WO2019237371A1 - Method for constructing cho cell strain with site-directed insertion of ct1.1 gene and use thereof - Google Patents

Method for constructing cho cell strain with site-directed insertion of ct1.1 gene and use thereof Download PDF

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WO2019237371A1
WO2019237371A1 PCT/CN2018/091701 CN2018091701W WO2019237371A1 WO 2019237371 A1 WO2019237371 A1 WO 2019237371A1 CN 2018091701 W CN2018091701 W CN 2018091701W WO 2019237371 A1 WO2019237371 A1 WO 2019237371A1
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site
gene
sgrna
expression
cho cell
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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 relates to the field of molecular biology, and in particular, to a method for constructing a CHO cell line with a CT1.1 gene inserted at a site and its use.
  • CRISPR Interspaced 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
  • Cancer / testis antigen is a group of tumor-associated antigens. It is not expressed in normal human tissues except testis, placenta, and trophoblast cells, but is highly expressed in a variety of tumor tissues, making it a recent year. Tumor immunotherapeutic target antigen has been widely concerned.
  • Melanin-associated antigen is a class of tumor-associated antigens isolated from melanoma cells. It is a subfamily of CTA. It can be processed into antigen peptides in tumor cells, combined with human leukocyte antigen I, and then Autocytotoxic T lymphocytes recognize and kill tumor cells.
  • CT1.1 is the earliest found member of the MAGE family. It is specifically expressed in malignant tumors and can bind to MHC molecules. It can be recognized by specific cell T-cell receptors and activated into cytotoxic T-lymph with specific T-cell receptors. Cells enable the body to produce an immune response that is dominated by cellular immunity. Therefore, CT1.1 is a tumor-specific ideal target antigen, which has become a hot topic in recent years. However, the lack of CHO cell lines with site-specific insertion of the CT1.1 gene in the prior art has caused certain obstacles to the progress of related research. .
  • the purpose of the present invention is to overcome the defects existing in the prior art, and provide a method for constructing a CHO cell line with the CT1.1 gene inserted at a site, laying a foundation for the subsequent study of the function of the human CT1.1 gene.
  • a method for constructing a CHO cell line inserted into the CT1.1 gene at a site by the present invention 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 sgRNA
  • the expression site containing the CT1.1 gene that can be integrated into the host genome is constructed at the action site, and the constructed sgRNA expression vector and expression frame are co-transfected into CHO cells to obtain a CHO cell line with the CT1.1 gene inserted at the site.
  • the sgRNA interaction site is located on chromosome 1 of CHO cells, and its nucleotide sequence is 5'-GAAGCGAGGTTTCCATTCTG-3 '.
  • the expression box contains the following sequential connected expression elements: 5 'end of the sgRNA interaction site, CAG promoter-CT1.1 gene, 3' end of the poly A-sgRNA interaction site Homology arm.
  • the 5 'and 3' homology arms of the sgRNA interaction site are both about 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 CHO cell line inserted with the CT1.1 gene at a fixed site in the expression of recombinant protein.
  • the object of the present invention can be further achieved by using the following technical measures.
  • step d) the CHO cell line with the CT1.1 gene inserted at the designated site is selected and screened by designing a PCR primer that spans the 5 'homology arm.
  • the PCR technique is used to amplify the sequence including the chromosome partial sequence, the entire 5' homology arm sequence,
  • the product of the CAG promoter partial sequence is designed to determine the integration of the CT1.1 gene into the expected site.
  • the method for constructing the CHO cell line with the CT1.1 gene inserted by the site provided by the present invention has significantly higher gene knockout efficiency and integration efficiency of foreign genes than conventional techniques, and realizes that the site-specific integration can be screened without adding any screening markers.
  • Gene-transgenic cell lines will play an important role in CT1.1-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 CHO cells, 2-normal CHO cells.
  • Embodiment one sgRNA Construction of expression vectors
  • SgRNA was designed based on the sequence of chromosome 1 of CHO cells, and its nucleotide sequence was 5’-CACCGAAGCGAGGTTTCCATTCTG -3 ’. This sequence and its reverse complement 5'-AAACCAGAATGGAAACCTCGCTTC-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. A 100 ⁇ g / ml ampicillin-containing LB medium was used to incubate the correctly sequenced E. coli at 37 ° C.
  • the sgRNA expression vector was extracted without endotoxin.
  • An expression cassette containing the CT1.1 gene 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 action site-CAG promoter-CT1.1 gene-poly A-sgRNA interaction site of the 3 ′ end homology arm, Its structure is shown in Figure 1 and its sequence is shown in SEQ ID NO 1.
  • CHO cells were seeded into six-well plates at a density of 50%. After 18-24 h of culture, the fusion degree of CHO 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 CHO 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 the CT1.1 gene fragment inserted at the site 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; 5 minutes at 72 ° C, 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 CT1.1 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 strains, there were 3 strains of CT1.1 gene-segmented cells.
  • the method for constructing the CHO cell line with the CT1.1 gene inserted by the site provided by the present invention has significantly higher gene knockout efficiency and integration efficiency of foreign genes than conventional techniques, and realizes that the site-specific integration can be screened without adding any screening markers.
  • Gene-transgenic cell lines will play an important role in CT1.1-related drug research and development.

