猪带绦虫Ts14-3-3.3蛋白的原核表达及多克隆抗体制备

doi:10.3969/j.issn.1002-2694.2019.00.179

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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。

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	罗波
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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 Ts14-3-3.3 gene of Taenia solium with gene-specific primers. Ts14-3-3.3 gene was cloned into the prokaryotic expression vector pCznⅠ and then expressed in Arctic Express with Isopropyl-1-thio-β-galactopyranoside (IPTG) induction. 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∶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

PACS:

R383.3

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

引用本文:

罗波, 李想, 周必英. 猪带绦虫Ts14-3-3.3蛋白的原核表达及多克隆抗体制备[J]. 中国人兽共患病学报, 2019, 35(12): 1100-1105. LUO Bo, LI Xiang, ZHOU Bi-ying. Prokaryotic expression and polyclonal antibody preparation of Ts14-3-3.3 protein of Taenia solium. Chinese Journal of Zoonoses, 2019, 35(12): 1100-1105.

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http://www.rsghb.cn/CN/10.3969/j.issn.1002-2694.2019.00.179 或 http://www.rsghb.cn/CN/Y2019/V35/I12/1100

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