猪带绦虫Ts14-3-3.3蛋白的原核表达及多克隆抗体制备

Prokaryotic expression and polyclonal antibody preparation of Ts14-3-3.3 protein of Taenia solium

目的建立猪带绦虫Ts14-3-3.3蛋白原核表达系统并制备兔多克隆抗体。方法通过逆转录PCR(RT-PCR)技术将猪囊尾蚴总RNA反转录为cDNA,利用特异性引物扩增猪带绦虫Ts14-3-3.3基因,将其克隆至原核表达质粒pCznⅠ中,在大肠杆菌Arctic Express中用异丙基硫代半乳糖苷(IPTG)诱导表达,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析其表达产物,用HisLink亲和层析柱进行纯化,免疫印迹(Western blot)法鉴定纯化后的Ts14-3-3.3重组蛋白。将纯化的Ts14-3-3.3重组蛋白免疫新西兰兔,制备Ts14-3-3.3多克隆抗体,Western blot和免疫组化法检测Ts14-3-3.3天然蛋白在猪带绦虫成虫和囊尾蚴的分布。结果成功构建猪带绦虫重组表达质粒pCznⅠ-Ts14-3-3.3,经IPTG诱导表达,获得可溶性形式高效表达的Ts14-3-3.3重组蛋白,分子质量约29.31 kD,纯化后带有His标签的Ts14-3-3.3重组蛋白能被抗血清所识别。用纯化的Ts14-3-3.3重组蛋白免疫新西兰兔,获得了Ts14-3-3.3多克隆抗体,其效价为1∶512000。Western blot和免疫组化结果显示,Ts14-3-3.3天然蛋白在猪带绦虫成虫和囊尾蚴中均有表达。结论成功制备猪带绦虫重组Ts14-3-3.3蛋白,并获得了高纯度、高效价的兔多克隆抗体。

To establish the prokaryotic expression system for Ts14-3-3.3 protein of Taenia solium and prepare its polyclonal antibodies.Reverse transcription PCR(RT-PCR)was used to prepare cDNA from cysticercus cellulosae total RNA,and then cDNA was used as a template to amplify the Ts14-3-3.3 gene of Taenia solium with gene-specific primers.Ts14-3-3.3 gene was cloned into the prokaryotic expression vector pCznⅠand then expressed in Arctic Express with Isopropyl-1-thio-β-galactopyranoside(IPTG)induction.Ts14-3-3.3 recombinant protein was detected by SDS-PAGE and purified by HisLink affinity chromatography.Ts14-3-3.3 recombinant protein was identified by Western blot.New Zealand rabbits were immunized with the purified expression products of Ts14-3-3.3 recombinant protein and Ts14-3-3.3 rabbit anti-serum was prepared.The tissue distribution of Ts14-3-3.3 nature protein at the stage of adult and cysticercus was analyzed by Western blot and immunohistochemistry.The results showed that the recombinant expression plasmid pCznⅠ-Ts14-3-3.3 was successfully constructed.After inducing with IPTG,Ts14-3-3.3 recombinant protein was obtained.The relative molecular weight was about 29.31 kilodalton and the protein was soluble.The purified Ts14-3-3.3 recombinant protein with His-tag was recognized by anti-serum.New Zealand rabbits were immunized with the purified Ts14-3-3.3 recombinant protein.Rabbit polyclonal antibody was successfully obtained,and the antibody titer was 1∶512000.The result of Western blot and immunohistochemical analysis showed that Ts14-3-3.3 nature protein was expressed at the stage of adult and cysticercus of Taenia solium.In conclusion,the recombinant Ts14-3-3.3 protein of Taenia solium was successfully prepared and polyclonal antibody with high-purity,high-efficiency was obtained.