HER-2阴性细胞MDA-MB-231和MCF-7，用作对照，通过在SK-BR-3，MDA-MB-231和MCF7细胞系上进行CCK-8分析以及细胞增殖分析，确定免疫细胞凋亡蛋白的体外细胞毒性作用。(1) Experimental grouping: HER-2 high-expressing cells SK-BR-3 were used as the experimental group, and HER-2 negative cells MDA-MB-231 and MCF-7 were used as controls. - CCK-8 assay as well as cell proliferation assay on MB-231 and MCF7 cell lines to determine in vitro cytotoxic effects of immune apoptotic proteins.

(二)细胞活力检测：(2) Cell viability detection:

(1)以104～105个细胞/孔的密度在96孔板中接种细胞，37℃，5％CO2培养箱中培养细胞24小时；(1) Seed cells in a 96-well plate at a density of 10 4 to 10 5 cells/well, and culture the cells in a 37°C, 5% CO 2 incubator for 24 hours;

(2)PBS洗涤细胞，设置空白孔(培养基+CCK8，不含细胞及蛋白)、对照孔(细胞+培养基+CCK8，不含蛋白)、实验孔(细胞+培养基+CCK8+蛋白)，实验孔分组加入不同浓度的目的蛋白，在培养箱中继续孵育48h；(每组设置3个副孔)(2) Wash the cells with PBS, set up blank wells (medium+CCK8, no cells and proteins), control wells (cells+medium+CCK8, no protein), and experimental wells (cells+medium+CCK8+protein), The experimental wells were grouped into different concentrations of the target protein, and incubated in the incubator for 48 hours; (3 sub-wells were set in each group)

(3)使用排枪将10μLCCK-8溶液添加到平板的每个孔中。(3) Add 10 μL of the LCCCK-8 solution to each well of the plate using a row gun.

(4)37℃孵育1～4小时，直至颜色发生明显变化；(4) Incubate at 37°C for 1 to 4 hours until the color changes significantly;

(5)取出平板，用锡纸包被避光，室温下在振荡器上轻轻混合1min，以确保颜色均匀分布；(5) Take out the plate, cover it with tin foil to protect from light, and mix gently on a shaker for 1 min at room temperature to ensure uniform color distribution;

(6)立即使用酶标仪测定450nm处的每孔吸光度值；(6) Immediately use a microplate reader to measure the absorbance value of each well at 450 nm;

(7)细胞活力计算公式：细胞活力＝[(As-Ab)/(Ac-Ab)]×100％(As为实验孔，Ac为对照孔，Ab为空白孔)。(7) Calculation formula of cell viability: cell viability=[(As-Ab)/(Ac-Ab)]×100% (As is an experimental well, Ac is a control well, and Ab is a blank well).

2、结晶紫染色实验2. Crystal violet staining experiment

(1)以104～105个细胞/孔的密度在6孔板中接种细胞，37℃，5％CO2培养箱中培养细胞24小时；(1) Seed cells in a 6-well plate at a density of 10 4 -10 5 cells/well, and culture the cells in a 37°C, 5% CO 2 incubator for 24 hours;

(2)PBS洗涤细胞，以scFv作用组为对照组，实验组每组加入不同浓度的凋亡蛋白融合型单链抗体蛋白(Cytc-scFv、3Cytc-scFv及DFF40-scFv)，在培养箱中继续孵育48h；(2) The cells were washed with PBS, and the scFv-treated group was used as the control group. Different concentrations of apoptotic protein fusion-type single-chain antibody proteins (Cytc-scFv, 3Cytc-scFv and DFF40-scFv) were added to each group in the experimental group. Continue to incubate for 48h;

(3)吸出培养基用PBS洗涤两次，并在室温下用4％多聚甲醛摇床固定10min，用蒸馏水洗涤2min，切换至新鲜蒸馏水，然后再洗涤2min；(3) Aspirate the medium and wash it twice with PBS, fix it with 4% paraformaldehyde on a shaker for 10 min at room temperature, wash with distilled water for 2 min, switch to fresh distilled water, and then wash for 2 min;

(4)每孔加入1ml 0.1％结晶紫染色液孵育10min，用蒸馏水洗涤完毕后即可进行观察并拍照。(4) Add 1 ml of 0.1% crystal violet staining solution to each well and incubate for 10 min. After washing with distilled water, observe and take pictures.

三、结果与分析3. Results and Analysis

1、细胞活力检测实验10-2 1. Cell Viability Detection Experiment 10 -2

如图5A，四种抗体构建体(scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv)与SK-BR-3细胞共孵育，浓度梯度为0.01～1000nM，我们可以直观地看出，与scFv相比，凋亡蛋白融合型单链抗体Cytc–scFv、3Cytc–scFv及DFF40-scFv可以有效抑制具有最高HER-2表达水平的SK-BR-3细胞的活力，而且其抑制效果呈浓度依赖性。在100nM相同浓度蛋白质刺激下细胞活力显著下降：DFF40-scFv毒性最高，细胞活力下降80％左右，其次是3Cytc-scFv，而Cytc-scFv是低毒性的，仅为3Cytc-scFv的一半左右，可见3Cytc-scFv由于多个Cytc的释放，毒性显著增加。此外，观察到与SK-BR-3细胞相比，表达低水平HER-2的MDA-MB-231和MCF7细胞的细胞活力几乎没有受到影响(图5B、图5C)，这清楚地表明，细胞表面存在较高水平的HER-2对于凋亡蛋白融合型单链抗体的特异性细胞毒性是必需的。As shown in Figure 5A, the four antibody constructs (scFv, Cytc–scFv, 3Cytc–scFv and DFF40-scFv) were incubated with SK-BR-3 cells at a concentration gradient of 0.01 to 1000 nM. In contrast, apoptotic protein fusion single-chain antibodies Cytc–scFv, 3Cytc–scFv and DFF40-scFv can effectively inhibit the viability of SK-BR-3 cells with the highest HER-2 expression level, and the inhibitory effect is concentration-dependent . The cell viability was significantly decreased under the stimulation of the same concentration of 100nM protein: DFF40-scFv was the most toxic, and the cell viability decreased by about 80%, followed by 3Cytc-scFv, while Cytc-scFv was low toxicity, only about half of that of 3Cytc-scFv, it can be seen that The toxicity of 3Cytc-scFv was significantly increased due to the release of multiple Cytc. Furthermore, it was observed that the cell viability of MDA-MB-231 and MCF7 cells expressing low levels of HER-2 was hardly affected compared to SK-BR-3 cells (Fig. 5B, Fig. 5C), which clearly indicated that the cells The presence of higher levels of HER-2 on the surface is necessary for the specific cytotoxicity of apoptotic protein-fusion scFv.

2、结晶紫染色实验2. Crystal violet staining experiment

分别使用四种抗体构建体(scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv)刺激HER-2高表达细胞SK-BR-3，类似地，从细胞染色结果图5D可以看出，SK-BR-3细胞在scFv作用后细胞数最多，细胞增殖活力基本不受影响；与scFv相比，凋亡蛋白融合型单链抗体Cytc–scFv、3Cytc–scFv及DFF40-scFv孵育的细胞集落数明显减少，这表明融合蛋白Cytc–scFv、3Cytc–scFv及DFF40-scFv可显着抑制细胞生长，并且活性差异与前述细胞活力检测实验结果一致：DFF40-scFv作用后细胞数最少，细胞毒性最强；3Cytc-scFv细胞数较Cytc-scFv更少，可见其活性更优；此外，用DFF40-scFv刺激两种对照细胞MDA-MB-231和MCF7，发现细胞增殖活力均不受影响。Four antibody constructs (scFv, Cytc–scFv, 3Cytc–scFv, and DFF40-scFv) were used to stimulate HER-2 high-expressing cells SK-BR-3. Similarly, it can be seen from the cell staining results in Figure 5D that SK-BR-3 BR-3 cells had the largest number of cells after the action of scFv, and the cell proliferation activity was basically unaffected; compared with scFv, the number of cell colonies incubated with apoptotic protein fusion single-chain antibodies Cytc-scFv, 3Cytc-scFv and DFF40-scFv was significantly higher This indicates that the fusion proteins Cytc-scFv, 3Cytc-scFv and DFF40-scFv can significantly inhibit cell growth, and the difference in activity is consistent with the previous cell viability test results: DFF40-scFv has the smallest number of cells and the strongest cytotoxicity; The number of 3Cytc-scFv cells was less than that of Cytc-scFv, which showed that its activity was better. In addition, two control cells, MDA-MB-231 and MCF7, were stimulated with DFF40-scFv, and it was found that the cell proliferation activity was not affected.

综上，我们可以得出：由于scFv的存在，DFF40或Cytc融合蛋白(Cytc–scFv、3Cytc–scFv及DFF40-scFv)对高表达HER-2的细胞产生特异性毒性。In conclusion, we can conclude that DFF40 or Cytc fusion proteins (Cytc–scFv, 3Cytc–scFv and DFF40-scFv) have specific toxicity to cells with high expression of HER-2 due to the presence of scFv.

实施例6：scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv融合型单链抗体对HER-2过表达型乳腺癌细胞诱导后Caspase3蛋白活性检测Example 6: Detection of Caspase3 protein activity after induction of HER-2 overexpressing breast cancer cells by scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv fusion single-chain antibodies

一、实验材料1. Experimental materials

Caspase3活性检测试剂盒购自南京建成生物工程研究所；人乳腺癌细胞系SK-BR-3、MDA-MB-231以及MCF-7来源同实施例4；The Caspase3 activity detection kit was purchased from Nanjing Jiancheng Bioengineering Institute; the sources of human breast cancer cell lines SK-BR-3, MDA-MB-231 and MCF-7 were the same as those in Example 4;

二、Caspase3蛋白活性检测2. Detection of Caspase3 protein activity

Caspase3是细胞凋亡执行蛋白，在凋亡通路中起到关键作用。Caspase3活性检测试剂盒就是将Caspase3序列特异性的多肽(Ac-DEVD-PNA)偶联至黄色基团pNA：当该底物被Caspase3剪切后，黄色基团pNA即游离出来，其在405nm附近有强吸收，可通过酶标仪检测，据此可间接考察Caspase3的活化程度。此外，凋亡执行蛋白Caspase3的特异性切割位点DEVD引入3Cytc-scFv蛋白构建体，一旦被细胞内化，Caspase3的激活和切割将使更多的Cytc从融合蛋白中释放出来。Caspase3 is an apoptosis executive protein and plays a key role in the apoptosis pathway. The Caspase3 activity detection kit is to couple the Caspase3 sequence-specific polypeptide (Ac-DEVD-PNA) to the yellow group pNA: when the substrate is cleaved by Caspase3, the yellow group pNA is released, which is near 405nm There is strong absorption, which can be detected by a microplate reader, based on which the activation degree of Caspase3 can be indirectly examined. In addition, the specific cleavage site DEVD of the apoptosis-executing protein Caspase3 was introduced into the 3Cytc-scFv protein construct, and once internalized by the cell, activation and cleavage of Caspase3 would release more Cytc from the fusion protein.

