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

摘要/Abstract

摘要： 【目的】原核截短表达猪细小病毒6型（Porcine parvovirus type 6，PPV6）ORF2基因，并制备PPV6 VP1_{（348 aa-675 aa）}蛋白多克隆抗体，为后续研究该蛋白的生物学功能提供材料。【方法】以PPV6分离毒株基因组为模板，PCR扩增获得ORF2截短基因片段，将其克隆至原核表达载体pET30a （+）中构建重组质粒pET30a-PPV6-ORF2。经酶切和测序鉴定后，将重组质粒转化大肠杆菌BL21（DE3）感受态细胞，IPTG诱导表达，Ni-NTA树脂亲和层析纯化，通过SDS-PAGE和Western blotting鉴定纯化后重组蛋白。将纯化后的重组蛋白与弗氏佐剂混匀乳化，免疫新西兰大耳白兔制备多克隆抗体，采用Western blotting、间接免疫荧光试验（IFA）和间接ELISA鉴定免疫兔血清特异性。【结果】成功构建了pET30a-PPV6-ORF2重组表达载体，原核截短表达了PPV6 VP1_{（348 aa-675 aa）}蛋白；SDS-PAGE结果显示，重组PPV6 VP1_{（348 aa-675 aa）}蛋白大小约为40 ku，主要以包涵体形式存在；Western blotting结果显示，该蛋白可与PPV6阳性猪血清发生特异性结合，具有良好的反应原性；Western blotting间接与ELISA结果显示，纯化后免疫兔血清与PPV6全病毒蛋白发生特异性反应，抗体效价为1：25 600；IFA鉴定结果表明，PPV6 VP1_{（348 aa-675 aa）}蛋白兔源多克隆抗体具有良好的特异性。【结论】本研究成功原核截短表达了PPV6 ORF2基因并制备了兔抗PPV6 VP1_{（348 aa-675 aa）}蛋白多克隆抗体，为PPV6血清学检测方法的建立和PPV6 VP1蛋白的进一步研究提供了参考。

关键词: 猪细小病毒6型(PPV6); ORF2基因; 截短表达; 多克隆抗体

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

中图分类号:

S852.65⁺1

欧云文, 潘琴, 汪洋, 代军飞, 任绍科, 张洋, 张杰. 猪细小病毒6型ORF2基因的截短表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2487-2495.

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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