猪腺病毒3型Protease蛋白在大肠杆菌中的表达及其抗体制备

doi:10.16431/j.cnki.1671-7236.2019.06.024

《中国畜牧兽医》 ›› 2019, Vol. 46 ›› Issue (6): 1764-1773.doi: 10.16431/j.cnki.1671-7236.2019.06.024

猪腺病毒3型Protease蛋白在大肠杆菌中的表达及其抗体制备

吴思雯, 胡聪, 唐青海, 韩涛涛, 刘祯珍, 向冬梅, 王姝姝, 李彦, 杨海, 王芳宇

衡阳师范学院生命科学与环境学院, 衡阳师范学院畜禽养殖生物工程技术研究所, 生物资源保护与利用衡阳市重点实验室, 衡阳 421008

收稿日期:2018-11-25 出版日期:2019-06-20 发布日期:2019-06-19
通讯作者: 唐青海 E-mail:qinghaitang109@126.com
作者简介:吴思雯(1997-),女,湖南邵阳人,本科生,研究方向:动物传染病病原学,E-mail:854813585@qq.com;胡聪(1998-),女,湖南浏阳人,本科生,研究方向:动物传染病病原学,E-mail:1004009172@qq.com
基金资助:
国家级大学生创新创业训练计划项目（201710546001）；湖南省教育厅优秀青年项目（17B041）；校企合作项目（ZXXQ201701、18H03）；衡阳师范学院引进人才专项项目（16D20）

Expression and Antibody Preparation of Porcine Adenovirus Type 3 Protease Protein in Escherichia coli

WU Siwen, HU Cong, TANG Qinghai, HAN Taotao, LIU Zhenzhen, XIANG Dongmei, WANG Shushu, LI Yan, YANG Hai, WANG Fangyu

Hengyang Key Laboratory of Protection and Utilization of Biological Resources, Institute of Bio-technology for Livestock, College of Life Sciences and Environment, Hengyang Normal University, Hengyang 421008, China

Received:2018-11-25 Online:2019-06-20 Published:2019-06-19

摘要/Abstract

摘要：

试验旨在研究猪腺病毒3型（PADV3） Protease蛋白在大肠杆菌中的表达，并制备该蛋白的多克隆抗体。利用PCR扩增PADV3 Protease基因，构建重组原核表达载体pET28a-PADV3-Protease和真核表达载体pEGFP-PADV3-Protease，采用双酶切和测序鉴定；将原核表达载体pET28a-PADV3-Protease转化大肠杆菌BL21（DE3）感受态细胞得到重组原核表达菌株，经IPTG诱导收集蛋白，采用SDS-PAGE和Western blotting鉴定，将目的蛋白纯化后与佐剂乳化制备免疫原免疫家兔，制备Protease蛋白多克隆抗体；将真核表达载体pEGFP-PADV3-Protease转染HEK293细胞经G418筛选建立稳定表达EGFP-Protease融合蛋白的细胞系，以该稳定表达细胞系为包被抗原，采用免疫过氧化物酶单层细胞染色法（IPMA）检测抗体的免疫活性与抗体滴度。结果显示，Protease基因开放阅读框（ORF）为615 bp，原核表达系统中Protease蛋白以包涵体形式存在，分子质量大小为23 ku，与真核细胞表达的Protease蛋白分子质量一致，该蛋白在细胞核与细胞质中均有分布，制备的Protease蛋白多克隆抗体能与EGFP-Protease融合蛋白稳定表达真核细胞系发生特异性的反应，与对照细胞无反应。本试验构建了Protease蛋白原核表达菌株和真核表达细胞株，制备的PADV3-Protease蛋白多克隆抗体免疫活性良好，为进一步研究Protease蛋白的生物学功能和PADV3的血清学诊断提供了基础材料。

关键词: 猪腺病毒3型(PADV3); Protease基因; 表达; 多克隆抗体

Abstract:

This study was aimed to investigate the expression of porcine adenovirus type 3 (PADV3) Protease protein in Escherichia coli (E.coli),and prepare the polyclonal antibody of Protease protein.PADV3 Protease gene was amplified by PCR,the recombinant prokaryotic expression vector pET28a-PADV3-Protease and eukaryotic expression vector pEGFP-PADV3-Protease were constructed and identified by double enzyme digestion and sequencing.The recombinant prokaryotic expression vector pET28a-PADV3-Protease was transformed into E.coli BL21(DE3),the positive recombinant prokaryotic expression strain was induced by IPTG,and identified by SDS-PAGE and Western blotting.The recombinant Protease protein was purified and emulsified with adjuvant to prepare the immunogen which was innoculated into rabbit to prepare a polyclonal antibody against Protease protein.The eukaryotic expression vector pEGFP-PADV3-Protease was transfected into HEK293 cells and the EGFP-protease fusion protein expression stable cell line was screened by G418.The immunological activity and antibody titer of the antibody were detected by immunoperoxidase monolayer staining (IPMA) base on the fusion protein expression stable cell line.The results showed that the length of Protease gene open reading frame (ORF) was 615 bp.Western blotting analysis result showed that Protease protein in the prokaryotic expression system existed as an inclusion body with a molecular weight of 23 ku which was similar to Protease protein expressed in eukaryotic cells.In the fusion protein expression stable cell line,Protease was distributed in both the nucleus and cytoplasm.The Protease specific polyclonal antibody specifically reacted with the EGFP-Protease fusion protein expression stable cell line.The Protease prokaryotic expression strain and the eukaryotic expression cell line were successfully constructed,the PADV3-Protease protein polyclonal antibody was prepared and there was wonderful immunoactivity,providing basic materials for further study of the biological function of Protease protein and the serological diagnostic of PADV3.

Key words: porcine adenovirus type 3(PADV3); Proteasegene; expression; polyclonal antibody

中图分类号:

S852.65⁺9.1

吴思雯, 胡聪, 唐青海, 韩涛涛, 刘祯珍, 向冬梅, 王姝姝, 李彦, 杨海, 王芳宇. 猪腺病毒3型Protease蛋白在大肠杆菌中的表达及其抗体制备[J]. 《中国畜牧兽医》, 2019, 46(6): 1764-1773.

WU Siwen, HU Cong, TANG Qinghai, HAN Taotao, LIU Zhenzhen, XIANG Dongmei, WANG Shushu, LI Yan, YANG Hai, WANG Fangyu. Expression and Antibody Preparation of Porcine Adenovirus Type 3 Protease Protein in Escherichia coli[J]. , 2019, 46(6): 1764-1773.

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猪腺病毒3型Protease蛋白在大肠杆菌中的表达及其抗体制备

Expression and Antibody Preparation of Porcine Adenovirus Type 3 Protease Protein in Escherichia coli

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