猪轮状病毒主要非结构蛋白的原核表达、抗体制备及应用  

Prokaryotic Expression,Antibody Preparation and Application of Major Non-Structural Proteins of Porcine Rotavirus

作　　者：卞贤宇 李素芬 王建新[2] 韩楠 卢洪婷 程曦 周金柱 陶然 朱雪蛟 董海龙 张雪寒 李彬[1,2] BIAN XianYu;LI SuFen;WANG JianXin;HAN Nan;LU HongTing;CHENG Xi;ZHOU JinZhu;TAO Ran;ZHU XueJiao;DONG HaiLong;ZHANG XueHan;LI Bin(College of Animal Science,Tibet Agriculture and Animal Husbandry University,Linzhi 860000,Xizang;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture and Rural Affairs,Nanjing 210014)

机构地区：[1]西藏农牧学院动物科学学院,西藏林芝860000 [2]江苏省农业科学院兽医研究所/农业农村部兽用生物制品工程技术重点实验室,南京210014

出　　处：《中国农业科学》2024年第17期3494-3506,共13页Scientia Agricultura Sinica

基　　金：江苏省农业科技自主创新资金(CX[23]1029)。

摘　　要：【背景】轮状病毒(Rotavirus,RV)是全球范围内幼儿和各种幼畜急性病毒性胃肠炎的主要原因之一,在公共卫生中具有重要意义。猪轮状病毒病是一种由猪轮状病毒(Porcine Rotavirus,PoRV)引起的一种急性肠道传染病,常造成仔猪消化道功能紊乱,引发严重的呕吐、腹泻和脱水症状,一旦爆发将对养猪业造成重大经济损失。目前,针对PoRV感染尚无特效药物治疗,疫苗接种是控制感染的最经济的途径。然而,PoRV基因型多且易变异,不同基因型之间的交叉保护很不理想。因此,亟需加强对PoRV的流行病学监测和致病机制研究,探索新型防控策略。【目的】利用大肠杆菌原核系统表达PoRV NSP2、NSP4和NSP5非结构蛋白并免疫家兔获得相应多克隆抗体,为PoRV的防控和致病机制研究提供技术手段。【方法】将PoRV的NSP2、NSP4和NSP5基因进行密码子优化后,克隆到pCold-sumo载体中。将测序正确的阳性重组质粒转化至大肠杆菌BL21(DE3),经IPTG诱导获得重组蛋白。利用SDS-PAGE和Western blot鉴定蛋白表达情况。对重组蛋白进行亲和层析柱纯化和定量后,采用皮下多点注射方法接种新西兰大白兔,每隔14 d加强免疫一次,经过3次免疫后制备多克隆抗体。采用间接ELISA方法测定多克隆抗体的效价、间接免疫荧光(IFA)和Western blot验证多克隆抗体与PoRV的反应性。再通过Western blot进一步检测PoRV感染过程中3个非结构蛋白的动态表达,以及鉴定3个重组真核质粒转染的效果。【结果】NSP2、NSP4和NSP5重组菌能够有效表达目的蛋白,SDS-PAGE分析可在目标位置清晰观察到明显条带,并且目的蛋白以可溶性形式存在。3个重组蛋白制备的多克隆抗体,ELISA效价均超过1﹕81000,表明重组蛋白免疫原性良好。IFA结果表明3个多克隆抗体均能够与优势流行基因型轮状病毒发生特异性反应,与其他常见腹泻病原无反应。Western blot结果也展示�【Background】Rotavirus(RV)is one of the main causes of acute viral gastroenteritis in young children and young animals worldwide,and it is of great significance in public health.Porcine rotavirus disease,caused by Porcine Rotavirus(PoRV),is an acute intestinal infectious disease that often results in gastrointestinal dysfunction in piglets,leading to severe symptoms,such as vomiting,diarrhea,and dehydration.Outbreaks of PoRV can result in significant economic losses in the pig industry.At present,there is no specific drug treatment for PoRV infection,so vaccination is the most economical way to control the infection.However,PoRV genotypes are various and easily mutable,and cross-protection between different genotypes is poor.Therefore,it is urgent to strengthen the epidemiological surveillance and pathogenic mechanism research of PoRV to explore new prevention and control strategies.【Objective】The prokaryotic system of Escherichia coli was employed to express the non-structural proteins(NSP),such as NSP2,NSP4,and NSP5 of PoRV.Subsequently,rabbits were immunized to produce polyclonal antibodies(pAbs)specific to these proteins,which could offer novel insights to the detection and prevention of PoRV.【Method】The NSP2,NSP4,and NSP5 genes of PoRV were codon-optimized and cloned into the pCold-sumo vector.The positive recombinant plasmids,with correct sequencing,were transformed into E.coli BL21(DE3),and the recombinant proteins were obtained with IPTG induction.Protein expression was identified using SDS-PAGE and Western blot assay.The recombinant proteins were then purified and quantified using affinity chromatography.New Zealand white rabbits were immunized with NSP2,NSP4,and NSP5 recombinant proteins by a subcutaneous multi-point injection method to prepare pAbs.The titers of pAbs were determined using indirect ELISA technology.The reactivity of pAbs with PoRV was verified by indirect immunofluorescence(IFA)and Western blot assay,and their applications in PoRV infection were also explored.【Result】SDS-

关 键 词：猪轮状病毒 非结构蛋白 原核表达 多克隆抗体 

分 类 号：S852.651[农业科学—基础兽医学]