CN1807450A - SIP gene and its coded protein and uses - Google Patents
SIP gene and its coded protein and uses Download PDFInfo
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- CN1807450A CN1807450A CN 200510011196 CN200510011196A CN1807450A CN 1807450 A CN1807450 A CN 1807450A CN 200510011196 CN200510011196 CN 200510011196 CN 200510011196 A CN200510011196 A CN 200510011196A CN 1807450 A CN1807450 A CN 1807450A
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Abstract
本发明涉及SIP基因，该基因编码的SIP蛋白，以及所述基因和蛋白在医学中的应用。所述蛋白能够抑制肿瘤的生长和增殖，对cyclinD1启动子启动荧光素酶表达起抑制作用。
The present invention relates to SIP gene, the SIP protein encoded by the gene, and the application of the gene and protein in medicine. The protein can inhibit tumor growth and proliferation, and can inhibit the expression of luciferase initiated by the cyclinD1 promoter.
Description
技术领域technical field
本发明涉及SIP基因，该基因编码的SIP蛋白，以及所述基因和蛋白在医学中的应用。所述蛋白能够抑制肿瘤的生长和增殖，对cyclinD1启动子启动荧光素酶表达起抑制作用。The present invention relates to SIP gene, the SIP protein encoded by the gene, and the application of the gene and protein in medicine. The protein can inhibit tumor growth and proliferation, and can inhibit the expression of luciferase initiated by the cyclinD1 promoter.
背景技术Background technique
某些蛋白可抑制肿瘤细胞的生长和增殖，发现新的抑制肿瘤生长和增殖的蛋白和编码它们的基因可为肿瘤的治疗提供新的方法和手段。Some proteins can inhibit the growth and proliferation of tumor cells, and the discovery of new proteins that inhibit tumor growth and proliferation and the genes encoding them can provide new methods and means for tumor treatment.
发明内容Contents of the invention
本发明发现了一种可抑制乳腺癌等的癌细胞的生长和增殖的蛋白，克隆了编码该蛋白的基因，构建了含有该基因的表达载体和含有该表达载体的宿主细胞，并且发现该蛋白可抑制乳腺癌等肿瘤细胞的生长和增殖，基于以上发现，完成了本发明。The present invention has discovered a protein that can inhibit the growth and proliferation of cancer cells such as breast cancer, cloned the gene encoding the protein, constructed an expression vector containing the gene and a host cell containing the expression vector, and found that the protein The present invention has been accomplished based on the above findings that the growth and proliferation of tumor cells such as breast cancer can be inhibited.
因此，在一个方面，本发明提供了可抑制肿瘤细胞的生长和增殖的蛋白，尤其重组蛋白，该蛋白具有序列表中序列2所示的氨基酸序列，或者通过所述氨基酸序列中的一个或多个氨基酸的缺失，取代或增加而得到的氨基酸序列。Therefore, in one aspect, the present invention provides a protein that can inhibit the growth and proliferation of tumor cells, especially a recombinant protein, which has the amino acid sequence shown in
在另一个方面，本发明提供了编码上述蛋白的基因，它是具有序列1所示的碱基序列的DNA或者与所述DNA在严格条件下杂交的DNA。In another aspect, the present invention provides a gene encoding the above protein, which is a DNA having the base sequence shown in
在另一个方面，本发明提供了含有上述基因或者编码上述蛋白的基因的表达载体。In another aspect, the present invention provides an expression vector containing the above-mentioned gene or a gene encoding the above-mentioned protein.
在又一个方面，本发明提供了由上述表达载体转化的宿主细胞。In yet another aspect, the present invention provides a host cell transformed with the above expression vector.
在另一个方面，本发明提供了生产本发明的重组蛋白的方法，该方法包括下列步骤：In another aspect, the present invention provides a method for producing the recombinant protein of the present invention, the method comprising the steps of:
培养上述转化的宿主细胞，使转化细胞产生本发明的重组蛋白；和cultivating the above-mentioned transformed host cells, so that the transformed cells produce the recombinant protein of the present invention; and
从培养物中分离出重组蛋白。Recombinant protein is isolated from the culture.
在另一个方面，本发明提供了针对上述蛋白的抗体，它是使用所述重组蛋白作为免疫原而产生的特异性抗体。它可以是单克隆或多克隆抗体。In another aspect, the present invention provides an antibody against the above protein, which is a specific antibody produced using the recombinant protein as an immunogen. It can be a monoclonal or polyclonal antibody.
在另一个方面，本发明提供了检测所述蛋白的试剂盒，它含有上述特异性抗体，可以进行抗原-抗体反应，用于检测具有序列2所示的蛋白。In another aspect, the present invention provides a kit for detecting the protein, which contains the above-mentioned specific antibody and can carry out antigen-antibody reaction for detecting the protein represented by
此外，本发明提供了用于检测序列1所示的核苷酸序列的探针诊断试剂盒，它含有与核苷酸序列1中的部分核苷酸序列互补的核苷酸序列。它可以是一个具有15-300个碱基的DNA片段。通过探针杂交的方法，用于检测含有所述核苷酸序列的mRNA的表达，该mRNA被翻译为含有序列2所示的氨基酸序列的蛋白。In addition, the present invention provides a probe diagnostic kit for detecting the nucleotide sequence shown in
此外，本发明提供了一个引物对，用于对进行PCR扩增，引物如下所示：Primer1：5’-tgc tct aga gcc acc atg gac tac aaggac gac gat gac aag gaa ttc atg gcc cag gtc ctg cac-3’In addition, the present invention provides a pair of primers for PCR amplification, the primers are as follows: Primer1: 5'-tgc tct aga gcc acc atg gac tac aaggac gac gat gac aag gaa ttc atg gcc cag gtc ctg cac- 3'
Primer2：5’-cgc gga tcc cta ctc ttc tgc cga gga-3’。Primer2: 5'-cgc gga tcc cta ctc ttc tgc cga gga-3'.
本发明还提供了上述基因、蛋白或抗体在制备治疗肿瘤的药物中的应用。The present invention also provides the application of the above-mentioned genes, proteins or antibodies in the preparation of drugs for treating tumors.
附图简述Brief description of the drawings
图1是Northern Blot结果表明：基因转录本与其全长大小相符。且在人类的8种组织中均有表达。与生物信息学预测相符。Figure 1 is the result of Northern Blot showing that the gene transcript is consistent with its full-length size. And it is expressed in 8 human tissues. Consistent with bioinformatics predictions.
图2为GST-SIP融合蛋白在大肠杆菌BL21中表达纯化的聚丙烯酰胺凝胶电泳图(SDS-PAGE)。90kDa为蛋白质的分子量。箭头所指为分离出的蛋白条带。Fig. 2 is a polyacrylamide gel electrophoresis pattern (SDS-PAGE) of GST-SIP fusion protein expressed and purified in Escherichia coli BL21. 90kDa is the molecular weight of the protein. Arrows indicate isolated protein bands.
图3为SIP蛋白在真核细胞MCF-7中的表达图(Western blot)。一抗为小鼠抗人Flag单克隆抗体，二抗为本实验室保存的兔抗鼠辣根过氧化物酶(HRP)偶联的二抗。1为阴性对照，2为细胞裂解液。MCF-7细胞用flag标签的pcDNA3.1(-)-SIP转染。蛋白水平用抗flag抗体测试。1-2道分别表示阴性对照和MCF-7的细胞溶解产物。Figure 3 is the expression map (Western blot) of SIP protein in eukaryotic cell MCF-7. The primary antibody was a mouse anti-human Flag monoclonal antibody, and the secondary antibody was a rabbit anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody preserved in our laboratory. 1 is negative control, 2 is cell lysate. MCF-7 cells were transfected with flag-tagged pcDNA3.1(-)-SIP. Protein levels were tested with anti-flag antibody. Lanes 1-2 represent negative control and MCF-7 cell lysates, respectively.
图4为SIP蛋白的亚细胞定位。绿色为GFP，蓝色为DAPI染的胞核。SIP的亚细胞定位于细胞质。Figure 4 shows the subcellular localization of SIP protein. Green is GFP, and blue is DAPI-stained nuclei. The subcellular localization of SIP is cytoplasmic.
图5为MTT法检测不同浓度的SIP对乳腺癌细胞增殖的影响。横坐标为转染时间，纵坐标为570nm吸光度值。在每组方柱中，从左至右依次是v-25ng，SIP-25ng，v-50ng，SIP-50ng，v-100ng，SIP-100ng。Figure 5 is the effect of different concentrations of SIP on the proliferation of breast cancer cells detected by MTT assay. The abscissa is the transfection time, and the ordinate is the absorbance value at 570nm. In each group of square columns, from left to right are v-25ng, SIP-25ng, v-50ng, SIP-50ng, v-100ng, SIP-100ng.
图6为SIP对cyclinD1启动子启动荧光素酶表达的作用。横坐标为cyclinDl启动子不同的缺失体。纵坐标为Firefly/Renilla比值。Figure 6 shows the effect of SIP on the expression of luciferase initiated by the cyclinD1 promoter. The abscissa represents different deletions of the cyclinD1 promoter. The ordinate is the Firefly/Renilla ratio.
图7是SIP的结构示意图，其中的两个灰色方框为由 Coils2 Figure 7 is a schematic diagram of the structure of SIP, where the two gray boxes are made by Coils2
<http:∥smart.embl-heidelberg.de/help/smart glossary.shtml>程序测定的卷曲螺旋区域和6个黑色方框是由 SEG The <http:∥smart.embl-heidelberg.de/help/smart glossary.shtml> program determines the coiled-coil region and the 6 black boxes are from the SEG
<http:∥smart.embl-heidelberg.de/help/smart glossary.shtml>测定的组成复杂性低的链段。 <http:∥smart.embl-heidelberg.de/help/smart glossary.shtml> Segments with low compositional complexity determined.
SIP(SRC-l interacting protein)是由p160家族成员之一的SRC-1通过酵母双杂交的方法筛选出来的新基因。我们从人乳腺cDNA文库中通过PCR的方法获取该基因的ORF。克隆到pcDNA3.1(-)真核表达载体中。为便于检测，在其N端接上Flag标签。SIP又叫KIAA1518，FLJ20004，DKFZp434N161。定位于染色体19p13.2。全长5358bp。表达蛋白～90kD。ORF：2580bp。编码859个氨基酸。SIP (SRC-l interacting protein) is a new gene selected from SRC-1, a member of the p160 family, through yeast two-hybrid method. We obtained the ORF of this gene from the human breast cDNA library by PCR. Cloned into pcDNA3.1(-) eukaryotic expression vector. To facilitate detection, a Flag label is attached to its N-terminus. SIP is also called KIAA1518, FLJ20004, DKFZp434N161. Located on chromosome 19p13.2. The full length is 5358bp. Expressed protein ~90kD. ORF: 2580bp. Encodes 859 amino acids.
