摘要
甲病毒属病毒E2结构蛋白是一种保守的受体结合蛋白,包含与免疫原性、组织噬性及宿主感染范围相关的重要残基。本试验以盖塔病毒(Getah virus,GETV)SD17/09株为模板设计特异性引物,利用RT-PCR方法扩增E2结构域基因(E2d),扩增产物E2d连接至原核表达载体pET-22b(+)融合表达,利用SDS-PAGE及Western blot方法分析表达产物。以His-Bind亲和层析柱纯化E2d蛋白,免疫新西兰兔获得阳性血清,通过血清中和方法检测血清效价,并通过Western blot分析其活性。结果显示,在预期38 kDa处出现目的条带,表明E2d基因成功表达,表达产物以包涵体形式大量存在;免疫后24 d多抗血清中和效价达1∶4096,能与重组蛋白E2d和GETV SD17/09株发生特异性反应。本试验成功应用大肠杆菌表达GETV E2d蛋白,表达的蛋白具有免疫原性,并获得兔源阳性血清,为开展GETV E2蛋白结构域的功能研究、诊断方法的建立和疫苗研发奠定基础。
The structural protein E2 of alphavirus is a conservative receptor binding protein,which contains critical residues related to immunogenicity,tissue tropism and host range.In this study,a pair of specific primers was designed using the Getah virus(GETV)SD17/09 strain as the template to amplify E2 domain(E2 d)by RT-PCR.The amplified product E2 d was cloned into the prokaryotic expression vector pET22 b(+)for expression,the expression product was verified by SDS-PAGE and Western blot.E2 d protein was purified by His-Bind affinity chromatography and the purified E2 d was used to immunize New Zealand white rabbits to obtain positive serum,the titer of serum was detected by serum neutralization test and the activity of positive serum was verified by Western blot.The results showed that the target bands appeared at the expected size of 38 kDa,indicating that E2 d gene was successfully expressed and the expression products were abundant in inclusion bodies.After 24 days of immunization,the neutralization titer of positive rabbit serum reached 1∶4096 and it reacted specifically with recombinant protein E2 d and GETV SD17/09strain.This study lays a foundation for the functional study,establishment of diagnostic methods and vaccine development.
作者
仇相书
史宁
朱翔宇
任世斌
张海森
李海
鲁会军
金宁一
QIU Xiangshu;SHI Ning;ZHU Xiangyu;REN Shibin;ZHANG Haisen;LI Hai;LU Hui-jun;JIN Ningyi(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi 712100,China;Military Veterinary Research Institute,Academy of Military Medicine,Academy of Military Sciences,Changchun 130122,China;College of Veterinary Medicine,Jilin Agricultural University,Changchun 130118,China;Zhaosu Country Xi-yu Horse Industry Co.Ltd,Yili,Xinjiang 835600,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2021年第8期1506-1511,共6页
Chinese Journal of Veterinary Science
基金
国家重点研发计划资助项目(2018YFD0500104,2018YFD0500803)。
