技术领域technical field
本发明涉及一种DNA甲基转移酶缺陷型中国仓鼠卵巢(CHO)细胞系的制备方法及应用，属于基因工程技术领域。The invention relates to a preparation method and application of a DNA methyltransferase-deficient Chinese hamster ovary (CHO) cell line, belonging to the technical field of genetic engineering.
背景技术Background technique
二十世纪八十年代组织纤溶酶原激活剂(tissue type plasminogen activator,t-PA)在重组中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞中首次成功表达并获得美国FDA批准用于临床，标志着以CHO细胞为基础的生物制药时代的到来。目前CHO细胞源生产的重组药物蛋白已占哺乳动物细胞生产的近70％。但现阶段CHO细胞表达系统存在表达量低、尤其是表达不稳定等问题，无法满足用于疫苗开发、临床治疗和基因疗法等方面日益增长的需求。因此，利用基因和细胞工程的方法进行CHO细胞重组表达研究，对于提高其转基因表达水平和表达稳定性、建立高产稳产的重组蛋白CHO细胞表达系统具有十分重要的意义。研究已证实，通过减少启动子中的CpG岛(CpG islands)而使转基因对甲基化不敏感有利于重组蛋白的高效和持续表达。例如，hCMV-MIE启动子转录起始位点上游的C-179突变为G能够显著提高稳定转染CHO细胞中重组蛋白的持续表达。因此，深入研究DNA甲基化对转基因表达的影响及其机制将有助于筛选获得高产稳产的重组CHO细胞系。In the 1980s, tissue plasminogen activator (tissue type plasminogen activator, t-PA) was successfully expressed in recombinant Chinese hamster ovary (CHO) cells for the first time and was approved by the US FDA for clinical use. The era of biopharmaceuticals based on CHO cells is coming. At present, the recombinant drug protein produced by CHO cells has accounted for nearly 70% of the production of mammalian cells. However, the current CHO cell expression system has problems such as low expression, especially unstable expression, and cannot meet the growing demand for vaccine development, clinical treatment, and gene therapy. Therefore, using gene and cell engineering methods to study the recombinant expression of CHO cells is of great significance for improving the expression level and stability of transgenes and establishing a high-yielding and stable-yielding recombinant protein CHO cell expression system. Studies have demonstrated that insensitivity of transgenes to methylation by reducing CpG islands in the promoter is beneficial for efficient and sustained expression of recombinant proteins. For example, the mutation of C-179 to G upstream of the transcription start site of hCMV-MIE promoter can significantly improve the sustained expression of recombinant proteins in stably transfected CHO cells. Therefore, an in-depth study of the effect of DNA methylation on the expression of transgenes and its mechanism will help to screen recombinant CHO cell lines with high and stable yields.
DNA甲基化(DNA methylation)是一种在原核和真核生物基因组中常见的表观遗传修饰，也是哺乳动物中基因表达的重要调控方式，在基因转录抑制方面起关键作用：基因启动子区域(包括转录因子结合位点)CpG岛的高甲基化状态能够直接阻止转录因子的结合而导致染色质结构的改变，从而限制转录因子与基因启动子结合，进而对基因转录、剪接、翻译及表达量等造成影响。有关DNA甲基化在CHO细胞中对重组蛋白表达的作用研究主要集中于启动子的改造，包括启动子甲基化位点点突变、无CpG岛启动子、合成性启动子的应用以及核心CpG岛元件的插入等。DNA甲基化需要DNA甲基转移酶(DNA methyltransferase,Dnmt)包括从头甲基转移酶Dnmt3a和Dnmt3b以及DNA甲基化维持酶Dnmt1的催化，特别是Dnmt3a介导的启动子甲基化与转基因的不稳定表达密切相关。DNA methylation (DNA methylation) is a common epigenetic modification in prokaryotic and eukaryotic genomes. It is also an important regulation method of gene expression in mammals and plays a key role in gene transcription repression: gene promoter region (Including transcription factor binding sites) The high methylation state of CpG islands can directly prevent the binding of transcription factors and lead to changes in chromatin structure, thereby limiting the binding of transcription factors to gene promoters, and further affecting gene transcription, splicing, translation and expression etc. have an impact. The research on the effect of DNA methylation on the expression of recombinant proteins in CHO cells mainly focuses on the modification of promoters, including point mutations of promoter methylation sites, CpG island-free promoters, the application of synthetic promoters, and core CpG islands. Component insertion, etc. DNA methylation requires the catalysis of DNA methyltransferases (DNA methyltransferase, Dnmt), including de novo methyltransferases Dnmt3a and Dnmt3b, and DNA methylation maintenance enzyme Dnmt1, especially Dnmt3a-mediated promoter methylation and transgene Unstable expression is closely related.
