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目的构建能同时独立表达双抗关节炎分子TNFR-Fc和CTLA4-FasL的重组腺相关病毒(AAV)载体,并对蛋白表达进行鉴定。方法应用furin-2A新型剪切策略,构建TNFR-Fc和CTLA4-FasL双融合基因重组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。结果成功构建双抗炎分子重组表达载体TFCF,在转染细胞上清中检测到目的蛋白TNFR-Fc和CTLA4-FasL的独立共表达。结论获得了可同时拮抗TNF和T细胞的新型双抗关节炎分子共表达系统,为今后动物体内研究奠定了基础。
Objective To construct a recombinant adeno-associated virus (AAV) vector capable of independent expression of dual anti-arthritic molecules TNFR-Fc and CTLA4-FasL, and to identify the protein expression. Methods The recombinant AAV vector of TNFR-Fc and CTLA4-FasL double fusion gene was constructed by using the furin-2A novel cleavage strategy. 293T cells were transfected in vitro. The supernatants were collected and the expression of the target protein was detected by ELISA and Western blot. Results TFCF was successfully constructed and the co-expression of TNFR-Fc and CTLA4-FasL was detected in the supernatant of transfected cells. Conclusions A novel double anti-arthritis molecular co-expression system that can simultaneously antagonize TNF and T cells was obtained, which laid the foundation for the in vivo study of animals in the future.