WO2019237373A1 - 定点插入btdc基因的293t细胞株的构建方法及其用途 - Google Patents

定点插入btdc基因的293t细胞株的构建方法及其用途 Download PDF

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WO2019237373A1
WO2019237373A1 PCT/CN2018/091703 CN2018091703W WO2019237373A1 WO 2019237373 A1 WO2019237373 A1 WO 2019237373A1 CN 2018091703 W CN2018091703 W CN 2018091703W WO 2019237373 A1 WO2019237373 A1 WO 2019237373A1
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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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    • 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
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  • the present invention relates to the field of molecular biology, in particular to a method for constructing a 293T cell line with a BTDC gene inserted at a site and its use.
  • CRISPR Clustered Regularly Interspaced Sequences Short Palindromic Repeats
  • the Cas gene encodes a protein that contains nucleases, polymerases, helicases, and domains that bind to ribonucleic acid.
  • the RNA transcribed by CRISPR combines with Cas protein to form a ribonucleoprotein complex that cooperates with the immune function of the CRISPR / Cas system to guide the Cas protein. Therefore, this RNA is also called single-stranded guide RNA (sgRNA).
  • sgRNA single-stranded guide RNA
  • the Cas protein in the complex can cut the invading virus DNA to achieve the purpose of defense. Therefore, you only need to synthesize a DNA sequence that guides RNA for the DNA sequence that needs to be edited.
  • the artificially constructed sgRNA can guide the Cas9 protein to accurately cut the specific DNA sequence of the host cell, making the DNA double Strand break (DSB), which in turn activates endogenous repair mechanisms, and there are usually two types of endogenous repair mechanisms, which are not very accurate when repaired under non-homologous end junction repair mechanisms, and are often randomly inserted or deleted at the break gap Bases; and in the presence of a homologous recombination repair mechanism and the presence of a repair template, site-specific insertion or deletion of single bases or long fragments can also be achieved to form gene knock-in and knock-out.
  • DSB DNA double Strand break
  • non-small cell lung cancer cell lung cancer is a common malignant tumor in the world, and its incidence is increasing year by year. It is of great theoretical significance and clinical application value to study the tumor recurrence factors and tumor immune escape mechanism of NSCLC.
  • T lymphocytes are important effector cells that mediate tumor immune responses. T cell activation requires TCR-mediated antigen-specific signals and costimulatory molecules-mediated costimulatory signals.
  • BTDC / B7-H1 is a negative T cell co-stimulatory molecule in the B7 family.
  • BTDC inhibits the proliferation and activation of CD4 and CD8 T cells by binding to its receptor PD-1, and negatively regulates the body's immune response process, thereby mediating tumors.
  • the purpose of the present invention is to overcome the defects existing in the prior art and provide a method for constructing a 293T cell line with a BTDC gene inserted at a site, laying a foundation for the subsequent study of the function of human BTDC genes.
  • a method for constructing a 293T cell line inserted into the BTDC gene at a site is to construct a sgRNA expression vector based on the CRISPER / Cas9 system based on the nucleotide sequence of the insertion site, and according to the sgRNA action site
  • the expression cassette containing the BTDC gene that can be integrated into the host genome was constructed by dots, and then the constructed sgRNA expression vector and expression cassette were co-transfected into 293T cells to obtain a 293T cell line with the BTDC gene inserted at the site.
  • the sgRNA interaction site is located on chromosome 10 of 293T cells, and its nucleotide sequence is 5'-CGATGATGGGAAGCGGGAAA-3 '.
  • the expression box contains the following sequential connected expression elements: 5 'homology arm of the sgRNA interaction site-CAG promoter-BTDC gene-poly The 3 'homology arm of the A-sgRNA interaction site.
  • the 5 'and 3' homology arms of the sgRNA interaction site are both about 300-500 bp in size.
  • nucleotide sequence of the expression element is shown in SEQ ID NO 1.
  • the invention also provides the application of the 293T cell line inserted into the BTDC gene in the expression of recombinant protein.
  • the object of the present invention can be further achieved by using the following technical measures.
  • sgRNA expression vector based on CRISPER / Cas9 system
  • d) Monoclonal cell lines were obtained by limiting dilution method, and 293T cell lines with BTDC gene inserted at the site were identified and screened by PCR.
