跨膜型TNF-alpha单克隆抗体腺病毒表达载体的构建和鉴

目的：应用AdEasy-1 系统构建包含tmTNF-alpha单克隆抗体轻、重链序列的重组腺病毒表达载体。方法：首先PCR 合成抗体轻、重链序列，分别将轻、重链序列插入经过改造的含有双启动子的穿梭质粒pShuttle-2CMV，将穿梭载体电转化转化AdEasy 系统BJ5183 感受态，挑取单克隆扩增质粒后酶切鉴定。结果：成功构建重组腺病毒表达载体pAdEasy-tmTNF-alpha，抗体重链、轻链序列酶切后经1%琼脂糖凝胶电泳证实条带片段大小正确，电转化BJ5183 后挑选重组克隆提取质粒，PacI酶切后重组片段位于4.5kb及3 kb位置，证明重组腺病毒质粒pAdeasy-1-tmTNF-alpha构建成功。结论：将腺病毒系统与单克隆抗体技术相结合，利用AdEasy-1系统成功构建腺病毒重组tmTNF-alpha单克隆抗体表达载体，为进一步开展肿瘤基因治疗的研究提供基础。

Construction and Identification of an Adenovirus-Mediated RecombinantMonoclonal Antibody Against Transmembrane TNF-alpha

Objective:To construct a recombinant adenovirus vector which contains the sequences of light and heavy chain oftmTNF-alpha antibody through AdEasy system.Methods:The heavy and light chain sequences of the antibody were firstly amplified byPCR, and these segments were respectively cloned into a reconstructed pShuttle vector that contains 2CMV. The vector waselectrotransformed within the AdEasy expression competent cell BJ5183. Monoclones were picked and then recombinant plasmid waspurified and identified.Results:The recombinant adenovirus pAdEasy-tmTNF-alpha was successfully constructed and confirmed by 1%agarose gel electrophoresis. The recombinant adenovirus plasmid pAdEasy-tmTNF-alpha, which electrotransformed into BJ5183 was alsoexamined by enzyme digestion.Conclusion:Recombinant adenovirus tmTNF-alpha monoclonal antibody expression vector was successfullyconstructed through AdEasy-1 systemby means of the integration of adenovirus systemand monoclonal antibody technology, which mayprovide the basis for further research of gene therapy of tumor.