本发明通过scFv、Cytc–scFv、3Cytc–scFv或DFF40-scFv融合型单链抗体分别刺激SK-BR-3，MDA-MB-231和MCF7细胞系，采用Caspase3活性检测试剂盒对诱导后Caspase3蛋白活性检测，具体步骤如下：In the present invention, SK-BR-3, MDA-MB-231 and MCF7 cell lines are stimulated respectively by scFv, Cytc-scFv, 3Cytc-scFv or DFF40-scFv fusion type single-chain antibody, and the Caspase3 activity detection kit is used to detect the induced Caspase3 protein. Activity detection, the specific steps are as follows:

(1)细胞进行抗体蛋白刺激后，使用胰酶消化，800g离心5min收集细胞；(1) After the cells were stimulated with antibody protein, they were digested with trypsin and centrifuged at 800 g for 5 min to collect the cells;

(2)PBS洗涤细胞沉淀一次，如上离心，吸弃上清后按照每200万细胞加入50μl裂解液的比例加入裂解液，重悬沉淀，冰浴裂解3min，其间涡旋振荡3-4次，每次10s，或冻融2-3次；(2) Wash the cell pellet once with PBS, centrifuge as above, aspirate and discard the supernatant, add lysate at a rate of 50 μl of lysate per 2 million cells, resuspend the pellet, lyse on ice for 3 minutes, and vortex for 3-4 times. 10s each time, or freeze and thaw 2-3 times;

(3)12000rpm，4℃离心15min，小心吸取上清转移至新的干净离心管中。按照Caspase3活性检测试剂盒要求设置对照组与实验组，并分别加入对应量的反应液、裂解液、待测样品、Caspase3酶切底物等，反应体系如表1；(3) Centrifuge at 12000rpm for 15min at 4°C, carefully transfer the supernatant to a new clean centrifuge tube. According to the requirements of the Caspase3 activity detection kit, the control group and the experimental group were set up, and corresponding amounts of reaction solution, lysis solution, sample to be tested, Caspase3 enzyme cleavage substrate, etc. were added respectively. The reaction system is shown in Table 1;

表1Table 1

Figure BDA0002585061130000181
Figure BDA0002585061130000181

(4)将反应体系混匀，继续在培养箱中孵育4h，颜色变化明显时即可在405nm处测定吸光度值；(4) Mix the reaction system and continue to incubate in the incubator for 4 hours. When the color changes obviously, the absorbance value can be measured at 405 nm;

(5)通过OD实验组/OD对照组来反映Caspase3活化的程度。(5) The degree of Caspase3 activation was reflected by OD experimental group /OD control group .

三、结果与分析3. Results and Analysis

如图6A所示，凋亡蛋白融合型scFv(Cytc–scFv、3Cytc–scFv及DFF40-scFv)与SK-BR-3细胞共孵育后Caspase3蛋白活性显著增强，与单纯scFv(scFv)相比，DFF40-scFv、Cytc-scFv及3Cytc-scFv作用后的Caspase3活化程度分别是其6倍、3倍和4.5倍左右；然而，对于HER-2阴性细胞MDA-MB-231和MCF-7，所有抗体构建体(scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv)作用后均未检测到Caspase3蛋白的激活(图6B、图6C)。As shown in Figure 6A, the apoptotic protein fusion scFv (Cytc–scFv, 3Cytc–scFv and DFF40-scFv) co-incubated with SK-BR-3 cells significantly enhanced the activity of Caspase3 protein, compared with pure scFv (scFv), DFF40-scFv, Cytc-scFv and 3Cytc-scFv had about 6-fold, 3-fold and 4.5-fold higher activation of Caspase3, respectively; however, for HER-2 negative cells MDA-MB-231 and MCF-7, all antibodies None of the constructs (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) could detect the activation of Caspase3 protein (Fig. 6B, Fig. 6C).

以上结果表明，单纯scFv没有促凋亡活性，将scFv与凋亡相关蛋白DFF40、Cytc融合表达后可在体外有效诱导HER-2过表达型乳腺癌细胞凋亡。The above results show that pure scFv has no pro-apoptotic activity, and the fusion expression of scFv with apoptosis-related proteins DFF40 and Cytc can effectively induce apoptosis of HER-2 overexpressing breast cancer cells in vitro.

实施例7：scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv融合型单链抗体对HER-2过表达型乳腺癌细胞SK-BR-3诱导后凋亡抑制蛋白Bcl-2与凋亡促进蛋白Bax的比值测定Example 7: Effect of scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv fusion single chain antibody on apoptosis inhibitory protein Bcl-2 and apoptosis promotion in HER-2 overexpressing breast cancer cells after induction of SK-BR-3 Ratio determination of protein Bax

一、实验材料1. Experimental materials

BCA浓度检测试剂盒购自美国CST；人乳腺癌细胞系SK-BR-3来源同实施例4；The BCA concentration detection kit was purchased from CST in the United States; the source of human breast cancer cell line SK-BR-3 was the same as that in Example 4;

二、凋亡蛋白表达量分析2. Analysis of apoptotic protein expression

Bcl-2基因全称为B淋巴细胞瘤-2，在调节凋亡相关基因中Bcl-2家族是最重要的调节因子之一。Bcl-2与Bax都是Bcl-2蛋白家族成员，前者为抗凋亡蛋白，后者为促凋亡蛋白，已证明两者主要在线粒体凋亡途径中发挥作用。Bcl-2通过控制线粒体膜通透性来调节细胞死亡，通过阻止细胞色素C从线粒体中释放和/或与凋亡激活因子(Apaf-1)结合来抑制半胱天冬酶活性，从而抑制细胞凋亡；而Bax可诱导细胞色素C释放，促进Caspase3的激活，从而形成凋亡。Bcl-2可与Bax形成二聚体，如果Bax表达量高于Bcl-2，则促进细胞凋亡；而如果Bcl-2表达量高于Bax，则抑制细胞凋亡。因此，我们通过检测Bcl-2与Bax的表达量变化来比较两者的比值变化，从而判断凋亡的发生。The full name of Bcl-2 gene is B-lymphoma-2, and the Bcl-2 family is one of the most important regulators in regulating apoptosis-related genes. Both Bcl-2 and Bax are members of the Bcl-2 protein family. The former is an anti-apoptotic protein and the latter is a pro-apoptotic protein. It has been proved that the two mainly play a role in the mitochondrial apoptosis pathway. Bcl-2 regulates cell death by controlling mitochondrial membrane permeability, inhibits caspase activity by preventing cytochrome c release from mitochondria and/or binding to apoptosis activator (Apaf-1), thereby inhibiting cellular Apoptosis; while Bax can induce the release of cytochrome C and promote the activation of Caspase3, resulting in apoptosis. Bcl-2 can form dimer with Bax, if the expression of Bax is higher than that of Bcl-2, it will promote apoptosis; if the expression of Bcl-2 is higher than that of Bax, it will inhibit apoptosis. Therefore, we compared the changes of the ratio of Bcl-2 and Bax by detecting the expression changes of Bcl-2 and Bax, so as to judge the occurrence of apoptosis.

1、总蛋白提取1. Total protein extraction

(1)配制细胞裂解液(RIPA：Cocktail：PMSF＝100:1:1)置于冰上待用；(1) Prepare a cell lysate (RIPA: Cocktail: PMSF=100:1:1) and place it on ice for later use;

(2)12孔板培养细胞，抗体蛋白刺激结束后，吸弃培养基，冰冷的PBS洗涤细胞2次，随即将板置于冰上，每孔加入约100μl配制好的裂解液，使用移液枪反复轻柔吹打几次，确保裂解液与底面充分接触，冰上静置裂解30min；(2) Culture cells in a 12-well plate. After the antibody protein stimulation is over, aspirate the culture medium, wash the cells twice with ice-cold PBS, then place the plate on ice, add about 100 μl of the prepared lysate to each well, and use pipetting. Repeatedly and gently pipetting the gun several times to ensure that the lysate is in full contact with the bottom surface, and let it stand on ice for 30min;

(3)到达规定时间后，使用细胞刮刮下含有细胞碎片的裂解液，转移至干净EP管中，4℃，12000rpm离心15min；(3) After reaching the specified time, use a cell scraper to scrape off the lysate containing cell debris, transfer it to a clean EP tube, and centrifuge at 12,000 rpm for 15 min at 4°C;

(4)小心吸取上清液，即为细胞浆总蛋白质。立即进行后续实验，剩余保存于-80℃冰箱。(4) Carefully aspirate the supernatant, which is the total protein in the cytoplasm. Follow-up experiments were performed immediately, and the remainder were stored in a -80°C refrigerator.

2、BCA蛋白浓度测定2. Determination of BCA protein concentration

(1)按照BCA浓度检测试剂盒说明书配制BCA工作液备用：A液：B液＝50:1，(1) Prepare BCA working solution according to the instructions of the BCA concentration detection kit for use: A solution: B solution = 50:1,

(2)稀释2mg/ml标准品至终浓度0.5mg/ml备用，(2) Dilute the 2mg/ml standard to a final concentration of 0.5mg/ml for later use,

(3)如下设置反应体系，包括标准曲线孔与实验样品孔；(3) The reaction system is set as follows, including the standard curve hole and the experimental sample hole;

表2Table 2

Figure BDA0002585061130000191
Figure BDA0002585061130000191

(4)在96孔板中按顺序依次加入各组试剂，并通过200μl移液枪轻轻混匀每孔试剂，样品组每组重复设置三个副孔；(4) Add each group of reagents in sequence to the 96-well plate, and gently mix the reagents in each well with a 200 μl pipette, and set three sub-wells for each sample group repeatedly;

(5)将孔板放入预热的37℃恒温孵箱孵育30min，结束后取出立即使用酶标仪检测570nm处的吸光度值；(5) Put the well plate into a preheated 37°C constant temperature incubator and incubate for 30min, take it out immediately after the end, and use a microplate reader to detect the absorbance value at 570nm;

(6)根据吸光度值建立标准曲线，根据标准曲线计算每孔蛋白浓度。(6) Establish a standard curve according to the absorbance value, and calculate the protein concentration per well according to the standard curve.

