大口黑鲈弹状病毒G蛋白胞外区原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2023.09.032

Abstract

Abstract: 【Objective】 The purpose of this experiment was to obtain polyclonal antibodies that could specifically recognize the G protein of Micropterus salmoides rhabdovirus (MSRV),and provide materials for the subsequent development of the MSRV double antibody sandwich colloidal gold strip.【Method】 Based on the genome sequence of MSRV (GenBank No.:OK272491.1),specific primers were designed to amplify the extracellular region of G protein of MSRV.The G gene was amplified via PCR using the cDNA from MSRV infected GS cells,and then ligated into the pET-28a(+) expression vector after double digestion to construct pET-28a-ΔG recombinant plasmid.The recombinant plasmid was transformed into Escherichia coli Trans5α competent cells and the positive clone was identified by DNA sequencing.Then,the plasmid pET-28a-ΔG was extracted and transformed into the Escherichia coli BL21(DE3) competent cells.Positive clones were selected again,and the expression was induced by IPTG after correct sequencing. The target protein was purified by Ni-NTA resin chromatography column and used as an immunogen to immunize female BALB/c mice.Polyclonal antibodies against MSRV G protein were obtained by orbital blood collection after four immunizations.Finally,the titer and specificity of the obtained polyclonal antibodies were characterized by ELISA,Western blotting and indirect immunofluorescence assay (IFA).【Result】 PCR results showed that a target band with a size of 1 323 bp was obtained.SDS-PAGE results showed that the recombinant plasmid could efficiently express the target protein,and a single target protein was obtained after Ni column purification,with a molecular weight of about 48.5 ku,which was consistent with expectations,and the purity met the immunogenic requirements.ELISA results showed that the titer of the prepared polyclonal antibody was 1∶204 800.Western blotting results showed that the prepared polyclonal antibodies could specifically recognize different batches of MSRV G proteins.IFA results showed that the prepared polyclonal antibody could specifically recognize G protein in its natural state. 【Conclusion】 In this study,the recombinant protein of the extracellular region of MSRV G protein was successfully used to prepare polyclonal antibodies that could specifically recognize MSRV G protein,which laid a foundation for subsequent MSRV immune detection and vaccine development.

Key words: Micropterus salmoides rhabdovirus; G protein; prokaryotic expression; polyclonal antibodies

CLC Number:

S852.65^＋9.5

LIU Haixiang, ZHANG Peipei, DENG Si, QIN Yinghui, YAO Lunguang. Prokaryotic Expression and Polyclonal Antibody Preparation of Extracellular Region of G Protein of Micropterus salmoides Rhabdovirus[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3762-3770.

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Prokaryotic Expression and Polyclonal Antibody Preparation of Extracellular Region of G Protein of Micropterus salmoides Rhabdovirus

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