当前，利用细胞工程技术对细胞进行改造已广泛应用，特别是通过基因敲除或基因沉默等影响与特定生物学功能相关基因的表达水平，以获得特定生物学功能发生改变的细胞。由于其具有操作简便、稳定、灵活及效率高等技术上的优势，将是目前及未来改造CHO细胞不断提升其重组蛋白生产能力的主要技术策略。At present, the use of cell engineering technology to modify cells has been widely used, especially through gene knockout or gene silencing to affect the expression level of genes related to specific biological functions, so as to obtain cells with changed specific biological functions. Due to its technical advantages such as easy operation, stability, flexibility and high efficiency, it will be the main technical strategy for the current and future transformation of CHO cells to continuously improve their recombinant protein production capacity.
发明内容Contents of the invention
为解决上述技术问题，本发明利用CRISPR/Cas9基因编辑技术敲除CHO细胞DNA甲基转移酶Dnmt3a基因，筛选并鉴定获得DNA甲基转移酶Dnmt3a缺陷型CHO细胞，通过转染真核表达载体并筛选稳定表达重组CHO细胞株而建立基于DNA甲基转移酶Dnmt3a缺陷型的新型无甲基化的CHO细胞表达系统。In order to solve the above technical problems, the present invention uses CRISPR/Cas9 gene editing technology to knock out the DNA methyltransferase Dnmt3a gene of CHO cells, screen and identify the DNA methyltransferase Dnmt3a-deficient CHO cells, and transfect the eukaryotic expression vector and A new methylation-free CHO cell expression system based on DNA methyltransferase Dnmt3a deficiency was established by screening stable recombinant CHO cell lines.
为了实现以上目的，本发明所采用的技术方案如下：In order to achieve the above object, the technical scheme adopted in the present invention is as follows:
一种DNA甲基转移酶Dnmt3a基因缺陷型的CHO细胞系。A CHO cell line deficient in the DNA methyltransferase Dnmt3a gene.
所述DNA甲基转移酶Dnmt3a基因组DNA序列为GenBank：NW_003613640.1，Dnmt3a缺陷型CHO细胞基因组DNA序列敲除缺失199个碱基即第41138-41336位碱基。The genomic DNA sequence of the DNA methyltransferase Dnmt3a is GenBank: NW_003613640.1, and the genomic DNA sequence of Dnmt3a-deficient CHO cells is knocked out and 199 bases are deleted, that is, bases 41138-41336.
进一步地，所述CHO细胞系选自CHO-K1、CHO-S、CHO-DG44中的任意一种。Further, the CHO cell line is selected from any one of CHO-K1, CHO-S, and CHO-DG44.
本发明还提供上述DNA甲基转移酶Dnmt3a基因缺陷型的CHO细胞系的制备方法，可利用CRISPR/Cas9基因编辑技术敲除CHO细胞DNA甲基转移酶Dnmt3a基因而获得；包括利用CRISPR/Cas9基因编辑技术敲除DNA甲基转移酶Dnmt3a基因，筛选并鉴定获得Dnmt3a缺陷型CHO细胞，通过细胞增殖、细胞凋亡等细胞生物学特性检测验证缺陷型细胞系能否进行正常的增殖和传代培养。The present invention also provides a method for preparing the above-mentioned DNA methyltransferase Dnmt3a gene-deficient CHO cell line, which can be obtained by knocking out the CHO cell DNA methyltransferase Dnmt3a gene by using CRISPR/Cas9 gene editing technology; including using CRISPR/Cas9 gene The editing technology knocks out the DNA methyltransferase Dnmt3a gene, screens and identifies Dnmt3a-deficient CHO cells, and verifies whether the defective cell lines can undergo normal proliferation and subculture through detection of cell biological characteristics such as cell proliferation and apoptosis.
