WO2019237377A1 - Method for site-directed integration of sleb2 gene into jurkat cell and use thereof - Google Patents
Method for site-directed integration of sleb2 gene into jurkat cell and use thereof Download PDFInfo
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- the invention belongs to the technical field of genetic engineering. More specifically, the present invention relates to a method for site-integrating SLEB2 gene into Jurkat cells and its application.
- SLEB2 is an important immunosuppressive molecule and is a member of the CD28 superfamily.
- the signaling pathway composed of SLEB2 and its ligand PDCD1L1 is one of the most important signaling pathways in the body's immune response. Its activation can lead to the formation of an immunosuppressive tumor microenvironment, allowing tumor cells to escape the body's immune surveillance and killing, and block SLEB2 / PDCD1L1 signaling pathway can reverse tumor immune microenvironment and enhance endogenous anti-tumor immune effects.
- Immunomodulation with SLEB2 as a target has important significance in the fight against tumors, anti-infection, anti-autoimmune diseases, and organ transplant survival. Its potential clinical transformation value is great, and solid research is required before it can be put into practical application. However, the lack of a means of knocking out SLEB2 gene expression in the prior art has caused a certain obstacle to the progress of related research.
- Adeno-associated virus is a non-enveloped single-stranded DNA virus. It has the advantages of good safety, wide tropism, infection of dividing or non-dividing cells, stable physical and chemical properties, and easy storage. Recombinant adeno-associated virus (rAAV) carrying a foreign gene can integrate the foreign gene into the host genome in a targeted manner to achieve long-term stable expression of the foreign gene in the host cell.
- the purpose of the present invention is to provide a method for site-specific integration of SLEB2 gene into Jurkat cells, so that the transformed Jurkat cells can stably overexpress SLEB2 protein.
- a method for site-directed integration of the SLEB2 gene into Jurkat cells includes the following steps:
- pRC-F and pRC-R as upstream and downstream primers, respectively, to amplify the Rep module and Cap module fusion sequences, and then insert them into the pFastBac1 vector to obtain the pFastBac1-RC vector.
- the sequence of the pRC-F primer is 5'-GACTAGTGCCACCATGCCGGGGTTTTACGAG-3 '
- the sequence of the pRC-R primer is 5'-TAGCATGCGCATTAAGCGCGGCGGGTGT-3';
- step 6) The small molecular weight DNA obtained in step 6) was transfected into Jurkat cells in logarithmic growth phase by electroporation. After 72 hours of incubation, the expression of SLEB2 and its insertion site were identified.
- the sequence of the AAV-ITR expression cassette containing the SLEB2 gene is shown in SEQ ID No.1.
- the site-specific integration site is the AAVS1 site on chromosome 19 of Jurkat cells.
- the ratio of the Bacmid-ITR-SLEB2 and Bacmid-RC vectors in step 5) is 3-10.
- the electrical conversion conditions described in step 7) are: the voltage is 600-900V, and the pulse time is 20-30 ms.
- the invention can realize the site-specific integration of SLEB2 gene in Jurkat cells at the AAVS1 site of chromosome 19, so that it can obtain the ability to continuously overexpress SLEB2 protein, and use the insect protein expression system to synthesize the elements necessary for AAV, avoiding the E. coli gene
- the risk of potential endotoxin contamination brought by the cloning system greatly enhances the safety and practicality of Jurkat cells for preclinical research.
- Figure 1 is a schematic diagram of the structure of the AAV-ITR expression cassette containing the SLEB2 gene
- FIG. 2 is a result chart of SLEB2 gene quantitative PCR
- FIG. 3 is a result of PCR identification of a SLEB2 gene insertion site, in which M-Marker, 1-control group, 2-experimental group.
- SpeI and SphI restriction enzymes were purchased from Fermentas, PCR Cleanup kits were purchased from Omega bio-tek, T4 DNA ligase was purchased from NEB, competent E. coli DH5 ⁇ and DH10Bac were purchased from Invitrogen, pFastBac1 and pAAV-RC vectors were purchased from BioVector NTCC Collection Center,
- Embodiment one pFastBac1-ITR-SLEB2 Construction of vectors
- an AAV-ITR expression cassette containing the SLEB2 gene was designed, whose sequence is as shown in SEQ. As shown in ID No. 1, SpeI and SphI digestion site sequences were added to the 5 'and 3' ends, respectively, and Shanghai Biotech was commissioned to synthesize the sequence by gene synthesis.
