抗人CD40人—鼠嵌合抗体的构建、表达及其生物学活性的初步研究
发布时间：2018-07-29 17:11
【摘要】：本研究在本科室自行研制成功的国内首株激发型抗人CD40单克隆抗体(5C11)的基础上，采用经典的嵌合抗体构建方法，对其进行基因工程改造，并在CHO细胞中实现真核表达，进而对其生物学活性进行初步的研究，以期获得能持续有效分泌特异性的抗人CD40人-鼠嵌合抗体的稳定细胞株，为靶向CD40分子的肿瘤免疫治疗提供必要的物质基础，也期在治疗性单抗的研发领域中取得突破。 第一部分首先采用RT-PCR从特异性分泌抗人CD40单克隆抗体的杂交瘤细胞株5C11中提取总RNA，常规逆转录，应用简并引物进行PCR扩增，并根据重、轻链基因序列分析结果设计引物，应用SMART-PCR方法扩增含信号肽序列的V_H和V_L基因，进行测序鉴定。同时采用RT-PCR从人脾脏细胞中抽提总RNA，分别设计特异性引物扩增人IgG1_(gamma)链huFc、huCH1基因和κappa链恒定区基因huCκ，再利用TP-PCR方法将huFc和huCH1进行拼接得到人IgG1_(gamma)(CH1-CH3)链恒定区基因，测序鉴定。将含信号肽序列的V_H序列和人的IgG1重链恒定区序列进行拼接获得嵌合重链，将含信号肽序列的V_L序列和人的κappa链恒定区序列进行拼接获得嵌合轻链，拼接产物测序鉴定，构建共表达嵌合重、轻链的重组质粒pIRES／hu5C11。pIRES／hu5C11重组质粒用脂质体法转染293T细胞，利用CD40-L929和mock-L929细胞株，通过流式细胞术对293T细胞瞬时表达上清中抗人CD40人-鼠嵌合抗体进行鉴定。结果显示，本研究成功构建了含人-鼠嵌合重链和嵌合轻链基因的抗人CD40人-鼠嵌合抗体真核表达载体pIRES／huSC11。 第二部分选择中国仓鼠卵巢细胞(CHO)作为表达宿主，经试剂盒抽提的pIRES／hu5C11嵌合抗体重组表达质粒用脂质体法转染CHO细胞，获得持续稳定分泌表达抗人CD40人-鼠嵌合抗体(ch-SC11)的基因转染细胞株(hu5C11-CHO)。RT-PCR鉴定结果表明ch-SC11表达基因成功整合在huSC11-CHO细胞中；FCM和Western blot结果表明，转染pIRES／hu5C11质粒的CHO培养上清中含有抗人CD40人-鼠嵌合抗体，，其不仅保留了特异识别CD40分子活性，并且含人免疫球蛋白Fc
[Abstract]:On the basis of the first domestic activated anti-human CD40 monoclonal antibody (5C11) developed by our department, the classical chimeric antibody construction method was used to carry out genetic engineering modification and eukaryotic expression in CHO cells. In order to obtain the stable cell line which can continuously secrete specific anti-human CD40 human-mouse chimeric antibody, and provide the necessary material basis for tumor immunotherapy targeting CD40 molecule, the biological activity of the cell line was studied preliminarily in order to obtain the stable cell line which can effectively secrete the specific anti-human CD40 human-mouse chimeric antibody. There is also a breakthrough in the research and development of therapeutic McAbs. In the first part, RT-PCR was used to extract total RNAs from hybridoma cell line 5C11 which specifically secreted monoclonal antibody against human CD40. Routine reverse transcription was used to amplify PCR with degenerate primers, and primers were designed according to the results of sequence analysis of heavy and light chain genes. The V _ (th) and V _ (th) L genes containing signal peptide sequences were amplified by SMART-PCR and sequenced. At the same time, RT-PCR was used to extract total RNAs from human spleen cells. Specific primers were designed to amplify the gene of huFchuCH1 and huC 魏 of human IgG1 _ (gamma) chain huFchuCH1 and 魏 appa chain. Then huFc and huCH1 were spliced to obtain the constant region gene of human IgG1 _ (gamma) (CH1-CH3 chain by TP-PCR and sequenced. The chimeric heavy chain was obtained by splicing the VSP sequence containing the signal peptide sequence and the constant region sequence of the human IgG1 heavy chain. The chimeric light chain was obtained by splicing the VSP sequence containing the signal peptide sequence and the constant region sequence of the human 魏 appa chain, and the splicing product was sequenced. The recombinant plasmid pIRES/hu5C11.pIRES/hu5C11 was constructed and transfected into 293T cells by liposome method. CD40-L929 and mock-L929 cell lines were used to transfect 293T cells. Human-mouse chimeric antibody against human CD40 in the supernatant of 293T cells was identified by flow cytometry. The results showed that the eukaryotic expression vector pIRES- rhuSC11 containing human mouse chimeric heavy chain and chimeric light chain gene against human CD40 human-mouse chimeric antibody was successfully constructed in this study. In the second part, Chinese hamster ovarian cell (CHO) was selected as the expression host. The recombinant expression plasmid of pIRES/hu5C11 chimeric antibody extracted by kit was transfected into CHO cells by liposome method. Gene transfection cell lines (hu5C11-CHO) expressing human chimeric antibody (ch-SC11) against human CD40 were obtained and identified by RT-PCR. The results of RT-PCR showed that ch-SC11 gene was successfully integrated into huSC11-CHO cells by FCM and Western blot. The supernatant of CHO transfected with pIRES/hu5C11 plasmid contains anti-human CD40 human-mouse chimeric antibody, which not only retains the activity of specifically recognizing CD40 molecule, but also contains human immunoglobulin FC.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392