对其进行生物信息学分析，发现其广谱表达。包含Ankyrin repeat结构域。It was analyzed by bioinformatics and found its broad-spectrum expression. Contains Ankyrin repeat domain.
(1)SIP表达信息：(1) SIP expression information:
cDNA来源：正常前列腺；睾丸；结肠；结肠肿瘤，RER+；Pooled-Skin；成神经细胞瘤COT 10 NORMALIZED；原发性肺囊性纤维化上皮细胞；脂肪；恶性黑色素瘤，淋巴结转移；白质；耳蜗；骨髓基质；恶性胶质瘤(pooled)；五种合并肉瘤，包括粘液样脂肉瘤，单发纤维瘤，恶性纤维组织细胞瘤，胃肠的基质肿瘤肿瘤，和间皮瘤；小细胞癌；胎盘；淋巴；三种合并脑膜瘤；全脑；中度分化子宫内膜腺癌，3种合并肿瘤；正常结肠；软骨肉瘤肺转移细胞系；成神经管细胞瘤；肿瘤；卵巢；B细胞，慢性淋巴细胞白血病；上皮癌；黑素瘤；乳房；浆液乳头状癌，高级，2种合并肿瘤；正常睾丸；整个胚胎，主要是躯体；卵巢肿瘤组织；前列腺；交感神经干；肝脏与脾；脑膜瘤；正常前列腺；肾肿瘤；虹膜；晶状体；嗅上皮；胎盘；颈；正常胎盘；正常子宫内膜，中分泌期，第23个周期日；黑素细胞；合并人黑素细胞，胎心，和妊娠子宫；肾；腺癌；具有印戒细胞特征的分化不良腺癌；合并；正常着色的视上皮；黑素瘤，高MDR(细胞系)；上皮癌细胞系；成神经细胞瘤；宫颈癌细胞系；海马；合并的脑，肺，睾丸；视神经；子宫肿瘤；胎儿胰腺(4 Pooled Donors，18-20星期，stratagene)；胎儿眼睛；衰老成纤维细胞；腹水；肺；初级肺上皮细胞；肺病灶纤维化；转移性软骨肉瘤；软骨肉瘤；不良分化子宫内膜腺癌，2种合并肿瘤；子宫；鳞状细胞癌；多发性硬化病变；胰岛瘤；脾；鳞状细胞癌，细胞系；成视网膜细胞瘤；混合(pool of 40 RNAs)；内生软骨瘤细胞系；软骨下骨；心脏；肺；良性肿瘤；十二指肠腺癌，细胞系；子宫内膜，腺癌细胞系；血；人肺上皮细胞；眼睛前段；脊神经后根神经节；畸胎癌，细胞系；RPE/脉络膜；细胞系；纯化郎格罕氏岛；平滑肌肉瘤；生发中心B细胞；中度分化腺癌；胰腺；滑膜；胸肌(在乳房切除术以后)；甲状腺；神经鞘瘤肿瘤；前列腺肿瘤。cDNA source: normal prostate; testis; colon; colon tumor, RER+; Pooled-Skin; neuroblastoma COT 10 NORMALIZED; primary lung cystic fibrosis epithelial cells; fat; malignant melanoma, lymph node metastasis; white matter; ; bone marrow stroma; malignant glioma (pooled); five associated sarcomas, including myxoid liposarcoma, solitary fibrous tumor, malignant fibrous histiocytoma, stromal tumor of the gastrointestinal tract, and mesothelioma; small cell carcinoma; Placenta; lymphoid; three combined meningiomas; whole brain; moderately differentiated endometrial adenocarcinoma, three combined tumors; normal colon; chondrosarcoma lung metastasis cell line; medulloblastoma; neoplasm; ovary; Chronic lymphocytic leukemia; epithelial carcinoma; melanoma; breast; serous papillary carcinoma, high-grade, 2 combined tumors; normal testis; whole embryo, mainly body; ovarian tumor tissue; prostate; sympathetic trunk; liver and spleen; Meningioma; normal prostate; kidney tumor; iris; lens; olfactory epithelium; placenta; neck; normal placenta; normal endometrium, mesocrine phase, cycle day 23; melanocytes; combined human melanocytes, fetal heart , and pregnant uterus; kidney; adenocarcinoma; poorly differentiated adenocarcinoma with signet-ring cell features; merged; normally pigmented optic epithelium; melanoma, high MDR (cell line); epithelial carcinoma cell line; neuroblastoma; Cervical cancer cell lines; hippocampus; combined brain, lung, testis; optic nerve; uterine tumor; fetal pancreas (4 Pooled Donors, 18-20 weeks, stratagene); fetal eye; senescent fibroblasts; cell; pulmonary fibrosis; metastatic chondrosarcoma; chondrosarcoma; poorly differentiated endometrial adenocarcinoma, 2 combined tumors; uterus; squamous cell carcinoma; multiple sclerosis lesions; Cell line; Retinoblastoma; Mixed (pool of 40 RNAs); Enchondroma cell line; Subchondral bone; Heart; Lung; Benign tumor; Duodenal adenocarcinoma, cell line; Endometrium, adenocarcinoma Cell line; blood; human lung epithelium; anterior segment of eye; dorsal root ganglion; teratoma, cell line; RPE/choroid; cell line; purified Islets of Langerhans; leiomyosarcoma; germinal center B cells; moderate Differentiated adenocarcinoma; pancreas; synovium; pectoralis (after mastectomy); thyroid; schwannoma tumors; prostate tumors.
(2)在氨基酸序列基础上通过ExPASy及interPro对其主要功能结构域预测，发现一个功能结构域：Ankyrin。具体细节见下图及表：图1是SIP的结构示意图，其中的两个灰色方框为由 Coils2 (2) Based on the amino acid sequence, the main functional domain was predicted by ExPASy and interPro, and a functional domain was found: Ankyrin. For details, see the figure and table below: Figure 1 is a schematic diagram of the SIP structure, and the two gray boxes in it are Coils2
<http:∥smart.embl-heidelberg.de/help/smart glossary.shtml>程序测定的卷曲螺旋区域和6个黑色方框是由 SEG The <http:∥smart.embl-heidelberg.de/help/smart glossary.shtml> program determines the coiled-coil region and the 6 black boxes are from the SEG
<http:∥smart.embl-heidelberg.de/help/smart glossary.shtml>测定的组成复杂性低的链段。
通过使用本发明的蛋白或者免疫学上等效的多肽，可以获得其抗体，抗体用于所述蛋白的检测和纯化。可以利用本发明的蛋白或其部分氨基酸序列作为免疫原来产生抗体。可以通过将抗体接种给宿主动物并回收血清的常规方法产生多克隆抗体。还可以通过常规杂交瘤方法产生单克隆抗体。By using the protein of the present invention or an immunologically equivalent polypeptide, its antibody can be obtained, and the antibody is used for detection and purification of the protein. The protein of the present invention or its partial amino acid sequence can be used as an immunogen to generate antibodies. Polyclonal antibodies can be produced by conventional methods of inoculating host animals with antibodies and recovering serum. Monoclonal antibodies can also be produced by conventional hybridoma methods.
利用MTT法检测不同浓度的SIP对MCF-7肿瘤细胞增殖的影响，结果发现，GIBC有抑制肿瘤细胞增殖的作用。MTT assay was used to detect the effect of different concentrations of SIP on the proliferation of MCF-7 tumor cells, and it was found that GIBC could inhibit the proliferation of tumor cells.
因此，本发明的蛋白可用于治疗肿瘤，尤其乳腺癌。Therefore, the protein of the present invention can be used in the treatment of tumors, especially breast cancer.
实施例Example
实施例1：用PCR方法从人乳腺癌cDNA文库克隆编码SIP蛋白的基因Embodiment 1: Cloning the gene encoding SIP protein from human breast cancer cDNA library by PCR method
用乳腺癌cDNA文库为模板，在SIP基因N端接上Flag标签，用下列引物进行PCR扩增：Using the breast cancer cDNA library as a template, a Flag tag was attached to the N-terminus of the SIP gene, and PCR amplification was performed with the following primers:
Primer1：5’-tgc tct aga gcc acc atg gac tac aag gacgac gat gac aag gaa ttc atg gcc cag gtcctg cac-3’Primer1: 5’-tgc tct aga gcc acc atg gac tac aag gacgac gat gac aag gaa ttc atg gcc cag gtcctg cac-3’
Primer2：5’-cgc gga tcc cta ctc ttc tgc cga gga-3’Primer2: 5'-cgc gga tcc cta ctc ttc tgc cga gga-3'
扩增反应的条件：在50μL的反应体积中含有50mmol/L KCl，10mmol/L Tris-Cl，(PH：8.5)，1.5mmol/L MgCl2，200μmol/L dNTP，10pmol引物，0.25U的pfu DNA聚合酶(Takara公司产品)。按下列条件反应30个周期：94℃ 30sec；59℃ 30sec；72℃ 2min 30sec。扩增产物用QIAGEN公司的试剂盒纯化，用XbaI及BamHI双酶切后，克隆到相同酶切的pcDNA3.1(-)真核表达载体中。DNA序列分析结果表明PCR产物的DNA序列与预期相符。Amplification reaction conditions: 50mmol/L KCl, 10mmol/L Tris-Cl, (PH: 8.5), 1.5mmol/L MgCl 2 , 200μmol/L dNTP, 10pmol primer, 0.25U pfu in a reaction volume of 50μL DNA polymerase (product of Takara Corporation). React for 30 cycles under the following conditions: 94°C 30sec; 59°C 30sec; 72°C 2min 30sec. The amplified product was purified with a kit from QIAGEN, digested with XbaI and BamHI, and cloned into the pcDNA3.1(-) eukaryotic expression vector with the same restriction enzymes. The results of DNA sequence analysis showed that the DNA sequence of the PCR product was consistent with the expectation.