- the synthetic AAV-ITR expression cassette containing the SLEB2 gene was integrated on the pUC19-ITR-SLEB2 vector.
- the pUC19-ITR-SLEB2 vector was digested with SpeI and SphI enzymes, and the ⁇ 1500 bp target fragment AAV-ITR-SLEB2 was recovered after agarose gel electrophoresis.
- the pFastBac1 vector was digested with SpeI and SphI enzymes, and the digested pFastBac1 vector was recovered by PCR Cleanup kit.
- the pAAV-RC vector was used as a template, and pRC-F and pRC-R were used as the upstream and downstream primers, respectively.
- the Rep module and Cap module fusion sequences were amplified, purified and recovered, and then digested with SpeI and SphI enzymes. In one step, it was inserted into the pFastBac1 vector to obtain the pFastBac1-RC vector.
- the sequence of the pRC-F primer is 5'-GACTAGTGCCACCATGCCGGGGTTTTACGAG-3 '
- the sequence of the pRC-R primer is 5'-TAGCATGCGCATTAAGCGCGGCGGGTGT-3'.
- the competent E. coli DH5 ⁇ was transformed, and ampicillin was screened and cultured. Monoclonal strains were selected and identified by sequencing. A large number of cultured and sequenced E. coli were cultured, and the recombinant vector pFastBac1-RC was extracted.
- the pFastBac1-ITR-SLEB2 vector and pFastBac1-RC vector were transformed into competent E. coli DH10Bac, respectively. Positive clones were selected from blue and white spots, and recombinant Bacmid was extracted to obtain Bacmid-ITR-SLEB2 and Bacmid-RC.
- Bacmid-ITR-SLEB2 and Bacmid-RC were transfected into sf9 cells in logarithmic growth phase using Cellfectin II Reagent, respectively. The culture supernatant was collected 120 hours after infection, which is P1. High-titer P3 viruses Bac-ITR-SLEB2 and Bac-RC were obtained after P1 was continuously infected with sf9 cells twice.
- the P3 virus Bac-ITR-SLEB2 and Bac-RC obtained in Example 3 were used to co-infect sf9 cells in the logarithmic growth phase, and continued to culture 72 After h, the cells were collected, the DNA was extracted separately and the small molecular weight DNA was isolated. The cells were transfected into Jurkat cells in logarithmic growth phase by electroporation, and culture was continued for 72 h.
- the 5′-end sequence of the AAVS1 site sequence in Jurkat cells was used as the upstream primer (sequence: 5’- GAATTCCTAACTGCCCCGGGGC -3 ’), using the 5 ′ end partial sequence of the SLEB2 gene as a downstream primer (sequence: 5’- GGAGTCTAAGAACCATCCTG -3 ').
- the results are shown in Figure 3. It can be seen that a band of ⁇ 1000 bp appeared in the cells of the experimental group, but no band appeared in the cells of the control group, indicating that the SLEB2 gene has been successfully integrated into the AAVS1 site.
- the invention can realize the site-specific integration of SLEB2 gene in Jurkat cells at the AAVS1 site of chromosome 19, so that it can obtain the ability to continuously overexpress SLEB2 protein, and use the insect protein expression system to synthesize the elements necessary for AAV, avoiding the E. coli gene
- the risk of potential endotoxin contamination brought by the cloning system greatly enhances the safety and practicality of Jurkat cells for preclinical research.
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Abstract
Disclosed is a method for the site-directed integration of the SLEB2 gene into a Jurkat cell, which method uses an insect protein expression system to synthesize the necessary components required for a recombinant adeno-associated virus (rAAV), and achieves the aim of integrating the SLEB2 gene in a site-directed manner into the AAVS1 locus of chromosome 19 in a Jurkat cell.