3、免疫印迹3. Immunoblotting

将各蛋白样品加热变性，根据浓度的不同计算上样体积，保证每孔上样量一致，按照如上Western blot方法完成蛋白质印迹相关实验步骤，并作灰度分析。The protein samples were heated and denatured, and the sample volume was calculated according to the different concentrations to ensure the same amount of samples loaded in each well. The relevant experimental steps of western blotting were completed according to the above Western blot method, and grayscale analysis was performed.

三、结果与分析3. Results and Analysis

如图7所示，四种融合蛋白(scFv、Cytc-scFv、3Cytc-scFv及DFF40-scFv)分别作用于SK-BR-3细胞后，提取总蛋白，检测凋亡抑制蛋白Bcl-2以及凋亡促进蛋白Bax的含量变化，以α-tubulin作为内参，结果如图7A所示；如图7B所示，四种融合蛋白(scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv)分别作用于SK-BR-3细胞，经ImageLab定量，Bcl-2/Bax比值：scFv>Cytc-scFv>3Cytc-scFv>DFF40-scFv。As shown in Figure 7, after the four fusion proteins (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) acted on SK-BR-3 cells respectively, the total proteins were extracted, and the apoptosis inhibitory proteins Bcl-2 and apoptosis were detected. The changes in the content of the apoptosis-promoting protein Bax, using α-tubulin as an internal reference, the results are shown in Figure 7A; as shown in Figure 7B, four fusion proteins (scFv, Cytc–scFv, 3Cytc–scFv and DFF40-scFv) acted on the SK-BR-3 cells, quantified by ImageLab, Bcl-2/Bax ratio: scFv>Cytc-scFv>3Cytc-scFv>DFF40-scFv.

由此可见，与凋亡蛋白DFF40融合的scFv(DFF40-scFv)具有最好的凋亡诱导活性，而三个Cytc串联融合的scFv(3Cytc-scFv)效果优于单个Cytc融合的scFv(Cytc-scFv)。It can be seen that the scFv fused with the apoptosis protein DFF40 (DFF40-scFv) has the best apoptosis-inducing activity, while the scFv fused in series with three Cytc (3Cytc-scFv) is more effective than the scFv fused with a single Cytc (Cytc-scFv) scFv).

实施例8：形态学观察与scFv、Cytc–scFv、3Cytc–scFv及DFF40-scFv融合型单链抗体共孵育后HER-2高表达型乳腺癌细胞SK-BR-3凋亡特征Example 8: Morphological observation of apoptosis characteristics of SK-BR-3 breast cancer cells with high expression of HER-2 after co-incubation with scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv fusion single-chain antibodies

一、实验材料1. Experimental materials

Hoechst染色液购自北京索莱宝；人乳腺癌细胞系SK-BR-3、MDA-MB-231以及MCF-7来源同实施例4；Hoechst staining solution was purchased from Beijing Soleibo; the sources of human breast cancer cell lines SK-BR-3, MDA-MB-231 and MCF-7 were the same as those in Example 4;

二、凋亡蛋白表达量分析2. Analysis of apoptotic protein expression

Hoechst是一种蓝色荧光染料，可标记DNA，由于细胞发生凋亡时，染色质会固缩，故凋亡细胞与正常细胞相比细胞核会有明显差异。Hoechst is a blue fluorescent dye that can label DNA. When cells undergo apoptosis, chromatin will shrink, so the nucleus of apoptotic cells will be significantly different from normal cells.

1、Hoechst核染色1. Hoechst nuclear staining

(1)将干净的爬片置于六孔板中，接种细胞并生长过夜，使其充满约50％-80％；(1) Place the clean slides in a six-well plate, inoculate cells and grow overnight to fill them with about 50%-80%;

(2)融合蛋白(scFv、Cytc-scFv、3Cytc-scFv或DFF40-scFv)刺激细胞凋亡后，吸出培养液，加入0.5ml固定液，固定10min；(2) After the fusion protein (scFv, Cytc-scFv, 3Cytc-scFv or DFF40-scFv) stimulates cell apoptosis, aspirate the culture medium, add 0.5 ml of fixative, and fix for 10 minutes;

(3)除去固定液并用PBS清洗两次，然后加入0.5ml Hoechst 33258染色溶液并室温摇床染色5min；(3) Remove the fixative and wash twice with PBS, then add 0.5ml Hoechst 33258 staining solution and shake at room temperature for 5min;

(4)除去染色液，用PBS洗涤，然后在载玻片上滴一滴抗荧光淬灭剂，盖上爬片，使细胞面接触载玻片，并尽可能避免气泡。随即在共聚焦荧光显微镜下进行观察，激发波长约为350nm。(4) Remove the staining solution, wash with PBS, then drop a drop of anti-fluorescence quencher on the slide, cover the slide, make the cell surface touch the slide, and avoid air bubbles as much as possible. It was then observed under a confocal fluorescence microscope with an excitation wavelength of about 350 nm.

2、细胞长时间成像观察2. Long-term imaging observation of cells

SK-BR-3细胞在活细胞工作站环境中与不同的蛋白构建体(scFv、Cytc-scFv、3Cytc-scFv及DFF40-scFv)相互作用48小时，在此期间使用20×放大倍数显微镜头连续记录细胞生长过程中的所有形态变化。比较0h，24h和48h细胞形态的差异，以评估细胞凋亡的程度并进行定量分析。SK-BR-3 cells interacted with different protein constructs (scFv, Cytc-scFv, 3Cytc-scFv, and DFF40-scFv) in a live cell workstation environment for 48 hours, during which time were continuously recorded using a 20× magnification microscope head All morphological changes during cell growth. The differences in cell morphology at 0h, 24h and 48h were compared to evaluate the degree of apoptosis and perform quantitative analysis.

三、结果与分析3. Results and Analysis

如图8A为SK-BR-3细胞在Hoechst染核后拍摄的荧光图像，可见scFv作用下细胞核仍然呈正常的蓝色，而DFF40-scFv作用下细胞核呈碎块状致密浓染，颜色发白(图中亮色)，相比之下，3Cytc-scFv颜色也出现发白但程度不及DFF40-scFv，Cytc-scFv则是稍有发白不是很明显；以上结果表明，凋亡蛋白融合型单链抗体作用后，由于每种蛋白诱导凋亡效价的不同，细胞出现了不同程度的核固缩。Figure 8A is the fluorescence image of SK-BR-3 cells after Hoechst staining. It can be seen that under the action of scFv, the nucleus is still normal blue, while under the action of DFF40-scFv, the nucleus is dense and densely stained, and the color is whitish. (bright color in the picture), in contrast, the color of 3Cytc-scFv also appears whitish but not as much as DFF40-scFv, while Cytc-scFv is slightly whitish, which is not very obvious; the above results show that the fusion-type single chain of apoptotic protein After the antibody was applied, the cells showed different degrees of nuclear pyknosis due to the different apoptosis-inducing potency of each protein.

图8B为活细胞长时间动态影像成像分析仪延时拍摄图像，记录了抗体蛋白与SK-BR-3细胞共孵育后48h内的生长变化情况，可以看到，scFv作用后，24h、48h细胞形态均完整，未出现固缩，表面不出泡，没有形成凋亡小体；相比之下，DFF40-scFv作用下，24h细胞体积明显减小，细胞固缩，48h再观察可见细胞表面起泡，形成凋亡小体，呈碎片状；3Cytc-scFv作用下，24h胞质致密，48h也起泡并且出现凋亡小体；最后，Cytc-scFv作用后的细胞24h形态完好，48h才观察到细胞固缩。Figure 8B is a time-lapse image of a long-term dynamic image imaging analyzer of living cells, which records the growth changes within 48 hours after co-incubating antibody protein with SK-BR-3 cells. The morphology was complete, no pyknosis appeared, no bleb on the surface, and no apoptotic bodies were formed; in contrast, under the action of DFF40-scFv, the volume of the cells was significantly reduced at 24 hours, and the cells were pyknotic. vesicles formed apoptotic bodies, which were fragmented; under the action of 3Cytc-scFv, the cytoplasm was dense at 24h, and blebbed and apoptotic bodies appeared at 48h; finally, cells treated with Cytc-scFv were in good shape at 24h, and only observed at 48h to cell pyknosis.

图9A-D为不同蛋白(scFv、Cytc-scFv、3Cytc-scFv及DFF40-scFv)与三种乳腺癌细胞(SK-BR-3、MDA-MB-231、MCF-7)共孵育48h后的凋亡率统计，SK-BR-3细胞的作用效果与图8B所描述一致；对于用作对照的HER-2阴性细胞MDA-MB-231以及MCF-7来说，在四种抗体蛋白诱导后均未发生显著凋亡。Figure 9A-D shows the co-incubation of different proteins (scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv) with three breast cancer cells (SK-BR-3, MDA-MB-231, MCF-7) for 48h Apoptosis rate statistics, the effect of SK-BR-3 cells is consistent with that described in Figure 8B; for HER-2 negative cells MDA-MB-231 and MCF-7 used as controls, after induction of four antibody proteins No significant apoptosis occurred.

综上，凋亡蛋白融合型scFv可有效诱导HER-2高表达的靶细胞凋亡，且DFF40-scFv活性最强，起效时间最快，3Cytc-scFv次之，Cytc-scFv活性较低，48h才检测到少量凋亡现象，而这些凋亡蛋白融合型scFv对于表面不含特异性抗原的细胞则没有作用。In conclusion, the apoptosis protein fusion scFv can effectively induce the apoptosis of target cells with high expression of HER-2, and DFF40-scFv has the strongest activity and the fastest onset time, followed by 3Cytc-scFv, and the activity of Cytc-scFv is lower. A small amount of apoptosis was detected only after 48h, and these apoptotic protein fusion scFv had no effect on cells without specific antigens on the surface.