实施例2：Northern BlotExample 2: Northern Blot
探针的标记和杂交操作参照Clontech公司的SpotlightTM RandomPrimer Labeling Kit试剂盒说明书。过程如下：Probe labeling and hybridization operations refer to the instructions of the Spotlight TM RandomPrimer Labeling Kit kit from Clontech Company. The process is as follows:
(1)探针标记：25ng-1μgDNA溶于20μL体积中，90-100℃ 2-3分钟，迅速冰浴5分钟，加5μL[α-32P]dCTP标记的RandomPrimers and Reaction Buffer Mix，补水至50μL，37℃孵育5-10min。(1) Probe labeling: Dissolve 25ng-1μg DNA in a volume of 20μL, incubate at 90-100°C for 2-3 minutes, quickly ice-bath for 5 minutes, add 5μL [α- 32 P]dCTP-labeled RandomPrimers and Reaction Buffer Mix, replenish water to 50μL, incubate at 37°C for 5-10min.
(2)杂交：68℃预热杂交液，将吸附有核酸的尼龙膜放入含有杂交液的杂交瓶中，加入含有100μg/mL的鲑精DNA的杂交液0.1mL/cm2，68℃预杂交30min。20ng/mL探针混入杂交液，95℃ 2-5分钟，冰浴5分钟。加入杂交瓶，68℃杂交1小时。(2) Hybridization: Preheat the hybridization solution at 68°C, put the nylon membrane adsorbed with nucleic acid into the hybridization bottle containing the hybridization solution, add 0.1 mL/cm 2 of the hybridization solution containing 100 μg/mL salmon sperm DNA, and preheat at 68°C. Hybridize for 30min. Mix 20ng/mL probe into the hybridization solution, incubate at 95°C for 2-5 minutes, and ice-bath for 5 minutes. Add to the hybridization flask and hybridize at 68°C for 1 hour.
(3)洗膜：用Wash Buffer 1室温洗膜30-40分钟，用Wash Buffer 250℃洗膜40分钟。(3) Membrane washing: wash the membrane with
(4)显色：用镊子取出膜，洗干Wash Buffer 2，用保鲜膜覆盖，-70℃曝光1-3天后显影，定影。(4) Color development: Take out the film with tweezers, wash and
实施例3：SIP在原核细胞中的表达及纯化Example 3: Expression and purification of SIP in prokaryotic cells
pGEX-4T-3-SIP可以在大肠杆菌BL21菌株中30℃诱导表达GST-SIP的融合蛋白，首先制备BL21的感受态细胞：挑取单克隆到适量培养基中，37℃ 250rpm培养至A600 0.4-0.5，2500g离心15分钟，备用。将1-50ng pGEX-4T-3-SIP转化到上述制备的感受态细胞中，用Glutathione Sepharose 4B进行纯化，纯化的步骤如下：pGEX-4T-3-SIP can induce the expression of GST-SIP fusion protein in Escherichia coli BL21 strain at 30°C, first prepare competent cells of BL21: pick a single clone into an appropriate amount of medium, and culture it at 37°C 250rpm to A 600 0.4-0.5, centrifuge at 2500g for 15 minutes, set aside. Transform 1-50ng pGEX-4T-3-SIP into the competent cells prepared above, and use Glutathione Sepharose 4B to purify. The purification steps are as follows:
(1)取单克隆到2-3mL 2YTA培养基中培养4-5小时。转接到100mL的锥形瓶中生长过夜。(1) Take a single clone and culture it in 2-3mL 2YTA medium for 4-5 hours. Transfer to a 100mL Erlenmeyer flask and grow overnight.
(2)转接到500mL的锥形瓶中培养3-5小时。(2) Transfer to a 500mL Erlenmeyer flask and cultivate for 3-5 hours.
(3)加0.5mL 1M IPTG 30℃，培养3-6小时。(3) Add 0.5mL 1M IPTG at 30°C and incubate for 3-6 hours.
(4)3000rpm 10分钟离心收集细胞。(4) Collect the cells by centrifugation at 3000rpm for 10 minutes.
(5)用25mL PBS重悬细胞。超声10分钟。(5) Resuspend the cells with 25mL PBS. Sonicate for 10 minutes.
(6)加1.25mL 20％Triton X-100，混匀1小时。(6) Add 1.25mL 20% Triton X-100 and mix for 1 hour.
(7)1000rpm 10分钟离心，加入0.5mL 50％slurry of GlutathioneSepharose 4B，室温30分钟。(7) Centrifuge at 1000rpm for 10 minutes, add 0.5mL 50% slurry of GlutathioneSepharose 4B, room temperature for 30 minutes.
(8)1000rpm 5分钟离心，PBS洗三遍。(8) Centrifuge at 1000rpm for 5 minutes, wash with PBS three times.
(9)用0.5mL洗脱Buffer洗脱，短暂离心后收集上清。(9) Elute with 0.5mL elution buffer, and collect the supernatant after brief centrifugation.
实施例4：SIP在真核细胞中的表达Example 4: Expression of SIP in eukaryotic cells
Western Blot是在蛋白质电泳和固相免疫测定的基础上发展起来的。其基本原理为抗原抗体反应。细胞蛋白提取物经聚丙烯酰胺凝胶电泳分离后转移到固相支持物上(本实验采用硝酸纤维素膜)。与特异的一抗反应(本实验用小鼠抗人Flag单克隆抗体，Sigma)，再与辣根过氧化物酶(HRP)偶联的二抗(本实验室保存兔抗鼠二抗)孵育，显色，可以得到特异蛋白分子的免疫印迹图。该方法具有从混杂抗原中检测出特定的抗原的优点，因此本实验用于检测带有Flag标签的SIP基因转染MCF-7细胞后的表达情况。Western Blot was developed on the basis of protein electrophoresis and solid-phase immunoassay. Its basic principle is antigen-antibody reaction. The cell protein extract was separated by polyacrylamide gel electrophoresis and transferred to a solid support (nitrocellulose membrane was used in this experiment). Reaction with specific primary antibody (mouse anti-human Flag monoclonal antibody, Sigma was used in this experiment), and then incubated with horseradish peroxidase (HRP)-coupled secondary antibody (rabbit anti-mouse secondary antibody kept in our laboratory) , color development, and the immunoblotting of specific protein molecules can be obtained. This method has the advantage of detecting specific antigens from mixed antigens, so this experiment is used to detect the expression of SIP gene with Flag tag after transfection into MCF-7 cells.
1)瞬时转染实验：1) Transient transfection experiment:
于转染前一天将MCF-7细胞接种于10cm培养板中，接种密度为2×106个细胞/孔，每孔加入培养液10ml。24小时后，细胞长至覆盖培养孔底面积约50-60％时，可准备转染。在EP管中配制下述溶液：溶液A：24μg pcDNA3.1(-)-SIP表达质粒，溶于1.5mL无血清无双抗DMEM培养液。溶液B：将48μL脂质体溶液(lipofectamine 2000reagent)加入到1.5m L无血清无双抗DMEM培养液中。室温静置5分钟。然后将溶液A、B混合，室温静置20分钟。用无血清和不含双抗的DMEM培养液洗涤细胞。将上述溶液A、B的混合液中加入12mL无血清无双抗的DMEM培养液，混匀后加至细胞表面。37℃，5％CO2条件下孵育4-6小时后，再用正常含10％胎牛血清的DMEM培养液取代前述培养液，继续培养细胞至48小时。The day before transfection, MCF-7 cells were seeded in a 10 cm culture plate at a seeding density of 2×10 6 cells/well, and 10 ml of culture medium was added to each well. After 24 hours, when the cells have grown to cover about 50-60% of the bottom area of the culture well, they are ready for transfection. Prepare the following solutions in EP tubes: Solution A: 24 μg pcDNA3.1(-)-SIP expression plasmid, dissolved in 1.5 mL serum-free and double-antibody-free DMEM culture medium. Solution B: Add 48 μL lipofectamine 2000 reagent to 1.5 mL serum-free DMEM culture medium without double antibody. Let stand at room temperature for 5 minutes. Then solutions A and B were mixed and allowed to stand at room temperature for 20 minutes. Cells were washed with serum-free and double-antibody-free DMEM medium. Add 12 mL of DMEM medium without serum and double antibody to the mixture of the above solutions A and B, mix well and add to the cell surface. After incubating for 4-6 hours at 37°C and 5% CO2, the culture medium was replaced with normal DMEM medium containing 10% fetal bovine serum, and the cells were continued to be cultured for 48 hours.
2)细胞总蛋白的提取：2) Extraction of total cell protein:
培养的细胞经0.25％胰酶消化使之分散，4℃低速离心去上清，将沉淀置于冰上30min，再用预冷的PBS洗2次，每106细胞加入裂解液100μL，使细胞充分裂解30min，再于4℃，20000×g离心15min，收集上清。The cultured cells were digested with 0.25% trypsin to disperse them, centrifuged at a low speed at 4°C to remove the supernatant, placed the precipitate on ice for 30 min, then washed twice with pre-cooled PBS, and added 100 μL of lysate per 106 cells to make the cells fully Lyse for 30 minutes, then centrifuge at 20,000×g for 15 minutes at 4°C, and collect the supernatant.
3)Bradford法测定细胞裂解液蛋白的浓度：3) Determination of protein concentration in cell lysate by Bradford method:
首先配制考马斯亮蓝染色液；将标准牛血清白蛋白(BSA)配制成1mg/ml的蛋白标准液，分别将10μg、20μg、30μg、40μg、50μg、60μg蛋白标准液和5μL待测蛋白质样品加入3mL考马斯亮蓝染液中，室温孵育10min，在595nm波长处测定吸收值。BSA标准样品测定标准曲线，在一定范围内OD595与蛋白浓度成正比。样品蛋白质的浓度经标准曲线拟合方程来计算。First prepare Coomassie Brilliant Blue staining solution; prepare standard bovine serum albumin (BSA) into 1mg/ml protein standard solution, add 10μg, 20μg, 30μg, 40μg, 50μg, 60μg protein standard solution and 5μL protein sample to be tested respectively Incubate in 3mL Coomassie Brilliant Blue staining solution at room temperature for 10min, and measure the absorbance at a wavelength of 595nm. The standard curve of BSA standard sample is determined, and the OD 595 is directly proportional to the protein concentration within a certain range. The concentration of sample protein was calculated by standard curve fitting equation.
