猪圆环病毒4型Cap蛋白的原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2025.10.035

Abstract

Abstract: 【Objective】 This experiment aimed to express the capsid protein (Cap) of Porcine circovirus type 4 (PCV4) through the prokaryotic expression system and prepare its polyclonal antibody,providing technical support for the prevention and control of PCV4.【Method】 In this study,a pair of specific primers was designed based on the immunogenic region of the PCV4 open reading frame 2(ORF2) gene.The PCV4 ORF2 gene sequence was obtained through PCR amplification and ligated to the pET-28a(+) vector to construct the recombinant expression plasmid pET-28a-ORF2. The recombinant plasmid pET-28a-ORF2 identified by PCR amplification and sequencing was transformed into E.coli BL21(DE3) competent cells,and 1 mmol/L IPTG was added to induce the expression of recombinant Cap protein.Then the induced recombinant protein products were analyzed by SDS-PAGE.The recombinant protein was purified using urea and identified by Western blotting.The purified recombinant Cap protein was used to immunize New Zealand White rabbits to prepare polyclonal antibodies and Western blotting was performed for identification.The titer of rabbit serum antibodies was detected by indirect ELISA.【Result】 PCV4 ORF2 gene fragment with a size of 414 bp was amplified by PCR,and the recombinant expression plasmid pET-28a-ORF2 was successfully constructed.SDS-PAGE analysis results showed that the recombinant Cap protein induced for expression was 16 ku.The results of Western blotting indicated that the prepared rabbit-derived polyclonal antibodies had good immunogenicity.Indirect ELISA detection showed that the titer of the prepared rabbit serum antibody reached 1∶12 800.【Conclusion】 In this study,the PCV4 Cap protein was successfully expressed using the prokaryotic expression system and polyclonal antibodies were prepared.The results provided a material basis for the in-depth study of PCV4 Cap protein and the subsequent development of PCV4 genetic engineering subunit vaccines,serological diagnostic reagents,etc.

Key words: Porcine circovirus type 4 (PCV4); Cap protein; prokaryotic expression; polyclonal antibody

CLC Number:

S852.65⁺9.2

WEN Yinghui, CUI Hongbo, CHEN Ximeng, CHEN Hongying, LI Jinlei, ZHAO Li, LIU Zhantong, YAN Zhihao. Prokaryotic Expression and Polyclonal Antibody Preparation of Cap Protein of Porcine Circovirus Type 4[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(10): 4904-4911.

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Prokaryotic Expression and Polyclonal Antibody Preparation of Cap Protein of Porcine Circovirus Type 4

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