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

Abstract

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

CLC Number:

S852.659.1

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

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