实施例9：流式细胞术检测到经融合蛋白作用后，HER-2高表达乳腺癌细胞SK-BR-3显著凋亡Example 9: Flow cytometry detected significant apoptosis of SK-BR-3 breast cancer cells with high expression of HER-2 after the effect of fusion protein

一、实验材料1. Experimental materials

Annexin V-FITC/PI双染凋亡检测试剂盒购自南京建成生物工程研究所；人乳腺癌细胞系SK-BR-3、MDA-MB-231以及MCF-7来源同实施例4；Annexin V-FITC/PI double staining apoptosis detection kit was purchased from Nanjing Jiancheng Bioengineering Institute; the sources of human breast cancer cell lines SK-BR-3, MDA-MB-231 and MCF-7 were the same as those in Example 4;

二、流式细胞术2. Flow cytometry

(1)以scFv作用组作为对照组，比较凋亡蛋白融合型单链抗体作用组(实验组，Cytc-scFv、3Cytc-scFv或DFF40-scFv)的促凋亡活性。在六孔板中培养细胞，融合蛋白(scFv、Cytc-scFv、3Cytc-scFv或DFF40-scFv)凋亡刺激48h后把细胞培养液吸出至一合适离心管内，PBS洗涤细胞3次，用不含EDTA的胰酶消化细胞；(1) The scFv-treated group was used as the control group to compare the pro-apoptotic activity of the apoptotic protein fusion single-chain antibody-treated group (experimental group, Cytc-scFv, 3Cytc-scFv or DFF40-scFv). The cells were cultured in a six-well plate, and the fusion protein (scFv, Cytc-scFv, 3Cytc-scFv or DFF40-scFv) was stimulated for apoptosis for 48 hours, and the cell culture medium was aspirated into a suitable centrifuge tube. Trypsinized cells with EDTA;

(2)细胞消化下来后，加入步骤(1)中收集的细胞培养液，稍混匀，转移到新的干净离心管内，1000g离心5min，弃上清，用PBS轻轻重悬细胞并计数；(2) After the cells are digested, add the cell culture solution collected in step (1), mix slightly, transfer to a new clean centrifuge tube, centrifuge at 1000 g for 5 min, discard the supernatant, and gently resuspend the cells with PBS and count;

(3)取1.5×105重悬的细胞，1000g离心5min，弃上清，加入500μl结合液(来自Annexin V-FITC/PI双染凋亡检测试剂盒)轻轻重悬细胞，(3) Take 1.5×10 5 resuspended cells, centrifuge at 1000g for 5 min, discard the supernatant, add 500 μl of binding solution (from Annexin V-FITC/PI double staining apoptosis detection kit) and gently resuspend the cells,

(4)先加入5μl Annexin V-FITC，轻轻混匀再加入5μl碘化丙啶(Propidiumiodide，PI)，轻轻混匀，(4) First add 5 μl Annexin V-FITC, mix gently, then add 5 μl propidium iodide (PI), mix gently,

(5)室温(20-25℃)避光(锡纸包被)孵育10min。随即进行流式细胞仪检测，Annexin V-FITC为绿色荧光，PI为红色荧光。流式象限图右侧两区域显示为凋亡/坏死细胞。(5) Incubate for 10 min at room temperature (20-25°C) in the dark (coated with tin foil). Then, flow cytometry was performed, Annexin V-FITC was green fluorescence, and PI was red fluorescence. The two regions on the right side of the flow quadrant show apoptotic/necrotic cells.

三、结果与分析3. Results and Analysis

如下图10A所示，以FITC为横坐标，PI为纵坐标生成流式图谱，第一纵列为scFv分别作用于三种乳腺癌细胞系的流式象限图，细胞类型基本都分布在左下象限，即AnnexinV-/PI-，细胞正常生长，鲜少细胞发生凋亡；第二纵列为DFF40-scFv作用结果，可见HER-2高表达细胞SK-BR-3在DFF40-scFv作用下右下象限Annexin V+/PI-以及右上象限Annexin V+/PI+的细胞总和达到50％，即50％的细胞出现了不同程度的凋亡；用于对照的HER-2阴性细胞MDA-MB-231、MCF-7没有出现凋亡；第三纵列为Cytc-scFv作用结果，Cytc-scFv作用下，除SK-BR-3细胞有大概15％的凋亡率外，用于对照的HER-2阴性细胞MDA-MB-231、MCF-7没有出现凋亡；最右一列为3Cytc-scFv作用结果，SK-BR-3细胞的凋亡比率约是Cytc-scFv的两倍，同样，MDA-MB-231、MCF-7细胞没有出现凋亡。As shown in Figure 10A below, the flow chart is generated with FITC as the abscissa and PI as the ordinate. The first column is the flow quadrant of scFv acting on three breast cancer cell lines, and the cell types are basically distributed in the lower left quadrant. , AnnexinV - /PI - , the cells grow normally, and few cells undergo apoptosis; the second column is the result of DFF40-scFv effect, it can be seen that the HER-2 high-expressing cells SK-BR-3 are under the effect of DFF40-scFv in the lower right The sum of the cells of Annexin V + /PI - and Annexin V + /PI + in the upper right quadrant reaches 50%, that is, 50% of the cells have different degrees of apoptosis; HER-2 negative cells used for control MDA-MB-231 , MCF-7 did not undergo apoptosis; the third column is the result of Cytc-scFv effect, under the effect of Cytc-scFv, except SK-BR-3 cells had about 15% apoptosis rate, the control HER-2 Negative cells MDA-MB-231 and MCF-7 did not undergo apoptosis; the rightmost column is the result of 3Cytc-scFv effect. The apoptosis rate of SK-BR-3 cells is about twice that of Cytc-scFv. Similarly, MDA-MB -231, MCF-7 cells did not appear apoptosis.

图10B为10A中的凋亡数据的量化结果图，与象限图所示结果一致。Figure 10B is a graph of the quantification results of the apoptosis data in 10A, consistent with the results shown in the quadrant plot.

综上，根据以上流式细胞术实验结果，我们可以得出与前述体外实验一致的结论，即凋亡蛋白融合型scFv相比于单纯scFv可有效诱导HER-2高表达的靶细胞凋亡，对于表面不含特异性抗原的细胞没有作用，且活性DFF40-scFv>3Cytc-scFv>Cytc-scFv。In summary, based on the above results of flow cytometry experiments, we can draw the same conclusion as the previous in vitro experiments, that is, compared with pure scFv, apoptotic protein fusion scFv can effectively induce apoptosis of target cells with high expression of HER-2, No effect on cells without specific antigen on the surface, and active DFF40-scFv>3Cytc-scFv>Cytc-scFv.

实施例10：凋亡蛋白融合型抗HER-2单链抗体的体内活性验证Example 10: In vivo activity verification of apoptotic protein fusion anti-HER-2 single chain antibody

前述多种体外实验充分验证了融合型单链抗体的靶向抗肿瘤活性，紧接着我们通过建立动物模型来探讨融合蛋白在体内是否能发挥与体外一致活性。The aforementioned various in vitro experiments have fully verified the targeted anti-tumor activity of the fusion single-chain antibody. Next, we established an animal model to explore whether the fusion protein can exert the same activity in vivo as in vitro.

一、材料与试剂1. Materials and reagents

Tunel细胞凋亡原位检测试剂盒购自南京建成生物工程研究所；人乳腺癌细胞系SK-BR-3购自美国模式培养物集存库(American type culture collection,ATCC)；动物：20只6周龄的BALB/C雌性裸鼠，体重16-18g，由南京大学模式动物研究中心提供；且其严格按照国家动物实验操作规范进行饲养与解剖，并获得南京中医药大学动物伦理委员会批准。Tunel cell apoptosis in situ detection kit was purchased from Nanjing Jiancheng Bioengineering Institute; human breast cancer cell line SK-BR-3 was purchased from American type culture collection (ATCC); animals: 20 6-week-old BALB/C female nude mice, weighing 16-18 g, were provided by the Model Animal Research Center of Nanjing University; they were reared and dissected in strict accordance with the National Animal Experimentation Operation Standard, and were approved by the Animal Ethics Committee of Nanjing University of Traditional Chinese Medicine.

二、实验方法2. Experimental method

1、建立裸鼠异种移植模型1. Establishment of nude mouse xenograft model

(1)75mm2培养瓶大量培养SK-BR-3乳腺癌细胞，消化后重悬于DMEM中，与基质胶(4℃)4:1混合用于肿瘤细胞种植，(1) SK-BR-3 breast cancer cells were cultured in large quantities in 75mm 2 culture flasks, resuspended in DMEM after digestion, mixed with Matrigel (4°C) 4:1 for tumor cell seeding,

(2)在六周龄的雌性裸鼠右上侧腋下皮下接种1×107个SK-BR-3细胞；(2) Subcutaneously inoculate 1×10 7 SK-BR-3 cells in the upper right armpit of six-week-old female nude mice;

(3)当肿瘤达到平均体积约50mm3时，将小鼠随机分为四组，分别为scFv作用组、DFF40-scFv作用组、3Cytc-scFv作用组和Cytc-scFv作用组，每组5只；( 3 ) When the tumor reached an average volume of about 50 mm, the mice were randomly divided into four groups, namely the scFv-treated group, the DFF40-scFv-treated group, the 3Cytc-scFv-treated group and the Cytc-scFv-treated group, with 5 mice in each group ;

(4)对上述各组小鼠每三天腹腔注射(10mg/kg)融合蛋白进行治疗，使用卡尺每三天测量肿瘤大小，并根据标准公式：V(mm3)＝width2(mm2)×length(mm)/2计算肿瘤体积；(4) The mice in the above groups were treated by intraperitoneal injection (10 mg/kg) of fusion protein every three days, and the tumor size was measured every three days using a caliper, and according to the standard formula: V (mm 3 )=width 2 (mm 2 ) ×length(mm)/2 to calculate tumor volume;

(5)在第30天终止实验，安乐死处死小鼠，并切除肿瘤拍照、称重。(5) The experiment was terminated on the 30th day, the mice were euthanized, and the tumor was excised to take pictures and weigh.

2、Tunel染色2. Tunel staining

将分离出来的肿瘤制作成石蜡切片，使用Tunel细胞凋亡原位检测试剂盒进行Tunel染色，然后于光学显微镜下观察拍照(凋亡细胞显示为深棕色)，分析肿瘤组织的凋亡情况。The isolated tumors were made into paraffin sections, which were stained with Tunel cell apoptosis in situ detection kit, and then observed and photographed under a light microscope (apoptotic cells were shown as dark brown) to analyze the apoptosis of tumor tissue.

三、结果与分析3. Results and Analysis

如图11A所示，每3天监测肿瘤体积变化，scFv作用组的小鼠肿瘤体积逐日增加，肿瘤生长没有受到抑制；DFF40-scFv作用组、3Cytc-scFv作用组和Cytc-scFv作用组作用下的小鼠肿瘤体积增长幅度出现不同程度的减小，其中，DFF40-scFv作用组肿瘤生长受到较大抑制，蛋白注射治疗期间肿瘤体积的变化微乎其微；3Cytc-scFv作用组肿瘤同样受到抑制，体积较DFF40-scFv作用组略大一点，而Cytc-scFv作用组肿瘤生长明显，相比对照组scFv，受到轻微抑制。实验结束后分离出瘤体，将每组肿瘤排列整齐留存照片进行对比，可以看出肿瘤体积与终质量的差异与实验期间记录的一致(图11B、图11C)。As shown in Figure 11A, the changes in tumor volume were monitored every 3 days. The tumor volume of the mice in the scFv-treated group increased day by day, but the tumor growth was not inhibited; The growth rate of tumor volume in mice treated with 3Cytc-scFv decreased to varying degrees. Among them, the tumor growth in the DFF40-scFv group was greatly inhibited, and the change in tumor volume during protein injection treatment was minimal; the tumor in the 3Cytc-scFv group was also inhibited, and the volume was smaller The DFF40-scFv-treated group was slightly larger, while the Cytc-scFv-treated group had obvious tumor growth, which was slightly inhibited compared with the control group scFv. After the experiment, the tumors were isolated, and the tumors in each group were arranged neatly and kept in photos for comparison. It can be seen that the difference between the tumor volume and the final mass was consistent with that recorded during the experiment ( FIG. 11B , FIG. 11C ).