具体地，上述方法包括如下步骤：Specifically, the above method includes the following steps:
1)针对候选基因确定打靶位点：根据NCBI的GenBank中中国仓鼠DNA甲基转移酶基因Dnmt3a基因组序列(No.NW_003613640.1)和mRNA序列(No.XM_016964036.1)设计引物进行相应基因组DNA基因片段PCR扩增，将PCR扩增基因片段进行克隆测序验证序列的准确性；应用在线工具(http://crispr.mit.edu/)辅助设计Dnmt3a基因的嵌合单链向导RNA(chimeric single-guide RNA,chimeric sgRNA)打靶位点，根据靶向序列分别设计两对引物以构建表达CRISPR/Cas9的打靶sgRNA载体；1) Determine the target site for the candidate gene: According to the genome sequence (No.NW_003613640.1) and mRNA sequence (No.XM_016964036.1) of the Chinese hamster DNA methyltransferase gene Dnmt3a in NCBI's GenBank, primers were designed for the corresponding genomic DNA gene Fragment PCR amplification, the PCR amplified gene fragment was cloned and sequenced to verify the accuracy of the sequence; the online tool (http://crispr.mit.edu/) was used to assist in the design of chimeric single-stranded guide RNA (chimeric single-stranded guide RNA) for the Dnmt3a gene. guide RNA, chimeric sgRNA) targeting site, respectively design two pairs of primers according to the target sequence to construct the targeting sgRNA vector expressing CRISPR/Cas9;
2)sgRNA载体的构建：使用Bbs I限制性内切酶线性化px330载体质粒并胶回收线性片段；sgRNA寡核苷酸单链退火形成双链；将线性化px330载体与双链sgRNA进行连接过夜；连接产物转化DH5α感受态细胞，氨苄抗性涂板筛选，挑取单菌落，37℃摇床摇菌过夜提取质粒并进行测序验证；选取测序正确的阳性克隆质粒进行细胞转染；2) Construction of sgRNA vector: use Bbs I restriction endonuclease to linearize the px330 vector plasmid and recover the linear fragment; the sgRNA oligonucleotide single strand anneals to form a double strand; the linearized px330 vector and the double-stranded sgRNA are ligated overnight ; The ligation product was transformed into DH5α competent cells, screened with ampicillin resistance, picked a single colony, and shaken overnight at 37°C to extract the plasmid and perform sequencing verification; select the correctly sequenced positive clone plasmid for cell transfection;
3)CHO细胞转染、单克隆挑选及细胞敲除鉴定3) CHO cell transfection, monoclonal selection and cell knockout identification
转染前将2.0×105个CHO细胞接种于24孔培养板中，当细胞融合度达到80％-90％时加入上述含有sgRNA的重组质粒共转染CHO细胞；将转染细胞进行梯度稀释和极限稀释，以达到挑选出具有独立敲除的单克隆突变型细胞；对获得的缺陷型细胞株提取其基因组DNA，利用PCR扩增Dnmt3a基因片段并测序以确定基因敲除成功。Before transfection, 2.0×105 CHO cellswere seeded in a 24-well culture plate, and when the cell confluence reached 80%-90%, the above-mentioned recombinant plasmid containing sgRNA was added to co-transfect CHO cells; the transfected cells were serially diluted and limiting dilution to achieve the selection of monoclonal mutant cells with independent knockout; the genomic DNA of the obtained defective cell lines was extracted, and the Dnmt3a gene fragment was amplified by PCR and sequenced to confirm the success of gene knockout.
本发明还提供包含上述DNA甲基转移酶Dnmt3a基因缺陷型的CHO细胞系的真核细胞表达系统。The present invention also provides a eukaryotic cell expression system comprising the above-mentioned DNA methyltransferase Dnmt3a gene-deficient CHO cell line.
本发明还提供上述真核细胞表达系统的制备方法，包括：将目的基因插入表达载体中，构建得到重组表达载体；将该重组表达载体转染进入所述缺陷型的CHO细胞系，经筛选得到表达系统。The present invention also provides a method for preparing the eukaryotic cell expression system, comprising: inserting the target gene into the expression vector to construct a recombinant expression vector; transfecting the recombinant expression vector into the defective CHO cell line, and obtaining expression system.
