非洲猪瘟病毒pS273R蛋白的原核表达及其多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2021.11.025

中国畜牧兽医 ›› 2021, Vol. 48 ›› Issue (11): 4154-4161.doi: 10.16431/j.cnki.1671-7236.2021.11.025

非洲猪瘟病毒pS273R蛋白的原核表达及其多克隆抗体的制备

赵改红, 李婷婷, 张涛清, 陈欣, 王晓, 李长尧, 张朝霞, 郑君, 翁长江

中国农业科学院哈尔滨兽医研究所, 兽医生物技术国家重点实验室, 基础免疫创新团队, 哈尔滨 150069

收稿日期:2021-04-07 出版日期:2021-11-20 发布日期:2021-11-01
通讯作者: 郑君, 翁长江 E-mail:zhengjun01@caas.cn;wengchangjiang@caas.cn
作者简介:赵改红(1993-),女,甘肃临夏人,硕士生,研究方向:兽医微生物及其分子生物学,E-mail:13359317177@163.com
基金资助:
国家自然科学基金项目（31941002）；中国农业科学院基本科研业务费专项（1610302020005）

Prokaryotic Expression of African Swine Fever Virus pS273R Protein and Preparation of Its Polyclonal Antibody

ZHAO Gaihong, LI Tingting, ZHANG Taoqing, CHEN Xin, WANG Xiao, LI Changyao, ZHANG Zhaoxia, ZHENG Jun, WENG Changjiang

Division of Fundamental Immunology, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China

Received:2021-04-07 Online:2021-11-20 Published:2021-11-01

摘要/Abstract

摘要： 本研究旨在表达非洲猪瘟病毒（ASFV） pS273R蛋白，并制备其多克隆抗体，为ASFV pS273R的功能研究提供材料。试验通过大肠杆菌表达系统表达重组pS273R蛋白，采用镍亲和层析柱和分子筛纯化重组pS273R蛋白并免疫BALB/c小鼠制备血清，经Protein G琼脂糖树脂纯化鼠抗pS273R蛋白多克隆抗体，通过Western blotting、间接免疫荧光（IFA）和免疫沉淀试验（IP）分析多克隆抗体的反应原性和特异性。结果显示，在16 ℃、1 mmol/L IPTG诱导条件下，pS273R蛋白以可溶形式高效表达，经纯化并免疫小鼠后，通过Protein G可以纯化出高质量的鼠抗pS273R的多克隆抗体。以制备的多克隆抗体为一抗，通过Western blotting、IFA和IP检测到HEK293T细胞中瞬时表达的Flag-pS273R蛋白和ASFV感染肺泡巨噬细胞（PAMs）中的pS273R蛋白。本研究在大肠杆菌中实现了ASFV pS273R蛋白的高效表达，制备并纯化的抗pS273R多克隆抗体具有较高的反应性和特异性，这为深入探讨ASFV pS273R蛋白的生物学功能奠定了基础。

关键词: 非洲猪瘟病毒(ASFV); pS273R蛋白; 多克隆抗体

Abstract: To provide the material for the functional study of ASFV pS273R, ASFV pS273R protein was expressed and polyclonal antibody against pS273R protein was prepared in present study. The recombinant pS273R protein expressed in E. coli expression system was purified by Ni-affinity column and molecular sieve, respectively. Specific polyclonal antibodies against the pS273R protein were obtained by the immunized BALB/c mice, and the anti-pS273R polyclonal antibody (pAb) was purified from the mouse serum by using a Protein G affinity chromatography column. The reactivity and specificity of polyclonal antibodies were evaluated by Western blotting, IFA and IP. The results showed recombinant pS273R protein was efficiently expressed in E. coli as when induced with 16 ℃ and the concentration of IPTG at 0.1 mmol/L. The Flag-tagged pS273R protein in HEK293T cells and the ASFV-encoded pS273R in porcine alveolar macrophage (PAMs) were detected by Western blotting, IFA and IP using the mouse anti-pS273R pAb. In summary, ASFV pS273R was successfully expressed in E. coli, preparation and purification of mouse anti-S273R pAb had good specificity and reactivity. The availability of anti-pS273R pAb laid an important foundation for further research studying the biological function of pS273R protein.

Key words: African swine fever virus (ASFV); pS273R protein; polyclonal antibody

中图分类号:

S855.3

赵改红, 李婷婷, 张涛清, 陈欣, 王晓, 李长尧, 张朝霞, 郑君, 翁长江. 非洲猪瘟病毒pS273R蛋白的原核表达及其多克隆抗体的制备[J]. 中国畜牧兽医, 2021, 48(11): 4154-4161.

ZHAO Gaihong, LI Tingting, ZHANG Taoqing, CHEN Xin, WANG Xiao, LI Changyao, ZHANG Zhaoxia, ZHENG Jun, WENG Changjiang. Prokaryotic Expression of African Swine Fever Virus pS273R Protein and Preparation of Its Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(11): 4154-4161.

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非洲猪瘟病毒pS273R蛋白的原核表达及其多克隆抗体的制备

Prokaryotic Expression of African Swine Fever Virus pS273R Protein and Preparation of Its Polyclonal Antibody

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