4)电泳及免疫反应：流程如下：A、制胶(5％浓缩胶，10％分离胶)；B、50μg蛋白样品加入上样缓冲液，煮沸，上样；C、电泳，浓缩胶80V电压，分离胶120V电压；D、转膜，100V，1.5h；E、封闭，1h；F、一抗反应，4℃，过夜；G、TBST洗膜，三次，每次10min；H、二抗反应，1.5h；I、TBST洗膜，三次，每次10min；J、显色、曝光。4) Electrophoresis and immune reaction: The process is as follows: A. Gel preparation (5% stacking gel, 10% separating gel); B. 50 μg protein sample is added to loading buffer, boiled, and loaded; C. Electrophoresis, stacking gel 80V voltage , separation gel 120V voltage; D, transfer membrane, 100V, 1.5h; E, blocking, 1h; F, primary antibody reaction, 4 ℃, overnight; G, TBST wash membrane, three times, each 10min; H, secondary antibody reaction , 1.5h; I, TBST washing, three times, each 10min; J, color development, exposure.
实施例5：SIP在细胞内的定位Example 5: Localization of SIP in cells
利用本实验室保存的pEGFP-C1 C末端蛋白融合载体构建了pEGFP-C1-SIP载体，它可以在真核细胞内表达GFP-SIP的融合蛋白，将该载体用LipofectinTM的方法转染进MCF-7细胞中，转染的细胞进行爬片，24小时后，用PBS洗三遍，用4％多聚甲醛/PBS进行固定，室温15分钟，然后用PBS洗三遍。用0.1％Triton X-100/PBS透化15分钟，然后用PBS洗三遍，加入200μL 2.5mg/mL的DAPI，室温作用30分钟，然后用PBS洗三遍，在荧光显微镜下分别用常规荧光波长和DAPI波长进行观察，并进行拍照。结果见图4，定位于细胞质。The pEGFP-C1-SIP vector was constructed using the pEGFP-C1 C-terminal protein fusion vector preserved in our laboratory, which can express the GFP-SIP fusion protein in eukaryotic cells, and the vector was transfected into MCF by Lipofectin TM In -7 cells, the transfected cells were slided, washed three times with PBS after 24 hours, fixed with 4% paraformaldehyde/PBS, room temperature for 15 minutes, and then washed three times with PBS. Permeabilize with 0.1% Triton X-100/PBS for 15 minutes, then wash three times with PBS, add 200 μL of 2.5 mg/mL DAPI, react at room temperature for 30 minutes, then wash three times with PBS, and use conventional fluorescence under a fluorescent microscope wavelength and DAPI wavelength were observed and photographed. The results are shown in Figure 4, localized in the cytoplasm.
实施例6：MTT法检测不同浓度的SIP对乳腺癌细胞增殖的影响Embodiment 6: MTT assay detects the influence of different concentrations of SIP on the proliferation of breast cancer cells
MTT(四甲基偶氮唑兰)可被细胞摄取，并被活细胞内线粒体脱氢酶还原成一种不溶于水的蓝紫色产物甲瓒，并沉淀于细胞中，而死细胞没有这种功能。甲瓒可溶于二甲基亚砜(DMSO)，溶液在570nm处有最大吸收。故该波段处吸收值越大代表存活细胞量越多。MTT法广泛应用于细胞毒性实验、细胞生长测定等。本实验利用MTT法观察SIP对MCF-7生长的影响。具体操作方法如下：MTT (Tetramethylazolane) can be taken up by cells and reduced by mitochondrial dehydrogenase in living cells to formazan, a water-insoluble blue-purple product, which is precipitated in cells, while dead cells do not have this function . Formazan is soluble in dimethyl sulfoxide (DMSO), and the solution has a maximum absorption at 570nm. Therefore, the greater the absorption value at this band, the greater the amount of viable cells. The MTT assay is widely used in cytotoxicity experiments, cell growth assays, etc. In this experiment, MTT method was used to observe the effect of SIP on the growth of MCF-7. The specific operation method is as follows:
将培养到对数生长期的MCF-7细胞接种于96孔板中，每孔接种细胞数为1×104个。次日按照浓度梯度加入SIP(25、50、100ng)，每浓度作用6孔细胞，37℃，5％CO2进行培养。分别培养1d、2d、3d、4d后，在每个细胞培养孔内加入50μlL 1mg/mL的MTT溶液，同样培养条件下作用4小时后取出，小心吸出每孔内液体，每孔加入二甲基亚砜150μL，轻微振荡10分钟，使蓝紫色结晶充分溶解在二甲基亚砜中。用自动酶标仪测量96孔板中每孔在570nm处的吸光值。参看图5，MTT实验表明SIP对MCF-7细胞生长增殖起抑制作用，并且与剂量有关。The MCF-7 cells cultured to the logarithmic growth phase were inoculated in 96-well plates, and the number of inoculated cells in each well was 1×10 4 . The next day, SIP (25, 50, 100 ng) was added according to the concentration gradient, and each concentration acted on the cells in 6 wells, and cultured at 37°C and 5% CO2. After culturing for 1d, 2d, 3d, and 4d respectively, add 50μlL of 1mg/mL MTT solution to each cell culture well, take it out after 4 hours under the same culture conditions, carefully suck out the liquid in each well, and add dimethyl Add 150 μL of sulfoxide and shake gently for 10 minutes to fully dissolve the blue-purple crystals in dimethyl sulfoxide. The absorbance value at 570 nm of each well in the 96-well plate was measured with an automatic microplate reader. Referring to Fig. 5, the MTT experiment showed that SIP inhibited the growth and proliferation of MCF-7 cells, and it was dose-related.
实施例7：瞬时转染及荧光素酶(Luciferase)实验：Embodiment 7: transient transfection and luciferase (Luciferase) experiment:
应用荧光素酶作为报告基因，为分析基因的调控元件和测量基因转录活性提供了便利的方法。本实验就是将cyclinD1基因启动子与荧光素酶读码框共同构建到真核表达载体中，将该融合表达质粒转染细胞，通过测定荧光素酶的活性，从而研究转录因子、调节蛋白对cyclinD1基因启动子的作用。实验采用萤火虫荧光素酶(Firefly)为报告基因，海三色堇(Renillar)荧光素酶为内参照。用Promega公司出产的双荧光报告基因分析系统试剂盒，在同一试管中测量两种酶的活性。并应用化学发光仪进行检测。The application of luciferase as a reporter gene provides a convenient method for analyzing the regulatory elements of genes and measuring gene transcriptional activity. In this experiment, the cyclinD1 gene promoter and the luciferase reading frame were jointly constructed into a eukaryotic expression vector, and the fusion expression plasmid was transfected into cells. By measuring the activity of luciferase, the effects of transcription factors and regulatory proteins on cyclinD1 were studied. The role of gene promoters. Firefly luciferase (Firefly) was used as the reporter gene, and sea pansy (Renillar) luciferase was used as the internal reference. The activities of the two enzymes were measured in the same test tube with the dual fluorescent reporter gene analysis system kit produced by Promega. And the application of chemiluminescence detection.
2)瞬时转染实验：2) Transient transfection experiment:
于转染前一天将MCF-7细胞接种于24孔培养板中，接种密度为1-2×105个细胞/孔，每孔加入培养液0.5mL。24小时后，细胞长至覆盖培养孔底面积约50-60％时，可准备转染。在EP管中配制下述溶液：溶液A：0.8μg DNA(cyclinD1报告基因质粒：200ng；内参Renillar质粒：10ng；SIP表达质粒：600ng或pcDNA3.1(-)空载体质粒：600ng)，溶于50μL无血清无双抗DMEM培养液。溶液B：将2μL脂质体溶液(lipofectamine 2000reagent)加入到50μL无血清无双抗DMEM培养液中。室温静置5分钟。然后将溶液A、B混合，室温静置20分钟。用无血清和不含双抗的DMEM培养液洗涤细胞。将上述溶液A、B的混合液中加入400μL无血清无双抗的DMEM培养液，混匀后加至细胞表面。每个实验组重复3孔。37℃，5％CO2条件下孵育4-6小时后，再用正常含10％胎牛血清的DMEM培养液取代前述培养液，继续培养细胞至48小时。裂解细胞，测量荧光素酶的活性。The day before transfection, MCF-7 cells were seeded in a 24-well culture plate at a seeding density of 1-2×10 5 cells/well, and 0.5 mL of culture solution was added to each well. After 24 hours, when the cells have grown to cover about 50-60% of the bottom area of the culture well, they are ready for transfection. Prepare the following solutions in EP tubes: solution A: 0.8 μg DNA (cyclinD1 reporter gene plasmid: 200ng; internal reference Renillar plasmid: 10ng; SIP expression plasmid: 600ng or pcDNA3.1(-) empty vector plasmid: 600ng), dissolved in 50 μL serum-free and double-antibody-free DMEM culture medium. Solution B: 2 μL lipofectamine 2000 reagent was added to 50 μL serum-free DMEM culture medium without double antibody. Let stand at room temperature for 5 minutes. Then solutions A and B were mixed and allowed to stand at room temperature for 20 minutes. Cells were washed with serum-free and double-antibody-free DMEM medium. Add 400 μL of serum-free and double-antibody-free DMEM culture solution to the mixture of the above solutions A and B, mix well and add to the cell surface. Each experimental group replicated 3 wells. After incubating for 4-6 hours at 37° C. and 5% CO 2 , replace the aforementioned culture medium with normal DMEM culture medium containing 10% fetal bovine serum, and continue culturing the cells for 48 hours. Cells were lysed and luciferase activity was measured.
2)用双荧光报告基因试剂盒测量双荧光素酶的活性：2) Measure the activity of dual luciferase with a dual fluorescent reporter gene kit:
(1)裂解细胞：(1) Lyse cells:
用冷PBS清洗细胞，将1×细胞裂解液(PLB)100μL加入每个细胞培养孔，室温孵育15分钟，使细胞充分裂解。所得到的细胞裂解液可直接用于荧光测量。Wash the cells with cold PBS, add 100 μL of 1× cell lysate (PLB) to each cell culture well, and incubate at room temperature for 15 minutes to fully lyse the cells. The resulting cell lysates can be used directly for fluorescence measurements.
(2)荧光素酶底物的配制：(2) Preparation of luciferase substrate:
将萤火虫荧光素酶底物冷冻干粉溶于10mL荧光素酶分析缓冲液中，所得溶液为LARII，分装，-70℃保存。Dissolve the lyophilized powder of firefly luciferase substrate in 10 mL of luciferase assay buffer, the resulting solution is LARII, aliquot and store at -70°C.