进一步地，可将所构建的表达载体转染Dnmt3a缺陷型和正常对照CHO细胞，筛选多克隆CHO重组细胞株并传代培养，检测分析目的基因在重组CHO细胞中瞬时表达和长期稳定表达情况。Further, the constructed expression vector can be transfected into Dnmt3a-deficient and normal control CHO cells, polyclonal CHO recombinant cell lines can be screened and subcultured, and the transient expression and long-term stable expression of the target gene in the recombinant CHO cells can be detected and analyzed.
本发明还提供上述DNA甲基转移酶Dnmt3a基因缺陷型的CHO细胞系、上述真核细胞表达系统在制备目的蛋白等方面的应用。The present invention also provides the application of the above-mentioned DNA methyltransferase Dnmt3a gene-deficient CHO cell line and the above-mentioned eukaryotic cell expression system in preparing the target protein and the like.
重组细胞株中目的基因不能持续高效表达甚至出现表达衰退是利用CHO细胞进行重组蛋白生产普遍和突出存在的问题。目前认为DNA甲基化是造成这种表达不稳定性的主要原因，特别是DNA甲基转移酶DNMT3a介导的启动子甲基化与转基因的不稳定表达密切相关。近年来，细胞工程改造策略即利用细胞工程技术特别是基因敲除、基因过表达或基因沉默等技术，改变与重组蛋白表达量或活性相关的生物学功能基因表达水平，表现出提高重组蛋白产量和(或)活性的巨大潜力。因此，本发明将宿主细胞遗传改造和表达载体优化相结合，利用CRISPR/Cas9基因编辑技术构建基于DNA甲基转移酶缺陷型的新型CHO细胞表达系统，能够明显提高重组蛋白的表达水平，克服了目前表达系统存在的表达水平较低、表达不稳定的问题。The inability to continuously and efficiently express the target gene in the recombinant cell line or even the expression decline is a common and prominent problem in the use of CHO cells for recombinant protein production. At present, it is believed that DNA methylation is the main cause of this expression instability, especially the promoter methylation mediated by DNA methyltransferase DNMT3a is closely related to the unstable expression of transgenes. In recent years, the cell engineering transformation strategy is to use cell engineering technology, especially gene knockout, gene overexpression or gene silencing, to change the expression level of biological function genes related to the expression or activity of recombinant proteins, and to improve the production of recombinant proteins. and/or great potential for activity. Therefore, the present invention combines host cell genetic modification and expression vector optimization, and uses CRISPR/Cas9 gene editing technology to construct a new CHO cell expression system based on DNA methyltransferase deficiency, which can significantly improve the expression level of recombinant proteins and overcome the The current expression system has the problems of low expression level and unstable expression.
附图说明Description of drawings
图1：CHO细胞Dnmt3a基因敲除单克隆Dnmt3a-30测序鉴定结果；其中，划删除线的为基因敲除缺失的199个碱基序列，箭头所指为缺失突变位点，下划线为Dnmt3a基因片段扩增引物，波浪线为sgRNA打靶位点。Figure 1: Sequencing identification results of Dnmt3a gene knockout monoclonal Dnmt3a-30 in CHO cells; among them, the deleted 199 base sequence is the gene knockout deletion, the arrow points to the deletion mutation site, and the underline is the Dnmt3a gene fragment Amplification primers, the wavy line is the sgRNA targeting site.
图2：CCK-8法检测Dnmt3a缺陷型CHO细胞单克隆细胞Dnmt3a-30和Dnmt3a-40及正常CHO-K1细胞增殖结果图。Figure 2: CCK-8 method to detect the proliferation of Dnmt3a-deficient CHO cell monoclonal cells Dnmt3a-30 and Dnmt3a-40 and normal CHO-K1 cells.
图3：流式细胞术检测Dnmt3a缺陷型CHO细胞及正常CHO细胞凋亡结果图。Fig. 3: Apoptotic results of Dnmt3a-deficient CHO cells and normal CHO cells detected by flow cytometry.
图4：CMV启动子驱动的真核表达载体pWTY-02示意图。Figure 4: Schematic diagram of eukaryotic expression vector pWTY-02 driven by CMV promoter.
