豆状带绦虫TPO18基因的原核表达及保护性分析  

Prokaryotic Expression and Protective Efficacy of TPO18 Gene from Taenia pisiformis

作　　者：韩进欢[1] 苟惠天[1] 尚清炎[1] 孙晓林[1] 

机构地区：[1]甘肃农业大学动物医学院,兰州730070

出　　处：《中国农业科学》2014年第15期3077-3084,共8页Scientia Agricultura Sinica

基　　金：甘肃省农业生物技术研究与应用开发项目(GNSW-2010-01);甘肃省科技支撑计划项目(1104NKCA082)

摘　　要：【目的】明确重组蛋白pGEX-TPO18的免疫保护效果。【方法】提取豆状带绦虫六钩蚴RNA,根据带科其他种属绦虫18 kD基因的保守区设计引物,5′端分别引入EcoR I、Xho I限制性酶切位点,RT-PCR扩增,产物克隆于pMD18-T中进行序列测定,并对序列进行生物信息学分析。将纯化后的PCR产物和质粒pGEX-4T-1分别用EcoRⅠ+XhoⅠ双酶切,回收目的片段,连接后转化至E.coli BL21感受态细胞,挑斑摇菌,通过PCR方法和重组质粒测序技术进行鉴定,将鉴定正确的重组表达质粒命名为pGEX-TPO18。用IPTG诱导表达重组质粒,收集菌体进行SDS-PAGE分析,用GST琼脂糖树脂纯化TPO18蛋白,进行Western blottting检测。将纯化后的重组蛋白分别乳化弗氏佐剂、206佐剂、氢氧化铝佐剂后免疫家兔,每次50μg/只,共免疫3次,末次免疫后34 d每只家兔感染1 500个虫卵,感染58 d后扑杀并计算各免疫组减囊率。免疫前后每隔7 d采血分离血清,ELISA法检测血清抗体水平,以40μg·mL-1重组蛋白包被酶标板,血清1﹕200稀释,检测血清样品的OD492nm值。【结果】TPO18基因的RT-PCR扩增产物为339 bp,与预期大小相符;测序结果表明无碱基突变,重组质粒pGEX-TPO18构建成功。pGEX-TPO18在大肠杆菌中获得高效表达,表达产物为38.6 kD的可溶性蛋白,Western blotting分析结果显示兔抗豆状囊尾蚴阳性血清与重组蛋白在38.6 kD处有一条明显的反应条带,表明该重组蛋白具有反应原性。经ELISA检测,免疫后7 d免疫组家兔抗体水平开始升高,第43天达到峰值。经口感染虫卵后,免疫组抗体水平开始降低,但仍保持一定水平。家兔免疫保护试验表明弗氏佐剂、206佐剂和氢氧化铝佐剂免疫组减囊率分别为79.13%、65.66%和50.43%。【结论】研究表明弗氏佐剂免疫效果较好,有望研制出抗豆状囊尾蚴病的高效疫苗。[Objective]The study aimed to investigate the protective efficacy of the recombinant antigen of TPO18 （pGEX-TPO18） from Taenia pisiformis. [Method] Total RNA was extracted from oncosphere of T. pisiformis. A pair of primers was designed based on the conserved region of other Taeniidae 18 kD gene. The EcoR I and Xho I restriction sites were introduced into 5′ end of primers. After RT-PCR, amplification product was cloned into pMD18-T, sequenced and the sequence analysis was made with bioinformatics. The products were ligated into the pGEX-4T-1 vector after digestion with EcoRⅠ＋XhoⅠ and purified. The recombinant pGEX-TPO18 plasmid was transformed into E. coli BL21 and spots were picked after shaking bacteria. The correctly identified recombinant plasmid, identified by PCR and sequenced, was named pGEX-TPO18. The recombinant plasmids were induced for expression with IPTG, cells were collected by SDS-PAGE analysis and purified using GST agarose resin. Then, TPO18 protein was detected by Western blotting. Rabbits were immunized with the purified recombinant protein emulsified with Freund’s adjuvant, 206 adjuvant, Al（OH）3 adjuvant, respectively, and rabbit anti-TPO18 serum was prepared. Each rabbit was injected with 50 μg recombinant protein for 3 times. On the 34th day after final inoculation, each rabbit was challenged by 1 500 eggs of T. pisiformis. On the 58th day after infection, rabbits were sacrificed and the number of cysts was counted. Before and after 7 days of every immunization, serum was separated. And serum antibody levels were detected using ELISA assay with 40 μg·mL-1 recombinant protein-coated microtiter plates, 1﹕200 dilution of serum and detection of the OD492nm value of serum samples was made.[Result] The product of RT-PCR was 339 bp and agree with expectations; The results of sequencing showed no mutation. So, the construction of recombinant plasmid pGEX-TPO18 was successful. The pGEX-TPO18 was transformed into E. coli BL21. SDS-PAGE showed that the 38.6 kD fusion protein was

关 键 词：豆状带绦虫 TPO18 原核表达 免疫保护性分析 

分 类 号：S858.291[农业科学—临床兽医学]