弓形虫ROP19蛋白生物信息学分析及真核表达载体的构建

Bioinformatics analysis and construction of an eukaryocyte vector of ROP19 protein in Toxoplasma gondii

目的构建刚地弓形虫PRU株ROP19蛋白的真核表达载体并检测其表达。方法利用生物信息学方法分析弓形虫ROP19蛋白的理化性质并比较弓形虫蛋白ROP19和SAG1的B细胞表位及T细胞表位,采用分子克隆技术构建重组真核表达质粒pEGFP-C1-ROP19(pROP19),经PCR扩增、酶切及测序鉴定正确后,体外转染HEK293T细胞,置于倒置荧光显微镜下观察荧光的表达情况。细胞转染质粒48 h后收集裂解细胞,提取蛋白后用Western blotting检测重组真核表达质粒pROP19在体外细胞中的表达。结果生物信息学分析显示ROP19主要位于膜上,具备比弓形虫SAG1更优异的抗原表位;RT-PCR结果表明重组真核载体pROP19成功构建,Western blotting结果显示ROP19蛋白可以被抗STAG小鼠血清识别。结论成功获得重组真核表达质粒pROP19,并能在真核细胞内表达。

Objective To construct and express the eukaryotic expression vector of ROP19 protein of Toxoplasma gondii PRU strain.Methods The physical and chemical characteristics of ROP19 protein were analyzed,and the B cell epitopes and T cell epitopes of ROP19 were compared with SAG1 using bioinformatics.The recombinant eukaryotic expression plasmid pROP19 was constructed by the molecular cloning technology.After being identified by PCR,restriction enzyme cleavage and sequencing,pROP19 was transfected into the HEK293 T cells.The cells from different groups（control,p EGFP-C1 and pROP19） were respectively detected with fluorescence microscope under blue laser.The ROP19 protein was detected by Western blotting.Results The ROP19 protein was mainly located in the membrane and had better antigenic index than SAG1.The plasmid was constructed successfully.Western blotting showed that the expressed proteins could be recognized by anti-STAg mouse sera.Conclusion The eukaryotic expression vector pROP19 is constructed and expressed in eukaryotic cells.