图5：由CMV启动子驱动的eGFP基因在Dnmt3a缺陷型CHO细胞和正常CHO细胞中表达阳性细胞率。Figure 5: The positive cell rate of eGFP gene driven by CMV promoter in Dnmt3a-deficient CHO cells and normal CHO cells.
图6：CMV启动子驱动表达的eGFP在Dnmt3a缺陷型CHO细胞和正常CHO细胞中表达稳定性实验结果。Figure 6: CMV promoter-driven eGFP expression stability experiment results in Dnmt3a-deficient CHO cells and normal CHO cells.
图7：重组EPO蛋白在Dnmt3a缺陷型CHO细胞和正常CHO细胞中的表达量检测分析结果。Figure 7: Detection and analysis results of recombinant EPO protein expression in Dnmt3a-deficient CHO cells and normal CHO cells.
具体实施方式Detailed ways
以下实施例用于说明本发明，但不用来限制本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW，Molecular Cloning:a Laboratory Manual，2001)，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购买得到的常规产品。The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Those who do not indicate specific techniques or conditions in the embodiments, according to the techniques or conditions described in documents in the art, such as Sambrook and other molecular cloning experiment manuals (Sambrook J&Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the product Instructions are carried out. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that can be purchased through formal channels.
实施例1CHO细胞Dnmt3a基因的敲除Example 1 Knockout of CHO cell Dnmt3a gene
1.1针对候选基因确定打靶位点1.1 Determine the target site for the candidate gene
根据NCBI的GenBank中中国仓鼠DNA甲基转移酶基因Dnmt3a基因组序列(No.NW_003613640.1)和mRNA序列(No.XM_016964036.1)设计引物(D3a-Ex1seq-L:5′-GACCACAAGAATTCCGGCTC-3′,D3a-Ex1seq-R:5′-CGTGTGTGAATCTGTGTGGG-3′)进行相应基因片段PCR扩增，将PCR扩增片段进行克隆测序验证序列的准确性。应用在线工具(http://crispr.mit.edu/)辅助设计的Dnmt3a基因的嵌合单链向导RNA打靶位点如下：Primers (D3a-Ex1seq-L:5′-GACCACAAGAATTCCGGCTC-3′, D3a-Ex1seq-R: 5'-CGTGTGTGAATCTGTGTGGG-3') PCR amplification of the corresponding gene fragment was carried out, and the accuracy of the sequence was verified by cloning and sequencing of the PCR amplified fragment. The chimeric single-stranded guide RNA targeting site of the Dnmt3a gene assisted by the online tool (http://crispr.mit.edu/) is as follows:
D3a-Ex1-98rev:5′-ATCATCCTCCCGCTCCAAAGTGG-3′；D3a-Ex1-98rev: 5'-ATCATCCTCCCGCTCCAAAGTGG-3';
D3a-Ex1-308fw:5′-TTTGAGGGGTCATCCTTGCAGGG3′D3a-Ex1-308fw:5′-TTTGAGGGGTCATCCTTGCAGGG3′
根据靶向序列分别设计两对引物以构建表达CRISPR/Cas9的打靶sgRNA载体。Two pairs of primers were designed according to the target sequence to construct the targeting sgRNA vector expressing CRISPR/Cas9.
1.2sgRNA载体的构建1.2 Construction of sgRNA vector
使用Bbs I限制性内切酶线性化px330载体质粒并胶回收线性片段。sgRNA寡核苷酸单链退火形成双链。将线性化px330载体与双链sgRNA进行连接过夜。连接产物转化DH5α感受态细胞，氨苄抗性涂板筛选，挑取单菌落，37℃摇床摇菌过夜提取质粒并进行测序验证。选取测序正确的阳性克隆质粒进行细胞转染。Use Bbs I restriction endonuclease to linearize the px330 vector plasmid and recover the linear fragment. The sgRNA oligonucleotide single strand anneals to form a double strand. The linearized px330 vector was ligated with double-stranded sgRNA overnight. The ligation product was transformed into DH5α competent cells, screened by ampicillin resistance, picked a single colony, and shaken overnight at 37°C to extract the plasmid and verify it by sequencing. The positive clone plasmids with correct sequencing were selected for cell transfection.