用Stop & Glo缓冲液将50×海三色堇荧光素酶底物稀释到1×，所得溶液为Stop & Glo，现用现配。Dilute 50× sea pansy luciferase substrate to 1× with Stop & Glo buffer solution, the resulting solution is Stop & Glo, which is ready for use.
(3)测量过程：(3) Measurement process:
将20μL细胞裂解液放入96孔荧光测量板，加入100μLLARII，测量萤火虫荧光素酶，测量条件为：延迟2秒，测量10秒。再加入100μLStop & Glo，测量海三色堇荧光素酶，测量条件同上。Put 20 μL of cell lysate into a 96-well fluorescence measurement plate, add 100 μLARII, and measure firefly luciferase. The measurement conditions are: delay for 2 seconds, and measure for 10 seconds. Then add 100 μL Stop & Glo to measure sea pansy luciferase, the measurement conditions are the same as above.
参看图6，SIP对cyclinD1启动子启动荧光素酶表达起抑制作用。Referring to Figure 6, SIP inhibits the expression of luciferase initiated by the cyclinD1 promoter.
SIP序~1SIP Sequence~1
SEQUENCE LISTINGSEQUENCE LISTING
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<120>SIP基因及其编码的蛋白和应用<120>SIP gene and its encoded protein and application
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<170>PatentIn version 3.1<170>PatentIn version 3.1
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atg gcc cag gtc ctg cac gtg cct gct ccc ttc cca ggg acc cct ggc 48atg gcc cag gtc ctg cac gtg cct gct ccc ttc cca ggg acc cct ggc 48
Met Ala Gln Val Leu His Val Pro Ala Pro Phe Pro Gly Thr Pro GlyMet Ala Gln Val Leu His Val Pro Ala Pro Phe Pro Gly Thr Pro Gly
1 5 10 151 5 10 15
cca gcc tcc cca cct gcc ttc cct gcc aag gac ccc gat cca ccc tac 96cca gcc tcc cca cct gcc ttc cct gcc aag gac ccc gat cca ccc tac 96
Pro Ala Ser Pro Pro Ala Phe Pro Ala Lys Asp Pro Asp Pro Pro TyrPro Ala Ser Pro Pro Ala Phe Pro Ala Lys Asp Pro Asp Pro Pro Tyr
20 25 3020 25 30
tcc gtg gag acc ccc tat ggc tac cgc ctg gac ctg gac ttc ctc aag 144tcc gtg gag acc ccc tat ggc tac cgc ctg gac ctg gac ttc ctc aag 144
Ser Val Glu Thr Pro Tyr Gly Tyr Arg Leu Asp Leu Asp Phe Leu LysSer Val Glu Thr Pro Tyr Gly Tyr Arg Leu Asp Leu Asp Phe Leu Lys
35 40 4535 40 45
tac gtg gat gac atc gag aag ggc cac acg ctg cga cgc gtg gca gtg 192tac gtg gat gac atc gag aag ggc cac acg ctg cga cgc gtg gca gtg 192
Tyr Val Asp Asp Ile Glu Lys Gly His Thr Leu Arg Arg Val Ala ValTyr Val Asp Asp Ile Glu Lys Gly His Thr Leu Arg Arg Val Ala Val
50 55 6050 55 60
cag cgc cgc ccc cgc ctg agc tcg ctg ccc cgt ggc cct ggc tcc tgg 240cag cgc cgc ccc cgc ctg agc tcg ctg ccc cgt ggc cct ggc tcc tgg 240
Gln Arg Arg Pro Arg Leu Ser Ser Leu Pro Arg Gly Pro Gly Ser TrpGln Arg Arg Pro Arg Leu Ser Ser Leu Pro Arg Gly Pro Gly Ser Trp
65 70 75 8065 70 75 80
tgg acg tcc act gag tcg ctg tgc tcc aat gcc agt ggg gac agc cgc 288tgg acg tcc act gag tcg ctg tgc tcc aat gcc agt ggg gac agc cgc 288
Trp Thr Ser Thr Glu Ser Leu Cys Ser Asn Ala Ser Gly Asp Ser ArgTrp Thr Ser Thr Glu Ser Leu Cys Ser Asn Ala Ser Gly Asp Ser Arg
85 90 9585 90 95
cac tca gcc tat tcc tac tgc ggc cgt ggc ttc tac cct cag tat ggt 336cac tca gcc tat tcc tac tgc ggc cgt ggc ttc tac cct cag tat ggt 336
His Ser Ala Tyr Ser Tyr Cys Gly Arg Gly Phe Tyr Pro Gln Tyr GlyHis Ser Ala Tyr Ser Tyr Cys Gly Arg Gly Phe Tyr Pro Gln Tyr Gly
100 105 110100 105 110
gct ctg gag acc cgc ggt ggc ttc aat ccg cgg gtg gag cgc acg ctg 384gct ctg gag acc cgc ggt ggc ttc aat ccg cgg gtg gag cgc acg ctg 384
Ala Leu Glu Thr Arg Gly Gly Phe Asn Pro Arg Val Glu Arg Thr LeuAla Leu Glu Thr Arg Gly Gly Phe Asn Pro Arg Val Glu Arg Thr Leu
115 120 125115 120 125
ctg gat gcc cgt cgc cgt ctc gag gac cag gcg gcc aca ccc acc ggc 432ctg gat gcc cgt cgc cgt ctc gag gac cag gcg gcc aca ccc acc ggc 432
SIP序~1SIP Sequence~1
Leu Asp Ala Arg Arg Arg Leu Glu Asp Gln Ala Ala Thr Pro Thr GlyLeu Asp Ala Arg Arg Arg Leu Glu Asp Gln Ala Ala Thr Pro Thr Gly
130 135 140130 135 140
ctg ggc tcc ctg acc ccc agt gcg gcc ggc tcg aca gcc tcc ctg gtg 480ctg ggc tcc ctg acc ccc agt gcg gcc ggc tcg aca gcc tcc ctg gtg 480
Leu Gly Ser Leu Thr Pro Ser Ala Ala Gly Ser Thr Ala Ser Leu ValLeu Gly Ser Leu Thr Pro Ser Ala Ala Gly Ser Thr Ala Ser Leu Val
145 150 155 160145 150 155 160
ggc gtg ggg ttg cca ccc ccg aca cca cgg agt tca gga ctg tcc aca 528ggc gtg ggg ttg cca ccc ccg aca cca cgg agt tca gga ctg tcc aca 528
Gly Val Gly Leu Pro Pro Pro Thr Pro Arg Ser Ser Gly Leu Ser ThrGly Val Gly Leu Pro Pro Pro Pro Thr Pro Arg Ser Ser Gly Leu Ser Thr
165 170 175165 170 175
ccg gtg cct ccc agt gcc ggg cac ctg gcc cac gtg cgg gag cag atg 576ccg gtg cct ccc agt gcc ggg cac ctg gcc cac gtg cgg gag cag atg 576
Pro Val Pro Pro Ser Ala Gly His Leu Ala His Val Arg Glu Gln MetPro Val Pro Pro Ser Ala Gly His Leu Ala His Val Arg Glu Gln Met
180 185 190180 185 190
gcg ggt gcc ctg cgg aag ctg cgg cag ctg gag gag cag gtg aag ctg 624gcg ggt gcc ctg cgg aag ctg cgg cag ctg gag gag cag gtg aag ctg 624
Ala Gly Ala Leu Arg Lys Leu Arg Gln Leu Glu Glu Gln Val Lys LeuAla Gly Ala Leu Arg Lys Leu Arg Gln Leu Glu Glu Gln Val Lys Leu
195 200 205195 200 205
atc cct gtg ctc cag gtg aag ctc tcg gtg ctc cag gag gaa aag cgg 672atc cct gtg ctc cag gtg aag ctc tcg gtg ctc cag gag gaa aag cgg 672
Ile Pro Val Leu Gln Val Lys Leu Ser Val Leu Gln Glu Glu Lys ArgIle Pro Val Leu Gln Val Lys Leu Ser Val Leu Gln Glu Glu Lys Arg
210 215 220210 215 220
cag ctc aca gta caa ctt aag agc cag aag ttc ctg ggc cac ccc aca 720cag ctc aca gta caa ctt aag agc cag aag ttc ctg ggc cac ccc aca 720
Gln Leu Thr Val Gln Leu Lys Ser Gln Lys Phe Leu Gly His Pro ThrGln Leu Thr Val Gln Leu Lys Ser Gln Lys Phe Leu Gly His Pro Thr
225 230 235 240225 230 235 240
gcg ggc cgg ggt cgc agc gag ctc tgc ctg gac ctc ccc gat ccc cca 768gcg ggc cgg ggt cgc agc gag ctc tgc ctg gac ctc ccc gat ccc cca 768
Ala Gly Arg Gly Arg Ser Glu Leu Cys Leu Asp Leu Pro Asp Pro ProAla Gly Arg Gly Arg Ser Glu Leu Cys Leu Asp Leu Pro Asp Pro Pro
245 250 255245 250 255
gag gac cca gtg gca ctg gag acc cgg agt gtg ggc acc tgg gtc cga 816gag gac cca gtg gca ctg gag acc cgg agt gtg ggc acc tgg gtc cga 816
Glu Asp Pro Val Ala Leu Glu Thr Arg Ser Val Gly Thr Trp Val ArgGlu Asp Pro Val Ala Leu Glu Thr Arg Ser Val Gly Thr Trp Val Arg
260 265 270260 265 270
gaa cgg gac ttg ggc atg cct gat ggg gag gct gcc ctc gcc gcc aag 864gaa cgg gac ttg ggc atg cct gat ggg gag gct gcc ctc gcc gcc aag 864
Glu Arg Asp Leu Gly Met Pro Asp Gly Glu Ala Ala Leu Ala Ala LysGlu Arg Asp Leu Gly Met Pro Asp Gly Glu Ala Ala Leu Ala Ala Lys
275 280 285275 280 285
gtc gct gtg ctg gag acc cag ctc aag aag gcg ctt cag gag ctg cag 912gtc gct gtg ctg gag acc cag ctc aag aag gcg ctt cag gag ctg cag 912
