猪带绦虫TSO45W-4B基因的克隆、表达和抗体制备

中国寄生虫学与寄生虫病杂志 ›› 2013, Vol. 31 ›› Issue (5): 9-372-375.

猪带绦虫TSO45W-4B基因的克隆、表达和抗体制备

周必英1 *，周泠2，刘美辰1，刘晖1，张曦1

1 遵义医学院寄生虫学教研室；2 遵义医学院附属第一医院中医科，遵义 563003

出版日期:2013-10-30 发布日期:2014-07-24

Cloning，Expression of TSO45W-4B Gene from Taenia solium and Preparation of Its Polyclonal Antibody

ZHOU Bi-ying1 *, ZHOU Ling2, LIU Mei-chen1, LIU Hui1, ZHANG Xi1

1 Department of Parasitology, Zunyi Medical College, Zunyi 563003, China; 2 Department of Traditional Chinese Medicine, the First Affiliated Hospital of Zunyi Medical College, Zunyi 563003, China

Online:2013-10-30 Published:2014-07-24

摘要/Abstract

摘要： 目的克隆、表达猪带绦虫TSO45W-4B基因，以重组蛋白TSO45W-4B免疫兔制备抗体。方法全基因合成猪带绦虫TSO45W-4B抗原编码基因，定向克隆至pGEX-1λT表达载体，构建重组质粒pGEX-TSO45W-4B。转化至大肠埃希菌ArcticExpress（DE3）后，异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表达情况。表达产物经谷胱甘肽-S-转移酶（GST）亲和层析柱纯化后，免疫新西兰大白兔，制备抗血清。GST亲和层析柱纯化获得血清中抗体后，ELISA测定抗体效价，蛋白质印迹法（Western blotting）鉴定抗体特异性。结果酶切和测序结果表明，重组质粒pGEX-TSO45W-4B构建成功。SDS-PAGE结果显示，重组蛋白相对分子质量（Mr）约为40 000，经GST亲和层析后可获得纯度为90%以上的重组蛋白。ELISA结果显示，抗体的效价为1 ∶ 512 000。Western blotting分析结果显示，该抗体能与纯化的重组蛋白发生特异性反应，在约Mr 40 000处出现一条带，与预期大小一致。结论猪带绦虫TSO45W-4B基因能在大肠埃希菌中表达，制备了TSO45W-4B重组蛋白和兔抗血清。

关键词: 猪带绦虫, TSO45W-4B, 表达, 纯化, 抗体

Abstract: Objective To clone and express TSO45W-4B gene of Taenia solium, and prepare the rabbit antiserum against the recombinant protein TSO45W-4B. Methods The gene encoding TSO45W-4B antigen was synthesized and cloned into the expression vector pGEX-1λT. The recombinant plasmid was transformed into Escherichia coli ArcticExpress（DE3）, and followed by the expression of the protein induced by IPTG. The recombinant protein was purified by GST affinity chromatography and analyzed by SDS-PAGE. The rabbits were immunized with the purified recombinant protein. The titer of the antibody against TSO45W-4B was detected by ELISA. The specificity of anti-TSO45W-4B antibody was determined by Western blotting. Results It was demonstrated that TSO45W-4B gene （351 bp） was synthesized. The gene was inserted into pGEX-1λT and confirmed by restriction enzyme digestion and DNA sequencing. SDS-PAGE analysis showed that the relative molecular mass （Mr） of the expressed recombinant protein was approximately 40 000. The purity of the recombinant protein with an affinity chromatography column was about 90%. The titer of the antibody against TSO45W-4B was 1 ∶ 512 000. The recombinant protein reacted with the anti-TSO45W-4B antibody by Western blotting. Conclusion The TSO45W-4B gene of Taenia solium is efficiently expressed in E. coli. The recombinant protein TSO45W-4B and its rabbit antiserum are successfully prepared.

Key words: Taenia solium, TSO45W-4B, Expression, Purification, Antibody Taenia solium, TSO45W-4B, Expression, Purification, Antibody

周必英1 *，周泠2，刘美辰1，刘晖1，张曦1. 猪带绦虫TSO45W-4B基因的克隆、表达和抗体制备[J]. 中国寄生虫学与寄生虫病杂志, 2013, 31(5): 9-372-375.

ZHOU Bi-ying1 *, ZHOU Ling2, LIU Mei-chen1, LIU Hui1, ZHANG Xi1. Cloning，Expression of TSO45W-4B Gene from Taenia solium and Preparation of Its Polyclonal Antibody[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2013, 31(5): 9-372-375.

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