1.3CHO细胞转染、单克隆挑选及细胞敲除鉴定1.3 CHO cell transfection, monoclonal selection and cell knockout identification
转染前将2.0×105个CHO-K1细胞接种于24孔培养板中，当细胞融合度达到80％-90％时加入上述含有sgRNA的重组质粒共转染CHO-K1细胞。将转染细胞进行梯度稀释和极限稀释，以达到挑选出具有独立敲除的单克隆突变型细胞。利用CRISPR/Cas9基因敲除方法和PCR扩增验证结果，我们最终筛选获得了6株Dnmt3a缺陷型CHO细胞单克隆(克隆30,31,32,33,40,41)。对其中Dnmt3a缺陷型细胞株克隆30的PCR扩增产物的测序结果表明，基因敲除造成Dnmt3a基因199个碱基的缺失突变，基因敲除成功(结果见图1)。Before transfection, 2.0×105 CHO-K1 cells were seeded in a 24-well culture plate, and when the cell confluence reached 80%-90%, the above-mentioned recombinant plasmid containing sgRNA was added to co-transfect CHO-K1 cells. Transfected cells were subjected to serial dilution and limiting dilution to achieve selection of monoclonal mutant cells with independent knockouts. Using the CRISPR/Cas9 gene knockout method and PCR amplification verification results, we finally screened and obtained six Dnmt3a-deficient CHO cell clones (clones 30, 31, 32, 33, 40, 41). The sequencing results of the PCR amplification product of the Dnmt3a-deficient cell line clone 30 showed that the gene knockout resulted in a deletion mutation of 199 bases in the Dnmt3a gene, and the gene knockout was successful (see Figure 1 for the results).
实施例2Dnmt3a缺陷型CHO细胞的生物学特性验证Example 2 Verification of biological characteristics of Dnmt3a-deficient CHO cells
以野生型CHO-K1细胞作为对照，对获得的Dnmt3a缺陷型CHO细胞单克隆进行细胞生长特性验证，包括细胞形态和生长状态观察、CCK-8法检测细胞增殖情况、流式细胞术(FCM)检测细胞凋亡情况，验证Dnmt3a缺陷型CHO细胞系能否进行正常的生长和传代。CCK-8试剂盒(Cell Counting Kit-8试剂盒，碧云天Beyotime生物公司)检测细胞增殖(结果见图2)和流式细胞术检测细胞凋亡结果(结果见图3)提示，获得的Dnmt3a缺陷型CHO细胞株能够正常进行生长和传代，细胞生长状态、形态、细胞增殖、细胞凋亡等生物学特性与正常CHO细胞无显著性差别。Using wild-type CHO-K1 cells as a control, the cell growth characteristics of the obtained Dnmt3a-deficient CHO cell clones were verified, including observation of cell morphology and growth status, detection of cell proliferation by CCK-8 method, flow cytometry (FCM) Cell apoptosis was detected to verify whether the Dnmt3a-deficient CHO cell line could undergo normal growth and passage. CCK-8 kit (Cell Counting Kit-8 kit, Beyotime Biological Company) detection of cell proliferation (results shown in Figure 2) and flow cytometry results of detection of cell apoptosis (results in Figure 3) suggest that the obtained Dnmt3a The defective CHO cell line can grow and pass on normally, and its biological characteristics such as cell growth state, shape, cell proliferation, and cell apoptosis are not significantly different from normal CHO cells.
实施例3不同启动子驱动的重组表达载体的构建Example 3 Construction of recombinant expression vectors driven by different promoters
本发明以真核表达载体pIRESneo2(Clontech公司)为母载体，构建由CMV启动子驱动表达绿色荧光蛋白(eGFP)的真核表达载体pWTY-02(见图4)。In the present invention, the eukaryotic expression vector pIRESneo2 (Clontech Company) is used as the parent vector to construct the eukaryotic expression vector pWTY-02 driven by the CMV promoter to express green fluorescent protein (eGFP) (see FIG. 4 ).