Description
本发明属于基因工程技术领域。更具体地说，本发明涉及一种将SLEB2基因定点整合至Jurkat细胞的方法及其应用。 The invention belongs to the technical field of genetic engineering. More specifically, the present invention relates to a method for site-integrating SLEB2 gene into Jurkat cells and its application.
SLEB2是一种重要的免疫抑制分子，为CD28超家族成员。SLEB2与及其配体PDCD1L1组成的信号通路是机体免疫反应中最重要的信号通路之一，其激活可导致免疫抑制性肿瘤微环境形成，使肿瘤细胞逃避机体免疫监视和杀伤，而阻断SLEB2/ PDCD1L1信号通路可以逆转肿瘤免疫微环境，增强内源性抗肿瘤免疫效应。SLEB2 is an important immunosuppressive molecule and is a member of the CD28 superfamily. The signaling pathway composed of SLEB2 and its ligand PDCD1L1 is one of the most important signaling pathways in the body's immune response. Its activation can lead to the formation of an immunosuppressive tumor microenvironment, allowing tumor cells to escape the body's immune surveillance and killing, and block SLEB2 / PDCD1L1 signaling pathway can reverse tumor immune microenvironment and enhance endogenous anti-tumor immune effects.
以SLEB2为靶点的免疫调节对抗肿瘤、抗感染、抗自身免疫性疾病及器官移植存活等均有重要的意义，其潜在的临床转化价值很大，需进行扎实的研究方可投入实际应用，但现有技术中缺乏敲除SLEB2基因表达的手段，对相关研究的进展造成了一定的阻碍。Immunomodulation with SLEB2 as a target has important significance in the fight against tumors, anti-infection, anti-autoimmune diseases, and organ transplant survival. Its potential clinical transformation value is great, and solid research is required before it can be put into practical application. However, the lack of a means of knocking out SLEB2 gene expression in the prior art has caused a certain obstacle to the progress of related research.
腺相关病毒（AAV）是一种无包膜的单链DNA 病毒，其具有安全性好、嗜性广泛、可感染分裂或不分裂的细胞、理化性质稳而易于保存等优点。携带外源基因的重组腺相关病毒（rAAV）可将外源基因定点整合到宿主基因组上，实现外源基因在宿主细胞体内的长期稳定表达。Adeno-associated virus (AAV) is a non-enveloped single-stranded DNA virus. It has the advantages of good safety, wide tropism, infection of dividing or non-dividing cells, stable physical and chemical properties, and easy storage. Recombinant adeno-associated virus (rAAV) carrying a foreign gene can integrate the foreign gene into the host genome in a targeted manner to achieve long-term stable expression of the foreign gene in the host cell.
本发明的目的在于提供一种将SLEB2基因定点整合至Jurkat细胞的方法，使改造后的Jurkat细胞稳定过表达SLEB2蛋白。The purpose of the present invention is to provide a method for site-specific integration of SLEB2 gene into Jurkat cells, so that the transformed Jurkat cells can stably overexpress SLEB2 protein.
