WO2019237382A1 - 用于人cd357基因编辑的修饰载体、其制备方法及应用 - Google Patents

用于人cd357基因编辑的修饰载体、其制备方法及应用 Download PDF

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WO2019237382A1
WO2019237382A1 PCT/CN2018/091712 CN2018091712W WO2019237382A1 WO 2019237382 A1 WO2019237382 A1 WO 2019237382A1 CN 2018091712 W CN2018091712 W CN 2018091712W WO 2019237382 A1 WO2019237382 A1 WO 2019237382A1
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vector
human
gene
gene editing
modified vector
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PCT/CN2018/091712
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French (fr)
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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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Publication of WO2019237382A1 publication Critical patent/WO2019237382A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the invention relates to the technical field of genetic engineering, in particular to a modified vector for human CD357 gene editing, a preparation method and application thereof.
  • the human anti-cancer immune mechanism includes two aspects of cellular immunity and humoral immunity. However, it is mainly based on cellular immunity.
  • the effector cells that play a major role in the cellular immune mechanism are T cells, of which CD8 + T cells are the most important effect in tumor immunity. Perform the cell.
  • T-cell activation requires dual signal recognition.
  • the tumor antigen first binds to MHC molecules on antigen-presenting cells or target cells, and then binds to T-lymphocyte surface antigen recognition receptors to provide the first signal for T-cell activation; CD28 on the T-cell surface Binding to B7 molecules on the surface of antigen presenting cells or on the surface of target cells provides a second signal.
  • the T cells are fully activated, and the activated T cells first proliferate, generating a large number of antigen-specific T cells, which migrate to the tumor site to play a killing role. After 2 to 3 days of activated T cells, the expression of negative regulatory molecules such as CD357 began to be up-regulated.
  • CD357 is a transmembrane protein and belongs to the CD28 family.
  • the ligand of CD357 is a member of the B7 family. It competes with CD28 molecules and binds to B7-1 and B7-2. It inhibits the proliferation of activated T cells and negatively regulates immunity. Under physiological conditions, it can prevent immune from being enlarged. It maintains a dynamic balance between health and disease, but is often used by tumor cells to achieve the purpose of immune escape. Therefore, the research on the role of CD357 in tumorigenesis and development can promote the development of tumor therapy, but the lack of targeted knockout of CD357 gene expression in the prior art has caused certain obstacles to the progress of related research.
  • a first object of the present invention is to provide a modified vector for human CD357 gene editing.
  • a second object of the present invention is to provide a method for preparing a modified vector for human CD357 gene editing.
  • the present invention provides a targeted sgRNA for human CD357 gene editing, and its sequence is 5′- AATTTCTACTCTTGTAGATCTGAAGAGCCCACAGCCAGTTGG -3 ’.
  • the invention also provides a method for preparing the modified vector for human CD357 gene editing, which includes the following steps:
  • step (3) ligating the sgRNA obtained in step (1) to a linearized core vector with T4 DNA ligase;
  • the ligation product is transformed into competent E. coli Stbl3. After a large amount of culture, the recombinant vector is extracted and commissioned for sequencing. The correct sequencing result is the modified vector for human CD357 gene editing.
  • the core vector is pLVX-ascpf1-puro.
  • the modified vector for human CD357 gene editing provided by the present invention has strong specificity, can edit human CD357 gene through the CRISPR / Cpf1 system very efficiently, and can be used in research and development of drugs related to abnormal expression of CD357 gene.
  • Figure 1 is a vector map of pLVX-AsCpf1-puro
  • FIG. 1 T7 Endonuclease I test results of Jurkat cells in the control group and the experimental group, wherein: 1-experimental group, 2-control group.
  • Cpf1 Is a Single The RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System
  • Cpf1 provides crRNA design rules and gRNA sequences that target the CD357 gene, designing crRNAs that target the CD357 gene, and according to the actual situation of the pLVX-AsCpf1-puro vector in its 5 '
  • the sticky ends of the BamHI and EcoRI digestion sites were added at the 3 'and 3' ends, respectively.
  • the forward sequence was 5'-GATCCTAATTTCTACTCTTGTAGATCTGAAGAGCCCACAGCCAGTTGG -3 'and the reverse sequence was 5'-GCCAACTGGCTGTGGGCTCTTCAGATCTACAAGAGTAGAAATTC-3'.
  • 5 ⁇ L of each was mixed, heated at 95 ° C for 5 minutes, and then naturally cooled to room temperature to form a double-stranded DNA with sticky ends of BamHI and EcoRI.
  • T4 DNA ligase was used to ligate a and b products.
  • the ligated product was then transformed into E. coli Stbl3 and identified by sequencing.
  • the recombinant vector contained in the correct strain was the pLVX-AsCpf1-puro-CD357 vector.
  • the correct strain was sequenced and identified in Example 1 and placed in an LB liquid medium having an ampicillin concentration of 100 ⁇ g / ml, and cultured with shaking at 250 rpm and 37 ° C. for 12-16 h. Collect the bacterial solution by centrifugation at 10,000 rpm at 4 ° C, discard the supernatant, collect the bacterial cells, and then extract the plasmid according to the instructions of the Endo-Free Plasmid Mini Kit kit to obtain the endotoxin-free pLVX-AsCpf1-puro-CD357 plasmid.
  • 293T cells were cultured and transfected after 2 passages of growth and culture: pLVX-AsCpf1-puro-CD357 vector was taken and transfected with the packaging plasmid and transfection reagent provided by the integrase-deficient Lenti-X HTX lentiviral packaging system Stained in 293T cells. 48 hours before transfection, inoculate cells into a well plate or petri dish for lentivirus production. During transfection, the confluence of cells is about 70% -80% is the best infection state, and the viability is ⁇ 95%. The staining time was the starting point. The supernatants were harvested after 48 h and 72 h, filtered through a 0.45 ⁇ m filter and stored at -80 ° C.
  • Untreated Jurkat cells (control group) and lentivirus-treated Jurkat cells (experimental group) were seeded into six-well plates. After the cells were full, genomic DNA was extracted, and then the high-fidelity PCR enzyme PrimeSTAR HS was used to expand The gene editing site is expected to be amplified, and the PCR product is recovered by electrophoresis.
  • the modified vector for human CD357 gene editing provided by the present invention has strong specificity, can edit human CD357 gene through the CRISPR / Cpf1 system very efficiently, and can be used in research and development of drugs related to abnormal expression of CD357 gene.

