大鼠TNFR-Fc重组腺相关病毒载体的构建、表达及中和活性鉴定

Cloning and expression of rat TNFR-Fc fusion gene and identification of its neutralization activity

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。

This study deigned to construct a recombinant type II adeno-associated virus vector harboring the fusion product of extracellular domain of rat tumor necrosis factor receptor （TNFR） with rat IgG Fc fragment, and detect the expression and biological activity of TNFR-Fc, preparing for further study of gene therapy for rheumatoid arthritis （RA）. The recombinant adeno-associated virus （AAV） vector containing rat TNFR-Fc fusion gene was constructed, and then the expression of target protein TNFR-Fc in the supernatants was identified by ELISA and Western blot after transfected with 293T cells. An in vitro TNF-α inhibition assay was performed using TNF-αsensitive mouse fibrosarcoma L929 cells to determine the neutralizing capacity of TNFR-Fc. The recombinant plasmid pAAV2/rat TNFR-Fc was constructed and the target protein was secretively expressed in the supernatants from transfected 293T cells, which could effectively neutralize the rat TNF-α cytotoxicity to L929 cells. In conclusion, the rat TNFR-Fc fusion gene was constructed and identified to be expressed secretively in serotype 2 AAV vector, which lay the foundations for future viral package and animal experiments.