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目的为了阐明血清中存在的抗精子抗体引起不孕不育的原因,构建重组人睾丸特异性抗原-1(recombinanthuman testis specific antigen-1,rhTSA-1)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TSA-1的cDNA,将其克隆入表达载体pGEX-4T-2,构建TSA-1的重组原核表达质粒pGEX/TSA-1。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TSA-1基因完全一致。SDS-PAGE电泳显示,表达产物的相对分子量与预期结果相符。结论获得了人TSA-1的编码基因,并在大肠杆菌中可表达人TSA-1。
Objective To elucidate the causes of infertility caused by anti-sperm antibodies in serum, a prokaryotic expression plasmid of recombinant human testis specific antigen-1 (rhTSA-1) was constructed and induced in E. coli . Methods The cDNA of human TSA-1 was amplified by RT-PCR from human testis tissue and cloned into the expression vector pGEX-4T-2 to construct recombinant prokaryotic expression vector pGEX / TSA-1 of TSA-1 . The recombinant plasmids were identified by restriction enzyme digestion and sequencing, then transformed into E. coli JM109 and expressed in Escherichia coli. Results sequencing showed that the recombinant gene sequence and human TSA-1 gene exactly. SDS-PAGE electrophoresis showed that the relative molecular weight of the expressed product was consistent with the expected result. Conclusion The human TSA-1 encoding gene was obtained and expressed in E. coli TSA-1.