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目的为了阐明免疫性不孕不育的原因,检测血清中存在的抗精子抗体的种类,构建重组人受精相关抗原-1(recombinant human Fertilization antigen-1,rhFA-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FA-1的cDNA,将其克隆入表达载体pBV220,构建人源性FA-1的重组原核表达质粒pBV220/FA-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FA-1基因完全一致。SDS-PAGE电泳显示,表达产物的相对分子量为14.6KDa与预期结果相符。结论获得了人FA-1的编码基因,并在大肠杆菌中表达了人的FA-1蛋白。
Objective To elucidate the causes of immune infertility, detect the types of anti-sperm antibodies present in serum and construct prokaryotic expression plasmids of recombinant human fertilization antigen-1 (rhFA-1) Bacillus induced expression. Methods cDNA of human FA-1 was amplified by RT-PCR from total human testis tissue and cloned into expression vector pBV220 to construct recombinant prokaryotic expression plasmid pBV220 / FA-1 of human FA-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 FA-1 gene exactly. SDS-PAGE electrophoresis showed that the relative molecular weight of the expressed product was 14.6KDa, which was consistent with the expected results. Conclusion The human FA-1 encoding gene was obtained and human FA-1 protein was expressed in Escherichia coli.