WO2019237378A1 - 一种将CD274基因定点整合至Jurkat细胞的方法及其应用 - Google Patents

一种将CD274基因定点整合至Jurkat细胞的方法及其应用 Download PDF

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WO2019237378A1
WO2019237378A1 PCT/CN2018/091708 CN2018091708W WO2019237378A1 WO 2019237378 A1 WO2019237378 A1 WO 2019237378A1 CN 2018091708 W CN2018091708 W CN 2018091708W WO 2019237378 A1 WO2019237378 A1 WO 2019237378A1
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毛吉炎
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深圳市博奥康生物科技有限公司
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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 the CD274 gene into Jurkat cells and its application.
  • CD274 is a negative T cell co-stimulatory molecule in the B7 family. By binding to its receptor SLEB2, it inhibits the proliferation and activation of CD4 and CD8 T cells, and negatively regulates the body's immune response process, thereby mediating tumor immune escape and promoting tumor growth. Therefore, the study of the role of CD274 in tumor escape has an important role in the prevention and treatment of tumors.
  • CD274 as a target for immune regulation against tumors, anti-infection, anti-autoimmune diseases, and organ transplant survival is of great significance. Its potential clinical transformation value is great, and solid research is required before it can be put into practical use. However, the lack of a method for site-directed integration of the CD274 gene into Jurkat cells in the prior art has caused certain obstacles 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-directed integration of the CD274 gene into Jurkat cells, so that the transformed Jurkat cells can stably overexpress the CD274 protein.
  • a method for site-directed integration of the CD274 gene into Jurkat cells which 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 CD274 and its insertion site were identified.
  • the sequence of the AAV-ITR expression cassette containing the CD274 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-CD274 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 the CD274 gene in Jurkat cells at the AAVS1 site of chromosome 19, so that it can obtain the ability to continuously overexpress the CD274 protein, and use the insect protein expression system to synthesize the elements necessary for AAV, thereby 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 CD274 gene
  • FIG. 2 is a result chart of CD274 gene quantitative PCR
  • Figure 3 is the result of PCR identification of the CD274 gene insertion site, where 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-CD274 Construction of vectors
  • an AAV-ITR expression cassette containing the CD274 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 CD274 gene was integrated on the pUC19-ITR-CD274 vector.
  • the pUC19-ITR-CD274 vector was digested with SpeI and SphI enzymes, and the ⁇ 1500 bp target fragment AAV-ITR-CD274 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-CD274 vector and pFastBac1-RC vector were transformed into competent E. coli DH10Bac, respectively. Positive clones were selected by blue and white spots, and recombinant Bacmid was extracted to obtain Bacmid-ITR-CD274 and Bacmid-RC.
  • Cellfectin II Reagent was used to transfect Bacmid-ITR-CD274 and Bacmid-RC into sf9 cells in logarithmic growth phase. The culture supernatant was collected 120 hours after infection, which is P1. High-titer P3 viruses Bac-ITR-CD274 and Bac-RC were obtained after P1 was continuously infected with sf9 cells twice.
  • the P3 virus Bac-ITR-CD274 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.
  • CD274 gene expression levels of Jurkat cells (experimental group) and normal Jurkat cells (control group) were detected by real-time quantitative PCR. The results are shown in Figure 2. It can be seen that the expression level of CD274 gene in the experimental group cells was significantly higher. In control cells, the CD274 gene sequence was successfully integrated into Jurkat cells.
  • 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 portion of the CD274 gene as a downstream primer (sequence: 5’- GAATATAAAGACAGCAAATATC -3 ') PCR was performed, and the results are shown in FIG. 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 CD274 gene has been successfully integrated into the AAVS1 site.
  • the invention can realize the site-specific integration of the CD274 gene in Jurkat cells at the AAVS1 site of chromosome 19, so that it can obtain the ability to continuously overexpress the CD274 protein, and use the insect protein expression system to synthesize the elements necessary for AAV, thereby 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

一种将CD274基因定点整合至Jurkat细胞的方法，其借助昆虫蛋白表达系统合成重组腺相关病毒(rAAV)所需的必要元件，并实现将CD274基因定点整合至Jurkat细胞19号染色体AAVS1位点的目的。可使改造后的Jurkat细胞稳定过表达CD274蛋白，且因rAAV所必需的元件均由昆虫蛋白表达系统合成，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。

