美洲型猪繁殖与呼吸综合征病毒ORF7的原核表达及免疫原性分析

摘要

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major pathogens causing reproductive and respiratory symptoms in pigs (Sus scrofa),and N protein is the main structure protein and antigen protein of the virus.Therefore,this experiment was aimed to study the immunogenicity of N protein of North American genotype Porcine reproductive and respiratory syrudrome virus (NA-PRRSV).The gene was amplified from the RNA of NA-PRRSV QH-08 strain by RT-PCR,whose product was approximately 372 bp.The product was cloned into pET30a(+)vector,RT-PCR,digestion and sequencing were used to identify positive plasmid.The identified recombined plasmid was expressed by Escherichia coli BL21 (DE3) and was induced by 1 mmol/L isopropy-[β-D-thiogalactoside (IPTG) at 37 ℃.The recombinant N protein was purified by Ni-NTA and refolding.The expressed recombinant N proteins was used as an immunogen for immunization of New Zealand white rabbits (Oryctolagus cuniculus) by subcutaneous injection in several sites on back.The specificity of the serum antibody was determined by Western blot.The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDA-PAGE) showed that the recombined protein was successfully expressed in E.coli BL21 (DE3) with a relative molecular weight of 24 kD.The results of Western blot showed that this recombined N protein was specifically reacted with NA-PRRSV positive pig serum;No cross reacted with Porcine parvovirus type 1 (PPV1) and PPV2 positive pig serum.The prepared lepus antisera was specifically reacted with recombined N protein.The recombinant vector pET30a-NA-PRRSV-ORF7 was successfully constructed,and the recombined N protein with excellent immunogenicity was successfully expressed in E.eoli which provided a theoretical basis for the diagnosis of NA-PRRSV.%猪(Sus scrofa)繁殖与呼吸障碍综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)是造成猪出现繁殖与呼吸障碍症状的主要病原之一,N蛋白作为该病毒主要的结构蛋白和抗原蛋白.为了研究美洲型猪繁殖与呼吸障碍综合征病毒(NA-PRRSV)N蛋白的免疫原性,本研究以NAPRRSV QH-08毒株的RNA为模板,扩增获得372 bp的目的片段,扩增产物克隆入pET30a(+)原核表达载体,构建pET30a-NA-PRRSV-ORF7重组质粒,转入大肠杆菌(Escherichia coli)BL21(DE3);在37℃,以1mmol/L异丙基-β-D-硫代半乳糖苷(isopropy-β-d-thiogalactoside,IPTG)诱导表达6 h;采用Ni-NTA树脂亲和层析纯化重组蛋白,并用不同浓度的尿素对纯化蛋白进行复性;复性后重组N蛋白作为免疫原,经背部多点免疫新西兰大耳白兔(Oryctolagus cuniculus),采用Western blot检测兔血清抗体特异性.十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)分析表明,该ORF7编码基因在大肠杆菌中得到表达,蛋白大小约为24 kD;Western blot检测结果表明,该重组N蛋白与NA-PRRSV阳性猪血清发生特异性反应,与猪细小病毒1型(Porcine parvovirus type1,PPVl)和PCV2猪血清不发生交叉反应,制各的NA-PRRSV兔抗血清与重组N蛋白发生特异性反应.该实验成功构建了NA-PRRSV-ORF7原核表达载体,实现了ORF7基因在大肠杆菌中的表达,纯化后的复性蛋白具有较好的免疫原性,为NA-PRRSV检测方法建立或试剂盒的开发提供了理论依据.