华支睾吸虫3-磷酸甘油酸激酶基因的扩增、克隆及表达

Amplification,Cloning and Expression of 3-Phosphoglycerate Kinase Gene from Clonorchis sinensis

目的 构建编码华支睾吸虫 3 磷酸甘油酸激酶基因的原核表达载体 ,并分析其在大肠埃希菌中的表达。 方法 用Trizol法从华支睾吸虫 3 成虫体内提取总RNA ,并将其反转录成cDNA。根据华支睾吸虫磷酸甘油酸激酶的已知基因 ,利用DNAclub和PCRdesign软件设计合成一对特异引物 ,从合成的cDNA中 ,用PCR技术扩增出目的片段。将目的片段和原核表达载体同时双酶切后连接 ,重组体转入JM10 9大肠埃希菌 ,用双酶切、PCR和测序筛选阳性克隆。挑选 1个阳性菌落 ,用异丙基 β D 硫代半乳糖苷诱导表达 ,表达产物进行十二烷基硫酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)鉴定。 结果 用逆转录 聚合酶链反应技术扩增出 1条 12 48bp大小的片段 ,PCR产物测序鉴定正确 ;转化后得到 10个克隆 ,双酶切、PCR扩增鉴定 ,其中 6个为阳性克隆 ;表达产物用SDS PAGE鉴定 ,在相对分子质量 70 0 0 0处有 1条与理论预测的融合蛋白大小相一致的特异条带。 结论 成功构建重组原核表达载体pGEX 4T 1 PGK 。

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 4T 1 vector. The recombinant plasmids pGEX 4T 1 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 (1 248 bp) 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.