Val Ala Val Leu Glu Thr Gln Leu Lys Lys Ala Leu Gln Glu Leu GlnVal Ala Val Leu Glu Thr Gln Leu Lys Lys Ala Leu Gln Glu Leu Gln
290 295 300290 295 300
gca gct cag gcc cgg cag gct gac ccc cag ccc cag gcc tgg cca ccg 960gca gct cag gcc cgg cag gct gac ccc cag ccc cag gcc tgg cca ccg 960
Ala Ala Gln Ala Arg Gln Ala Asp Pro Gln Pro Gln Ala Trp Pro ProAla Ala Gln Ala Arg Gln Ala Asp Pro Gln Pro Gln Ala Trp Pro Pro
305 310 315 320305 310 315 320
ccg gac agc ccg gtc cgc gtg gat aca gtc cgg gtg gta gaa ggg cca 1008ccg gac agc ccg gtc cgc gtg gat aca gtc cgg gtg gta gaa ggg cca 1008
Pro Asp Ser Pro Val Arg Val Asp Thr Val Arg Val Val Glu Gly ProPro Asp Ser Pro Val Arg Val Asp Thr Val Arg Val Val Glu Gly Pro
325 330 335325 330 335
cgg gag gtg gag gtg gtg gcc agc aca gcc gct ggc gcc ccc gca cag 1056cgg gag gtg gag gtg gtg gcc agc aca gcc gct ggc gcc ccc gca cag 1056
Arg Glu Val Glu Val Val Ala Ser Thr Ala Ala Gly Ala Pro Ala GlnArg Glu Val Glu Val Val Ala Ser Thr Ala Ala Gly Ala Pro Ala Gln
340 345 350340 345 350
cgg gcc cag agc ctg gag cct tac ggc aca ggg ctg agg gcc ctg gca 1104cgg gcc cag agc ctg gag cct tac ggc aca ggg ctg agg gcc ctg gca 1104
SIP序~1SIP Sequence~1
Arg Ala Gln Ser Leu Glu Pro Tyr Gly Thr Gly Leu Arg Ala Leu AlaArg Ala Gln Ser Leu Glu Pro Tyr Gly Thr Gly Leu Arg Ala Leu Ala
355 360 365355 360 365
atg cct ggt agg cct gag agc cca cct gtg ttc cgc agc cag gag gtg 1152atg cct ggt agg cct gag agc cca cct gtg ttc cgc agc cag gag gtg 1152
Met Pro Gly Arg Pro Glu Ser Pro Pro Val Phe Arg Ser Gln Glu ValMet Pro Gly Arg Pro Glu Ser Pro Pro Val Phe Arg Ser Gln Glu Val
370 375 380370 375 380
gtg gag aca atg tgc cca gtg ccc gct gca gct acc agc aac gtc cat 1200gtg gag aca atg tgc cca gtg ccc gct gca gct acc agc aac gtc cat 1200
Val Glu Thr Met Cys Pro Val Pro Ala Ala Ala Thr Ser Asn Val HisVal Glu Thr Met Cys Pro Val Pro Ala Ala Ala Thr Ser Asn Val His
385 390 395 400385 390 395 400
atg gtg aag aag att agc atc aca gag cga agc tgc gat gga gca gca 1248atg gtg aag aag att agc atc aca gag cga agc tgc gat gga gca gca 1248
Met Val Lys Lys Ile Ser Ile Thr Glu Arg Ser Cys Asp Gly Ala AlaMet Val Lys Lys Ile Ser Ile Thr Glu Arg Ser Cys Asp Gly Ala Ala
405 410 415405 410 415
ggc ctc cca gaa gtt cct gcc gaa tcg tct tcg tca ccc ccg ggg tcc 1296ggc ctc cca gaa gtt cct gcc gaa tcg tct tcg tca ccc ccg ggg tcc 1296
Gly Leu Pro Glu Val Pro Ala Glu Ser Ser Ser Ser Pro Pro Gly SerGly Leu Pro Glu Val Pro Ala Glu Ser Ser Ser Ser Pro Pro Gly Ser
420 425 430420 425 430
gag gta gcc tcc ctt aca cag cct gag aag agc aca ggc cga gtg ccc 1344gag gta gcc tcc ctt aca cag cct gag aag agc aca ggc cga gtg ccc 1344
Glu Val Ala Ser Leu Thr Gln Pro Glu Lys Ser Thr Gly Arg Val ProGlu Val Ala Ser Leu Thr Gln Pro Glu Lys Ser Thr Gly Arg Val Pro
435 440 445435 440 445
acc cag gag ccc acc cac agg gag ccc acc agg caa gca gcc tcc caa 1392acc cag gag ccc acc cac agg gag ccc acc agg caa gca gcc tcc caa 1392
Thr Gln Glu Pro Thr His Arg Glu Pro Thr Arg Gln Ala Ala Ser GlnThr Gln Glu Pro Thr His Arg Glu Pro Thr Arg Gln Ala Ala Ser Gln
450 455 460450 455 460
gag tcc gag gag gcc ggg ggc acc gca cct ccg ctg tcc tcc cct cca 1440gag tcc gag gag gcc ggg ggc acc gca cct ccg ctg tcc tcc cct cca 1440
Glu Ser Glu Glu Ala Gly Gly Thr Ala Pro Pro Leu Ser Ser Pro ProGlu Ser Glu Glu Ala Gly Gly Thr Ala Pro Pro Leu Ser Ser Pro Pro
465 470 475 480465 470 475 480
ggc ggg ccc ccg gca ggc gtg cga tct atc atg aaa cgg aaa gag gag 1488ggc ggg ccc ccg gca ggc gtg cga tct atc atg aaa cgg aaa gag gag 1488
Gly Gly Pro Pro Ala Gly Val Arg Ser Ile Met Lys Arg Lys Glu GluGly Gly Pro Pro Ala Gly Val Arg Ser Ile Met Lys Arg Lys Glu Glu Glu
485 490 495485 490 495
gtt gca gac ccc acg gcc cac cgg agg agc ctc cag ttc gtg ggg gtc 1536gtt gca gac ccc acg gcc cac cgg agg agc ctc cag ttc gtg ggg gtc 1536
Val Ala Asp Pro Thr Ala His Arg Arg Ser Leu Gln Phe Val Gly ValVal Ala Asp Pro Thr Ala His Arg Arg Ser Leu Gln Phe Val Gly Val
500 505 510500 505 510
aac ggc ggg tat gag tcg tca tcc gag gac tcc agc aca gca gag aac 1584aac ggc ggg tat gag tcg tca tcc gag gac tcc agc aca gca gag aac 1584
Asn Gly Gly Tyr Glu Ser Ser Ser Glu Asp Ser Ser Thr Ala Glu AsnAsn Gly Gly Tyr Glu Ser Ser Ser Ser Glu Asp Ser Ser Thr Ala Glu Asn
515 520 525515 520 525
atc tca gac aac gac agc aca gag aac gag gcc cca gag ccg agg gag 1632atc tca gac aac gac agc aca gag aac gag gcc cca gag ccg agg gag 1632
Ile Ser Asp Asn Asp Ser Thr Glu Asn Glu Ala Pro Glu Pro Arg GluIle Ser Asp Asn Asp Ser Thr Glu Asn Glu Ala Pro Glu Pro Arg Glu
530 535 540530 535 540
agg gtt ccg agt gtg gcc gaa gcc ccc cag ctc agg cct gca ggg acg 1680agg gtt ccg agt gtg gcc gaa gcc ccc cag ctc agg cct gca ggg acg 1680
Arg Val Pro Ser Val Ala Glu Ala Pro Gln Leu Arg Pro Ala Gly ThrArg Val Pro Ser Val Ala Glu Ala Pro Gln Leu Arg Pro Ala Gly Thr
545 550 555 560545 550 555 560
gca gcg gcc aag acc agc cgg cag gag tgt cag ctg tct cga gaa tct 1728gca gcg gcc aag acc agc cgg cag gag tgt cag ctg tct cga gaa tct 1728
Ala Ala Ala Lys Thr Ser Arg Gln Glu Cys Gln Leu Ser Arg Glu SerAla Ala Ala Lys Thr Ser Arg Gln Glu Cys Gln Leu Ser Arg Glu Ser
565 570 575565 570 575
cag cac ata ccc act gct gag ggg gca tca gga tca aac acg gag gag 1776cag cac ata ccc act gct gag ggg gca tca gga tca aac acg gag gag 1776
SIP序~1SIP Sequence~1
Gln His Ile Pro Thr Ala Glu Gly Ala Ser Gly Ser Asn Thr Glu GluGln His Ile Pro Thr Ala Glu Gly Ala Ser Gly Ser Asn Thr Glu Glu
580 585 590580 585 590
gag atc agg atg gag cta agc cct gac ctc atc tca gcc tgc ttg gcc 1824gag atc agg atg gag cta agc cct gac ctc atc tca gcc tgc ttg gcc 1824
Glu Ile Arg Met Glu Leu Ser Pro Asp Leu Ile Ser Ala Cys Leu AlaGlu Ile Arg Met Glu Leu Ser Pro Asp Leu Ile Ser Ala Cys Leu Ala
595 600 605595 600 605
ctg gaa aag tac ctg gac aat ccc aac gcc ctc aca gag cgg gag ctg 1872ctg gaa aag tac ctg gac aat ccc aac gcc ctc aca gag cgg gag ctg 1872
Leu Glu Lys Tyr Leu Asp Asn Pro Asn Ala Leu Thr Glu Arg Glu LeuLeu Glu Lys Tyr Leu Asp Asn Pro Asn Ala Leu Thr Glu Arg Glu Leu
610 615 620610 615 620
aaa gtg gcc tac acc aca gtg ctg cag gag tgg ctg cgc ctg gcc tgc 1920aaa gtg gcc tac acc aca gtg ctg cag gag tgg ctg cgc ctg gcc tgc 1920
Lys Val Ala Tyr Thr Thr Val Leu Gln Glu Trp Leu Arg Leu Ala CysLys Val Ala Tyr Thr Thr Val Leu Gln Glu Trp Leu Arg Leu Ala Cys
625 630 635 640625 630 635 640
cgc agc gac gca cac ccc gag ctg gtg cgg cgg cac ctg gtc acg ttc 1968cgc agc gac gca cac ccc gag ctg gtg cgg cgg cac ctg gtc acg ttc 1968
Arg Ser Asp Ala His Pro Glu Leu Val Arg Arg His Leu Val Thr PheArg Ser Asp Ala His Pro Glu Leu Val Arg Arg His Leu Val Thr Phe
