人精子相关抗原7的基因克隆及原核表达

Cloning and expression of recombinant human fertilization antigen-1 in E.coli

目的为了阐明免疫性不孕不育的原因,研制人特异性精子抗原避孕疫苗,构建重组人精子相关抗原7(recombinant human sperm associated antigen 7,rhSPAG7)的原核表达质粒并在大肠杆菌中诱导表达。方法采用RT-PCR法,从人睾丸组织总RNA中扩增获得人SPAG7的c DNA,将其克隆入表达载体p BV220,构建人源性SPAG7的重组原核表达质粒p BV220/SPAG7。重组质粒经酶切和测序鉴定后,转化大肠杆菌JM109并在大肠杆菌中诱导表达目的蛋白。结果测序表明重组基因序列与人SPAG7基因完全一致。SDS-PAGE电泳显示,表达产物的相对分子量为25.8KDa与预期结果相符。结论获得了人SPAG7的编码基因,并在大肠杆菌中表达了人的SPAG7蛋白。

Objective：To elucidate the cause of immunological infertility,and develop the contraceptive vaccine of human specific sperm antigen,a recombinant prokaryotic expression plasmid of human sperm associated antigen 7 was constructed and the rhSPAG7 protein was expressed in E.coli. Methods：The code gene of human SPAG7 was amplified by reverse transcript PCR from human testes organism and cloned into a prokaryotic expression vector p BV220. The recombinant plasmid was identified by restriction enzyme digestion and DNA sequencing. The recombinant plasmid was transfected into E.coli JM109,then the recombinant protein was expressed in E.coli and detected by using SDS-PAGE. Results：The sequence of recombinant human SPAG7 was correct. The relative molecular mass of the expressed product was identical to that of expectation of 25.8KDa. Conclusion：The code gene of human FA-1 has been successfully cloned and expressed in E.coli.