旋毛虫ES抗原P49基因的体外表达分析

In vitro expression of P49 gene from ES antigen of Trichinella spiralis

目的克隆和表达旋毛虫ES抗原P49基因,并分析表达产物的免疫原性。方法以重组质粒pCDNA3.1-P49为模板,PCR扩增旋毛虫ES抗原P49基因,将扩增片段双酶切产物连接到质粒pEGFP-N1中,构建重组真核表达载体pEG-FP-N1-P49,利用脂质体法将其转染哺乳动物细胞COS-7。于转染24h后在荧光显微镜下观察发绿色荧光的转基因细胞;通过Western-blot分析证实P49基因表达产物的免疫原性。结果成功构建旋毛虫ES抗原P49基因的绿色荧光蛋白表达载体pEGFP-N1-P49,并在COS-7细胞中表达了目的蛋白,并且表达的蛋白可被感染旋毛虫小鼠阳性血清特异地识别。结论成功获得了重组蛋白,并证实该融合蛋白具有免疫原性,其结果为研究其生物学功能奠定了基础。

The P49 gene from ES antigen of Trichinella spiralis was amplified by PCR with the recombinant plasmid pCDNA3.1-P49 as the template and the fragment was combined with plasmid pEGFP-N1.The recombinant expression vector,pEGFP-N1-P49,was transfected to COS-7 using liposome.The transgenic cells appeared bright fluorescence 24 h after transfection under fluorescence microscope.Western-blot analysis of the expression product of P49 gene confirmed its immunogenicity as well.The results indicated that P49 and EGFP gene co-expression vector pEGFP-N1-P49 has been successfully constructed and it could express the target protein in COS-7 cells,and the immunogenicity of fusion protein has been confirmed.