645 650 655645 650 655
cgg gcc atg tct gcg cgg ctg ctg gac tac gtg gtc aac atc gcc gac 2016cgg gcc atg tct gcg cgg ctg ctg gac tac gtg gtc aac atc gcc gac 2016
Arg Ala Met Ser Ala Arg Leu Leu Asp Tyr Val Val Asn Ile Ala AspArg Ala Met Ser Ala Arg Leu Leu Asp Tyr Val Val Asn Ile Ala Asp
660 665 670660 665 670
agc aac ggc aac aca gcc ctg cac tac tcc gtg tct cat gcc aac ttc 2064agc aac ggc aac aca gcc ctg cac tac tcc gtg tct cat gcc aac ttc 2064
Ser Asn Gly Asn Thr Ala Leu His Tyr Ser Val Ser His Ala Asn PheSer Asn Gly Asn Thr Ala Leu His Tyr Ser Val Ser His Ala Asn Phe
675 680 685675 680 685
ccc gtg gtg cag cag ctg ctc gac agc ggt gtc tgc aag gtg gac aaa 2112ccc gtg gtg cag cag ctg ctc gac agc ggt gtc tgc aag gtg gac aaa 2112
Pro Val Val Gln Gln Leu Leu Asp Ser Gly Val Cys Lys Val Asp LysPro Val Val Gln Gln Leu Leu Asp Ser Gly Val Cys Lys Val Asp Lys
690 695 700690 695 700
cag aac cgt gct ggc tac agc cct att atg ctc acc gcc ctg gcc acc 2160cag aac cgt gct ggc tac agc cct att atg ctc acc gcc ctg gcc acc 2160
Gln Asn Arg Ala Gly Tyr Ser Pro Ile Met Leu Thr Ala Leu Ala ThrGln Asn Arg Ala Gly Tyr Ser Pro Ile Met Leu Thr Ala Leu Ala Thr
705 710 715 720705 710 715 720
ctg aag acc cag gac gac atc gag act gtc ctt cag ctc ttc cgg ctt 2208ctg aag acc cag gac gac atc gag act gtc ctt cag ctc ttc cgg ctt 2208
Leu Lys Thr Gln Asp Asp Ile Glu Thr Val Leu Gln Leu Phe Arg LeuLeu Lys Thr Gln Asp Asp Ile Glu Thr Val Leu Gln Leu Phe Arg Leu
725 730 735725 730 735
ggc aac atc aat gcc aaa gcc agc cag gca gga cag acg gcc ctg atg 2256ggc aac atc aat gcc aaa gcc agc cag gca gga cag acg gcc ctg atg 2256
Gly Asn Ile Asn Ala Lys Ala Ser Gln Ala Gly Gln Thr Ala Leu MetGly Asn Ile Asn Ala Lys Ala Ser Gln Ala Gly Gln Thr Ala Leu Met
740 745 750740 745 750
ctg gcc gtc agc cac ggg cgg gtg gac gtt gtc aaa gcc ctg ctg gcc 2304ctg gcc gtc agc cac ggg cgg gtg gac gtt gtc aaa gcc ctg ctg gcc 2304
Leu Ala Val Ser His Gly Arg Val Asp Val Val Lys Ala Leu Leu AlaLeu Ala Val Ser His Gly Arg Val Asp Val Val Lys Ala Leu Leu Ala
755 760 765755 760 765
tgt gag gca gat gtc aac gtg caa gat gat gac ggc tcc acg gcc ctc 2352tgt gag gca gat gtc aac gtg caa gat gat gac ggc tcc acg gcc ctc 2352
Cys Glu Ala Asp Val Asn Val Gln Asp Asp Asp Gly Ser Thr Ala LeuCys Glu Ala Asp Val Asn Val Gln Asp Asp Asp Gly Ser Thr Ala Leu
770 775 780770 775 780
atg tgc gcc tgt gag cac ggc cac aag gag atc gcg ggg ctg ctg ctg 2400atg tgc gcc tgt gag cac ggc cac aag gag atc gcg ggg ctg ctg ctg 2400
Met Cys Ala Cys Glu His Gly His Lys Glu Ile Ala Gly Leu Leu LeuMet Cys Ala Cys Glu His Gly His Lys Glu Ile Ala Gly Leu Leu Leu
785 790 795 800785 790 795 800
gcc gtg ccc agc tgt gac atc tca ctc aca gat cgc gat ggg agc aca 2448gcc gtg ccc agc tgt gac atc tca ctc aca gat cgc gat ggg agc aca 2448
SIP序~1SIP Sequence~1
Ala Val Pro Ser Cys Asp Ile Ser Leu Thr Asp Arg Asp Gly Ser ThrAla Val Pro Ser Cys Asp Ile Ser Leu Thr Asp Arg Asp Gly Ser Thr
805 810 815805 810 815
gct ctg atg gtg gcc ttg gac gca ggg cag agt gag att gcg tcc atg 2496gct ctg atg gtg gcc ttg gac gca ggg cag agt gag att gcg tcc atg 2496
Ala Leu Met Val Ala Leu Asp Ala Gly Gln Ser Glu Ile Ala Ser MetAla Leu Met Val Ala Leu Asp Ala Gly Gln Ser Glu Ile Ala Ser Met
820 825 830820 825 830
ctg tat tcc cgc atg aac atc aag tgc tcg ttt gcc cca atg tca gat 2544ctg tat tcc cgc atg aac atc aag tgc tcg ttt gcc cca atg tca gat 2544
Leu Tyr Ser Arg Met Asn Ile Lys Cys Ser Phe Ala Pro Met Ser AspLeu Tyr Ser Arg Met Asn Ile Lys Cys Ser Phe Ala Pro Met Ser Asp
835 840 845835 840 845
gac gag agc cct aca tca tcc tcg gca gaa gag tag 2580gac gag agc cct aca tca tcc tcg gca gaa gag tag 2580
Asp Glu Ser Pro Thr Ser Ser Ser Ala Glu GluAsp Glu Ser Pro Thr Ser Ser Ser Ser Ala Glu Glu
850 855850 855
<210>2<210>2
<211>859<211>859
<212>PRT<212>PRT
<213>人<213> people
<400>2<400>2
Met Ala Gln Val Leu His Val Pro Ala Pro Phe Pro Gly Thr Pro GlyMet Ala Gln Val Leu His Val Pro Ala Pro Phe Pro Gly Thr Pro Gly
1 5 10 151 5 10 15
Pro Ala Ser Pro Pro Ala Phe Pro Ala Lys Asp Pro Asp Pro Pro TyrPro Ala Ser Pro Pro Ala Phe Pro Ala Lys Asp Pro Asp Pro Pro Tyr
20 25 3020 25 30
Ser Val Glu Thr Pro Tyr Gly Tyr Arg Leu Asp Leu Asp Phe Leu LysSer Val Glu Thr Pro Tyr Gly Tyr Arg Leu Asp Leu Asp Phe Leu Lys
35 40 4535 40 45
Tyr Val Asp Asp Ile Glu Lys Gly His Thr Leu Arg Arg Val Ala ValTyr Val Asp Asp Ile Glu Lys Gly His Thr Leu Arg Arg Val Ala Val
50 55 6050 55 60
Gln Arg Arg Pro Arg Leu Ser Ser Leu Pro Arg Gly Pro Gly Ser TrpGln Arg Arg Pro Arg Leu Ser Ser Leu Pro Arg Gly Pro Gly Ser Trp
65 70 75 8065 70 75 80
Trp Thr Ser Thr Glu Ser Leu Cys Ser Asn Ala Ser Gly Asp Ser ArgTrp Thr Ser Thr Glu Ser Leu Cys Ser Asn Ala Ser Gly Asp Ser Arg
85 90 9585 90 95
His Ser Ala Tyr Ser Tyr Cys Gly Arg Gly Phe Tyr Pro Gln Tyr GlyHis Ser Ala Tyr Ser Tyr Cys Gly Arg Gly Phe Tyr Pro Gln Tyr Gly
100 105 110100 105 110
Ala Leu Glu Thr Arg Gly Gly Phe Asn Pro Arg Val Glu Arg Thr LeuAla Leu Glu Thr Arg Gly Gly Phe Asn Pro Arg Val Glu Arg Thr Leu
115 120 125115 120 125
Leu Asp Ala Arg Arg Arg Leu Glu Asp Gln Ala Ala Thr Pro Thr GlyLeu Asp Ala Arg Arg Arg Leu Glu Asp Gln Ala Ala Thr Pro Thr Gly
SIP序~1SIP Sequence~1
130 135 140130 135 140
Leu Gly Ser Leu Thr Pro Ser Ala Ala Gly Ser Thr Ala Ser Leu ValLeu Gly Ser Leu Thr Pro Ser Ala Ala Gly Ser Thr Ala Ser Leu Val
145 150 155 160145 150 155 160
Gly Val Gly Leu Pro Pro Pro Thr Pro Arg Ser Ser Gly Leu Ser ThrGly Val Gly Leu Pro Pro Pro Pro Thr Pro Arg Ser Ser Gly Leu Ser Thr
165 170 175165 170 175
Pro Val Pro Pro Ser Ala Gly His Leu Ala His Val Arg Glu Gln MetPro Val Pro Pro Ser Ala Gly His Leu Ala His Val Arg Glu Gln Met
180 185 190180 185 190
Ala Gly Ala Leu Arg Lys Leu Arg Gln Leu Glu Glu Gln Val Lys LeuAla Gly Ala Leu Arg Lys Leu Arg Gln Leu Glu Glu Gln Val Lys Leu
195 200 205195 200 205
Ile Pro Val Leu Gln Val Lys Leu Ser Val Leu Gln Glu Glu Lys ArgIle Pro Val Leu Gln Val Lys Leu Ser Val Leu Gln Glu Glu Lys Arg
210 215 220210 215 220
Gln Leu Thr Val Gln Leu Lys Ser Gln Lys Phe Leu Gly His Pro ThrGln Leu Thr Val Gln Leu Lys Ser Gln Lys Phe Leu Gly His Pro Thr
225 230 235 240225 230 235 240
Ala Gly Arg Gly Arg Ser Glu Leu Cys Leu Asp Leu Pro Asp Pro ProAla Gly Arg Gly Arg Ser Glu Leu Cys Leu Asp Leu Pro Asp Pro Pro
245 250 255245 250 255
Glu Asp Pro Val Ala Leu Glu Thr Arg Ser Val Gly Thr Trp Val ArgGlu Asp Pro Val Ala Leu Glu Thr Arg Ser Val Gly Thr Trp Val Arg
260 265 270260 265 270
Glu Arg Asp Leu Gly Met Pro Asp Gly Glu Ala Ala Leu Ala Ala LysGlu Arg Asp Leu Gly Met Pro Asp Gly Glu Ala Ala Leu Ala Ala Lys