如图12所示的肿瘤组织石蜡切片的Tunel染色结果，scFv作用组小鼠肿瘤细胞没有检测到凋亡，相比之下，DFF40-scFv作用组小鼠凋亡显著，3Cytc-scFv作用组凋亡率次之，而Cytc-scFv仅出现少量凋亡。As shown in Figure 12, the results of Tunel staining of paraffin sections of tumor tissue showed that no apoptosis was detected in the tumor cells of the mice in the scFv-treated group. The mortality rate was second, and Cytc-scFv showed only a small amount of apoptosis.

综上，裸鼠异种移植模型在动物水平上成功验证了凋亡蛋白融合型抗HER-2单链抗体体内能有效靶向HER-2过表达型乳腺癌肿瘤并诱导肿瘤细胞凋亡，且三种凋亡蛋白融合型抗HER-2单链抗体(DFF40-scFv、3Cytc-scFv和Cytc-scFv)的活性与体外试验一致；由此我们可以得出：将促凋亡蛋白与单链抗体融合表达用于癌症的治疗可实现靶向性与抗肿瘤活性的良好结合，为癌症的靶向免疫治疗提供新的思路。In conclusion, the nude mouse xenograft model successfully verified that the apoptotic protein fusion anti-HER-2 single-chain antibody can effectively target HER-2 overexpressing breast cancer tumors and induce tumor cell apoptosis at the animal level. The activities of the anti-HER-2 single-chain antibodies (DFF40-scFv, 3Cytc-scFv and Cytc-scFv) fused to apoptotic proteins were consistent with the in vitro experiments; from this we can conclude that the fusion of pro-apoptotic proteins with single-chain antibodies Expression for cancer therapy can achieve a good combination of targeting and anti-tumor activity, providing new ideas for cancer-targeted immunotherapy.

以上仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，应视为本发明的保护范围。The above are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions that belong to the idea of the present invention belong to the protection scope of the present invention. It should be pointed out that for those skilled in the art, some improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlyGlu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 151 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp ThrSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30 20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValTyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45 35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser ValAla Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60 50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala TyrLys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 8065 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr CysLeu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95 85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly GlnSer Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110 100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly GlyGly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125 115 120 125

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro SerGly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser

130 135 140 130 135 140

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg AlaSer Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala

145 150 155 160145 150 155 160

Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro GlySer Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly

165 170 175 165 170 175

Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser GlyLys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly

180 185 190 180 185 190

Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr LeuVal Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu

195 200 205 195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys GlnThr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln

210 215 220 210 215 220

Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val GluGln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu

225 230 235 240225 230 235 240

Ile Lys ArgIle Lys Arg

<210> 5<210> 5

<211> 729<211> 729

<212> DNA<212> DNA

<213> 人工序列 (Artificial Sequence)<213> Artificial Sequence

<400> 5<400> 5

gaagttcagc tggttgaatc tggtggtggt ctggttcagc cgggtggttc tctgcgtctg 60gaagttcagc tggttgaatc tggtggtggt ctggttcagc cgggtggttc tctgcgtctg 60

tcttgcgctg cttctggttt caacatcaaa gacacctaca tccactgggt tcgtcaggct 120tcttgcgctg cttctggttt caacatcaaa gacacctaca tccactgggt tcgtcaggct 120

ccgggtaaag gtctggaatg ggttgctcgt atctacccga ccaacggtta cacccgttac 180ccgggtaaag gtctggaatg ggttgctcgt atctacccga ccaacggtta cacccgttac 180

gctgactctg ttaaaggtcg tttcaccatc tctgctgaca cctctaaaaa caccgcttac 240gctgactctg ttaaaggtcg tttcaccatc tctgctgaca cctctaaaaa caccgcttac 240

ctgcagatga actctctgcg tgctgaagac accgctgttt actactgctc tcgttggggt 300ctgcagatga actctctgcg tgctgaagac accgctgttt actactgctc tcgttggggt 300

ggtgacggtt tctacgctat ggactactgg ggtcagggta ccctggttac cgtttcttct 360ggtgacggtt tctacgctat ggactactgg ggtcagggta ccctggttac cgtttcttct 360

gggggcgggg gctctggggg cgggggctct gggggcgggg gctctgatat tcagatgacc 420gggggcgggg gctctggggg cgggggctct gggggcgggg gctctgatat tcagatgacc 420

cagagcccga gcagcctgag cgcgagcgtg ggcgatcgcg tgaccattac ctgccgcgcg 480cagagcccga gcagcctgag cgcgagcgtg ggcgatcgcg tgaccattac ctgccgcgcg 480

agccaggatg tgaacaccgc ggtggcgtgg tatcagcaga aaccgggcaa agcgccgaaa 540agccaggatg tgaacaccgc ggtggcgtgg tatcagcaga aaccgggcaa agcgccgaaa 540

ctgctgattt atagcgcgag ctttctgtat agcggcgtgc cgagccgctt tagcggcagc 600ctgctgattt atagcgcgag ctttctgtat agcggcgtgc cgagccgctt tagcggcagc 600

cgcagcggca ccgattttac cctgaccatt agcagcctgc agccggaaga ttttgcgacc 660cgcagcggca ccgattttac cctgaccatt agcagcctgc agccggaaga ttttgcgacc 660

tattattgcc agcagcatta taccaccccg ccgacctttg gccagggcac caaagtggaa 720tattattgcc agcagcatta taccaccccg ccgacctttg gccagggcac caaagtggaa 720

attaaacgc 729attaaacgc 729

<210> 6<210> 6

<211> 105<211> 105

<212> PRT<212> PRT

<213> 人工序列 (Artificial Sequence)<213> Artificial Sequence

<400> 6<400> 6

Met Gly Asp Val Glu Lys Gly Lys Lys Ile Phe Ile Met Lys Cys SerMet Gly Asp Val Glu Lys Gly Lys Lys Ile Phe Ile Met Lys Cys Ser

1 5 10 151 5 10 15

Gln Cys His Thr Val Glu Lys Gly Gly Lys His Lys Thr Gly Pro AsnGln Cys His Thr Val Glu Lys Gly Gly Lys His Lys Thr Gly Pro Asn

20 25 30 20 25 30

Leu His Gly Leu Phe Gly Arg Lys Thr Gly Gln Ala Pro Gly Tyr SerLeu His Gly Leu Phe Gly Arg Lys Thr Gly Gln Ala Pro Gly Tyr Ser

35 40 45 35 40 45

Tyr Thr Ala Ala Asn Lys Asn Lys Gly Ile Ile Trp Gly Glu Asp ThrTyr Thr Ala Ala Asn Lys Asn Lys Gly Ile Ile Trp Gly Glu Asp Thr

50 55 60 50 55 60

Leu Met Glu Tyr Leu Glu Asn Pro Lys Lys Tyr Ile Pro Gly Thr LysLeu Met Glu Tyr Leu Glu Asn Pro Lys Lys Tyr Ile Pro Gly Thr Lys

65 70 75 8065 70 75 80

Met Ile Phe Val Gly Ile Lys Lys Lys Glu Glu Arg Ala Asp Leu IleMet Ile Phe Val Gly Ile Lys Lys Lys Glu Glu Arg Ala Asp Leu Ile

85 90 95 85 90 95

Ala Tyr Leu Lys Lys Ala Thr Asn GluAla Tyr Leu Lys Lys Ala Thr Asn Glu

100 105 100 105

<210> 7<210> 7

<211> 315<211> 315

<212> DNA<212> DNA

<213> 人工序列 (Artificial Sequence)<213> Artificial Sequence

<400> 7<400> 7

atgggtgatg ttgagaaagg caagaagatt tttattatga agtgttccca gtgccacacc 60atgggtgatg ttgagaaagg caagaagatt tttattatga agtgttccca gtgccacacc 60

gttgaaaagg gaggcaagca caagactggg ccaaatctcc acggtctctt tgggcggaag 120gttgaaaagg gaggcaagca caagactggg ccaaatctcc acggtctctt tgggcggaag 120

acaggtcagg cccctggata ctcttacaca gccgccaata agaacaaagg catcatctgg 180acaggtcagg cccctggata ctcttacaca gccgccaata agaacaaagg catcatctgg 180

ggagaggata cactgatgga gtatttggag aatcccaaga agtacatccc tggaacaaaa 240ggagaggata cactgatgga gtatttggag aatcccaaga agtacatccc tggaacaaaa 240

atgatctttg tcggcattaa gaagaaggaa gaaagggcag acttaatagc ttatctcaaa 300atgatctttg tcggcattaa gaagaaggaa gaaagggcag acttaatagc ttatctcaaa 300

aaagctacta atgag 315aaagcacta atgag 315

<210> 8<210> 8

<211> 338<211> 338

<212> PRT<212> PRT

<213> 人工序列 (Artificial Sequence)<213> Artificial Sequence

<400> 8<400> 8

Met Leu Gln Lys Pro Lys Ser Val Lys Leu Arg Ala Leu Arg Ser ProMet Leu Gln Lys Pro Lys Ser Val Lys Leu Arg Ala Leu Arg Ser Pro

1 5 10 151 5 10 15

Arg Lys Phe Gly Val Ala Gly Arg Ser Cys Gln Glu Val Leu Arg LysArg Lys Phe Gly Val Ala Gly Arg Ser Cys Gln Glu Val Leu Arg Lys

20 25 30 20 25 30

Gly Cys Leu Arg Phe Gln Leu Pro Glu Arg Gly Ser Arg Leu Cys LeuGly Cys Leu Arg Phe Gln Leu Pro Glu Arg Gly Ser Arg Leu Cys Leu

35 40 45 35 40 45

Tyr Glu Asp Gly Thr Glu Leu Thr Glu Asp Tyr Phe Pro Ser Val ProTyr Glu Asp Gly Thr Glu Leu Thr Glu Asp Tyr Phe Pro Ser Val Pro

