中国农业科学 ›› 2007, Vol. 40 ›› Issue (2): 385-390 .

• 畜牧·兽医·资源昆虫 • 上一篇    下一篇

猪带绦虫不同阶段45W-4BX和18kD基因联合表达及保护性分析

骆学农,郑亚东,窦永喜,郭爱疆,侯俊琳,景志忠,才学鹏   

  1. 中国农业科学院兰州兽医研究所 / 家畜疫病病原生物学国家重点实验室/甘肃省动物寄生虫病重点实验室
  • 收稿日期:2005-09-21 修回日期:1900-01-01 出版日期:2007-02-10 发布日期:2007-02-10
  • 通讯作者: 才学鹏

Combining expression and protective efficacy of 45W-4BX and 18ku from different stages of Taenia solium

  

  1. 中国农业科学院兰州兽医研究所 / 家畜疫病病原生物学国家重点实验室/甘肃省动物寄生虫病重点实验室
  • Received:2005-09-21 Revised:1900-01-01 Online:2007-02-10 Published:2007-02-10

摘要: 【目的】获得不同发育阶段基因联合表达的具有良好免疫保护性的重组抗原,为研制高效的猪囊虫基因工程疫苗奠定基础。【方法】PCR扩增截去45W-4B基因的信号肽和C端17个疏水氨基酸序列,经BamH I和EcoR I酶切后与表达载体pGEX-4T-1连接转化BL21感受态细胞,酶切及PCR扩增鉴定阳性克隆。测序正确的质粒经EcoR I和Not I酶切处理后与截去信号肽的18 kD基因连接,构建双基因融合表达载体pGEX-4BX/18。用IPTG诱导的表达产物,进行SDS-PAGE电泳、Western blot分析活性。分别用300μg重组GST-4BX、GST-4BX/18蛋白免疫猪,间接ELISA测定抗体水平。感染后90 d剖检计算各组的减虫率,比较评价重组抗原的免疫保护性。【结果】4BX/18 kD在大肠杆菌中获得高效表达,表达产物为50 kD的融合蛋白,并能被人囊虫和感染初期的猪囊虫阳性血清所识别。重组抗原免疫猪后45 d抗体达到峰值,联合表达重组抗原的减虫率为97%,GST-4BX免疫组的减虫率为95%。【结论】重组抗原4BX 和4BX/18kD均具有较好的免疫保护效果,有望利用它们研制出抗猪囊尾蚴病的高效疫苗。

关键词: 猪带绦虫, 45W-4BX, 18kD, 联合表达, 免疫保护性分析

Abstract: Truncated 45W-4B with the deletion of signal peptide sequence and C?terminal nucleotides encoding 17 hydrophobic amino acids, cloned into BamH I and EcoR I pre-digested pGEX-4T-1 and transformed into competent BL21 and the positive transformants were determined by enzyme restriction and PCR and sequencing, followed by ligating with the signal sequence-removed 18ku fragment, the positive designed as pGEX-4BX/18. Results of SDS-PAGE and Western blot showed that the 50ku fusion protein was expressed at a high level in E. coli and recognized by sera from either patients or pigs early infected by Cysticercus cellulosae. In order to evaluate the efficacy of 4BX/18 and 4BX antigens, vaccination trail was performed with 300礸 dose per pig. Statistic data indicated that the efficacy of the group immunized with 4BX/18 was 99%, a little higher than that of the GST-4BX group (95%), supporting the bases of development of highly efficient recombinant vaccines against cysticercosis jointly using different protective antigens from different developmental stages of parasites.