猪带绦虫Tsl4-3-3.6蛋白多克隆抗体制备及在不同发育阶段的表达

Preparation of a polyclonal antibody against Taenia solium Ts14-3-3.6 protein and expression of Ts14-3-3.6 protein at different stages

目的制备猪带绦虫Ts14-3-3.6蛋白多克隆抗体,并检测Ts14-3-3.6蛋白在猪带绦虫成虫及囊尾蚴阶段的表达情况。方法合成Ts14-3-3.6基因全长,然后克隆至pCznI原核表达质粒,转化至大肠埃希菌ArctieEx-press中并诱导表达,采用SDS-PAGE和Western blot对表达产物进行分析,通过Ni柱亲和纯化获得Ts14-3-3.6重组蛋白。将该重组蛋白免疫新西兰兔,制备抗Ts14-3-3.6多克隆抗体,以此为一抗采用Western blot检测Ts14-3-3.6蛋白在猪带绦虫成虫和囊尾蚴阶段的表达。结果pCznI一Ts14-3-3.6重组表达质粒构建成功,诱导表达的Ts14-3-3.6重组蛋白存在于包涵体中,相对分子质量约29.43X102。Western blot检测纯化带有His标签的Ts14-3-3.6重组蛋白能被His标签抗体识别;用该蛋白免疫新西兰兔获得效价为1:512000的多克隆抗体;分别以提取的猪带绦虫成虫和囊尾蚴总蛋白为抗原进行Westernblot,均出现与Ts14-3-3.6多克隆抗体、囊虫病人血清及囊虫病猪血清反应条带,即均有该蛋白的表达。结论成功制备猪带绦虫Ts14-3-3.6重组蛋白并获得较高纯度的特异性高效价多克隆抗体。Ts14-3-3.6蛋白在猪带绦虫成虫和囊尾蚴阶段均有表达。

Objective To prepare polyclonal antibody against Taenia solium Ts14-3-3.6 protein and detect the ex-pression of Ts14-3-3.6 protein at T.solium adult and cysticercus stages.Methods The full length of Ts14-3-3.6 gene was synthesized and cloned into pCznI prokaryotic expression plasmid,transformed into E.coli Arctic Express and induced expression.The expression products were analyzed by SDS-PAGE and Western blot.The recombinant protein Ts14-3-3.6 was obtained by Ni-affinity chromatography.New Zealand rabbits were immunized with the recombinant protein to prepare the anti-Ts14-3-3.6 polyclonal antibody which was used as a primary antibody,Western blot was used to detect the expression of Ts14-3-3.6 protein at T.solium adult and cysticercus stages.Results The pCznl-Ts14-3-3.6 recombinant expression plasmid was successfully constructed.The induced Ts14-3-3.6 recombinant protein existed in the inclusion body with relative molecular mass of about 29.43×103.Western blot indicated that the purified Ts14-3-3.6 recombinant protein with His-tag could be recognized by His-tag antibody.Polyclonal antibody with titer of 1:512000 was obtained by immunizing New Zealand rabbits with the recombinant protein.Western blot showed that the extracted proteins of T.solium adult and cysticercus reacted with Ts14-3-3.6 polyclonal antibody,se-rum from patient with cysticercosis and serum from pig with cysticercosis,which indicated the expression of the protein.Conclusion The recombinant protein Ts14-3-3.6 of T.solium was successfully prepared and the specific polyclonal antibody with relatively higher purity and titer was obtained.The protein Ts14-3-3.6 was expressed at both adult and cysticercus stages of T.solium.