275 280 285275 280 285
Val Ala Val Leu Glu Thr Gln Leu Lys Lys Ala Leu Gln Glu Leu GlnVal Ala Val Leu Glu Thr Gln Leu Lys Lys Ala Leu Gln Glu Leu Gln
290 295 300290 295 300
Ala Ala Gln Ala Arg Gln Ala Asp Pro Gln Pro Gln Ala Trp Pro ProAla Ala Gln Ala Arg Gln Ala Asp Pro Gln Pro Gln Ala Trp Pro Pro
305 310 315 320305 310 315 320
Pro Asp Ser Pro Val Arg Val Asp Thr Val Arg Val Val Glu Gly ProPro Asp Ser Pro Val Arg Val Asp Thr Val Arg Val Val Glu Gly Pro
325 330 335325 330 335
Arg Glu Val Glu Val Val Ala Ser Thr Ala Ala Gly Ala Pro Ala GlnArg Glu Val Glu Val Val Ala Ser Thr Ala Ala Gly Ala Pro Ala Gln
340 345 350340 345 350
Arg Ala Gln Ser Leu Glu Pro Tyr Gly Thr Gly Leu Arg Ala Leu AlaArg Ala Gln Ser Leu Glu Pro Tyr Gly Thr Gly Leu Arg Ala Leu Ala
SIP序~1SIP Sequence~1
355 360 365355 360 365
Met Pro Gly Arg Pro Glu Ser Pro Pro Val Phe Arg Ser Gln Glu ValMet Pro Gly Arg Pro Glu Ser Pro Pro Val Phe Arg Ser Gln Glu Val
370 375 380370 375 380
Val Glu Thr Met Cys Pro Val Pro Ala Ala Ala Thr Ser Asn Val HisVal Glu Thr Met Cys Pro Val Pro Ala Ala Ala Thr Ser Asn Val His
385 390 395 400385 390 395 400
Met Val Lys Lys Ile Ser Ile Thr Glu Arg Ser Cys Asp Gly Ala AlaMet Val Lys Lys Ile Ser Ile Thr Glu Arg Ser Cys Asp Gly Ala Ala
405 410 415405 410 415
Gly Leu Pro Glu Val Pro Ala Glu Ser Ser Ser Ser Pro Pro Gly SerGly Leu Pro Glu Val Pro Ala Glu Ser Ser Ser Ser Pro Pro Gly Ser
420 425 430420 425 430
Glu Val Ala Ser Leu Thr Gln Pro Glu Lys Ser Thr Gly Arg Val ProGlu Val Ala Ser Leu Thr Gln Pro Glu Lys Ser Thr Gly Arg Val Pro
435 440 445435 440 445
Thr Gln Glu Pro Thr His Arg Glu Pro Thr Arg Gln Ala Ala Ser GlnThr Gln Glu Pro Thr His Arg Glu Pro Thr Arg Gln Ala Ala Ser Gln
450 455 460450 455 460
Glu Ser Glu Glu Ala Gly Gly Thr Ala Pro Pro Leu Ser Ser Pro ProGlu Ser Glu Glu Ala Gly Gly Thr Ala Pro Pro Leu Ser Ser Pro Pro
465 470 475 480465 470 475 480
Gly Gly Pro Pro Ala Gly Val Arg Ser Ile Met Lys Arg Lys Glu GluGly Gly Pro Pro Ala Gly Val Arg Ser Ile Met Lys Arg Lys Glu Glu Glu
485 490 495485 490 495
Val Ala Asp Pro Thr Ala His Arg Arg Ser Leu Gln Phe Val Gly ValVal Ala Asp Pro Thr Ala His Arg Arg Ser Leu Gln Phe Val Gly Val
500 505 510500 505 510
Asn Gly Gly Tyr Glu Ser Ser Ser Glu Asp Ser Ser Thr Ala Glu AsnAsn Gly Gly Tyr Glu Ser Ser Ser Ser Glu Asp Ser Ser Thr Ala Glu Asn
515 520 525515 520 525
Ile Ser Asp Asn Asp Ser Thr Glu Asn Glu Ala Pro Glu Pro Arg GluIle Ser Asp Asn Asp Ser Thr Glu Asn Glu Ala Pro Glu Pro Arg Glu
530 535 540530 535 540
Arg Val Pro Ser Val Ala Glu Ala Pro Gln Leu Arg Pro Ala Gly ThrArg Val Pro Ser Val Ala Glu Ala Pro Gln Leu Arg Pro Ala Gly Thr
545 550 555 560545 550 555 560
Ala Ala Ala Lys Thr Ser Arg Gln Glu Cys Gln Leu Ser Arg Glu SerAla Ala Ala Lys Thr Ser Arg Gln Glu Cys Gln Leu Ser Arg Glu Ser
565 570 575565 570 575
Gln His Ile Pro Thr Ala Glu Gly Ala Ser Gly Ser Asn Thr Glu GluGln His Ile Pro Thr Ala Glu Gly Ala Ser Gly Ser Asn Thr Glu Glu
SIP序~1SIP Sequence~1
580 585 590580 585 590
Glu Ile Arg Met Glu Leu Ser Pro Asp Leu Ile Ser Ala Cys Leu AlaGlu Ile Arg Met Glu Leu Ser Pro Asp Leu Ile Ser Ala Cys Leu Ala
595 600 605595 600 605
Leu Glu Lys Tyr Leu Asp Asn Pro Asn Ala Leu Thr Glu Arg Glu LeuLeu Glu Lys Tyr Leu Asp Asn Pro Asn Ala Leu Thr Glu Arg Glu Leu
610 615 620610 615 620
Lys Val Ala Tyr Thr Thr Val Leu Gln Glu Trp Leu Arg Leu Ala CysLys Val Ala Tyr Thr Thr Val Leu Gln Glu Trp Leu Arg Leu Ala Cys
625 630 635 640625 630 635 640
Arg Ser Asp Ala His Pro Glu Leu Val Arg Arg His Leu Val Thr PheArg Ser Asp Ala His Pro Glu Leu Val Arg Arg His Leu Val Thr Phe
645 650 655645 650 655
Arg Ala Met Ser Ala Arg Leu Leu Asp Tyr Val Val Asn Ile Ala AspArg Ala Met Ser Ala Arg Leu Leu Asp Tyr Val Val Asn Ile Ala Asp
660 665 670660 665 670
Ser Asn Gly Asn Thr Ala Leu His Tyr Ser Val Ser His Ala Asn PheSer Asn Gly Asn Thr Ala Leu His Tyr Ser Val Ser His Ala Asn Phe
675 680 685675 680 685
Pro Val Val Gln Gln Leu Leu Asp Ser Gly Val Cys Lys Val Asp LysPro Val Val Gln Gln Leu Leu Asp Ser Gly Val Cys Lys Val Asp Lys
690 695 700690 695 700
Gln Asn Arg Ala Gly Tyr Ser Pro Ile Met Leu Thr Ala Leu Ala ThrGln Asn Arg Ala Gly Tyr Ser Pro Ile Met Leu Thr Ala Leu Ala Thr
705 710 715 720705 710 715 720
Leu Lys Thr Gln Asp Asp Ile Glu Thr Val Leu Gln Leu Phe Arg LeuLeu Lys Thr Gln Asp Asp Ile Glu Thr Val Leu Gln Leu Phe Arg Leu
725 730 735725 730 735
Gly Asn Ile Asn Ala Lys Ala Ser Gln Ala Gly Gln Thr Ala Leu MetGly Asn Ile Asn Ala Lys Ala Ser Gln Ala Gly Gln Thr Ala Leu Met
740 745 750740 745 750
Leu Ala Val Ser His Gly Arg Val Asp Val Val Lys Ala Leu Leu AlaLeu Ala Val Ser His Gly Arg Val Asp Val Val Lys Ala Leu Leu Ala
755 760 765755 760 765
Cys Glu Ala Asp Val Asn Val Gln Asp Asp Asp Gly Ser Thr Ala LeuCys Glu Ala Asp Val Asn Val Gln Asp Asp Asp Gly Ser Thr Ala Leu
770 775 780770 775 780
Met Cys Ala Cys Glu His Gly His Lys Glu Ile Ala Gly Leu Leu LeuMet Cys Ala Cys Glu His Gly His Lys Glu Ile Ala Gly Leu Leu Leu
785 790 795 800785 790 795 800
Ala Val Pro Ser Cys Asp Ile Ser Leu Thr Asp Arg Asp Gly Ser ThrAla Val Pro Ser Cys Asp Ile Ser Leu Thr Asp Arg Asp Gly Ser Thr
SIP序~1SIP Sequence~1
805 810 815805 810 815
Ala Leu Met Val Ala Leu Asp Ala Gly Gln Ser Glu Ile Ala Ser MetAla Leu Met Val Ala Leu Asp Ala Gly Gln Ser Glu Ile Ala Ser Met
820 825 830820 825 830
Leu Tyr Ser Arg Met Asn Ile Lys Cys Ser Phe Ala Pro Met Ser AspLeu Tyr Ser Arg Met Asn Ile Lys Cys Ser Phe Ala Pro Met Ser Asp
835 840 845835 840 845
Asp Glu Ser Pro Thr Ser Ser Ser Ala Glu GluAsp Glu Ser Pro Thr Ser Ser Ser Ser Ala Glu Glu
850 855850 855
Claims (9)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100111965A CN100354302C (en) | 2005-01-18 | 2005-01-18 | SIP gene and its coded protein and uses |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100111965A CN100354302C (en) | 2005-01-18 | 2005-01-18 | SIP gene and its coded protein and uses |
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| CN1807450A true CN1807450A (en) | 2006-07-26 |
| CN100354302C CN100354302C (en) | 2007-12-12 |
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| US20020019005A1 (en) * | 1999-02-18 | 2002-02-14 | Arcaris, Inc. | Process for identification of genes encoding proteins having cell proliferation-promoting activity |
| AU2001280443A1 (en) * | 2000-06-22 | 2002-01-02 | Corixa Corporation | Compositions and methods for the therapy and diagnosis of breast cancer |
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