Abstract

提供一种用于人CD357基因编辑的修饰载体、其制备方法及应用。通过CRISPR/Cpf1系统在细胞水平上对人CD357基因进行编辑，用于获得的CD357基因编辑阳性细胞。

Description

用于人CD357基因编辑的修饰载体、其制备方法及应用 技术领域
本发明涉及基因工程技术领域，尤其是涉及一种用于人CD357基因编辑的修饰载体、其制备方法及应用。
 背景技术
人体的抗癌免疫机制包括细胞免疫和体液免疫两个方面，但是以细胞免疫为主，在细胞免疫机制中起主要作用的效应细胞为T细胞，其中CD8+ T细胞是肿瘤免疫中最主要的效应执行细胞。T细胞活化需要双信号识别，肿瘤抗原首先与抗原提呈细胞或者靶细胞上的MHC分子结合后，与T淋巴细胞表面抗原识别受体结合为T细胞活化提供第一信号；T细胞表面的CD28与抗原提呈细胞表面或者靶细胞表面B7分子结合提供第二信号。双信号识别后，T细胞完全活化，活化的T细胞首先自我增殖，产生大量的抗原特异性T细胞，迁移到肿瘤局部起到杀伤作用。活化的T细胞2~3天后CD357等负调控分子开始表达上调。
 技术问题
CD357为跨膜蛋白，属于CD28家族成员。CD357的配体为B7家族成员，其与CD28分子竞争性地与B7-1和B7-2结合，抑制活化的T细胞增殖，对免疫起负调控作用，在生理情况下，可以防止免疫过度放大，维持着健康与疾病的动态平衡，但却常常会被肿瘤细胞利用，达到免疫逃逸的目的。因此对CD357在肿瘤发生发展中作用的研究可以很好地促进肿瘤治疗领域的发展，但现有技术中缺乏靶向敲除CD357基因表达的手段，对相关研究的进展造成了一定的阻碍。
 技术解决方案
本发明的第一个目的在于提供一种用于人CD357基因编辑的修饰载体。
本发明的第二个目的在于提供一种用于人CD357基因编辑的修饰载体的制备方法。
本发明提供的一种用于人CD357基因编辑的靶向sgRNA，其序列为5’- AATTTCTACTCTTGTAGATCTGAAGAGCCCACAGCCAGTTGG -3’。
本发明还提供了上述的用于人CD357基因编辑的修饰载体的制备方法，包括以下步骤：
（1）将所述靶向CD357基因的sgRNA的正义链和反义链进行退火互补配对；
（2）使用BamHI和EcoRI内切酶对核心载体进行酶切，琼脂糖凝胶电泳回收线性化的核心载体；
（3）将步骤（1）获得的sgRNA用T4 DNA连接酶连接至线性化的核心载体中；
（4）连接产物转化感受态大肠杆菌Stbl3中，大量培养后提取重组载体并委托测序，测序结果正确的即为所述用于人CD357基因编辑的修饰载体。
进一步地，在步骤（2）和步骤（3）中，所述核心载体为pLVX-ascpf1-puro。
 有益效果
本发明提供的用于人CD357基因编辑的修饰载体，特异性强，能非常高效地通过CRISPR/Cpf1系统对人CD357基因进行编辑，可用于与CD357基因表达异常相关的药物研究和开发中。
 附图说明
图1为pLVX-AsCpf1-puro的载体图谱；
图2对照组和实验组Jurkat细胞的T7 Endonuclease I试验结果图，其中：1-实验组，2-对照组。
 本发明的实施方式
以下所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明的保护范围。
下述实施例中所用的方法如无特别说明均为常规方法。所用引物及DNA 序列均由上海 生工公司合成。
实施例 1 ： 靶向人 CD357 基因的 pLVX-AsCpf1-puro-CD357 载体的制备
