共表达2个独立的抗关节炎分子TNFR-Fc和CTLA4-FasL重组表达系统的建立与鉴定

Construction and identification of the recombinant system expressing two separate anti-arthritic molecules of TNFR-Fc and CTLA4-FasL

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。

This study performed to construct the recombinant adeno-associated virus （AAV） vector expressing simultaneously two separate anti-arthritic molecules TNFR-Fc and CTLA4-FasL and identify the expressions of them. Applying a novel furin-2A cleavage strategy, the recombinant AAV vector cloning with rat TNFR-Fc and CTLA4-FasL dual fusion genes was constructed, and then the expressions of target proteins in the supernatants were identified by ELISA and Western blot after transfected with 293T cells. The recombinant expression plasmid TFCF harboring two anti-inflammatory molecules was constructed and the target proteins of TNFR-Fc and CTLA4- FasL were expressed simultaneously and separately in the transfected supernatants. In conclusion, a novel dual anti-arthritic molecule expression system is obtained, which can antagonize TNF and T cells simultaneously, and thus lays a foundation for future animal experiments in vivo.