尼罗罗非鱼Hsc70的原核表达和多克隆抗体制备

doi:10.3969/j.issn.1004-1524.2019.08.07

浙江农业学报 ›› 2019, Vol. 31 ›› Issue (8): 1272-1279.DOI: 10.3969/j.issn.1004-1524.2019.08.07

尼罗罗非鱼Hsc70的原核表达和多克隆抗体制备

潘传燕, 林勇, 冯鹏霏, 张永德, 罗洪林^*

广西水产科学研究院广西水产遗传育种与健康养殖重点实验室,广西南宁 530021

收稿日期:2019-01-22 出版日期:2019-08-25 发布日期:2019-08-30
通讯作者: 罗洪林,E-mail: 541365548@qq.com
作者简介:潘传燕(1988-),女,云南昭通人,硕士,工程师,研究方向为水生动物遗传育种。E-mail: 723870955@qq.com
基金资助:
国家自然科学基金(31372553,31760765); 广西重点研发计划(桂科AB16380074)

Prokaryotic expression and polyclonal antibody preparation of Hsc70 in Nile tilapia

PAN Chuanyan, LIN Yong, FENG Pengfei, ZHANG Yongde, LUO Honglin^*

Guangxi Academy of Fishery Sciences, Guangxi Key Laboratory of Aquatic Genetic Breeding and Healthy Aquaculture, Nanning 530021, China

Received:2019-01-22 Online:2019-08-25 Published:2019-08-30

摘要/Abstract

摘要： 通过原核表达系统表达尼罗罗非鱼热休克蛋白Hsc70,纯化该蛋白并制备其多克隆抗体。对尼罗罗非鱼热休克蛋白进行生物信息学分析,设计特异性引物,扩增出Hsc70基因,通过双酶切与pET-28a构建重组质粒表达载体,并转入大肠埃希菌B21中通过诱导剂IPTG进行原核诱导表达,表达产物用SDS-PAGE鉴定并用镍柱进行纯化。将纯化后的重组蛋白免疫日本大耳兔制备多克隆抗体,用ELISA测定抗体效价,Western blot检测抗体特异性。结果表明,原核表达系统成功表达了尼罗罗非鱼热休克蛋白Hsc70,重组蛋白分子量约为75 ku,主要以包涵体形式表达。经镍柱纯化得到高纯度的Hsc70;该蛋白免疫日本大耳兔获得特异性多克隆抗体,效价为1∶2 048 000;Western blot检测到一条75 ku的特异性条带,表明该抗体能特异性地识别尼罗罗非鱼Hsc70蛋白。尼罗罗非鱼Hsc70蛋白在大肠埃希菌中成功表达,制备的重组蛋白和多克隆抗体为深入研究尼罗罗非鱼Hsc70的功能提供了分子工具。

关键词: 尼罗罗非鱼, Hsc70, 原核表达, 多克隆抗体

Abstract: To express Hsc70 (heat shock cognate 70) of Nile tilapia via a prokaryotic expression system, the recombinant protein was purified and polyclonal antibody was prepared. Bioinformatics analysis of Hsc70 was analyzed, primers were designed, and then Hsc70 gene was amplified. A prokaryotic expression vector of Hsc70 was constructed and the Hsc70 recombinant protein was induced by IPTG.The recombinant protein was identified by SDS-PAGE and purified with a Ni-NTA. To obtain the polyclonal antibody, the purified Hsc70 recombinant protein was used to immunize Japanese white rabbits, the titer was determined by ELISA, and the specificity was analyzed by Western blot. The Hsc70 recombinant protein was mainly obtained from the inclusion body with a molecular weight of 75 ku. After the purified recombinant protein was immunized with Japanese white rabbits, the rabbit anti-Hsc70 polyclonal antibody was successfully obtained, whose titer was 1∶2 048 000 and the specificity was good. Hsc70 of Nile Tilapia expressed successfully in E. coli B21, and the recombinant protein and polyclonal antibody obtained in this study provided a molecular tool for further study the function of Hsc70.

Key words: Nile tilapia, Hsc70, prokaryotic expression, polyclonal antibody

中图分类号:

S917.4

潘传燕, 林勇, 冯鹏霏, 张永德, 罗洪林. 尼罗罗非鱼Hsc70的原核表达和多克隆抗体制备[J]. 浙江农业学报, 2019, 31(8): 1272-1279.

PAN Chuanyan, LIN Yong, FENG Pengfei, ZHANG Yongde, LUO Honglin. Prokaryotic expression and polyclonal antibody preparation of Hsc70 in Nile tilapia[J]. , 2019, 31(8): 1272-1279.

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尼罗罗非鱼Hsc70的原核表达和多克隆抗体制备

Prokaryotic expression and polyclonal antibody preparation of Hsc70 in Nile tilapia

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