实施例4目的基因eGFP在重组CHO细胞中的表达分析Example 4 Expression analysis of target gene eGFP in recombinant CHO cells
4.1CHO细胞转染4.1 CHO cell transfection
培养CHO-K1细胞，细胞转染前1天，以1×105细胞密度传代到新鲜的无血清培养基，待细胞融合度达80％-90％时即可用脂质体3000(Invitrogen，USA)进行转染。分别转染两组CHO细胞：Dnmt3a缺陷型和正常对照CHO-K1细胞。每个质粒平行转染3个复孔。CHO-K1 cells were cultured, and one day before cell transfection, they were subcultured to fresh serum-free medium at a cell density of 1×105 , and when the cell confluence reached 80%-90%, Liposome 3000 (Invitrogen, USA ) for transfection. Two groups of CHO cells were transfected: Dnmt3a-deficient and normal control CHO-K1 cells. Three replicate wells were transfected in parallel with each plasmid.
4.2转染细胞株瞬时表达观察4.2 Transient expression observation of transfected cell lines
转染24h，倒置荧光显微镜观察瞬时转染细胞中eGFP的表达情况。结果表明，真核表达载体在Dnmt 3a缺陷型和正常CHO-K1细胞转染效率相当，两者eGFP表达阳性细胞率无明显差别(见图5)。这说明Dnmt 3a基因敲除对细胞转染效率没有影响。After 24 hours of transfection, the expression of eGFP in transiently transfected cells was observed with an inverted fluorescence microscope. The results showed that the transfection efficiency of the eukaryotic expression vector in Dnmt 3a-deficient and normal CHO-K1 cells was equivalent, and there was no significant difference in the positive cell rate of eGFP expression between the two (see Figure 5). This shows that the knockout of Dnmt3a gene has no effect on cell transfection efficiency.
4.3稳定表达多克隆CHO细胞株筛选及eGFP长期稳定表达分析4.3 Screening of stable expression polyclonal CHO cell lines and analysis of long-term stable expression of eGFP
加G418筛选，筛选培养两周后获得稳定转化的多克隆细胞株并在有筛选压力(加G418)和无筛选压力(不加G418)下传代培养50代，每10代分别将各实验组CHO细胞按照每个样本106个细胞进行流式细胞仪检测分析，测定eGFP平均荧光强度(Mean fluorescenceintensity,MFI)。流式检测结果表明，无论是否存在G418筛选压力，由CMV启动子驱动表达的eGFP可在Dnmt3a缺陷型CHO细胞长期稳定表达(见图6)。Add G418 for selection, obtain stable transformed polyclonal cell lines after two weeks of selection and culture, and subculture for 50 generations with selection pressure (with G418) and without selection pressure (without G418), and each experimental group was divided into CHO cells for every 10 generations. The cells were analyzed by flow cytometry according to 106 cells in each sample, and the mean fluorescence intensity (MFI) of eGFP was measured. The results of flow cytometry showed that regardless of the G418 screening pressure, eGFP expressed by the CMV promoter could be stably expressed in Dnmt3a-deficient CHO cells for a long time (see Figure 6).
实施例5促红细胞生成素(EPO)目的蛋白在重组CHO细胞中的表达分析Example 5 Expression Analysis of Erythropoietin (EPO) Target Protein in Recombinant CHO Cells
为进一步测试Dnmt3a缺陷型CHO细胞对重组蛋白表达稳定性的作用，我们以质粒pWTY-02为基础，构建了由CMV启动子驱动促红细胞生成素(Erythropoietin,EPO)基因表达的真核表达载体。将构建的含EPO基因表达载体质粒分别转染Dnmt3a缺陷型和正常CHO-K1细胞。转染的细胞在含有G418(800μg/mL)的培养基中培养15天以筛选稳定转染的重组细胞池(cell pool)，每隔3天将重组CHO细胞进行传代培养至50代。随后将重组CHO细胞在125mL培养摇瓶中用25mL无蛋白、无血清、化学成分确定的CD CHO培养基(Life Technologies公司，培养基中含有8mM的L-谷氨酰胺)培养5天至细胞数达到8×106，离心收集上清液用于重组EPO蛋白表达检测分析。In order to further test the effect of Dnmt3a-deficient CHO cells on the stability of recombinant protein expression, we constructed a eukaryotic expression vector driven by CMV promoter to express erythropoietin (EPO) gene based on plasmid pWTY-02. The constructed plasmid containing EPO gene expression vector was transfected into Dnmt3a-deficient and normal CHO-K1 cells respectively. The transfected cells were cultured in a medium containing G418 (800 μg/mL) for 15 days to select a stably transfected recombinant cell pool, and the recombinant CHO cells were subcultured every 3 days to passage 50. The recombinant CHO cells were then cultured in 125 mL culture shake flasks with 25 mL of protein-free, serum-free, chemically defined CD CHO medium (Life Technologies, containing 8 mM L-glutamine) for 5 days until the cell count When the concentration reached 8×106 , the supernatant was collected by centrifugation for detection and analysis of recombinant EPO protein expression.