  • step d the 293T cell line that is inserted into the BTDC gene at the designated site is identified and designed by PCR primers that span the 5 'homology arm.
  • the PCR technique is used to amplify the chromosome partial sequence, the entire 5' homology arm sequence, and CAG activation.
  • the product of the sub-sequence is intended to determine the integration of the BTDC gene into the desired site.
  • the method for constructing a 293T cell line with a BTDC gene inserted at the site provided by the present invention has significantly higher gene knock-out efficiency and integration efficiency of a foreign gene than conventional techniques, and realizes that a site-integrated foreign gene can be selected without adding any screening markers.
  • the transgenic cell line will play an important role in BTDC-related drug research and development.
  • FIG. 1 is a schematic structural diagram of the expression box
  • Figure 2 shows the results of PCR identification of transfected cells, where M-Marker, 1-transfected 293T cells, 2-normal 293T cells.
  • Embodiment one sgRNA Construction of expression vectors
  • SgRNA was designed based on the sequence of chromosome 10 of 293T cells, and its nucleotide sequence was 5’-CACCCGATGATGGGAAGCGGGAAA -3 ’. This sequence and its reverse complement 5'-AAACTTTCCCGCTTCCCATCATCG-3 'were synthesized.
  • the two nucleotide sequences were each formulated with 100 ⁇ mol / L of deionized bacteria water, placed in 600 mL of boiling water, and naturally cooled to room temperature for annealing to form a double-stranded sgRNA sequence.
  • the px330 plasmid was double-digested by Bbs I, mixed with the double-stranded sgRNA sequence 1: 3 after recovery, and T4 DNA ligase was ligated at 16 ° C overnight. Escherichia coli NEBStable was transformed, and ampicillin was screened and cultured. Monoclonal strains were selected and identified by sequencing. With 100 ⁇ g / ml ampicillin LB medium, cultured at 37 ° C in large quantities and sequenced correctly E. coli, and sgRNA expression vector was extracted without endotoxin.
  • An BTDC gene-containing expression cassette was constructed based on the sgRNA interaction site.
  • the expression box contains the following sequential connected expression elements: the 5 ′ end homology arm of the sgRNA interaction site-CAG promoter-BTDC gene-poly A-sgRNA interaction site of the 3 ′ end homology arm, its structure As shown in Figure 1, the sequence is shown in SEQ ID NO 1.
  • ID NO 1 The sequence shown in ID NO 1 is provided with an EcoRV digestion site at each of its 5 'and 3' ends.
  • the synthetic sequence was loaded into the pUC19 vector.
  • the vector was digested with EcoRV enzyme, and the target fragment was recovered after electrophoresis to obtain the expression frame.
  • 293T cells were seeded into six-well plates at a density of 50%. After 18-24 h of culture, the fusion degree of 293T cells reached 60% -80%. According to the instructions of Lipofectamine 2000 transfection reagent, 2 ⁇ g sgRNA expression vector and 2 ⁇ g expression frame were co-transfected into 293T cells by liposome method. .
  • the genomic DNA of the cells collected in Example 3 was extracted, primers were designed across the homologous arms, and the presence of cells with site-inserted BTDC gene fragments was identified by PCR.
  • the reaction conditions of the PCR are: 1 minute at 98 ° C, 1 cycle; 10 seconds at 98 ° C, 10s at 57 ° C, 1 minute at 72 ° C, 30 cycles; 72 ° C 5min, 1 cycle.
  • 1% agarose gel electrophoresis was performed, and the results are shown in FIG. 2. It can be seen that there are cells in the total cell line that insert the BTDC gene fragment at a site, as expected.
  • Example 4 Take the remaining transfected cells in Example 4 and dilute them to three 96-well plates by limiting dilution method to ensure that the average number of cells in each well is between 1-2. After these cells are continuously cultured until they form a monoclonal cell population, a part of cells are taken from each well to extract genomic DNA, and identification is performed according to the method of Example 4. The final results showed that of the 288 cells, 5 cells were inserted into the BTDC gene fragment.
  • the method for constructing a 293T cell line with a BTDC gene inserted at the site provided by the present invention has significantly higher gene knock-out efficiency and integration efficiency of a foreign gene than conventional techniques, and realizes that a site-integrated foreign gene can be selected without adding any screening markers.
  • the transgenic cell line will play an important role in BTDC-related drug research and development.

