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目的 :构建表达人 鼠嵌合抗体的通用真核表达载体 ,用于以嵌合抗体的形式表达PCR获取的小鼠抗体可变区基因 ,以便将人 鼠嵌合抗体应用于临床治疗。方法 :用人Tac抗原信号肽以及人IgCκ基因和γ1CH基因 ,构建人 鼠嵌合抗体的表达载体 ,并转染真核细胞 2 93T进行表达。用RT PCR、FACS和ELISA进行抗体表达的鉴定。结果 :利用人Tac抗原信号肽以及人IgCκ基因和γ1CH基因 ,构建了用于表达人 鼠嵌合抗体的通用真核表达载体。用本研究设计的引物 ,扩增小鼠抗人HER2抗体的V区基因片段 ,酶切后先后插入所构建的载体的相应克隆位点 ,转染 2 93T细胞可将PCR获得的小鼠抗体V区基因片段表达为人 鼠嵌合抗体。用RT PCR、FACS和ELISA证实 ,本系统可表达嵌合抗体。结论 :构建了人 鼠嵌合抗体的真核表达载体 ,并证实了其可在 2 93T细胞中表达。
OBJECTIVE: To construct a universal eukaryotic expression vector expressing chimeric antibody of human and mouse, and to express the variable region of mouse antibody obtained by PCR in the form of chimeric antibody, so as to apply human chimeric antibody to clinical treatment. Methods: The human Tac antigen signal peptide, human IgCκ gene and γ1CH gene were used to construct the chimeric antibody expression vector of human and mouse, and then transfected into 293T cells for expression. Antibody expression was identified by RT PCR, FACS and ELISA. Results: The universal eukaryotic expression vector for human chimeric antibody was constructed by using human Tac antigen signal peptide, human IgCκ gene and γ1CH gene. The primers designed in this study were used to amplify the V region of mouse anti-human HER2 antibody and inserted into the corresponding cloning sites of the constructed vector after digestion. The 293T cells transfected with the mouse antibody V Region gene fragments are expressed as human mouse chimeric antibodies. RT-PCR, FACS and ELISA confirmed that the system can express chimeric antibodies. Conclusion: The eukaryotic expression vector of human chimeric antibody was constructed and confirmed that it can be expressed in 293T cells.