为了实现根据本发明的这些目的和其它优点，提供了一种将SLEB2基因定点整合至Jurkat细胞的方法，其包括以下步骤：In order to achieve these objects and other advantages according to the present invention, a method for site-directed integration of the SLEB2 gene into Jurkat cells is provided, which includes the following steps:
1）设计包含SLEB2基因的AAV-ITR表达盒，并委托合成；1) Design the AAV-ITR expression cassette containing the SLEB2 gene and commission the synthesis;
2）将所述包含SLEB2基因的AAV-ITR表达盒插入pFastBac1载体中，构建得到质粒pFastBac1-ITR-SLEB2；2) Insert the AAV-ITR expression cassette containing the SLEB2 gene into the pFastBac1 vector to construct a plasmid pFastBac1-ITR-SLEB2;
3）以pAAV-RC载体为模板，分别以pRC-F和pRC-R为上下游引物，扩增Rep组件和Cap组件融合序列，然后将其插入pFastBac1载体中，获得pFastBac1-RC载体。其中，pRC-F引物的序列为5’- GACTAGTGCCACCATGCCGGGGTTTTACGAG-3’， pRC-R引物的序列为5’-TAGCATGCGCATTAAGCGCGGCGGGTGT -3’；3) Using the pAAV-RC vector as a template, pRC-F and pRC-R as upstream and downstream primers, respectively, to amplify the Rep module and Cap module fusion sequences, and then insert them into the pFastBac1 vector to obtain the pFastBac1-RC vector. The sequence of the pRC-F primer is 5'-GACTAGTGCCACCATGCCGGGGTTTTACGAG-3 ', and the sequence of the pRC-R primer is 5'-TAGCATGCGCATTAAGCGCGGCGGGTGT-3';
4）将步骤2）得到的pFastBac1-ITR-SLEB2载体和步骤3）得到的pFastBac1-RC载体分别转化感受态大肠杆菌DH10Bac，蓝白斑筛选出阳性克隆，抽提重组Bacmid，获得Bacmid-ITR-SLEB2和Bacmid-RC；4) The pFastBac1-ITR-SLEB2 vector obtained in step 2) and the pFastBac1-RC vector obtained in step 3) were respectively transformed into competent E. coli DH10Bac. Blue and white spots were selected for positive clones, and recombinant Bacmid was extracted to obtain Bacmid-ITR-SLEB2 And Bacmid-RC;
5）用Cellfectin
II Reagent分别将Bacmid-ITR-SLEB2和Bacmid-RC转染处于对数生长期的sf9细胞。感染120
h后收集培养上清，即为P1。取P1连续感染sf9细胞两次后获得高滴度的P3病毒Bac-ITR-SLEB2和Bac-RC；5) Use Cellfectin
II Reagent transfected Bacmid-ITR-SLEB2 and Bacmid-RC into sf9 cells in logarithmic growth phase, respectively. Infection 120
The culture supernatant was collected after h, which is P1. High-titer P3 virus Bac-ITR-SLEB2 and Bac-RC were obtained after P1 was continuously infected with sf9 cells twice.
6）用Bac-ITR-SLEB2和Bac-RC共同感染处于对数生长期的sf9细胞，继续培养72
h后，收集细胞，分别提取DNA并分离出其中的小分子量DNA；6) Co-infection of sf9 cells in logarithmic growth phase with Bac-ITR-SLEB2 and Bac-RC, and continue to culture 72
After h, the cells were collected, the DNA was extracted separately and the small molecular weight DNA was isolated;
7）通过电转将步骤6）获得的小分子量DNA转染至处于对数生长期的Jurkat细胞中，继续培养72 h后，对SLEB2的表达情况及其插入位点进行鉴定。7) The small molecular weight DNA obtained in step 6) was transfected into Jurkat cells in logarithmic growth phase by electroporation. After 72 hours of incubation, the expression of SLEB2 and its insertion site were identified.
其中，所述包含SLEB2基因的AAV-ITR表达盒的序列如SEQ ID No.1所示。The sequence of the AAV-ITR expression cassette containing the SLEB2 gene is shown in SEQ ID No.1.
其中，所述定点整合位点为Jurkat细胞的19号染色体AAVS1位点。The site-specific integration site is the AAVS1 site on chromosome 19 of Jurkat cells.
优选的，步骤5）所述Bacmid-ITR-SLEB2和Bacmid-RC载体的比例为3-10。Preferably, the ratio of the Bacmid-ITR-SLEB2 and Bacmid-RC vectors in step 5) is 3-10.
优选的，步骤7）所述的电转条件为：电压为600～900V、脉冲时间为20～30
ms。Preferably, the electrical conversion conditions described in step 7) are: the voltage is 600-900V, and the pulse time is 20-30
ms.
本发明可实现SLEB2基因在Jurkat细胞内的定点整合于19号染色体AAVS1位点，使其获得持续过表达SLEB2蛋白的能力，并且借助昆虫蛋白表达系统合成AAV所必需的元件，避免了大肠杆菌基因克隆系统带来的潜在内毒素污染的风险，极大地提升了Jurkat细胞用于临床前研究的安全性和实用性。The invention can realize the site-specific integration of SLEB2 gene in Jurkat cells at the AAVS1 site of chromosome 19, so that it can obtain the ability to continuously overexpress SLEB2 protein, and use the insect protein expression system to synthesize the elements necessary for AAV, avoiding the E. coli gene The risk of potential endotoxin contamination brought by the cloning system greatly enhances the safety and practicality of Jurkat cells for preclinical research.