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Abstract

一种定点插入BTDC基因的293T细胞株的构建方法及其用途。该细胞株是根据插入位点的核苷酸序列，构建基于CRISPER/Cas9系统的sgRNA表达载体，并根据sgRNA作用位点构建含有BTDC基因的且可以整合至宿主基因组中的表达框，然后将构建的sgRNA表达载体和线性化的表达框共转染至293T细胞中，筛选后获得定点插入BTDC基因的293T细胞株。获得的细胞株可长期维持BTDC蛋白的高效和稳定表达。

Description

定点插入BTDC基因的293T细胞株的构建方法及其用途 技术领域
本发明涉及分子生物学领域，具体来说，涉及一种定点插入BTDC基因的293T细胞株的构建方法及其用途。
 背景技术
规律间隔成簇短回文重复序列（Clustered Regularly Interspaced Short Palindromic Repeats，CRISPR）是一系列成簇排列的DNA 序列，是细菌用以保护自身对抗病毒的一个系统，也是一种对付攻击者的基因武器。Cas基因编码的蛋白包含核酸酶、聚合酶、解旋酶以及与核糖核酸结合的结构域。CRISPR转录出的RNA与Cas蛋白结合形成核糖核蛋白复合物协同行使CRISPR/Cas系统的免疫功能，来对Cas蛋白起到导向作用，因此这段RNA也被称为单链导向RNA（sgRNA）。当入侵的病毒DNA和sgRNA序列一致时，复合物中的Cas蛋白就能够切割入侵的病毒DNA，达到防御的目的。因此，只需针对需要编辑的DNA序列合成一段导向RNA的DNA序列，在转入宿主细胞后，产生的人工构建的sgRNA就能指导Cas9蛋白精准地切割宿主细胞特定的DNA序列，使DNA发生双链断裂（DSB），进而激活内源性修复机制，而内源修复机制通常有两种，在非同源末端连接修复机制下修复时并不是非常精确，在断裂缺口处往往随机的插入或删除碱基；而在同源重组修复机制以及修复模板存在的条件下，也可以实现定点的单个碱基或者长片段的插入、删除或者突变，形成基因的敲入与敲除。
非小细胞肺癌（non-small cell lung cancer，NSCLC）是当前世界的常见恶性肿瘤，其发病率有逐年增加的趋势。研究NSCLC肿瘤复发因素及肿瘤免疫逃逸机制，具有重要的理论意义和临床应用价值。T淋巴细胞是介导肿瘤免疫应答的重要效应细胞，T细胞活化需要TCR介导的抗原特异性信号和共刺激分子介导的共刺激信号。
 技术问题
BTDC/B7-H1是B7家族中一个负性T细胞共刺激分子，BTDC通过与其受体PD-1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BTDC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，但现有技术中缺乏定点插入BTDC基因的293T细胞株，对相关研究的进展造成了一定的阻碍。
 技术解决方案
本发明的目的在于克服现有技术中的存在的缺陷，提供一种定点插入BTDC基因的293T细胞株的构建方法，为后续研究人BTDC基因功能奠定基础。
为了实现本发明目的，本发明提供的一种定点插入BTDC基因的293T细胞株的构建方法是根据插入位点的核苷酸序列，构建基于CRISPER/Cas9系统的sgRNA表达载体，并根据sgRNA作用位点构建含有BTDC基因的且可以整合至宿主基因组中的表达框，然后将构建的sgRNA表达载体和表达框共转染至293T细胞中，获得定点插入BTDC基因的293T细胞株。
前述的方法，所述sgRNA作用位点位于293T细胞10号染色体上，其核苷酸序列为5’- CGATGATGGGAAGCGGGAAA -3’。
前述的方法，所述表达框中含有如下顺次连接的表达元件：sgRNA作用位点的5’端同源臂-CAG启动子-BTDC基因-poly A- sgRNA作用位点的3’端同源臂。其中sgRNA作用位点的5’端同源臂和3’同源臂的大小均为约300-500 bp。
前述的方法，所述表达元件的核苷酸序列如SEQ ID NO 1所示。
本发明还提供定点插入BTDC基因的293T细胞株在重组蛋白表达中的应用。
本发明的目的还可以采用以下的技术措施来进一步实现。
a) 构建基于CRISPER/Cas9系统的sgRNA表达载体；b）根据sgRNA作用位点构建含有BTDC基因的且可以整合至宿主基因组中的表达框；c）将上述sgRNA表达载体和线性化的表达框共转染至293T细胞中；d）通过有限稀释法获得单克隆细胞系，通过PCR技术鉴定筛选出定点插入BTDC基因的293T细胞株。
步骤d）中鉴定筛选定点插入BTDC基因的293T细胞株主要采用设计跨5’同源臂的PCR引物的方法，通过PCR技术扩增出包含染色体部分序列、5’同源臂全部序列、CAG启动子部分序列的产物，目的是确定BTDC基因整合到预期位点。