50 55 60 50 55 60

Asp Asn Ala Glu Leu Val Leu Leu Thr Leu Gly Gln Ala Trp Gln GlyAsp Asn Ala Glu Leu Val Leu Leu Thr Leu Gly Gln Ala Trp Gln Gly

65 70 75 8065 70 75 80

Tyr Val Ser Asp Ile Arg Arg Phe Leu Ser Ala Phe His Glu Pro GlnTyr Val Ser Asp Ile Arg Arg Phe Leu Ser Ala Phe His Glu Pro Gln

85 90 95 85 90 95

Val Gly Leu Ile Gln Ala Ala Gln Gln Leu Leu Cys Asp Glu Gln AlaVal Gly Leu Ile Gln Ala Ala Gln Gln Leu Leu Cys Asp Glu Gln Ala

100 105 110 100 105 110

Pro Gln Arg Gln Arg Leu Leu Ala Asp Leu Leu His Asn Val Ser GlnPro Gln Arg Gln Arg Leu Leu Ala Asp Leu Leu His Asn Val Ser Gln

115 120 125 115 120 125

Asn Ile Ala Ala Glu Thr Arg Ala Glu Asp Pro Pro Trp Phe Glu GlyAsn Ile Ala Ala Glu Thr Arg Ala Glu Asp Pro Pro Trp Phe Glu Gly

130 135 140 130 135 140

Leu Glu Ser Arg Phe Gln Ser Lys Ser Gly Tyr Leu Arg Tyr Ser CysLeu Glu Ser Arg Phe Gln Ser Lys Ser Gly Tyr Leu Arg Tyr Ser Cys

145 150 155 160145 150 155 160

Glu Ser Arg Ile Arg Ser Tyr Leu Arg Glu Val Ser Ser Tyr Pro SerGlu Ser Arg Ile Arg Ser Tyr Leu Arg Glu Val Ser Ser Tyr Pro Ser

165 170 175 165 170 175

Thr Val Gly Ala Glu Ala Gln Glu Glu Phe Leu Arg Val Leu Gly SerThr Val Gly Ala Glu Ala Gln Glu Glu Phe Leu Arg Val Leu Gly Ser

180 185 190 180 185 190

Met Cys Gln Arg Leu Arg Ser Met Gln Tyr Asn Gly Ser Tyr Phe AspMet Cys Gln Arg Leu Arg Ser Met Gln Tyr Asn Gly Ser Tyr Phe Asp

195 200 205 195 200 205

Arg Gly Ala Lys Gly Gly Ser Arg Leu Cys Thr Pro Glu Gly Trp PheArg Gly Ala Lys Gly Gly Ser Arg Leu Cys Thr Pro Glu Gly Trp Phe

210 215 220 210 215 220

Ser Cys Gln Gly Pro Phe Asp Met Asp Ser Cys Leu Ser Arg His SerSer Cys Gln Gly Pro Phe Asp Met Asp Ser Cys Leu Ser Arg His Ser

225 230 235 240225 230 235 240

Ile Asn Pro Tyr Ser Asn Arg Glu Ser Arg Ile Leu Phe Ser Thr TrpIle Asn Pro Tyr Ser Asn Arg Glu Ser Arg Ile Leu Phe Ser Thr Trp

245 250 255 245 250 255

Asn Leu Asp His Ile Ile Glu Lys Lys Arg Thr Ile Ile Pro Thr LeuAsn Leu Asp His Ile Ile Glu Lys Lys Arg Thr Ile Ile Pro Thr Leu

260 265 270 260 265 270

Val Glu Ala Ile Lys Glu Gln Asp Gly Arg Glu Val Asp Trp Glu TyrVal Glu Ala Ile Lys Glu Gln Asp Gly Arg Glu Val Asp Trp Glu Tyr

275 280 285 275 280 285

Phe Tyr Gly Leu Leu Phe Thr Ser Glu Asn Leu Lys Leu Val His IlePhe Tyr Gly Leu Leu Phe Thr Ser Glu Asn Leu Lys Leu Val His Ile

290 295 300 290 295 300

Val Cys His Lys Lys Thr Thr His Lys Leu Asn Cys Asp Pro Ser ArgVal Cys His Lys Lys Thr Thr His Lys Leu Asn Cys Asp Pro Ser Arg

305 310 315 320305 310 315 320

Ile Tyr Lys Pro Gln Thr Arg Leu Lys Arg Lys Gln Pro Val Arg LysIle Tyr Lys Pro Gln Thr Arg Leu Lys Arg Lys Gln Pro Val Arg Lys

325 330 335 325 330 335

Arg GlnArg Gln

<210> 9<210> 9

<211> 1014<211> 1014

<212> DNA<212> DNA

<213> 人工序列 (Artificial Sequence)<213> Artificial Sequence

<400> 9<400> 9

atgctccaga agcccaagag cgtgaagctg cgggccctgc gcagcccgag gaagttcggc 60atgctccaga agcccaagag cgtgaagctg cgggccctgc gcagcccgag gaagttcggc 60

gtggctggcc ggagctgcca ggaggtgctg cgcaagggct gtctccgctt ccagctccct 120gtggctggcc ggagctgcca ggaggtgctg cgcaagggct gtctccgctt ccagctccct 120

gagcgcggtt cccggctgtg cctgtacgag gatggcacgg agctgacgga agattacttc 180gagcgcggtt cccggctgtg cctgtacgag gatggcacgg agctgacgga agattacttc 180

cccagtgttc ccgacaacgc cgagctggtg ctgctcacct tgggccaggc ctggcagggc 240cccagtgttc ccgacaacgc cgagctggtg ctgctcacct tgggccaggc ctggcagggc 240

tatgtgagcg acatcaggcg cttcctcagt gcatttcacg agccacaggt ggggctcatc 300tatgtgagcg acatcaggcg cttcctcagt gcatttcacg agccacaggt ggggctcatc 300

caggccgccc agcagctgct gtgtgatgag caggccccac agaggcagag gctgctggct 360caggccgccc agcagctgct gtgtgatgag caggccccac agaggcagag gctgctggct 360

gacctcctgc acaacgtcag ccagaacatc gcggccgaga cccgggctga ggacccgccg 420gacctcctgc acaacgtcag ccagaacatc gcggccgaga cccgggctga ggacccgccg 420

tggtttgaag gcttggagtc ccgatttcag agcaagtctg gctatctgag atacagctgt 480tggtttgaag gcttggagtc ccgatttcag agcaagtctg gctatctgag atacagctgt 480

gagagccgga tccggagtta cctgagggag gtgagctcct acccctccac ggtgggtgcg 540gagagccgga tccggagtta cctgagggag gtgagctcct acccctccac ggtgggtgcg 540

gaggctcagg aggaattcct gcgggtcctc ggctccatgt gccagaggct ccggtccatg 600gaggctcagg aggaattcct gcgggtcctc ggctccatgt gccagaggct ccggtccatg 600

cagtacaatg gcagctactt cgacagagga gccaagggcg gcagccgcct ctgcacaccg 660cagtacaatg gcagctactt cgacagagga gccaagggcg gcagccgcct ctgcacaccg 660

gaaggctggt tctcctgcca gggtcccttt gacatggaca gctgcttatc aagacactcc 720gaaggctggt tctcctgcca gggtcccttt gacatggaca gctgcttatc aagacactcc 720

atcaacccct acagtaacag ggagagcagg atcctcttca gcacctggaa cctggatcac 780atcaacccct acagtaacag ggagagcagg atcctcttca gcacctggaa cctggatcac 780

ataatagaaa agaaacgcac catcattcct acactggtgg aagcaattaa ggaacaagat 840ataatagaaa agaaacgcac catcattcct acactggtgg aagcaattaa ggaacaagat 840

ggaagagaag tggactggga gtatttttat ggcctgcttt ttacctcaga gaacctaaaa 900ggaagagaag tggactggga gtatttttat ggcctgcttt ttacctcaga gaacctaaaa 900

ctagtgcaca ttgtctgcca taagaaaacc acccacaagc tcaactgtga cccaagcaga 960ctagtgcaca ttgtctgcca taagaaaacc acccacaagc tcaactgtga cccaagcaga 960

atctacaaac cccagacaag gttgaagcgg aagcagcctg tgcggaaacg ccag 1014atctacaaac cccagacaag gttgaagcgg aagcagcctg tgcggaaacg ccag 1014

<210> 10<210> 10

<211> 351<211> 351

<212> PRT<212> PRT

<213> 人工序列 (Artificial Sequence)<213> Artificial Sequence

<400> 10<400> 10

Met Gly Asp Val Glu Lys Gly Lys Lys Ile Phe Ile Met Lys Cys SerMet Gly Asp Val Glu Lys Gly Lys Lys Ile Phe Ile Met Lys Cys Ser

1 5 10 151 5 10 15

Gln Cys His Thr Val Glu Lys Gly Gly Lys His Lys Thr Gly Pro AsnGln Cys His Thr Val Glu Lys Gly Gly Lys His Lys Thr Gly Pro Asn

20 25 30 20 25 30

Leu His Gly Leu Phe Gly Arg Lys Thr Gly Gln Ala Pro Gly Tyr SerLeu His Gly Leu Phe Gly Arg Lys Thr Gly Gln Ala Pro Gly Tyr Ser

35 40 45 35 40 45

Tyr Thr Ala Ala Asn Lys Asn Lys Gly Ile Ile Trp Gly Glu Asp ThrTyr Thr Ala Ala Asn Lys Asn Lys Gly Ile Ile Trp Gly Glu Asp Thr

50 55 60 50 55 60

Leu Met Glu Tyr Leu Glu Asn Pro Lys Lys Tyr Ile Pro Gly Thr LysLeu Met Glu Tyr Leu Glu Asn Pro Lys Lys Tyr Ile Pro Gly Thr Lys

65 70 75 8065 70 75 80

Met Ile Phe Val Gly Ile Lys Lys Lys Glu Glu Arg Ala Asp Leu IleMet Ile Phe Val Gly Ile Lys Lys Lys Glu Glu Arg Ala Asp Leu Ile

85 90 95 85 90 95

Ala Tyr Leu Lys Lys Ala Thr Asn Glu Thr Met Ala Glu Val Gln LeuAla Tyr Leu Lys Lys Ala Thr Asn Glu Thr Met Ala Glu Val Gln Leu

100 105 110 100 105 110

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg LeuVal Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

115 120 125 115 120 125

Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His TrpSer Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp

130 135 140 130 135 140

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile TyrVal Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr

145 150 155 160145 150 155 160

Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg PhePro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe

165 170 175 165 170 175

Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met AsnThr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn

180 185 190 180 185 190

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp GlySer Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly

195 200 205 195 200 205

Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu ValGly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val

210 215 220 210 215 220

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly GlyThr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