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Abstract

Disclosed are a method for constructing a CHO cell strain with site-directed insertion of the CT1.1 gene and a use thereof. The cell strain is obtained by constructing an sgRNA expression vector based on CRISPR/Cas9 system according to the nucleotide sequence of an insertion site; constructing, according to an sgRNA action site, an expression cassette that contains the CT1.1 gene and can be integrated into a host genome; then co-transfecting the constructed sgRNA expression vector and a linearized expression cassette into CHO cells; and screening to obtain the CHO cell strain with site-directed insertion of the CT1.1 gene. The cell strain can maintain the efficient and stable expression of the CT1.1 protein for a long period of time.

Description

定点***CT1.1基因的CHO细胞株的构建方法及其用途Construction method and application of CHO cell line inserted with CT1.1 gene at fixed site 技术领域Technical field
本发明涉及分子生物学领域，具体来说，涉及一种定点***CT1.1基因的CHO细胞株的构建方法及其用途。The invention relates to the field of molecular biology, and in particular, to a method for constructing a CHO cell line with a CT1.1 gene inserted at a site and its use.
背景技术Background technique
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***，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***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***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***、删除或者突变，形成基因的敲入与敲除。1. Regularly spaced clustered short palindrome repeats Interspaced Short Palindromic Repeats (CRISPR) is a series of clustered DNA sequences, a system used by bacteria to protect themselves against viruses, and a genetic weapon against attackers. 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). When the invading virus DNA and sgRNA sequences are identical, 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. After being transferred into the host cell, 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.
癌睾丸抗原（cancer/testis antigen，CTA）是一组肿瘤相关抗原，它在除睾丸、胎盘及滋养层细胞以外的正常人体组织不表达，而在多种肿瘤组织高表达，使其成为近年来被广泛关注的肿瘤免疫治疗靶抗原。黑色素相关抗原（MAGE）是从黑色素瘤细胞中分离出来的一类肿瘤相关抗原，是CTA中的一个亚家族，能在肿瘤细胞内被加工为抗原肽，与人类白细胞抗原Ⅰ相结合，继而被自身细胞毒性 T 淋巴细胞所识别而杀伤肿瘤细胞。Cancer / testis antigen (CTA) is a group of tumor-associated antigens. It is not expressed in normal human tissues except testis, placenta, and trophoblast cells, but is highly expressed in a variety of tumor tissues, making it a recent year. Tumor immunotherapeutic target antigen has been widely concerned. Melanin-associated antigen (MAGE) is a class of tumor-associated antigens isolated from melanoma cells. It is a subfamily of CTA. It can be processed into antigen peptides in tumor cells, combined with human leukocyte antigen Ⅰ, and then Autocytotoxic T lymphocytes recognize and kill tumor cells.
技术问题technical problem
CT1.1是最早发现的MAGE家族成员，特异性表达于恶性肿瘤，且能够与MHC分子结合，可被特定个体的T细胞受体识别并激活成为具有特异性T细胞受体的细胞毒性T淋巴细胞，使机体产生以细胞免疫为主的免疫反应。因此，CT1.1是一种肿瘤特异性的理想靶抗原，成为近年来研究的热点，但现有技术中缺乏定点***CT1.1基因的CHO细胞株，对相关研究的进展造成了一定的阻碍。CT1.1 is the earliest found member of the MAGE family. It is specifically expressed in malignant tumors and can bind to MHC molecules. It can be recognized by specific cell T-cell receptors and activated into cytotoxic T-lymph with specific T-cell receptors. Cells enable the body to produce an immune response that is dominated by cellular immunity. Therefore, CT1.1 is a tumor-specific ideal target antigen, which has become a hot topic in recent years. However, the lack of CHO cell lines with site-specific insertion of the CT1.1 gene in the prior art has caused certain obstacles to the progress of related research. .
技术解决方案Technical solutions
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***CT1.1基因的CHO细胞株的构建方法，为后续研究人CT1.1基因功能奠定基础。The purpose of the present invention is to overcome the defects existing in the prior art, and provide a method for constructing a CHO cell line with the CT1.1 gene inserted at a site, laying a foundation for the subsequent study of the function of the human CT1.1 gene.
为了实现本发明目的，本发明提供的一种定点***CT1.1基因的CHO细胞株的构建方法是根据***位点的核苷酸序列，构建基于CRISPER/Cas9***的sgRNA表达载体，并根据sgRNA作用位点构建含有CT1.1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CHO细胞中，获得定点***CT1.1基因的CHO细胞株。In order to achieve the purpose of the present invention, a method for constructing a CHO cell line inserted into the CT1.1 gene at a site by the present invention 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 sgRNA The expression site containing the CT1.1 gene that can be integrated into the host genome is constructed at the action site, and the constructed sgRNA expression vector and expression frame are co-transfected into CHO cells to obtain a CHO cell line with the CT1.1 gene inserted at the site. .
前述的方法，所述sgRNA作用位点位于CHO细胞1号染色体上，其核苷酸序列为5’- GAAGCGAGGTTTCCATTCTG -3’。In the foregoing method, the sgRNA interaction site is located on chromosome 1 of CHO cells, and its nucleotide sequence is 5'-GAAGCGAGGTTTCCATTCTG-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-CT1.1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500 bp。In the foregoing method, the expression box contains the following sequential connected expression elements: 5 'end of the sgRNA interaction site, CAG promoter-CT1.1 gene, 3' end of the poly A-sgRNA interaction site Homology arm. The 5 'and 3' homology arms of the sgRNA interaction site are both about 500 bp in size.
前述的方法，所述表达元件的核苷酸序列如SEQ ID NO 1所示。In the aforementioned method, the nucleotide sequence of the expression element is shown in SEQ ID NO 1.
本发明还提供定点***CT1.1基因的CHO细胞株在重组蛋白表达中的应用。The invention also provides the application of the CHO cell line inserted with the CT1.1 gene at a fixed site in the expression of recombinant protein.
本发明的目的还可以采用以下的技术措施来进一步实现。The object of the present invention can be further achieved by using the following technical measures.
