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目的构建能稳定表达人CTLA4 Ig融合基因的减毒鼠伤寒沙门菌真核表达载体,探讨其在哺乳动物细胞中的表达并对表达产物进行鉴定。方法用touchdown PCR法扩增CTLA-4 Ig融合基因,将PCR产物连接真核表达载体pcDNA3.1(+),构建pcDNA3.1(+)-CTLA-4 Ig,用电穿孔仪转入减毒鼠伤寒沙门菌SL7207。将表达质粒转染COS-7细胞,SDS-PAGE、W estern b lot检测转染细胞裂解上清中目的蛋白的表达。结果酶切鉴定和基因序列测定显示重组质粒构建成功。在pcDNA3.1(+)-CTLA-4 Ig质粒转染后48 h细胞裂解上清中,检测到CTLA-4 Ig融合蛋白的表达,该蛋白能与抗人CTLA-4单抗特异结合。结论成功构建了能稳定表达人CTLA4 Ig融合基因的减毒鼠伤寒沙门菌真核表达载体,并在哺乳动物细胞中成功表达有生物学活性的重组人CTLA-4 Ig蛋白。
Objective To construct an eukaryotic expression vector of attenuated Salmonella typhimurium that can stably express human CTLA4 Ig fusion protein and investigate its expression in mammalian cells and identify its expression product. Methods The CTLA-4 Ig fusion gene was amplified by touchdown PCR. The PCR product was ligated into the eukaryotic expression vector pcDNA3.1 (+) to construct pcDNA3.1 (+) - CTLA-4 Ig. Salmonella typhimurium SL7207. The expression plasmids were transfected into COS-7 cells, and the expression of the target protein in the supernatant of the transfected cells was detected by SDS-PAGE and Western blot. Results Enzyme digestion and sequencing showed that the recombinant plasmid was successfully constructed. The expression of CTLA-4 Ig fusion protein was detected in the cell lysate supernatant 48 h after transfection with pcDNA3.1 (+) - CTLA-4 Ig plasmid, which specifically bound to anti-human CTLA-4 mAb. Conclusion The eukaryotic expression vector of attenuated Salmonella typhimurium, which can stably express human CTLA4 Ig fusion gene, was successfully constructed and the recombinant human CTLA-4 Ig protein was successfully expressed in mammalian cells.