猪细小病毒6型ORF2基因的截短表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2023.06.033

Abstract

Abstract: 【Objective】 This study was aimed to truncatly express ORF2 gene of Porcine parvovirus type 6 (PPV6) prokaryotic expression system and prepare its polyclonal antibody, so as to provide materials for the follow-up study of the biological function of PPV6 VP1 protein.【Method】 Using the genome of the PPV6 isolate as a template, a truncated ORF2 gene fragment was obtained by PCR amplification and inserted into the prokaryotic expression vector pET30a(+) to construct the recombinant expression plasmid pET30a-PPV6-ORF2.After enzyme digestion and sequencing, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, then the recombinant protein was induced by IPTG, purified by Ni-NTA resin affinity chromatography, and identified by SDS-PAGE and Western blotting.The purified recombinant protein was emulsified with Freund's adjuvant and immunized with New Zealand White rabbits to prepare polyclonal antibodies.Western blotting, indirect immunofluorescence assay (IFA) and indirect ELISA were used to identify the specificity of the immunized rabbit serum.【Result】 The recombinant expression vector pET30a-PPV6-ORF2 was successfully constructed, and the prokaryotic truncated expression of PPV6 VP1_{(348 aa-675 aa)} protein was obtained.SDS-PAGE results showed that the recombinant PPV6 VP1_{(348 aa-675 aa)} protein was about 40 ku in size and existed mainly in the form of inclusion bodies.Western blotting results showed that the protein could specifically bind to PPV6 positive pig serum, and had good immunogenicity.Western blotting and indirect ELISA results showed that the purified immunized rabbit serum reacted specifically with the PPV6 whole virus protein, and the antibody titer was 1:25 600.The IFA identification results showed that the rabbit derived polyclonal antibody against PPV6 VP1_{(348 aa-675 aa)} protein had good specificity.【Conclusion】 In this study, ORF2 gene of PPV6 was successfully truncately expressed, and the polyclonal antibody of PPV6 VP1_{(348 aa-675 aa)} protein was successfully prepared, laid a foundation for further study of VP1 protein and the establishment of serological detection method for PPV6.

Key words: Porcine parvovirus type 6 (PPV6); ORF2 gene; truncated expression; polyclonal antibody

CLC Number:

S852.65⁺1

OU Yunwen, PAN Qin, WANG Yang, DAI Junfei, REN Shaoke, ZHANG Yang, ZHANG Jie. Truncated Expression of ORF2 Gene of Porcine Parvovirus Type 6 and Preparation of Its Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2487-2495.

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Truncated Expression of ORF2 Gene of Porcine Parvovirus Type 6 and Preparation of Its Polyclonal Antibody

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