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目的构建携带大鼠肿瘤坏死因子II型受体胞外区(TNFR)融合大鼠IgG Fc片段的重组2型腺相关病毒载体(pAAV2/TNFR-Fc),并对目的蛋白TNFR-Fc的表达和生物学活性进行初步鉴定,为后续的类风湿关节炎基因治疗研究做准备。方法构建大鼠TNFR-Fc融合基因重组腺相关病毒(AAV)载体,体外转染293T细胞,收集转染上清,以ELISA和Western blot等方法检测目的蛋白的表达,并应用L929细胞测定重组表达产物的TNF-α细胞毒中和活性。结果成功构建重组表达载体pAAV2/TNFR-Fc,在转染细胞上清中检测到目的蛋白TNFR-Fc的表达,其可有效中和大鼠TNF-α的L929细胞毒活性。结论成功构建了大鼠TNFR-Fc融合基因,并验证了其在2型AAV载体上的分泌性表达,为进一步病毒包装及动物实验奠定了基础。
Objective To construct a recombinant adeno-associated virus vector (pAAV2 / TNFR-Fc) carrying rat Fc fragment of rat TNF-α fusion rat IgG and to express TNFR-Fc Biological activity preliminary identification, for the follow-up of rheumatoid arthritis gene therapy research preparation. Methods The recombinant adeno-associated virus (AAV) vector containing rat TNFR-Fc fusion gene was constructed and transfected into 293T cells in vitro. The supernatant was collected and the expression of the target protein was detected by ELISA and Western blot. The expression of the recombinant protein was detected by L929 cells TNF-alpha cytotoxic activity of the product. Results The recombinant expression vector pAAV2 / TNFR-Fc was successfully constructed and the expression of TNFR-Fc protein was detected in the supernatant of the transfected cells. It could effectively neutralize L929 cytotoxicity of TNF-α in rats. Conclusion The rat TNFR-Fc fusion gene was successfully constructed and its secreted expression on type 2 AAV vector was verified, which laid the foundation for further virus packaging and animal experiments.