a. 根据Bernd Zetsche等在其文章（Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System）中提供crRNA设计规则及靶向CD357基因的gRNA序列，设计靶向CD357基因的crRNA，并根据pLVX-AsCpf1-puro载体的实际情况在其5’端和3’端分别添加BamHI和EcoRI酶切位点的粘性末端，其正向序列为5’-GATCCTAATTTCTACTCTTGTAGATCTGAAGAGCCCACAGCCAGTTGG -3’，反向序列为5’-GCCAACTGGCTGTGGGCTCTTCAGATCTACAAGAGTAGAAATTC-3’，合成这两段序列。溶解后，各取5 μL混匀后，95℃加热5 min，然后自然冷却至室温，形成带有BamHI和EcoRI粘性末端的DNA双链。
b. 用BamHI和EcoRI酶切pLVX-AsCpf1-puro载体，PCR Cleanup试剂盒回收线性化的pLVX-AsCpf1-puro载体。
c. 用T4 DNA连接酶连接a、b两步获得的产物。然后将连接产物转化至大肠杆菌Stbl3中，测序鉴定。鉴定正确的菌株所含的重组载体即为pLVX-AsCpf1-puro-CD357载体。
实施例 2 ：无内毒素质粒 DNA 的制备
取实施例1中测序鉴定正确的菌株，置于氨苄青霉素浓度为100μg/ml的LB液体培养基中，250 rpm、37℃振荡培养12-16 h。4℃，10000 rpm离心收集菌液，弃上清，收集菌体，然后按照Endo-Free Plasmid Mini Kit试剂盒说明书操作步骤提取质粒，得无内毒素的pLVX-AsCpf1-puro-CD357质粒。
实施例 3 ：慢病毒的包装
培养293T细胞，待生长培养传代2次后，进行转染操作：取pLVX-AsCpf1-puro-CD357载体，用整合酶缺陷型的Lenti-X HTX慢病毒包装系统提供的包装质粒和转染试剂转染293T细胞中。转染前48小时，接种细胞至备用生产慢病毒的孔板或是培养皿中，转染时，细胞汇合度约为70%-80%为最佳感染状态，活力≥95%以上；以转染时间为起始点，分别于48 h和72 h后收获上清，0.45 μm滤膜过滤后，保存于-80℃下。
实施例 4 ： Jurkat 细胞的慢病毒感染及嘌呤霉素筛选
培养Jurkat细胞至其汇合度约为70%-80%时，加入慢病毒与培养基的混合液（含4 μg/mL polybrene）处理24 h后，将慢病毒液换成含1 μg/mL嘌呤霉素的完全培养基，开始进行筛选培养，筛选时间为7 d。隔天换液一次。被慢病毒感染的细胞将形成单细胞克隆，此时即完成了细胞的筛选。
实施例 5 ： T7 Endonuclease I 试验检测转导效果
分别取未经处理的Jurkat细胞（对照组）和经慢病毒处理后的Jurkat细胞（实验组）接种至六孔板，待细胞长满后，提取基因组DNA，然后应用高保真PCR酶PrimeSTAR HS扩增基因编辑预期发生的位点，电泳回收PCR产物。
PCR产物经重退火后，用T7 Endonuclease I在37℃酶切1 h，然后进行琼脂糖凝胶电泳，结果如图2所示，与对照组相比，实验组出现了2条明显的切割条带，表明pLVX-AsCpf1-puro-CD357载体包装成的慢病毒可对人CD357基因进行编辑，说明pLVX-AsCpf1-puro载体可用于细胞的基因编辑研究中。
 工业实用性
本发明提供的用于人CD357基因编辑的修饰载体，特异性强，能非常高效地通过CRISPR/Cpf1系统对人CD357基因进行编辑，可用于与CD357基因表达异常相关的药物研究和开发中。