有益效果
本发明提供的定点插入BTDC基因的293T细胞株的构建方法，基因敲除效率和外源基因整合效率均显著高于常规技术，并且实现了不加任何筛选标记即可筛选出定点整合外源基因的转基因细胞系，可在BTDC相关的药物研究和开发中将起重要作用。
 附图说明
图1为所述表达框的结构示意图；
图2为转染细胞的PCR鉴定结果图，其中M-Marker，1-经转染的293T细胞，2-正常293T细胞。
 本发明的实施方式
以下实施例用于说明本发明，但不对本发明的范围造成限制。若未特别指明，实施例均按照常规实验条件或按照制造厂商说明书建议的条件。
实施例一： sgRNA 表达载体的构建
根据293T细胞10号染色体的序列设计sgRNA，其核苷酸序列为5’-CACCCGATGATGGGAAGCGGGAAA -3’。合成该序列及其反向互补序列5’-AAACTTTCCCGCTTCCCATCATCG -3’。将两种核苷酸序列各自用去离子菌水配成100 μmol/L，置于600 mL沸水中，自然冷却至室温退火，形成双链sgRNA序列。
Bbs I双酶切px330质粒，回收后将其与所述双链sgRNA序列按1:3混合后，T4 DNA连接酶16℃连接过夜。转化大肠杆菌NEBStable，氨苄青霉素筛选培养并挑单克隆菌株，测序鉴定。用含100 μg/ml氨苄青霉素的LB培养基，37℃大量培养测序正确的大肠杆菌，无内毒素提取sgRNA表达载体。
实施例二 ： 表达框的构建
根据sgRNA作用位点构建含有BTDC基因的表达框。所述表达框中含有如下顺次连接的表达元件：sgRNA作用位点的5’端同源臂-CAG启动子-BTDC基因-poly A- sgRNA作用位点的3’端同源臂，其结构如图1所示，序列如SEQ ID NO 1所示。
委托上海生工以基因合成的方式合成SEQ ID NO 1所示序列，并在其5’和3’端各加上一个EcoRV酶切位点。合成的序列装载于pUC19载体中。用EcoRV酶对该载体进行酶切，电泳后胶回收目的片段，即可获得表达框。
实施例三 ： 293T 细胞转染
转染前一天将293T胞接种至六孔板，接种密度50%。培养18-24 h至293T细胞融合度达到60%-80%后，参照Lipofectamine 2000转染试剂说明，采用脂质体法将2 μg  sgRNA表达载体和2 μg 表达框各共转染至293T细胞中。
实施例四：转染细胞总细胞株鉴定
提取实施例三中收集细胞的基因组DNA，设计跨同源臂的引物，通过PCR鉴定是否存在定点插入BTDC基因片段的细胞。所述PCR的反应条件为：98℃ 1 min，1个循环；98℃ 10s，57℃ 10s，72℃ 1min，30个循环；72℃ 5min，1个循环。PCR反应完成后进行1%琼脂糖凝胶电泳，其结果如图2所示。可以看到，总细胞株中存在定点插入BTDC基因片段的细胞，与预期相符。
实施例五：单克隆细胞株的筛选与鉴定
取实施例四中剩余的转染细胞，通过有限稀释法将其稀释至3个96孔板中，确保每个孔的平均细胞数在1-2个之间。持续培养这些细胞，至其形成单克隆细胞群后，每个孔取部分细胞提取基因组DNA，并按照实施例四的方法进行鉴定。最终结果表明，288株细胞中，定点插入BTDC基因片段的细胞共有5株。
 工业实用性
本发明提供的定点插入BTDC基因的293T细胞株的构建方法，基因敲除效率和外源基因整合效率均显著高于常规技术，并且实现了不加任何筛选标记即可筛选出定点整合外源基因的转基因细胞系，可在BTDC相关的药物研究和开发中将起重要作用。

Claims (6)

  1. 定点插入BTDC基因的293T细胞株的构建方法，其特征在于，其是根据插入位点的核苷酸序列，构建基于CRISPER/Cas9系统的sgRNA表达载体，并根据sgRNA作用位点构建含有BTDC基因的且可以整合至宿主基因组中的表达框，然后将构建的sgRNA表达载体和表达框共转染至293T细胞中，获得定点插入BTDC基因的293T细胞株。
  2. 根据权利要求1所述的方法，其特征在于，所述sgRNA作用位点位于293T细胞10号染色体上。
  3. 根据权利要求2所述的方法，其特征在于，所述sgRNA作用位点的核苷酸序列为5’- CGATGATGGGAAGCGGGAAA -3’。
  4. 根据权利要求1所述的方法，其特征在于，所述表达框中含有如下顺次连接的表达元件：sgRNA作用位点的5’端同源臂-CAG启动子-BTDC基因-poly A- sgRNA作用位点的3’端同源臂。
  5. 根据权利要求4所述的表达元件，其特征在于，其序列如SEQ ID NO 1所示。
  6. 根据权利要求1所述的定点插入BTDC基因的293T细胞株在重组蛋白表达中的应用。
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