图1为包含SLEB2基因的AAV-ITR表达盒结构示意图；Figure 1 is a schematic diagram of the structure of the AAV-ITR expression cassette containing the SLEB2 gene;
图2为SLEB2基因荧光定量PCR的结果图；FIG. 2 is a result chart of SLEB2 gene quantitative PCR;
图3为PCR鉴定SLEB2基因插入位点的结果图，其中M-Marker，1-对照组，2-实验组。FIG. 3 is a result of PCR identification of a SLEB2 gene insertion site, in which M-Marker, 1-control group, 2-experimental group.
SpeI和SphI限制酶购自Fermentas，PCR Cleanup试剂盒购自Omega bio-tek，T4 DNA连接酶购自NEB，感受态大肠杆菌DH5α和DH10Bac购自Invitrogen，pFastBac1和pAAV-RC载体购自BioVector
NTCC保藏中心， SpeI and SphI restriction enzymes were purchased from Fermentas, PCR Cleanup kits were purchased from Omega bio-tek, T4 DNA ligase was purchased from NEB, competent E. coli DH5α and DH10Bac were purchased from Invitrogen, pFastBac1 and pAAV-RC vectors were purchased from BioVector
NTCC Collection Center,
实施例一：Embodiment one:
pFastBac1-ITR-SLEB2pFastBac1-ITR-SLEB2
载体的构建Construction of vectors
根据GenBank中提供的人SLEB2基因的序列，设计包含SLEB2基因的AAV-ITR表达盒，其序列如SEQ
ID No.1所示，在其5’端和3’端分别加上SpeI和SphI酶切位点序列，委托上海生工以基因合成的方式合成该序列。According to the sequence of the human SLEB2 gene provided in GenBank, an AAV-ITR expression cassette containing the SLEB2 gene was designed, whose sequence is as shown in SEQ.
As shown in ID No. 1, SpeI and SphI digestion site sequences were added to the 5 'and 3' ends, respectively, and Shanghai Biotech was commissioned to synthesize the sequence by gene synthesis.
合成所得的包含SLEB2基因的AAV-ITR表达盒整合在pUC19-ITR-SLEB2载体上。使用SpeI和SphI酶对pUC19-ITR-SLEB2载体进行酶切，琼脂糖凝胶电泳后回收~1500 bp的目的片段AAV-ITR-SLEB2。The synthetic AAV-ITR expression cassette containing the SLEB2 gene was integrated on the pUC19-ITR-SLEB2 vector. The pUC19-ITR-SLEB2 vector was digested with SpeI and SphI enzymes, and the ~ 1500 bp target fragment AAV-ITR-SLEB2 was recovered after agarose gel electrophoresis.
使用SpeI和SphI酶对pFastBac1载体进行酶切，PCR Cleanup试剂盒回收经酶切的pFastBac1载体。The pFastBac1 vector was digested with SpeI and SphI enzymes, and the digested pFastBac1 vector was recovered by PCR Cleanup kit.
取pFastBac1载体50 μg，按pFastBac1载体与AAV-ITR-SLEB2的摩尔比为1:5量取AAV-ITR-SLEB2，混匀后用T4 DNA连接酶，16℃连接过夜。转化感受态大肠杆菌DH5α，氨苄青霉素筛选培养并挑单克隆菌株，测序鉴定。大量培养测序正确的大肠杆菌，提取重组载体pFastBac1-ITR-SLEB2。Take 50 μg of pFastBac1 vector, and measure AAV-ITR-SLEB2 according to the molar ratio of pFastBac1 vector and AAV-ITR-SLEB2 1: 5. After mixing, use T4 DNA ligase and ligate at 16 ° C overnight. The competent E. coli DH5α was transformed, and ampicillin was screened and cultured. Monoclonal strains were selected and identified by sequencing. A large number of cultured and sequenced E. coli were cultured, and the recombinant vector pFastBac1-ITR-SLEB2 was extracted.
