弓形虫复合抗原基因P30-ROP2在毕赤酵母中的表达、纯化与鉴定

目的构建含弓形虫主要表面抗原P30与致密颗粒蛋白ROP2复合基因重组质粒,并在毕赤酵母中表达、纯化与鉴定。方法用亚克隆技术把P30-ROP2复合基因克隆入表达载体,构建酵母表达载体pGAPZαA-P30-ROP2;线性化重组酵母表达载体,电穿孔法导入毕赤酵母GS115中,抗生素Zeocin筛选、PCR法鉴定阳性转化子;酵母工程菌大量摇瓶表达,纯化产物,免疫活性鉴定。结果获得pGAPZαA-P30-ROP2重组表达载体,SDS-PAGE和Western Blot结果显示P30-ROP2复合基因表达蛋白产物分子量约为66kD,具有一定的免疫活性。结论弓形虫复合抗原基因P30-ROP2在毕赤酵母中成功分泌表达,表达产物具有免疫活性,为弓形虫病诊断抗原及疫苗研制奠定基础。

Expression, purification and characterization of the multi-antigenic gene encoding recombinant protein P30-ROP2 in Pichia pastoris

In the present study,the expression,purification and characterization of the multi-antigenic gene encoding recombinant protein P30-ROP2 in Pichia pastoris were performed,in which the P30-ROP2 gene was subcloned into expression vector pGAPZαA and then the recombinants were transferred to yeast cell GS115 by electroporation.The Pichia transformants were identified by PCR and the expression products were analyzed by SDS-PAGE and Western blotting.It was found that the positive recombinant plasmid pGAPZaA-P30-ROP2 was successfully constructed through PCR.Enzyme digestion and sequence analysis.and this recombinant plasmid expressed a specific 66 kDa fusion protein in Pichia pastoris with certain degree of immunogenicity as demonstrated by Western blotting.These results provide basis for the researches on the diagnostic antigens and preparation of vaccine in Toxoplasma infections.