非洲猪瘟病毒p72蛋白原核表达及单克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2023.11.032

中国畜牧兽医 ›› 2023, Vol. 50 ›› Issue (11): 4646-4654.doi: 10.16431/j.cnki.1671-7236.2023.11.032

非洲猪瘟病毒p72蛋白原核表达及单克隆抗体制备

陈桂娥¹, 容芳¹, 冯夏宁¹, 孙荣航¹, 马绍钊², 郝文茜^1,2, 叶子安², 刘郁夫^3,4, 李作生¹, 陈瑞爱^1,2,4,5

1. 岭南现代农业科学与技术广东省实验室肇庆分中心, 肇庆 526238;
2. 华南农业大学兽医学院, 广州 510642;
3. 肇庆学院生命科学学院, 肇庆 526060;
4. 华农(肇庆)生物产业技术研究院有限公司, 肇庆 526238;
5. 肇庆大华农生物药品有限公司, 肇庆 526238

收稿日期:2023-03-24 出版日期:2023-11-05 发布日期:2023-10-27
通讯作者: 陈瑞爱 E-mail:chensa727@vip.126.com
作者简介:陈桂娥,E-mail:guieChan@163.com。
基金资助:
岭南现代农业科学与技术广东省实验室肇庆分中心自主项目（P20211154-0201）；肇庆市重点领域研发计划"揭榜制"项目（2021C001）

Prokaryotic Expression of p72 Protein of African Swine Fever Virus and Preparation of Its Monoclonal Antibodies

CHEN Guie¹, RONG Fang¹, FENG Xianing¹, SUN Ronghang¹, MA Shaozhao², HAO Wenqian^1,2, YE Zian², LIU Yufu^3,4, LI Zuosheng¹, CHEN Ruiai^1,2,4,5

1. Zhaoqing Branch Center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology, Zhaoqing 526238, China;
2. College of Veterinary Medicne, South China Agricultural University, Guangzhou 510642, China;
3. School of Life Sciences, Zhaoqing University, Zhaoqing 526060, China;
4. Zhaoqing Institute of Biotechnology Co., Ltd., Zhaoqing 526238, China;
5. Zhaoqing Dahuanong Biological Medicine Co., Ltd., Zhaoqing 526238, China

Received:2023-03-24 Online:2023-11-05 Published:2023-10-27

摘要/Abstract

摘要： 【目的】表达与纯化非洲猪瘟病毒（African swine fever virus，ASFV）p72蛋白，并制备其单克隆抗体。【方法】构建原核表达载体pET-28b-p72，转化大肠杆菌BL21（DE3）感受态细胞并进行诱导表达和蛋白纯化；用纯化后的p72蛋白免疫BALB/c小鼠，将免疫小鼠的脾细胞与骨髓瘤细胞进行细胞融合并制备单克隆抗体，通过间接ELISA法和亚克隆筛选出分泌抗体的阳性杂交瘤细胞；通过小鼠抗体类型检测试剂盒检测抗体类型；通过体内诱生法制备抗ASFV p72蛋白的单克隆抗体腹水，使用饱和硫酸铵沉淀法进行抗体纯化，通过间接ELISA法、间接免疫荧光试验（IFA）和Western blotting检测单克隆抗体效价和特异性。【结果】ASFV p72重组蛋白在大肠杆菌中以包涵体形式表达，将纯化的p72蛋白制备单克隆抗体，获得4株分泌IgG1抗体的单克隆细胞株，分别命名为6F8、7C3、8H7和9G2。间接ELISA结果显示，6F8与8H7细胞株分泌的抗体效价均为1：64 00，7C3与9G2细胞株的抗体效价均为1：12 800，且4株细胞株分泌的抗体与p72蛋白反应结果均呈阳性，与猪伪狂犬病病毒（Pseudorabies virus，PRV）、猪瘟病毒（Classical swine fever virus，CSFV）、猪细小病毒（Porcine parvovirus，PPV）、猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus，PRRSV）和猪流行性腹泻病毒（Porcine epidemic diarrhea virus，PEDV）反应均呈阴性。4株单克隆细胞株分泌的抗体均能与p72真核蛋白发生特异性反应。【结论】本研究成功制备了4株能稳定分泌与ASFV p72蛋白特异性结合的单克隆抗体的细胞株，为进一步探究ASFV p72蛋白的功能以及非洲猪瘟的防控和诊断建立了基础。

关键词: 非洲猪瘟病毒; p72蛋白; 原核表达; 单克隆抗体

Abstract: 【Objective】 The aim of this experiment was to express and purify African swine fever virus (ASFV) p72 protein and prepare the monoclonal antibodies.【Method】 The prokaryotic expression vector pET-28b-p72 was constructed and transformed into Escherichia coli BL21(DE3) competent cells for inducing expression and protein purification.The purified p72 protein was used to immunize BALB/c mice, and the spleen of the immunized mice was fused with myeloma cells to prepare monoclonal antibodies.The positive hybridoma cells secreting antibodies were screened by indirect ELISA and subcloning.Antibody types were detected by mouse antibody type detection kit.Ascites of monoclonal antibody against ASFV p72 protein was prepared by in vivo induction method, purified by saturated ammonium sulfate precipitation method, and the titer and specificity of monoclonal antibody were detected by indirect ELISA, indirect immunofluorescence assay (IFA) and Western blotting.【Result】 The recombinant protein of ASFV p72 was expressed in the form of inclusion bodies in Escherichia coli.Four monoclonal cell lines secreting IgG1 antibody were obtained, named 6F8, 7C3, 8H7 and 9G2, respectively.Indirect ELISA results showed that the titer of the antibody secreted by 6F8 and 8H7 cell lines was 1:6 400, and the titer of the antibody by 7C3 and 9G2 cell lines was 1:28 000.In addition, the antibody secreted by four cell lines was positive in reaction with p72 protein, but negative for Pseudorabies virus (PRV), Classical swine fever virus (CSFV), Porcine parvovirus (PPV), Porcine reproductive and respiratory syndrome virus (PRRSV) and Porcine epidemic diarrhea virus (PEDV). The antibodies secreted by four monoclonal cell lines could react specifically with p72 eukaryotic protein.【Conclusion】 Four cell lines that could secrete monoclonal antibodies specifically bound to ASFV p72 protein were successfully prepared, which laid a foundation for further investigation of the function of ASFV p72 protein and the prevention and diagnosis of African swine fever.

Key words: African swine fever virus; p72 protein; prokaryotic expression; monoclonal antibody

中图分类号:

S852.65⁺1

陈桂娥, 容芳, 冯夏宁, 孙荣航, 马绍钊, 郝文茜, 叶子安, 刘郁夫, 李作生, 陈瑞爱. 非洲猪瘟病毒p72蛋白原核表达及单克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(11): 4646-4654.

CHEN Guie, RONG Fang, FENG Xianing, SUN Ronghang, MA Shaozhao, HAO Wenqian, YE Zian, LIU Yufu, LI Zuosheng, CHEN Ruiai. Prokaryotic Expression of p72 Protein of African Swine Fever Virus and Preparation of Its Monoclonal Antibodies[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(11): 4646-4654.

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非洲猪瘟病毒p72蛋白原核表达及单克隆抗体制备

Prokaryotic Expression of p72 Protein of African Swine Fever Virus and Preparation of Its Monoclonal Antibodies

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