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

作　　者：陈爱华[1] 王春仁[1] 陈佳[1] 王宇[1] 朱兴全[2] 程玉楠 倪宏波[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319 [2]华南农业大学兽医学院,广东广州510642 [3]大庆市气象局,黑龙江大庆163000

出　　处：《中国预防兽医学报》2009年第2期94-98,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：教育部“长江学者和创新团队发展计划”创新团队项目(IRT0723);广东省科技计划项目(2005B20201003)

摘　　要：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.

关 键 词：华支睾吸虫 28 ku谷胱甘肽S-转移酶 原核表达 反应原性 犬 

分 类 号：S852.735[农业科学—基础兽医学]