225 230 235 240225 230 235 240

Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser AlaGly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala

245 250 255 245 250 255

Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp ValSer Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val

260 265 270 260 265 270

Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro LysAsn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

275 280 285 275 280 285

Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser ArgLeu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg

290 295 300 290 295 300

Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser SerPhe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

305 310 315 320305 310 315 320

Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr ThrLeu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr

325 330 335 325 330 335

Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys ArgThr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

340 345 350 340 345 350

<210> 11<210> 11

<211> 12<211> 12

<212> DNA<212> DNA

<213> 人工序列 (Artificial Sequence)<213> Artificial Sequence

<400> 11<400> 11

gatgaagttg at 12gatgaagttg at 12

<210> 12<210> 12

<211> 584<211> 584

<212> PRT<212> PRT

<213> 人工序列 (Artificial Sequence)<213> Artificial Sequence

<400> 12<400> 12

Met Leu Gln Lys Pro Lys Ser Val Lys Leu Arg Ala Leu Arg Ser ProMet Leu Gln Lys Pro Lys Ser Val Lys Leu Arg Ala Leu Arg Ser Pro

1 5 10 151 5 10 15

Arg Lys Phe Gly Val Ala Gly Arg Ser Cys Gln Glu Val Leu Arg LysArg Lys Phe Gly Val Ala Gly Arg Ser Cys Gln Glu Val Leu Arg Lys

20 25 30 20 25 30

Gly Cys Leu Arg Phe Gln Leu Pro Glu Arg Gly Ser Arg Leu Cys LeuGly Cys Leu Arg Phe Gln Leu Pro Glu Arg Gly Ser Arg Leu Cys Leu

35 40 45 35 40 45

Tyr Glu Asp Gly Thr Glu Leu Thr Glu Asp Tyr Phe Pro Ser Val ProTyr Glu Asp Gly Thr Glu Leu Thr Glu Asp Tyr Phe Pro Ser Val Pro

50 55 60 50 55 60

Asp Asn Ala Glu Leu Val Leu Leu Thr Leu Gly Gln Ala Trp Gln GlyAsp Asn Ala Glu Leu Val Leu Leu Thr Leu Gly Gln Ala Trp Gln Gly

65 70 75 8065 70 75 80

Tyr Val Ser Asp Ile Arg Arg Phe Leu Ser Ala Phe His Glu Pro GlnTyr Val Ser Asp Ile Arg Arg Phe Leu Ser Ala Phe His Glu Pro Gln

85 90 95 85 90 95

Val Gly Leu Ile Gln Ala Ala Gln Gln Leu Leu Cys Asp Glu Gln AlaVal Gly Leu Ile Gln Ala Ala Gln Gln Leu Leu Cys Asp Glu Gln Ala

100 105 110 100 105 110

Pro Gln Arg Gln Arg Leu Leu Ala Asp Leu Leu His Asn Val Ser GlnPro Gln Arg Gln Arg Leu Leu Ala Asp Leu Leu His Asn Val Ser Gln

115 120 125 115 120 125

Asn Ile Ala Ala Glu Thr Arg Ala Glu Asp Pro Pro Trp Phe Glu GlyAsn Ile Ala Ala Glu Thr Arg Ala Glu Asp Pro Pro Trp Phe Glu Gly

130 135 140 130 135 140

Leu Glu Ser Arg Phe Gln Ser Lys Ser Gly Tyr Leu Arg Tyr Ser CysLeu Glu Ser Arg Phe Gln Ser Lys Ser Gly Tyr Leu Arg Tyr Ser Cys

145 150 155 160145 150 155 160

Glu Ser Arg Ile Arg Ser Tyr Leu Arg Glu Val Ser Ser Tyr Pro SerGlu Ser Arg Ile Arg Ser Tyr Leu Arg Glu Val Ser Ser Tyr Pro Ser

165 170 175 165 170 175

Thr Val Gly Ala Glu Ala Gln Glu Glu Phe Leu Arg Val Leu Gly SerThr Val Gly Ala Glu Ala Gln Glu Glu Phe Leu Arg Val Leu Gly Ser

180 185 190 180 185 190

Met Cys Gln Arg Leu Arg Ser Met Gln Tyr Asn Gly Ser Tyr Phe AspMet Cys Gln Arg Leu Arg Ser Met Gln Tyr Asn Gly Ser Tyr Phe Asp

195 200 205 195 200 205

Arg Gly Ala Lys Gly Gly Ser Arg Leu Cys Thr Pro Glu Gly Trp PheArg Gly Ala Lys Gly Gly Ser Arg Leu Cys Thr Pro Glu Gly Trp Phe

210 215 220 210 215 220

Ser Cys Gln Gly Pro Phe Asp Met Asp Ser Cys Leu Ser Arg His SerSer Cys Gln Gly Pro Phe Asp Met Asp Ser Cys Leu Ser Arg His Ser

225 230 235 240225 230 235 240

Ile Asn Pro Tyr Ser Asn Arg Glu Ser Arg Ile Leu Phe Ser Thr TrpIle Asn Pro Tyr Ser Asn Arg Glu Ser Arg Ile Leu Phe Ser Thr Trp

245 250 255 245 250 255

Asn Leu Asp His Ile Ile Glu Lys Lys Arg Thr Ile Ile Pro Thr LeuAsn Leu Asp His Ile Ile Glu Lys Lys Arg Thr Ile Ile Pro Thr Leu

260 265 270 260 265 270

Val Glu Ala Ile Lys Glu Gln Asp Gly Arg Glu Val Asp Trp Glu TyrVal Glu Ala Ile Lys Glu Gln Asp Gly Arg Glu Val Asp Trp Glu Tyr

275 280 285 275 280 285

Phe Tyr Gly Leu Leu Phe Thr Ser Glu Asn Leu Lys Leu Val His IlePhe Tyr Gly Leu Leu Phe Thr Ser Glu Asn Leu Lys Leu Val His Ile

290 295 300 290 295 300

Val Cys His Lys Lys Thr Thr His Lys Leu Asn Cys Asp Pro Ser ArgVal Cys His Lys Lys Thr Thr His Lys Leu Asn Cys Asp Pro Ser Arg

305 310 315 320305 310 315 320

Ile Tyr Lys Pro Gln Thr Arg Leu Lys Arg Lys Gln Pro Val Arg LysIle Tyr Lys Pro Gln Thr Arg Leu Lys Arg Lys Gln Pro Val Arg Lys

325 330 335 325 330 335

Arg Gln Thr Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly LeuArg Gln Thr Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

340 345 350 340 345 350

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly PheVal Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

355 360 365 355 360 365

Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly LysAsn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys

370 375 380 370 375 380

Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr ArgGly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg

385 390 395 400385 390 395 400

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr SerTyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser

405 410 415 405 410 415

Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp ThrLys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

420 425 430 420 425 430

Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala MetAla Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met

435 440 445 435 440 445

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly GlyAsp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

450 455 460 450 455 460

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln MetGly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

465 470 475 480465 470 475 480

Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val ThrThr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

485 490 495 485 490 495

Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp TyrIle Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr

500 505 510 500 505 510

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala SerGln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser

515 520 525 515 520 525

Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser GlyPhe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly

530 535 540 530 535 540

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe AlaThr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

545 550 555 560545 550 555 560

Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly GlnThr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln

565 570 575 565 570 575

Gly Thr Lys Val Glu Ile Lys ArgGly Thr Lys Val Glu Ile Lys Arg

580 580

<210> 13<210> 13

<211> 569<211> 569

<212> PRT<212> PRT

<213> 人工序列 (Artificial Sequence)<213> Artificial Sequence

<400> 13<400> 13

Met Gly Asp Val Glu Lys Gly Lys Lys Ile Phe Ile Met Lys Cys SerMet Gly Asp Val Glu Lys Gly Lys Lys Ile Phe Ile Met Lys Cys Ser

1 5 10 151 5 10 15

Gln Cys His Thr Val Glu Lys Gly Gly Lys His Lys Thr Gly Pro AsnGln Cys His Thr Val Glu Lys Gly Gly Lys His Lys Thr Gly Pro Asn

20 25 30 20 25 30

Leu His Gly Leu Phe Gly Arg Lys Thr Gly Gln Ala Pro Gly Tyr SerLeu His Gly Leu Phe Gly Arg Lys Thr Gly Gln Ala Pro Gly Tyr Ser

35 40 45 35 40 45

Tyr Thr Ala Ala Asn Lys Asn Lys Gly Ile Ile Trp Gly Glu Asp ThrTyr Thr Ala Ala Asn Lys Asn Lys Gly Ile Ile Trp Gly Glu Asp Thr

50 55 60 50 55 60

Leu Met Glu Tyr Leu Glu Asn Pro Lys Lys Tyr Ile Pro Gly Thr LysLeu Met Glu Tyr Leu Glu Asn Pro Lys Lys Tyr Ile Pro Gly Thr Lys

65 70 75 8065 70 75 80

Met Ile Phe Val Gly Ile Lys Lys Lys Glu Glu Arg Ala Asp Leu IleMet Ile Phe Val Gly Ile Lys Lys Lys Glu Glu Arg Ala Asp Leu Ile

85 90 95 85 90 95

Ala Tyr Leu Lys Lys Ala Thr Asn Glu Asp Glu Val Asp Met Gly AspAla Tyr Leu Lys Lys Ala Thr Asn Glu Asp Glu Val Asp Met Gly Asp

100 105 110 100 105 110

Val Glu Lys Gly Lys Lys Ile Phe Ile Met Lys Cys Ser Gln Cys HisVal Glu Lys Gly Lys Lys Lys Ile Phe Ile Met Lys Cys Ser Gln Cys His

115 120 125 115 120 125

Thr Val Glu Lys Gly Gly Lys His Lys Thr Gly Pro Asn Leu His GlyThr Val Glu Lys Gly Gly Lys His Lys Thr Gly Pro Asn Leu His Gly

130 135 140 130 135 140

Leu Phe Gly Arg Lys Thr Gly Gln Ala Pro Gly Tyr Ser Tyr Thr AlaLeu Phe Gly Arg Lys Thr Gly Gln Ala Pro Gly Tyr Ser Tyr Thr Ala

145 150 155 160145 150 155 160

Ala Asn Lys Asn Lys Gly Ile Ile Trp Gly Glu Asp Thr Leu Met GluAla Asn Lys Asn Lys Gly Ile Ile Trp Gly Glu Asp Thr Leu Met Glu

165 170 175 165 170 175

Tyr Leu Glu Asn Pro Lys Lys Tyr Ile Pro Gly Thr Lys Met Ile PheTyr Leu Glu Asn Pro Lys Lys Tyr Ile Pro Gly Thr Lys Met Ile Phe

