精子蛋白SP22在毕赤氏酵母中的表达、纯化和鉴定

Expression of Human Sperm Protein SP22 in Pichia Pastoris Yeast and Purification, Identification of Expression Product

目的:克隆人睾丸精子蛋白SP22基因在毕赤氏酵母中表达,并对表达产物进行纯化和鉴定。方法:RT-PCR克隆出睾丸SP22基因,导入pGEM-T克隆载体。再亚克隆到酵母真核分泌型表达载体pHIL-S1。电击转化将重组质粒pHIL-S1/SP22导入GS115酵母菌株。筛选出阳性克隆,以甲醇诱导分泌表达。镍离子亲和层析法从培养上清中纯化目的蛋白。结果:SP22编码序列与人致癌基因DJ1基本相同。重组质粒正确插入酵母基因组后,甲醇诱导SP22表达并分泌至培养上清中,表达量约占上清总蛋白的20%。纯化出的目的蛋白为糖蛋白,能被免疫印迹证实。结论:成功构建了酵母GS115/pHIL-S1/SP22重组型表达菌株,并纯化获得糖基化的蛋白,为进一步研究该蛋白的结构及生理功能打下了基础。

Objective： To colne the cDNA of human testis SP22 which was expressed in Pichza Pastoris yeast and to purify and identify the expression product. Methods： SP22 cDNA was cloned by RT-PCR. After being linked to pGEM-T vector and DNA sequencing, human testis SP22 was obtained from pGEM-T/htSP22 and inserted into pHIL- S1 to construct secretory yeast expression vector, pHIL-S1/SP22 was transformed into Pichia Pastoris GS115 yeast genome by electroporation. Positive clone was selected and expression of recombinant protein SP22 was induced by methnol. The expressed recombinant protein htSP22 was purified with Ni^2＋-NTA resin.Results： SP22 coding sequences was almost the same to oncogene D J1. After the recombinant plasmid pHIL-S1/ SP22 was correctly inserted into GS115 genome, was methnol induced the recombinant gene to express secretory htSP22 into supernatant. The expressed protein was identified by Western blotting and the protein htSP22 had 20% expression level in total supernatant proteins. Conclusion： Recombinant GS115/pHIL-S1/SP22 was constructed successfully and purified glycosylated protein was obtocined. This study offers solid basis for further research on the structure activity and function of human testis SP22.