猪带绦虫TSOL18基因的表达、纯化和兔抗血清的制备

目的 构建猪带绦虫重组质粒pGEX-TSOL18,表达纯化TSOL18重组蛋白,制备兔抗重组蛋白血清。方法 全基因合成猪带绦虫TSOL18抗原编码基因,定向克隆到pGEX-1λT表达载体,构建重组质粒pGEX-TSOL18;转化大肠埃希菌ArcticExpress(DE3), IPTG诱导表达,SDS-PAGE电泳分析表达情况;亲和层析纯化获得重组蛋白,免疫兔制备抗血清;ELISA测定抗血清的效价,Western blot鉴定抗血清的特异性。结果 全基因扩增出393 bp的TSOL18抗原编码基因,酶切和测序鉴定证实TSOL18基因成功插入pGEX-1λT中;SDS-PAGE分析显示表达重组蛋白相对分子质量(Mr)约为41 kDa,经亲和层析后可获得纯度为75%的重组蛋白;ELISA测定抗血清的效价为1∶256 000,Western blot证实该抗血清能与重组蛋白发生特异性反应。结论 猪带绦虫TSOL18基因能在大肠埃希菌中高效表达,成功制备了高纯度的TSOL18重组蛋白和高滴度的兔抗重组蛋白血清,为猪带绦虫的疫苗研制奠定了基础。

Expression,purification and preparation of rabbit antiserum of the gene TSOL18 of Taenia solium

To construct the recombinant plasmid pGEX-TSOL18 of Taenia solium for the purified recombinant protein TSOL18 and prepare the rabbit antiserum against the recombinant protein, TSOL18 antigen encoding gene was synthesized and cloned into the expression vector pGEX-1λT. The positive recombinants were transformed into Escherichia coli ArcticExpress(DE3) and the recombinant bacteria pGEX-TSOL18 were induced with isopropyl-β-D-thiogalactopyranosid (IPTG), then the expressed products were analyzed by SDS-PAGE. The expressed recombinant protein was purified through affinity chromatography. Meanwhile, the rabbit was immunized with the purified recombinant protein to prepare the antiserum. The titer of the antiserum was detected by ELISA and the specificity of the antiserum was determined by Western blot assay. It was demonstrated that the 393 bp TSOL18 gene was synthesized and successfully inserted into pGEX-1λT by restriction enzyme digestion and DNA sequencing. As demonstrated by SDS-PAGE, the relative molecular mass (Mr) of the expressed recombinant protein was approximately 41 kDa, and the purity of the recombinant protein was 75% after purification with affinity chromatography. The titer of the antiserum against the recombinant protein was 1∶256 000 in ELISA assay and the specific rabbit antiserum against the purified recombinant protein TSOL18 could bind to the recombinant protein in Western blot assay. It is demonstrated that the gene TSOL18 of Taenia solium could be efficiently expressed in E. coli. The high quality recombinant protein TSOL18 and the high titer rabbit antiserum is successfully prepared, which would lay the foundation for developing vaccine of Taenia solium.