180 185 190 180 185 190

Val Gly Ile Lys Lys Lys Glu Glu Arg Ala Asp Leu Ile Ala Tyr LeuVal Gly Ile Lys Lys Lys Glu Glu Arg Ala Asp Leu Ile Ala Tyr Leu

195 200 205 195 200 205

Lys Lys Ala Thr Asn Glu Asp Glu Val Asp Met Gly Asp Val Glu LysLys Lys Ala Thr Asn Glu Asp Glu Val Asp Met Gly Asp Val Glu Lys

210 215 220 210 215 220

Gly Lys Lys Ile Phe Ile Met Lys Cys Ser Gln Cys His Thr Val GluGly Lys Lys Ile Phe Ile Met Lys Cys Ser Gln Cys His Thr Val Glu

225 230 235 240225 230 235 240

Lys Gly Gly Lys His Lys Thr Gly Pro Asn Leu His Gly Leu Phe GlyLys Gly Gly Lys His Lys Thr Gly Pro Asn Leu His Gly Leu Phe Gly

245 250 255 245 250 255

Arg Lys Thr Gly Gln Ala Pro Gly Tyr Ser Tyr Thr Ala Ala Asn LysArg Lys Thr Gly Gln Ala Pro Gly Tyr Ser Tyr Thr Ala Ala Asn Lys

260 265 270 260 265 270

Asn Lys Gly Ile Ile Trp Gly Glu Asp Thr Leu Met Glu Tyr Leu GluAsn Lys Gly Ile Ile Trp Gly Glu Asp Thr Leu Met Glu Tyr Leu Glu

275 280 285 275 280 285

Asn Pro Lys Lys Tyr Ile Pro Gly Thr Lys Met Ile Phe Val Gly IleAsn Pro Lys Lys Tyr Ile Pro Gly Thr Lys Met Ile Phe Val Gly Ile

290 295 300 290 295 300

Lys Lys Lys Glu Glu Arg Ala Asp Leu Ile Ala Tyr Leu Lys Lys AlaLys Lys Lys Glu Glu Arg Ala Asp Leu Ile Ala Tyr Leu Lys Lys Lys Ala

305 310 315 320305 310 315 320

Thr Asn Glu Thr Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly GlyThr Asn Glu Thr Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly

325 330 335 325 330 335

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser GlyLeu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

340 345 350 340 345 350

Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro GlyPhe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly

355 360 365 355 360 365

Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr ThrLys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr

370 375 380 370 375 380

Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp ThrArg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr

385 390 395 400385 390 395 400

Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu AspSer Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

405 410 415 405 410 415

Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr AlaThr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala

420 425 430 420 425 430

Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly GlyMet Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

435 440 445 435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile GlnGly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Asp Ile Gln

450 455 460 450 455 460

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg ValMet Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

465 470 475 480465 470 475 480

Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala TrpThr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp

485 490 495 485 490 495

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser AlaTyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala

500 505 510 500 505 510

Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg SerSer Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser

515 520 525 515 520 525

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp PheGly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

530 535 540 530 535 540

Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe GlyAla Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly

545 550 555 560545 550 555 560

Gln Gly Thr Lys Val Glu Ile Lys ArgGln Gly Thr Lys Val Glu Ile Lys Arg

565 565

<210> 14<210> 14

<211> 48<211> 48

<212> DNA<212> DNA

<213> 人工序列 (Artificial Sequence)<213> Artificial Sequence

<400> 14<400> 14

cgacgacgac gacaaggcca tggctgaagt tcagctggtt gaatctgg 48cgacgacgac gacaaggcca tggctgaagt tcagctggtt gaatctgg 48

<210> 15<210> 15

<211> 49<211> 49

<212> DNA<212> DNA

<213> 人工序列 (Artificial Sequence)<213> Artificial Sequence

<400> 15<400> 15

cgacggagct cgaattcgga tccttagcgt ttaatttcca ctttggtgc 49cgacggagct cgaattcgga tccttagcgt ttaatttcca ctttggtgc 49

<210> 16<210> 16

<211> 46<211> 46

<212> DNA<212> DNA

<213> 人工序列 (Artificial Sequence)<213> Artificial Sequence

<400> 16<400> 16

ccagcacatg gacagcccag atctcatggg tgatgttgag aaaggc 46ccagcacatg gacagcccag atctcatggg tgatgttgag aaaggc 46

<210> 17<210> 17

<211> 52<211> 52

<212> DNA<212> DNA

<213> 人工序列 (Artificial Sequence)<213> Artificial Sequence

<400> 17<400> 17

caaccagctg aacttcagcc atggtctcat tagtagcttt tttgagataa gc 52caaccagctg aacttcagcc atggtctcat tagtagcttt tttgagataa gc 52

<210> 18<210> 18

<211> 46<211> 46

<212> DNA<212> DNA

<213> 人工序列 (Artificial Sequence)<213> Artificial Sequence

<400> 18<400> 18

ccagcacatg gacagcccag atctcatgct ccagaagccc aagagc 46ccagcacatg gacagcccag atctcatgct ccagaagccc aagagc 46

<210> 19<210> 19

<211> 46<211> 46

<212> DNA<212> DNA

<213> 人工序列 (Artificial Sequence)<213> Artificial Sequence

<400> 19<400> 19

caaccagctg aacttcagcc atggtctggc gtttccgcac aggctg 46caaccagctg aacttcagcc atggtctggc gtttccgcac aggctg 46

Claims (9)

1. The apoptosis protein fusion type anti-HER-2 single-chain antibody is characterized in that the anti-HER-2 single-chain antibody is coupled with apoptosis proteins in different tandem modes, the anti-HER-2 single-chain antibody comprises a heavy chain variable region VH shown in a sequence SEQ ID NO.1 and a light chain variable region VL shown in a sequence SEQ ID NO.2, and the heavy chain variable region and the light chain variable region are respectively connected with a flexible peptide (G) shown in a sequence SEQ ID NO.34S)3And the VH upstream of the heavy chain variable region contains a 6 x his tag, and the amino acid sequence of the anti-HER-2 single-chain antibody is shown as SEQ ID NO.4, the cDNA sequence of the anti-HER-2 single-chain antibody is shown in SEQ ID NO.5, and the apoptosis protein is cytochrome C or DNA fragmentation factor 40.
2. The apoptotic protein fusion-type anti-HER-2 single chain antibody of claim 1, wherein: the amino acid sequence of the cytochrome C is shown as SEQ ID NO.6, and the cDNA sequence of the cytochrome C is shown as SEQ ID NO. 7.
3. The apoptotic protein fusion-type anti-HER-2 single chain antibody of claim 1, wherein: the amino acid sequence of the DNA fragmentation factor 40 is shown as SEQ ID NO.8, and the cDNA sequence for coding the DNA fragmentation factor 40 is shown as SEQ ID NO. 9.
4. The apoptotic protein fusion-type anti-HER-2 single chain antibody of claim 2, wherein: the apoptosis protein fusion type anti-HER-2 single-chain antibody is a Cytc-scFv fusion type single-chain antibody, and is formed by coupling the anti-HER-2 single-chain antibody of claim 1 and cytochrome C of claim 3, wherein the cytochrome C is connected between a 6 x his tag and the anti-HER-2 single-chain antibody, and the amino acid sequence of the Cytc-scFv fusion type single-chain antibody is shown as SEQ ID NO. 10.
5. The apoptotic protein fusion-type anti-HER-2 single chain antibody of claim 2, wherein: the apoptosis protein fusion type anti-HER-2 single-chain antibody is an nCytc-scFv fusion type single-chain antibody, and is formed by coupling the anti-HER-2 single-chain antibody of claim 1 and a cytochrome C tandem aggregate, wherein the cytochrome C tandem aggregate is connected between a 6 x his tag and the anti-HER-2 single-chain antibody in series, the cytochrome C tandem aggregate is formed by connecting a plurality of cytochrome C of claim 3 through a connecting peptide, the connecting peptide is Caspase-3 enzyme cutting site DEVD, and the cDNA sequence of the DEVD is shown in SEQ ID NO. 11.
6. The apoptotic protein fusion-type anti-HER-2 single chain antibody of claim 3, wherein: the apoptin-fused anti-HER-2 single-chain antibody is a DFF 40-scFv-fused single-chain antibody, and is formed by connecting the anti-HER-2 single-chain antibody of claim 1 with the DNA fragmentation factor 40 of claim 4, wherein the DNA fragmentation factor 40 is connected between a 6 x his tag and the anti-HER-2 single-chain antibody, and the amino acid sequence of the DFF 40-scFv-fused single-chain antibody is shown as SEQ ID NO. 12.
7. The method for producing an apoptotic protein fusion-type anti-HER-2 single chain antibody according to any one of claims 1 to 6, comprising the steps of:
(1) synthesizing a cDNA sequence of the anti-HER-2 single-chain antibody and a cDNA sequence of the apoptosis protein shown in SEQ ID NO. 5;
(2) constructing a recombinant plasmid: respectively connecting the cDNA sequence of the anti-HER-2 single-chain antibody coded in the step (1) and the cDNA sequence of the apoptosis protein coded in the step (1) into a pET-32a (+) expression vector to construct a recombinant plasmid;
(3) transforming the recombinant plasmid obtained in the step (2) into DH5 alpha competent cells, screening recombinant plasmid with correct sequencing into BL21(DE3) competent cells, and inducing and expressing the apoptosis protein fusion type anti-HER-2 single-chain antibody.
8. The method of claim 7, wherein: in the step (2), a cDNA fragment encoding the anti-HER-2 single-chain antibody is inserted between the Nco I and BamH I cleavage sites of the pET-32a (+) expression vector, the apoptosis protein is cytochrome C as claimed in claim 3 or DNA fragmentation factor 40 as claimed in claim 4, a cDNA fragment encoding the apoptosis protein is inserted between Bgl II and Nco I cleavage sites of pET-32a (+) expression vector, and the restriction enzyme Nco I enzyme cutting site adopted on the pET-32a (+) expression vector when inserting the cDNA segment of the anti-HER-2 single-chain antibody and the cDNA segment of the coding apoptosis protein is the same enzyme cutting site, when the apoptosis protein is cytochrome C and the number of cytochrome C is more than one, the individual cytochrome C are connected in series by Caspase-3 cleavage site DEVD as shown in SEQ ID NO. 11.
9. Use of the anti-HER-2 single-chain antibody of claim 1 or the apoptotic protein fusion-type anti-HER-2 single-chain antibody of any one of claims 1 to 6 in the preparation of an anti-cancer medicament for the targeted treatment of HER-2 high-expression cancer.
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