a) 构建基于CRISPER/Cas9***的sgRNA表达载体；b）根据sgRNA作用位点构建含有CT1.1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CHO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***CT1.1基因的CHO细胞株。a) Construction of a sgRNA expression vector based on the CRISPER / Cas9 system; b) Construction of an expression frame containing the CT1.1 gene that can be integrated into the host genome based on the sgRNA interaction site; c) The above sgRNA expression vector and linearized expression Box co-transfection into CHO cells; d) Monoclonal cell lines were obtained by limiting dilution method, and CHO cell lines with the CT1.1 gene inserted at the site were identified and screened by PCR.
步骤d）中鉴定筛选定点***CT1.1基因的CHO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CT1.1基因整合到预期位点。 In step d), the CHO cell line with the CT1.1 gene inserted at the designated site is selected and screened by designing a PCR primer that spans the 5 'homology arm. The PCR technique is used to amplify the sequence including the chromosome partial sequence, the entire 5' homology arm sequence, The product of the CAG promoter partial sequence is designed to determine the integration of the CT1.1 gene into the expected site.
有益效果Beneficial effect
本发明提供的定点***CT1.1基因的CHO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CT1.1相关的药物研究和开发中将起重要作用。The method for constructing the CHO cell line with the CT1.1 gene inserted by the site provided by the present invention has significantly higher gene knockout efficiency and integration efficiency of foreign genes than conventional techniques, and realizes that the site-specific integration can be screened without adding any screening markers. Gene-transgenic cell lines will play an important role in CT1.1-related drug research and development.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为所述表达框的结构示意图；FIG. 1 is a schematic structural diagram of the expression box; FIG.
图2为转染细胞的PCR鉴定结果图，其中M-Marker，1-经转染的CHO细胞，2-正常CHO细胞。Figure 2 shows the results of PCR identification of transfected cells, where M-Marker, 1-transfected CHO cells, 2-normal CHO cells.
本发明的实施方式Embodiments of the invention
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。The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the examples are in accordance with conventional experimental conditions or conditions recommended by the manufacturer's instructions.
实施例一：Embodiment one: sgRNAsgRNA 表达载体的构建Construction of expression vectors
根据CHO细胞1号染色体的序列设计sgRNA，其核苷酸序列为5’-CACCGAAGCGAGGTTTCCATTCTG -3’。合成该序列及其反向互补序列5’-AAACCAGAATGGAAACCTCGCTTC-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。SgRNA was designed based on the sequence of chromosome 1 of CHO cells, and its nucleotide sequence was 5’-CACCGAAGCGAGGTTTCCATTCTG -3 ’. This sequence and its reverse complement 5'-AAACCAGAATGGAAACCTCGCTTC-3 'were synthesized. The two nucleotide sequences were each formulated with 100 μmol / 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.
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。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. A 100 μg / ml ampicillin-containing LB medium was used to incubate the correctly sequenced E. coli at 37 ° C. The sgRNA expression vector was extracted without endotoxin.
实施例二：表达框的构建Example 2: Construction of the expression box
根据sgRNA作用位点构建含有CT1.1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-CT1.1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。An expression cassette containing the CT1.1 gene 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 action site-CAG promoter-CT1.1 gene-poly A-sgRNA interaction site of the 3 ′ end homology arm, Its structure is shown in Figure 1 and its sequence is shown in SEQ ID NO 1.
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。Shanghai Shenggong was commissioned to synthesize the sequence shown in SEQ ID NO 1 by gene synthesis, and add an EcoRV restriction 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.
实施例三：Example three: CHOCHO 细胞转染Cell transfection
转染前一天将CHO胞接种至六孔板，接种密度50%。培养18-24 h至CHO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CHO细胞中。One day before transfection, CHO cells were seeded into six-well plates at a density of 50%. After 18-24 h of culture, the fusion degree of CHO cells reached 60% -80%. According to the instructions of Lipofectamine 2000 transfection reagent, 2 μg sgRNA expression vector and 2 μg expression frame were co-transfected into CHO cells by liposome method. .
实施例四：转染细胞总细胞株鉴定Example 4: Identification of total cell lines of transfected cells
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***CT1.1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***CT1.1基因片段的细胞，与预期相符。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 the CT1.1 gene fragment inserted at the site 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; 5 minutes at 72 ° C, 1 cycle. After the PCR reaction was completed, 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 CT1.1 gene fragment at a site, as expected.
实施例五：单克隆细胞株的筛选与鉴定Example 5: Screening and identification of monoclonal cell lines
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***CT1.1基因片段的细胞共有3株。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 strains, there were 3 strains of CT1.1 gene-segmented cells.
工业实用性Industrial applicability
本发明提供的定点***CT1.1基因的CHO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CT1.1相关的药物研究和开发中将起重要作用。The method for constructing the CHO cell line with the CT1.1 gene inserted by the site provided by the present invention has significantly higher gene knockout efficiency and integration efficiency of foreign genes than conventional techniques, and realizes that the site-specific integration can be screened without adding any screening markers. Gene-transgenic cell lines will play an important role in CT1.1-related drug research and development.

