猪带绦虫六钩蚴TSOL16基因的表达及免疫原性分析

Expression of TSOL16 gene of Taenia solium oncosphere

本研究采用RT-PCR技术扩增猪带绦虫六钩蚴TSOL16免疫原基因,将扩增产物与pMD18-T载体连接,重组质粒经PCR、酶切鉴定后进行测序;构建TSOL16基因的pGEX-4T-1原核表达载体,用IPTG诱导表达后进行SDS-PAGE、Western blot分析;将所表达的蛋白免疫小鼠,经ELISA检测血清抗体验证其免疫原性。结果显示:所克隆的TSOL16基因片段长度为559bp,含有1个402bp的开放阅读框架,编码134个氨基酸,与已报道的猪带绦虫六钩蚴TSOL16基因核苷酸序列同源性为99%;表达的融合蛋白大小为40Ku,并能被猪带绦虫六钩蚴阳性血清所识别;免疫小鼠在1周后即检测到血清抗体,第30d即达到较高水平,说明该融合蛋白具有较好的免疫原性。

The TSOL16 immunogen gene of Taenia solium oncosphere was amplified by RT-PCR and sequenced. The TSOL16 gene comprised 559 bp and had an ORF of 402 bp in length encoding 134 amino acids. There was 99.2 % nucleotide sequence homology between TSOL16 gene and the published sequence. The TSOL16 gene was cloned into a prokaryotic expression vector pGEX-4T-1 and expressed in E.coli by IPTG induction. The expressed products were analyzed by SDS-PAGE and Western blot. The results showed that the fusion protein was 40 Ku in size and reacted strongly with serum of Taenia solium oncosphere. The fusion protein was used to immunize mice and antibody against the TSOL16 protein was tested by ELISA. Antibody could be tested on day 7 post-immunization, and reached peak on day 30, indicating that the fusion protein had strong immunogenicity.