Description

一种将CD274基因定点整合至Jurkat细胞的方法及其应用 技术领域
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CD274基因定点整合至Jurkat细胞的方法及其应用。
 背景技术
CD274是B7家族中一个负性T细胞共刺激分子，通过与其受体SLEB2结合，抑制CD4和CD8T细胞的增殖和活化，负性调控机体免疫应答过程，从而介导肿瘤免疫逃逸，促进肿瘤生长，因此对CD274在肿瘤逃逸中作用的研究对于肿瘤的防治具有重要的作用。
 技术问题
以CD274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，但现有技术中缺乏将CD274基因定点整合至Jurkat细胞的手段，对相关研究的进展造成了一定的阻碍。
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。
 技术解决方案
本发明的目的在于提供一种将CD274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CD274蛋白。
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CD274基因定点整合至Jurkat细胞的方法，其包括以下步骤：
1）设计包含CD274基因的AAV-ITR表达盒，并委托合成；
2）将所述包含CD274基因的AAV-ITR表达盒插入pFastBac1载体中，构建得到质粒pFastBac1-ITR-CD274；
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’；
4）将步骤2）得到的pFastBac1-ITR-CD274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-CD274和Bacmid-RC；
5）用Cellfectin II Reagent分别将Bacmid-ITR-CD274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-CD274和Bac-RC；
6）用Bac-ITR-CD274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；
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CD274的表达情况及其插入位点进行鉴定。
其中，所述包含CD274基因的AAV-ITR表达盒的序列如SEQ ID No.1所示。
其中，所述定点整合位点为Jurkat细胞的19号染色体AAVS1位点。
优选的，步骤5）所述Bacmid-ITR-CD274和Bacmid-RC载体的比例为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。
 有益效果
本发明可实现CD274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CD274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。
 附图说明
图1为包含CD274基因的AAV-ITR表达盒结构示意图；
图2为CD274基因荧光定量PCR的结果图；
图3为PCR鉴定CD274基因插入位点的结果图，其中M-Marker，1-对照组，2-实验组。
 本发明的实施方式
下面结合附图与具体实施例对本发明做进一步的说明。
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，
实施例一： pFastBac1-ITR-CD274 载体的构建
根据GenBank中提供的人CD274基因的序列，设计包含CD274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。
合成所得的包含CD274基因的AAV-ITR表达盒整合在pUC19-ITR-CD274载体上。使用SpeI和SphI酶对pUC19-ITR-CD274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-CD274。
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。
取pFastBac1载体50 μg，按pFastBac1载体与AAV-ITR-CD274的摩尔比为1:5量取AAV-ITR-CD274，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-CD274。
实施例二 ： pFastBac1-RC 载体的构建
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’。
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。
实施例三 ：高滴度杆状病毒 Bac-ITR-CD274 和 Bac -RC 的制备
将pFastBac1-ITR-CD274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-CD274和Bacmid-RC。
用Cellfectin II Reagent分别将Bacmid-ITR-CD274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-CD274和Bac-RC。
实施例四 ：定点插入 CD274 基因的 Jurkat 细胞的构建及鉴定
用实施例三获得的P3病毒Bac-ITR-CD274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。
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CD274基因表达水平，其结果如图2所示，可以看到，实验组细胞的CD274基因表达水平显著高于对照组细胞，说明CD274基因序列被成功整合到了Jurkat细胞中。
接下来对CD274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CD274基因5’端部分序列为下游引物（序列为：5’- GAATATAAAGACAGCAAATATC -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CD274基因已被成功整合至AAVS1位点中。
 工业实用性
本发明可实现CD274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CD274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。

Claims (5)

  1. 一种将CD274基因定点整合至Jurkat细胞的方法，其特征在于，包括以下步骤：
    1）设计包含CD274基因的AAV-ITR表达盒，并委托合成；
    2）将所述包含CD274基因的AAV-ITR表达盒插入pFastBac1载体中，构建得到质粒pFastBac1-ITR-CD274；
    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’；
    4）将步骤2）得到的pFastBac1-ITR-CD274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-CD274和Bacmid-RC；
    5）用Cellfectin II Reagent分别将Bacmid-ITR-CD274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-CD274和Bac-RC；
    6）用Bac-ITR-CD274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；
    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CD274的表达情况及其插入位点进行鉴定。
  2. 如权利要求1所述的一种将CD274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CD274基因的AAV-ITR表达盒的序列如SEQ ID No.1所示。
  3. 如权利要求1所述的一种将CD274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。
  4. 如权利要求1所述的一种将CD274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-CD274和Bacmid-RC载体的比例为3-10。
  5. 如权利要求1所述的一种将CD274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。
PCT/CN2018/091708 2018-06-16 2018-06-16 一种将CD274基因定点整合至Jurkat细胞的方法及其应用 WO2019237378A1 (zh)

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