Claims (6)

  1. 定点***CT1.1基因的CHO细胞株的构建方法，其特征在于，其是根据***位点的核苷酸序列，构建基于CRISPER/Cas9***的sgRNA表达载体，并根据sgRNA作用位点构建含有CT1.1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CHO细胞中，获得定点***CT1.1基因的CHO细胞株。A method for constructing a CHO cell line with a CT1.1 gene inserted at a site, which is characterized in that it constructs an sgRNA expression vector based on the CRISPER / Cas9 system based on the nucleotide sequence of the insertion site, and constructs a CT1 containing the sgRNA action site. .1 gene expression cassette that can be integrated into the host genome, and then the sgRNA expression vector and expression cassette constructed are co-transfected into CHO cells to obtain a CHO cell line with site-specific insertion of the CT1.1 gene.
  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CHO细胞1号染色体上。The method according to claim 1, wherein the sgRNA interaction site is located on chromosome 1 of CHO cells.
  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’- GAAGCGAGGTTTCCATTCTG -3’。The method according to claim 2, wherein the nucleotide sequence of the sgRNA interaction site is 5'- GAAGCGAGGTTTCCATTCTG-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-CT1.1基因-poly A- sgRNA作用位点的3’端同源臂。The method according to claim 1, characterized in that the expression box contains sequentially connected expression elements: the 5 'end homology arm of the sgRNA action site-CAG promoter-CT1.1 gene-poly A -The 3 'homology arm of the sgRNA site.
  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。The expression element according to claim 4, wherein the sequence is as shown in SEQ ID NO 1.
  6. 根据权利要求1所述的定点***CT1.1基因的CHO细胞株在重组蛋白表达中的应用。The use of the CHO cell line inserted with the CT1.1 gene at a site according to claim 1 in the expression of a recombinant protein.
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