关键词: 3-磷酸甘油酸激酶, 华支睾吸虫, 克隆, 原核表达

Abstract: 　Objective To construct a prokaryotic expression plasmid encoding 3 phosphoglycerate kinase(PGK) gene of Clonorchis sinensis and analyze its expression in E.coli JM109. Methods Total RNA was extracted with Trizol. The gene encoding PGK was amplified by RT PCR from the total RNA, and was cloned into the pGEX 4T1 vector. The recombinant plasmids pGEX4T1 PGK constructed were transferred into JM109, positive recombinants were screened and identified by tool enzyme digestion, PCR technique and sequencing. The recombinant was induced with IPTG to express the target protein. The expression product was characterized by SDS PAGE. Results There were three clear bands from extracted total RNA, the PGK gene (1 248bp) was amplified by RT PCR technique, the sequencing result was consistent with the known sequence. Ten clones were obtained by screening after transferring, six of which were positive recombinants. The positive recombinant was induced to express, and the SDS PAGE showed the expression products was about Mr 70 000. Conclusion The recombination prokaryotic expression vector pGEX 4T 1 PGK has been reconstructed and a fused protein was expressed which contained Sj26 GST.

Key words: 3-Phosphoglycerate kinase, Clonorchis sinensis, Cloning, Prokaryotic expression