本文编号：2153401
[Abstract]:On the basis of the first domestic activated anti-human CD40 monoclonal antibody (5C11) developed by our department, the classical chimeric antibody construction method was used to carry out genetic engineering modification and eukaryotic expression in CHO cells. In order to obtain the stable cell line which can continuously secrete specific anti-human CD40 human-mouse chimeric antibody, and provide the necessary material basis for tumor immunotherapy targeting CD40 molecule, the biological activity of the cell line was studied preliminarily in order to obtain the stable cell line which can effectively secrete the specific anti-human CD40 human-mouse chimeric antibody. There is also a breakthrough in the research and development of therapeutic McAbs. In the first part, RT-PCR was used to extract total RNAs from hybridoma cell line 5C11 which specifically secreted monoclonal antibody against human CD40. Routine reverse transcription was used to amplify PCR with degenerate primers, and primers were designed according to the results of sequence analysis of heavy and light chain genes. The V _ (th) and V _ (th) L genes containing signal peptide sequences were amplified by SMART-PCR and sequenced. At the same time, RT-PCR was used to extract total RNAs from human spleen cells. Specific primers were designed to amplify the gene of huFchuCH1 and huC 魏 of human IgG1 _ (gamma) chain huFchuCH1 and 魏 appa chain. Then huFc and huCH1 were spliced to obtain the constant region gene of human IgG1 _ (gamma) (CH1-CH3 chain by TP-PCR and sequenced. The chimeric heavy chain was obtained by splicing the VSP sequence containing the signal peptide sequence and the constant region sequence of the human IgG1 heavy chain. The chimeric light chain was obtained by splicing the VSP sequence containing the signal peptide sequence and the constant region sequence of the human 魏 appa chain, and the splicing product was sequenced. The recombinant plasmid pIRES/hu5C11.pIRES/hu5C11 was constructed and transfected into 293T cells by liposome method. CD40-L929 and mock-L929 cell lines were used to transfect 293T cells. Human-mouse chimeric antibody against human CD40 in the supernatant of 293T cells was identified by flow cytometry. The results showed that the eukaryotic expression vector pIRES- rhuSC11 containing human mouse chimeric heavy chain and chimeric light chain gene against human CD40 human-mouse chimeric antibody was successfully constructed in this study. In the second part, Chinese hamster ovarian cell (CHO) was selected as the expression host. The recombinant expression plasmid of pIRES/hu5C11 chimeric antibody extracted by kit was transfected into CHO cells by liposome method. Gene transfection cell lines (hu5C11-CHO) expressing human chimeric antibody (ch-SC11) against human CD40 were obtained and identified by RT-PCR. The results of RT-PCR showed that ch-SC11 gene was successfully integrated into huSC11-CHO cells by FCM and Western blot. The supernatant of CHO transfected with pIRES/hu5C11 plasmid contains anti-human CD40 human-mouse chimeric antibody, which not only retains the activity of specifically recognizing CD40 molecule, but also contains human immunoglobulin FC.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392
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