实施例二Example two
：:
pFastBac1-RCpFastBac1-RC
载体的构建Construction of vectors
以pAAV-RC载体为模板，分别以pRC-F和pRC-R为上下游引物，扩增Rep组件和Cap组件融合序列，纯化回收后使用SpeI和SphI酶进行酶切，然后将其按照实施例一中的步骤将其插入pFastBac1载体中，获得pFastBac1-RC载体。其中，pRC-F引物的序列为5’- GACTAGTGCCACCATGCCGGGGTTTTACGAG-3’， pRC-R引物的序列为5’-TAGCATGCGCATTAAGCGCGGCGGGTGT -3’。The pAAV-RC vector was used as a template, and pRC-F and pRC-R were used as the upstream and downstream primers, respectively. The Rep module and Cap module fusion sequences were amplified, purified and recovered, and then digested with SpeI and SphI enzymes. In one step, it was inserted into the pFastBac1 vector to obtain the pFastBac1-RC vector. The sequence of the pRC-F primer is 5'-GACTAGTGCCACCATGCCGGGGTTTTACGAG-3 ', and the sequence of the pRC-R primer is 5'-TAGCATGCGCATTAAGCGCGGCGGGTGT-3'.
转化感受态大肠杆菌DH5α，氨苄青霉素筛选培养并挑单克隆菌株，测序鉴定。大量培养测序正确的大肠杆菌，提取重组载体pFastBac1-RC。The competent E. coli DH5α was transformed, and ampicillin was screened and cultured. Monoclonal strains were selected and identified by sequencing. A large number of cultured and sequenced E. coli were cultured, and the recombinant vector pFastBac1-RC was extracted.
实施例三Example three
：高滴度杆状病毒: High titer baculovirus
Bac-ITR-SLEB2Bac-ITR-SLEB2
和with
BacBac
-RC-RC
的制备Preparation
将pFastBac1-ITR-SLEB2载体和pFastBac1-RC载体分别转化感受态大肠杆菌DH10Bac，蓝白斑筛选出阳性克隆，抽提重组Bacmid，获得Bacmid-ITR-SLEB2和Bacmid-RC。The pFastBac1-ITR-SLEB2 vector and pFastBac1-RC vector were transformed into competent E. coli DH10Bac, respectively. Positive clones were selected from blue and white spots, and recombinant Bacmid was extracted to obtain Bacmid-ITR-SLEB2 and Bacmid-RC.
用Cellfectin II Reagent分别将Bacmid-ITR-SLEB2和Bacmid-RC转染处于对数生长期的sf9细胞。感染120 h后收集培养上清，即为P1。取P1连续感染sf9细胞两次后获得高滴度的P3病毒Bac-ITR-SLEB2和Bac-RC。Bacmid-ITR-SLEB2 and Bacmid-RC were transfected into sf9 cells in logarithmic growth phase using Cellfectin II Reagent, respectively. The culture supernatant was collected 120 hours after infection, which is P1. High-titer P3 viruses Bac-ITR-SLEB2 and Bac-RC were obtained after P1 was continuously infected with sf9 cells twice.
实施例四Example 4
：定点插入: Fixed-point insertion
SLEB2SLEB2
基因的genetic
JurkatJurkat
细胞的构建及鉴定Construction and identification of cells
用实施例三获得的P3病毒Bac-ITR-SLEB2和Bac-RC共同感染处于对数生长期的sf9细胞，继续培养72
h后，收集细胞，分别提取DNA并分离出其中的小分子量DNA，并通过电转将其转染至处于对数生长期的Jurkat细胞中，继续培养72 h。The P3 virus Bac-ITR-SLEB2 and Bac-RC obtained in Example 3 were used to co-infect sf9 cells in the logarithmic growth phase, and continued to culture 72
After h, the cells were collected, the DNA was extracted separately and the small molecular weight DNA was isolated. The cells were transfected into Jurkat cells in logarithmic growth phase by electroporation, and culture was continued for 72 h.
