鼠痘病毒EVM135和EVM085蛋白免疫原性及抗体中和活性的鉴定

doi:10.11843/j.issn.0366-6964.2023.06.025

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (6): 2468-2477.doi: 10.11843/j.issn.0366-6964.2023.06.025

鼠痘病毒EVM135和EVM085蛋白免疫原性及抗体中和活性的鉴定

高真贞¹, 蒙泽菁^1,2, 何小兵¹, 房永祥¹, 田慧慧¹, 陈国华^1*, 景志忠^1,2*

1. 中国农业科学院兰州兽医研究所/动物疫病防控全国重点实验室/农业部兽医公共卫生重点实验室, 兰州 730046;
2. 兰州大学公共卫生学院, 兰州 730000

收稿日期:2022-10-10 出版日期:2023-06-23 发布日期:2023-06-16
通讯作者: 景志忠,主要从事动物疫苗与分子免疫学研究,E-mail:zhizhongj@163.com;陈国华,主要从事痘病毒病诊断、疫苗及免疫防控技术研究,E-mail:chengguohua78@163.com
作者简介:高真贞(1982-),女,河北张家口人,博士生,主要从事动物疫苗与分子免疫学研究,E-mail:hbgzz20@sina.com
基金资助:
中国农业科学院创新工程(CAAS-ASTIP-2019-LVRI-06)

Identification of Immunogenicity and Neutralizing Activity of Ectromelia Virus EVM135 and EVM085 Proteins

GAO Zhenzhen¹, MENG Zejing^1,2, HE Xiaobing¹, FANG Yongxiang¹, TIAN Huihui¹, CHEN Guohua^1*, JING Zhizhong^1,2*

1. State Key Laboratory for Animal Disease Control and Prevention, Key Laboratory of Veterinary Public Health of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;
2. School of Public Health, Lanzhou University, Lanzhou 730000, China

Received:2022-10-10 Online:2023-06-23 Published:2023-06-16

摘要/Abstract

摘要： 正痘病毒属病毒的A33和H3L蛋白是其成员共有的中和抗体的两个主要靶标。为检测鼠痘病毒(ectromelia virus,ECTV)A33和H3L蛋白同源物的免疫原性及抗体中和活性,以ECTV-Moscow株基因组DNA为模板,PCR扩增A33和H3L同源基因EVM135、EVM085目的序列,分别构建pET30a-EVM135、pET30-EVM085原核表达载体,转化Rosetta感受态细胞,IPTG诱导表达,利用镍层析柱纯化并进行逐步透析复性,免疫兔制备多克隆抗体。建立间接ELISA方法测定其抗体效价分别达1∶120 000和1∶360 000,Western blot和IFA证实该多克隆抗体具有良好的特异性和反应性;ECTV病毒中和试验表明抗EVM135多克隆抗体中和效价为1∶16,而抗EVM085多克隆抗体效价为1∶128。本研究通过高效表达EVM135和EVM085蛋白,并分析其免疫原性及抗体中和活性,为深入研究痘病毒致病和免疫机理,进而快速建立其诊断方法,为猴痘防控技术措施的研究奠定基础。

关键词: A33, H3L, 原核表达, 多克隆抗体, 中和活性

Abstract: The A33 and H3L proteins of orthopoxviruse are the two major targets of neutralizing antibodies. In order to detect the immunogenicity and neutralizing activity of A33 and H3L homologous proteins of ectromelia virus (ECTV), we amplified target sequence of EVM135 and EVM085 from the genome of ECTV-Moscow strain, and constructed the pET30a-EVM135 and pET30a-EVM085 recombinant prokaryotic expression plasmids, which were then transformed into Rosetta competent cells and induced by IPTG, the recombinant proteins were purified by Ni-chelating affinity chromatography and renatured by stepwise dialysis, and antiserum were prepared by immunized rabbits. The indirect ELISA detection showed that the antibody titers of EVM135 and EVM085 reached 1∶120 000 and 1∶360 000, respectively. Western blot and immunofluorescence tests revealed that the two polyclonal antibodies had great specificity and reactivity. The neutralization assay of ECTV showed that the titer of anti-EVM135 polyclonal antibody was 1∶16, while the EVM085 was 1∶128. Through the high expression of EVM135 and EVM085 proteins, and subsequent analysis of their immunogenicity and neutralizing activity, a thorough investigation of the pathogenic and immune mechanisms of orthopoxviruses can be conducted. This research can pave the way for the establishment of a diagnostic method, which can contribute to the development of effective prevention and control measures against monkeypox.

Key words: A33, H3L, prokaryotic expression, polyclonal antibody, neutralizing activity

中图分类号:

S852.659.1

高真贞, 蒙泽菁, 何小兵, 房永祥, 田慧慧, 陈国华, 景志忠. 鼠痘病毒EVM135和EVM085蛋白免疫原性及抗体中和活性的鉴定[J]. 畜牧兽医学报, 2023, 54(6): 2468-2477.

GAO Zhenzhen, MENG Zejing, HE Xiaobing, FANG Yongxiang, TIAN Huihui, CHEN Guohua, JING Zhizhong. Identification of Immunogenicity and Neutralizing Activity of Ectromelia Virus EVM135 and EVM085 Proteins[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2468-2477.

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鼠痘病毒EVM135和EVM085蛋白免疫原性及抗体中和活性的鉴定

Identification of Immunogenicity and Neutralizing Activity of Ectromelia Virus EVM135 and EVM085 Proteins

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