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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 causes of immunological infertility, a human-specific sperm antigen contraceptive vaccine was developed to construct a prokaryotic expression plasmid of recombinant human sperm associated antigen 7 (rhSPAG7) and induced in E. coli. Methods The c DNA of human SPAG7 was amplified by RT-PCR from total human testis tissue and cloned into expression vector p BV220 to construct recombinant prokaryotic expression plasmid p BV220 / SPAG7 of human SPAG7. 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 was completely identical with human SPAG7 gene. SDS-PAGE electrophoresis showed that the relative molecular weight of the expressed product was 25.8KDa, which was in accordance with the expected results. Conclusion The gene encoding human SPAG7 was obtained and human SPAG7 protein was expressed in E. coli.