采用Western blot检测分析重组EPO蛋白在Dnmt3a缺陷型和正常CHO-K1细胞池中的表达。具体地，将含有重组EPO的上清液加入5×SDS样品缓冲液煮水浴煮10分钟充分变性。分别取10μL蛋白样品经10％SDS-聚丙烯酰胺凝胶电泳分离后，湿转法转移至硝酸纤维素膜。5％BSA封闭膜2小时，1:1000稀释的抗EPO一抗(Santa Cruz,CA)4℃孵育过夜，TBST洗膜后加1:5000稀释的HRP标记的抗小鼠IgG二抗(Santa Cruz,CA)37℃孵育1小时，TBST洗膜后加化学发光显色液显影，在凝胶成像仪上观察并采集图像结果，分析目的蛋白的灰度值表示待测蛋白的表达水平。Western blot检测结果表明，重组EPO蛋白在Dnmt3a缺陷型CHO细胞池中的平均表达量(860.5±42.9mg/L)显著高于在未缺陷型CHO细胞池中的平均表达量(373.7±29.6mg/L)。重组EPO蛋白在稳定培养50代的重组Dnmt3a缺陷型CHO细胞池和未缺陷型CHO细胞池中的表达量分别为：672.7±35.1mg/L和157.9±16.2mg/mL。表达量差异均具有统计学意义(见图7，P<0.05)。这些结果说明，Dnmt3a缺陷型CHO细胞可明显提高重组EPO蛋白的表达水平和长期表达稳定性。Western blot was used to analyze the expression of recombinant EPO protein in Dnmt3a-deficient and normal CHO-K1 cell pools. Specifically, the supernatant containing recombinant EPO was added to 5×SDS sample buffer and boiled in a water bath for 10 minutes to fully denature. 10 μL of protein samples were separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membrane by wet transfer method. Block the membrane with 5% BSA for 2 hours, incubate overnight at 4°C with 1:1000 diluted anti-EPO primary antibody (Santa Cruz, CA), and add 1:5000 diluted HRP-labeled anti-mouse IgG secondary antibody (Santa Cruz, CA) after washing the membrane with TBST , CA) incubate at 37°C for 1 hour, wash the membrane with TBST and add chemiluminescence color development solution to develop, observe and collect image results on a gel imager, and analyze the gray value of the target protein to indicate the expression level of the protein to be tested. The results of Western blot detection showed that the average expression level (860.5 ± 42.9 mg/L) of recombinant EPO protein in the Dnmt3a-deficient CHO cell pool was significantly higher than the average expression level (373.7 ± 29.6 mg/L) in the non-deficient CHO cell pool. L). The expression levels of recombinant EPO protein in the recombinant Dnmt3a-deficient CHO cell pool and non-defective CHO cell pool stably cultured for 50 passages were 672.7±35.1mg/L and 157.9±16.2mg/mL, respectively. The differences in expression levels were statistically significant (see Figure 7, P<0.05). These results indicated that Dnmt3a-deficient CHO cells could significantly increase the expression level and long-term expression stability of recombinant EPO protein.
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.
序列表sequence listing
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<120> 一种DNA甲基转移酶缺陷型CHO细胞系及其制备方法及应用<120> A DNA methyltransferase-deficient CHO cell line and its preparation method and application
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| CN201711211535.3APendingCN107828738A (en) | 2017-11-28 | 2017-11-28 | A kind of dnmt rna deficiency Chinese hamster ovary celI system and preparation method and application |
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| RJ01 | Rejection of invention patent application after publication | Application publication date:20180323 |