猪带绦虫不同阶段45W-4BX和18kD基因联合表达及保护性分析  

Combining Expression and Protective Efficacy of 45W-4BX and 18kD from Different Stages of Taenia solium

作　　者：骆学农[1] 郑亚东[1] 窦永喜[1] 郭爱疆[1] 侯俊琳[1] 景志忠[1] 才学鹏[1] 

机构地区：[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,兰州730046

出　　处：《中国农业科学》2007年第2期385-390,共6页Scientia Agricultura Sinica

基　　金：国家重大基础研究发展规划"973"项目(G1999011906)

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。[Objective]The study aimed looked at the efficacy of the recombinant antigen of co-expressed 45W-4BX and 18kD from Taenia solium different development stages. [Method] Truncated 45W-4B with the signal peptide sequence deletion and C＇-terminal nucleotides encoding, 17 hydrophobic amino acids were cloned into BamH I and EcoR I pre-digested pGEX-4T-1 and transformed into competent BL21 .The positive transformants were determined by enzyme restriction, PCR, and sequencing. This was followed by ligating with the signal sequence. It removed the 18kD fragment and identified pGEX-4BX/18. In order to evaluate the protective efficacy of4BX/18 and 4BX antigens, vaccination trail was performed with 300μg dose per pig. [Result] SDS-PAGE and Westem blot showed that the 50kD fusion protein was expressed at a high level in E. coli and recognized by sera from either cysticercosis patients or in pigs infected by Cysticercus cellulosae. Statistical data indicated that the efficacy of the group immunized with 4BX/l 8 was 97% reduction in the number of cysticerci, a little higher than that of the GST-4BX group （95%）. [ Conclusion ] Recombinant antigens GST-4BX and GST-4BX/18 have high protection, supporting the development of highly efficient recombinant vaccines against cysticercosis, using protective antigens from different developmental stages of the parasite.

关 键 词：猪带绦虫 45W-4BX 18kD 联合表达 免疫保护性分析 

分 类 号：S858.28[农业科学—临床兽医学]