猪带绦虫Ts14-3-3.3蛋白的原核表达及多克隆抗体制备
罗波, 李想, 周必英
遵义医科大学寄生虫学教研室,遵义 563000
Prokaryotic expression and polyclonal antibody preparation of Ts14-3-3.3 protein of Taenia solium
LUO Bo, LI Xiang, ZHOU Bi-ying
Department of Parasitology,Zunyi Medical University,Zunyi 563000,China
摘要 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∶512 000。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。
关键词 :
猪带绦虫 ,
Ts14-3-3.3蛋白 ,
原核表达 ,
多克隆抗体
Abstract :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 Ts 14-3-3.3 gene of Taenia solium with gene-specific primers. Ts 14-3-3.3 gene was cloned into the prokaryotic expression vector pCznⅠ and then expressed in Arctic Express with Isopropyl-1-thio-β-galactopyranoside (IPTG) induction. Ts 14-3-3.3 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∶512 000. 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.
Key words :
Taenia solium
Ts14-3-3.3 protein
prokaryotic expression
polyxlonal antibody
收稿日期: 2019-04-03
基金资助: 贵州省科技计划项目(黔科合基础[2018]1190)和省市科技合作专项资金项目(省市科合[2015]52)联合资助
通讯作者:
周必英,Email: zbyzl01@126.com; ORCID: 0000-0002-1832-5204
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