HIV-1膜蛋白基因gp120和gp41的合成及在毕赤酵母中的表达

Expression of Human Immunodeficiency Virus-1 gp120 and gp41 Gene Synthesized in Pichia pastoris

目的合成适合酵母表达的HIV-1膜蛋白gp120和gp41基因,在毕赤酵母中高效表达分泌型重组HIV-1 gp120和gp41抗原。方法选用酵母偏爱的同义密码子替换野生型gp120和gp41基因的酵母稀有密码子,采取基因搭桥法及聚合酶链反应(PCR),合成HIV-1 gp120和gp41基因,构建分泌型重组质粒并转化毕赤酵母菌株GS115,经甲醇诱导表达HIV-1外膜糖蛋白gp120和gp41。结果DNA测序证实合成的HIV-1 gp120和gp41基因正确克隆到表达载体中;聚丙烯酰胺凝胶电泳及免疫印迹显示gp120和gp41在毕赤酵母中高效分泌表达。结论gp120和gp41合成基因在毕赤酵母表达系统高效分泌表达。

Objective, To synthesize HIV - 1 envelope gene gp120 and gp41 which was suitable for yeast protein expression and to express synthetic genes in Pichia pastoris. Methods Using synonymous cedons preferred by yeast usage on protein expression to replace some native cedons of wildtype HIV genes, the synthetic gp120 and gp41genes were achieved by a recursive PCR （rPCR）. The genes synthesized were inserted into the yeast expression vector pPICZαA. The recombinant plasmids were transformed into GS115 yeast by electroporation. The yeast transformants induced by methanol expressed gp120 and gp41 protein. Results The restriction analysis and DNA sequencing confirmed that the synthetic genes were inserted to yeast pPICZαA in correct orientation. SDS -PAGE and immunoblotting indicated that the secreted forms of gp120 and gp41 were expressed by yeast transformants. Conclusion The secreted forms of recombinant gp120 and gp41 were expressed efficiently in Pichia pastoris by using synthetic genes.