猪传染性胃肠炎病毒M、sM基因重组杆状病毒的构建及病毒样颗粒的组装  

Expression of M Gene and sM Gene of Porcine Transmissible Gastroenteritis Virus by Baculovirus Expression System and VLPs Assmbly of TGEV

作　　者：冷勇[1] 宋振辉[1,2] 卿家超[1] 翟少华[2] 买买提.艾孜子 

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

出　　处：《畜牧兽医学报》2013年第9期1432-1437,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：中央高校基本科研业务基金(XDJK2010C094);重庆自然科学基金(2008BB5243);乌鲁木齐市科技计划项目(Y121210005)

摘　　要：利用RT-PCR方法扩增猪传染性胃肠炎病毒（TGEV）M和sM结构蛋白基因，将其分别克隆人载体pFastBac^TM Dual，获得转移质粒pFastBac^TM Dual-M和pFastBac^TM Dual-M-sM，将重组质粒转化至DHl0Bac感受态细胞，经抗性与蓝白斑筛选，获得杆状病毒重组质粒Bacmid-M和Bacmid-M-sM，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-M-sM。通过WesternBlot、间接免疫荧光试验（IFA）进行检测，结果显示：重组蛋白在杆状病毒感染的sf9昆虫细胞中获得正确表达。将重组杆状病毒rBac-M和rBae-M-sM分别感染sf9昆虫细胞，进行TGEV病毒样颗粒（VLPs）的体外组装试验。结果表明，M蛋白在sf9细胞内单独表达，M蛋白和sM在sf9细胞内共同表达，都可形成TGEV病毒样颗粒，而且病毒粒子大小不等，直径在61～101nm。试验结果为进一步研究TGEV结构蛋白在病毒装配过程中的作用提供了理论依据。Recombinant baculovirus containing M and sM gene of transmissible gastroenteritis vi- rus （TGEV） were constructed using Bac-To-Bae Baculovirus expression system, and recombinant M, sM proteins were expressed by infecting the sf9 cell with recombinant baculovirus. Firstly, M gene and sM gene of TGEV were amplified, then were cloned and inserted into donor plasmid pFastBacTMDual to obtain recombinant donor plamid, pFastBacTMDual-M and pFastBacTMDual-M- sM. Plasmid pFastBacTM Dual-M and pFastBacTM Dual-M-sM were transformed into E. coli DH10Bac respectively. Recombinant baculovirus rBac-M and rBac-M-sM were obtained by trans- fection of the sf9 cells with Bacmid-M and Bacmid-M-sM. Western blot and indirect immunofluo- rescence assay （IFA） were conducted, and the results showed that recombinant M protein and sM protein was expressed successfully, two proteins had a positive reaction with antiserum. In vitro experiments of virus-like particles （VLP） assembly of TGEV was performed by infection of insect cell with rBac-M and rBac-M-sM. It is showed that the expressed M proteins alone and co-expres- sion of M and sM proteins in insect cell Sf9 infected by recombinant baculovirus could form VLPsmorphologically similar to virion of TGEV. Diameter of VI.P is variable from 61 nm to 101 nm. This study provides the foundation for researching the VLP assembly of TGEV.

关 键 词：猪传染性胃肠炎病毒（TGEV） M基因 sM基因 SF9细胞 病毒样颗粒 

分 类 号：S852.659.6[农业科学—基础兽医学]