猪传染性胃肠炎病毒M、N基因重组杆状病毒的构建及其口服免疫小鼠诱导抗体水平测定  

Expression of M gene and N gene of porcine transmissible gastroenteritis virus by baculovirus expression system and determination of specific antibodies in orally immunized mice

作　　者：宋振辉[1,2] 卿家超[1] 冷勇[1] 买买提.艾孜子 齐晓娟[2] 叶翠芳[2] 

机构地区：[1]西南大学荣昌校区动物医学系,重庆荣昌402460 [2]新疆农业大学动物医学学院,乌鲁木齐830052

出　　处：《中国兽医学报》2015年第1期5-10,共6页Chinese Journal of Veterinary Science

基　　金：中央高校基本科研业务基金重点资助项目(XDJK2014B039);新疆维吾尔自治区教育厅高校科研计划资助项目(20120984);乌鲁木齐市科技计划资助项目(Y121210005)

摘　　要：利用RT-PCR方法扩增猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)M和N结构蛋白基因,将其分别克隆入载体FastBacTM Dual,获得转移质粒pFastBacTM Dual-M和pFastBacTM Dual-N,将重组质粒转化至DH10Bac感受态细胞,获得杆状病毒重组质粒Bacmid-M和Bacmid-N;在Cellfection作用下,将杆状病毒重组质粒转染昆虫细胞sf9,获得重组杆状病毒rBac-M和rBac-N。间接免疫荧光试验(IFA)检测表明,杆状病毒表达的M蛋白和N蛋白能够被特异性阳性血清识别,重组蛋白具有较好的反应原性。将重组杆状病毒rBac-M和rBac-N口服免疫小鼠,收集小鼠粪便检测抗TGEV sIgA抗体水平,采集血液检测血清中抗TGEV IgG。结果显示,重组杆状病毒(rBac-M和rBac-N)可诱导小鼠产生粘膜免疫和体液免疫应答。试验结果初步预示了杆状病毒经口服途径作为抗原递呈载体的可行性,也为开展TGEV口服免疫研究奠定了基础。M gene and N gene of transmissible gastroenteritis was amplified,then cloned and inserted into donor plasmid pFastBacTMDual to obtain recombinant donor plamid pFastBacTM DualM and pFastBacTMDual-N.Plasmid pFastBacTM Dual-M and pFastBacTM Dual-N was transformed into E.coli DH10 Bac,respectively.Recombinant baculovirus rBac-M and rBac-N were obtained by transfection of the sf9 cells with Bacmid-M and Bacmid-N.Indirect immunofluorescence assay(IFA)showed that recombinant M protein and N protein were expressed successfully,two proteins had a positive reaction with antiserum.Spcific anti-TGEV secret immunoglobulin A(sIgA)antibody was detected by indirect ELISA in the feces,anti-TGEV IgG antibody was detected by indirect ELISA in the serum of immunized mices after oral administration.The results indicated that the mice immunized with recombinant baculovirus rBac-M and rBac-N could evoke both mucosal and systemic immune responses,which demonstrated primarily a feasibility of using baculoviruses for vector of oral vaccine and established material basement for the research of oral immunization of TGEV.

关 键 词：猪传染性胃肠炎病毒(TGEV) M基因 N基因 口服免疫 抗体 

分 类 号：S852.651[农业科学—基础兽医学]