猪细小病毒6型ORF 2基因的截短表达及多克隆抗体制备  

Truncated Expression of ORF 2 Gene of Porcine Parvovirus Type 6 and Preparation of Its Polyclonal Antibody

作　　者：欧云文 潘琴 汪洋[2] 代军飞 任绍科 张洋 张杰[2,3] OU Yunwen;PAN Qin;WANG Yang;DAI Junfei;REN Shaoke;ZHANG Yang;ZHANG Jie(Animal Disease Prevention and Control Center of Kaijiang County,Dazhou 636250,China;State Key Laboratory of Veterinary Etiological Biology,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;Hebei Key Laboratory of Preventive Veterinary Medicine,Hebei Normal University of Science&Technology,Qinhuangdao 066004,China)

机构地区：[1]开江县动物疫病预防控制中心,达州636250 [2]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,兰州730046 [3]河北科技师范学院,河北省预防兽医学重点实验室,秦皇岛066004

出　　处：《中国畜牧兽医》2023年第6期2487-2495,共9页China Animal Husbandry & Veterinary Medicine

基　　金：四川省科技计划资助项目(2023 JDRC0126);达州市科技计划资助项目(22CYRC0016)。

摘　　要：【目的】原核截短表达猪细小病毒6型(Porcine parvovirus type 6,PPV6)ORF 2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ORF 2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∶25600;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 ORF 2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。【Objective】This study was aimed to truncatly express ORF 2 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 ORF 2 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∶25600.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,ORF 2 gene of PPV6 was successfully truncately expressed,and the polyclonal antibody of PPV6 VP1(348 aa-675 aa)protein was successfully prepared,lai

关 键 词：猪细小病毒6型(PPV6) ORF 2基因 截短表达 多克隆抗体 

分 类 号：S852.651[农业科学—基础兽医学]