Claims (5)

  1. 一种用于人CD357基因编辑的修饰载体，其特征在于，包括核心载体及靶向人CD357基因的sgRNA。
  2. 根据权利要求1所述的一种用于人CD357基因编辑的修饰载体，其特征在于，所述靶向CD357基因的sgRNA的序列为5’-AATTTCTACTCTTGTAGATCTGAAGAGCCCACAGCCAGTTGG -3’。
  3. 如权利要求2所述的一种用于人CD357基因编辑的修饰载体的制备方法，其特征在于，包括以下步骤：
    （1）将所述靶向CD357基因的sgRNA的正义链和反义链进行退火互补配对；
    （2）使用BamHI和EcoRI内切酶对核心载体进行酶切，琼脂糖凝胶电泳回收线性化的核心载体；
    （3）将步骤（1）获得的sgRNA用T4 DNA连接酶连接至线性化的核心载体中；
    （4）连接产物转化感受态大肠杆菌Stbl3中，大量培养后提取重组载体并委托测序，测序结果正确的即为所述用于人CD357基因编辑的修饰载体。
  4. 根据权利要求3所述的一种用于人CD357基因编辑的修饰载体的制备方法，其特征在于，在步骤（2）和步骤（3）中，所述核心载体为pLVX-ascpf1-puro。
  5. 根据权利要求1所述任意一种用于人CD357基因编辑的修饰载体，其特征在于，所述载体可用于人的CD357基因敲除。
PCT/CN2018/091712 2018-06-16 2018-06-16 用于人cd357基因编辑的修饰载体、其制备方法及应用 WO2019237382A1 (zh)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
CN106459203A (zh) * 2014-06-06 2017-02-22 百时美施贵宝公司 抗糖皮质激素诱导肿瘤坏死因子受体(gitr)的抗体及其用途
CN107815468A (zh) * 2016-08-31 2018-03-20 北京百奥赛图基因生物技术有限公司 人源化基因改造动物模型的制备方法及应用

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CN106459203A (zh) * 2014-06-06 2017-02-22 百时美施贵宝公司 抗糖皮质激素诱导肿瘤坏死因子受体(gitr)的抗体及其用途
CN107815468A (zh) * 2016-08-31 2018-03-20 北京百奥赛图基因生物技术有限公司 人源化基因改造动物模型的制备方法及应用

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