关键词: 猪伪狂犬病病毒, gE蛋白, 生物信息学, 原核表达, 蛋白纯化, 多克隆抗体

Abstract: [Objective] To investigate the immunogenicity of the gE protein of porcine pseudorabies virus and to prepare murine polyclonal antibodies. [Methods] Bioinformatics software was used to analyze the gE protein of porcine pseudorabies virus, including restriction enzyme sites, transmembrane regions, signal peptides, physicochemical properties, and antigenicity index, to select a gene fragment suitable for expression. After the target gene fragment was amplified by PCR and verified by sequencing, the recombinant plasmid pCold-I-gE was constructed. This plasmid was transformed into BL21 competent cells to obtain recombinant expression bacteria. Following IPTG-induced expression, the recombinant protein was subjected to solubility analysis, identification, and purification. The purified recombinant protein was used to immunize mice for the preparation of polyclonal antibodies. Antibody titers, reactivity with the recombinant protein, and specific binding to the native protein in virus-infected cells were evaluated using indirect ELISA, Western blot, and immunofluorescence assay （IFA）. [Results] The induced recombinant bacteria showed an expected target band at 26.4 kDa. The recombinant protein was predominantly expressed in inclusion bodies and could bind specifically to antibodies in PRV-positive serum, with a single band observed after purification. IFA confirmed that the prepared polyclonal antibodies specifically recognized the gE protein in porcine pseudorabies virus-infected PK-15 cells. The antibody titer reached 1:102,400. [Conclusion] The gE protein of PRV was successfully expressed with good immunogenicity. The prepared murine polyclonal antibodies exhibited high titers and good specificity.

Key words: porcine pseudorabies virus, gE protein, bioinformatics, prokaryotic expression, protein purification, polyclonal antibody