中国农业科学 ›› 2014, Vol. 47 ›› Issue (15): 3077-3084.doi: 10.3864/j.issn.0578-1752.2014.15.017

• 畜牧·兽医·资源昆虫 • 上一篇    下一篇

豆状带绦虫TPO18基因的原核表达及保护性分析

 韩进欢, 苟惠天, 尚清炎, 孙晓林   

  1. 甘肃农业大学动物医学院,兰州 730070
  • 收稿日期:2013-11-06 出版日期:2014-08-01 发布日期:2014-05-15
  • 通讯作者: 孙晓林,E-mail:sunxl@gasu.edu.cn
  • 作者简介:韩进欢,Tel:13359408308;E-mail:hjh513162325@126.com
  • 基金资助:

    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)

Prokaryotic Expression and Protective Efficacy of TPO18 Gene from Taenia pisiformis

 HAN  Jin-Huan, GOU  Hui-Tian, SHANG  Qing-Yan, SUN  Xiao-Lin   

  1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070
  • Received:2013-11-06 Online:2014-08-01 Published:2014-05-15

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 50 μg/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 40 µg•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。

关键词: 豆状带绦虫 , TPO18 , 原核表达 , 免疫保护性分析

Abstract: 【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 expressed at a high level in E. coli. Western blotting analysis showed that a significant response band at 38.6 kD was displayed between rabbit against Cysticercus pisiformis positive serum and recombinant protein. This result indicated that the recombinant protein has reactogenicity and recognized by sera. On the 43th day after final immunization, the levels of antibody from tested group showed a peak. The reduction rate of cysts in immunized sheep (Freund’s adjuvant, 206 adjuvant and Al(OH)3 adjuvant) was 79.13%, 65.66%, and 50.43% respectively. 【Conclusion】Freund’s adjuvant emulsified recombinant antigens showed high protective activity, thus providing valuable information for the development of highly efficient recombinant vaccines against cysticercoids.

Key words: Taenia pisiformis , TPO18 , prokaryotic expression , protective efficacy