荧光定量PCR检测经转染的Jurkat细胞（实验组）和正常Jurkat细胞（对照组）的SLEB2基因表达水平，其结果如图2所示，可以看到，实验组细胞的SLEB2基因表达水平显著高于对照组细胞，说明SLEB2基因序列被成功整合到了Jurkat细胞中。The quantitative expression of SLEB2 gene in Jurkat cells (experimental group) and normal Jurkat cells (control group) was detected by real-time PCR. The results are shown in Figure 2. It can be seen that the SLEB2 gene expression level in the experimental group cells was significantly higher. In control cells, the SLEB2 gene sequence was successfully integrated into Jurkat cells.
接下来对SLEB2基因的插入位点进行鉴定。以Jurkat细胞AAVS1位点部分序列（登录号：S51329.1）5’端序列为上游引物（序列为：5’-
GAATTCCTAACTGCCCCGGGGC -3’），以SLEB2基因5’端部分序列为下游引物（序列为：5’-
GGAGTCTAAGAACCATCCTG -3’）进行PCR，其结果如图3所示。可以看到，实验组细胞出现了~1000 bp的条带，而对照组细胞无任何条带出现，说明SLEB2基因已被成功整合至AAVS1位点中。Next, the insertion sites of the SLEB2 gene were identified. The 5′-end sequence of the AAVS1 site sequence in Jurkat cells (accession number: S51329.1) was used as the upstream primer (sequence: 5’-
GAATTCCTAACTGCCCCGGGGC -3 ’), using the 5 ′ end partial sequence of the SLEB2 gene as a downstream primer (sequence: 5’-
GGAGTCTAAGAACCATCCTG -3 '). The results are shown in Figure 3. It can be seen that a band of ~ 1000 bp appeared in the cells of the experimental group, but no band appeared in the cells of the control group, indicating that the SLEB2 gene has been successfully integrated into the AAVS1 site.
本发明可实现SLEB2基因在Jurkat细胞内的定点整合于19号染色体AAVS1位点，使其获得持续过表达SLEB2蛋白的能力，并且借助昆虫蛋白表达系统合成AAV所必需的元件，避免了大肠杆菌基因克隆系统带来的潜在内毒素污染的风险，极大地提升了Jurkat细胞用于临床前研究的安全性和实用性。 The invention can realize the site-specific integration of SLEB2 gene in Jurkat cells at the AAVS1 site of chromosome 19, so that it can obtain the ability to continuously overexpress SLEB2 protein, and use the insect protein expression system to synthesize the elements necessary for AAV, avoiding the E. coli gene The risk of potential endotoxin contamination brought by the cloning system greatly enhances the safety and practicality of Jurkat cells for preclinical research.
Claims (5)
- 一种将SLEB2基因定点整合至Jurkat细胞的方法，其特征在于，包括以下步骤：A method for site-specific integration of SLEB2 gene into Jurkat cells, which comprises the following steps:1）设计包含SLEB2基因的AAV-ITR表达盒，并委托合成；1) Design the AAV-ITR expression cassette containing the SLEB2 gene and commission the synthesis;2）将所述包含SLEB2基因的AAV-ITR表达盒插入pFastBac1载体中，构建得到质粒pFastBac1-ITR-SLEB2；2) Insert the AAV-ITR expression cassette containing the SLEB2 gene into the pFastBac1 vector to construct a plasmid pFastBac1-ITR-SLEB2;3）以pAAV-RC载体为模板，分别以pRC-F和pRC-R为上下游引物，扩增Rep组件和Cap组件融合序列，然后将其插入pFastBac1载体中，获得pFastBac1-RC载体。