犬源华支睾吸虫28ku谷胱甘肽S-转移酶的原核表达与反应原性分析

Expression of 28 ku glutathione S-transferse gene from Clonorchis sinensis

本研究提取犬源华支睾吸虫总RNA,应用RT-RCR技术扩增28ku谷胱甘肽S-转移酶(Cs28GST)编码基因,PCR产物经T/A克隆后,亚克隆入pET-30a(+)原核表达载体,构建重组质粒pET-Cs28GST,转化大肠埃希菌BL21(DE3),异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达;表达产物经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白质印迹分析和鉴定,研究结果显示犬源Cs28GST编码区含有639个碱基,编码212个氨基酸残基,表达的蛋白以包涵体形式存在,大小约为30.6ku,可被自然感染华支睾吸虫病犬的阳性血清识别,证明Cs28GST基因可在原核表达系统中高效表达并具有反应原性。

The gene of 28 ku Glutathione S-transferse （Cs28GST） was amplified by RT-PCR from total RNA of Clonorchis sinensis was extracted from dogs. The PCR product was cloned into pET-30a （＋） vector, and the recombinant plasmids were transformed into E.coli BL21 （DE3）. The expressed protein induced by IPTG was purified and identified by SDS-PAGE and western blot. The results showed that the Cs28GST contained 639 base pairs encoding 212 amino acids. The protein was expressed in the form of inclusion body and had a MW about 30.6 ku. The recombinant protein could react with positive serum from dogs naturally infected with C.sinensis, indicating that the Cs28GST expressed in prokaryotic expression system retained antigenicity of the native protein.