其中，pRC-F引物的序列为5’- GACTAGTGCCACCATGCCGGGGTTTTACGAG-3’， pRC-R引物的序列为5’-TAGCATGCGCATTAAGCGCGGCGGGTGT -3’；3) Using the pAAV-RC vector as a template, pRC-F and pRC-R as upstream and downstream primers, respectively, to amplify the Rep module and Cap module fusion sequences, and then insert them into the pFastBac1 vector to obtain the pFastBac1-RC vector. The sequence of the pRC-F primer is 5'-GACTAGTGCCACCATGCCGGGGTTTTACGAG-3 ', and the sequence of the pRC-R primer is 5'-TAGCATGCGCATTAAGCGCGGCGGGTGT-3';4）将步骤2）得到的pFastBac1-ITR-SLEB2载体和步骤3）得到的pFastBac1-RC载体分别转化感受态大肠杆菌DH10Bac，蓝白斑筛选出阳性克隆，抽提重组Bacmid，获得Bacmid-ITR-SLEB2和Bacmid-RC；4) The pFastBac1-ITR-SLEB2 vector obtained in step 2) and the pFastBac1-RC vector obtained in step 3) were respectively transformed into competent E. coli DH10Bac. Blue and white spots were screened for positive clones, and recombinant Bacmid was extracted to obtain Bacmid-ITR-SLEB2. And Bacmid-RC;5）用Cellfectin II Reagent分别将Bacmid-ITR-SLEB2和Bacmid-RC转染处于对数生长期的sf9细胞。感染120 h后收集培养上清，即为P1。取P1连续感染sf9细胞两次后获得高滴度的P3病毒Bac-ITR-SLEB2和Bac-RC；5) Bacmid-ITR-SLEB2 and Bacmid-RC were transfected into sf9 cells in logarithmic growth phase with Cellfectin II Reagent. The culture supernatant was collected 120 hours after infection, which is P1. High-titer P3 virus Bac-ITR-SLEB2 and Bac-RC were obtained after P1 was continuously infected with sf9 cells twice.6）用Bac-ITR-SLEB2和Bac-RC共同感染处于对数生长期的sf9细胞，继续培养72 h后，收集细胞，分别提取DNA并分离出其中的小分子量DNA；6) Co-infection of sf9 cells in logarithmic growth phase with Bac-ITR-SLEB2 and Bac-RC. After continuing to culture for 72 hours, collect cells, extract DNA and isolate small molecular weight DNA;7）通过电转将步骤6）获得的小分子量DNA转染至处于对数生长期的Jurkat细胞中，继续培养72 h后，对SLEB2的表达情况及其插入位点进行鉴定。7) The small molecular weight DNA obtained in step 6) was transfected into Jurkat cells in logarithmic growth phase by electroporation. After 72 hours of incubation, the expression of SLEB2 and its insertion site were identified.
- 如权利要求1所述的一种将SLEB2基因定点整合至Jurkat细胞的方法，其特征在于，步骤1）所述包含SLEB2基因的AAV-ITR表达盒的序列如SEQ ID No.1所示。The method for site-integrating the SLEB2 gene into Jurkat cells according to claim 1, wherein, in step 1), the sequence of the AAV-ITR expression cassette containing the SLEB2 gene is shown in SEQ ID No.1.
- 如权利要求1所述的一种将SLEB2基因定点整合至Jurkat细胞的方法，其特征在于，所述定点整合位点为Jurkat细胞的19号染色体AAVS1位点。The method for site-integrating the SLEB2 gene into Jurkat cells according to claim 1, wherein the site-specific integration site is the AAVS1 site on chromosome 19 of Jurkat cells.
- 如权利要求1所述的一种将SLEB2基因定点整合至Jurkat细胞的方法，其特征在于，步骤5）所述Bacmid-ITR-SLEB2和Bacmid-RC载体的比例为3-10。The method for site-integrating the SLEB2 gene into Jurkat cells according to claim 1, wherein, in step 5), the ratio of the Bacmid-ITR-SLEB2 and Bacmid-RC vectors is 3-10.
- 如权利要求1所述的一种将SLEB2基因定点整合至Jurkat细胞的方法，其特征在于，步骤7）所述的电转条件为：电压为600～900V、脉冲时间为20～30 ms。The method according to claim 1, wherein the SLEB2 gene is site-integrated into Jurkat cells, wherein the electrotransformation conditions in step 7) are: a voltage of 600 to 900 V and a pulse time of 20 to 30 ms.
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