猪TGEV HN-2012株ORF3a和ORF3b基因遗传变异分析及其真核表达研究  

Genetic variation and eukaryotic expression of ORF3a and ORF3b from porcine TGEV HN-2012 strain

作　　者：曹贝贝[1] 兰培英[1,2] 韩丽[1] 刘玲玲[1] 韦学雷 胡慧[1,2] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]河南省动物性食品安全重点实验室,河南郑州450002

出　　处：《西北农林科技大学学报（自然科学版）》2016年第4期10-16,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目"Ⅰ型干扰素在TGEV与树突状细胞相互作用中的免疫功能及调控机制研究"(31101796);河南省基础与前沿技术研究计划项目"猪传染性胃肠炎病毒感染对ST细胞免疫生物学特性的影响"(112300410161)

摘　　要：[目的] 研究猪传染性胃肠炎病毒（TGEV）HN-2012株ORF3a和ORF3b基因的遗传变异情况。[方法] 根据GenBank公布的猪传染性胃肠炎病毒ORF3a和ORF3b基因序列,分别设计合成1对特异性引物,通过RTPCR从猪传染性胃肠炎病毒HN-2012株的cDNA中扩增ORF3a和ORF3b基因,经克隆测序后,将其基因序列与NCBI中不同来源的TGEV毒株的相应序列进行同源性分析,构建系统进化树并进行遗传变异分析,然后将ORF3a和ORF3b基因亚克隆至真核表达载体,构建pCAGGS-ORF3a-flag和pCAGGS-ORF3b-flag载体,转染293T细胞进行表达,利用flag标签抗体对2个基因的蛋白表达情况进行Western blot分析。[结果] TGEV HN-2012株的ORF3a基因与其他毒株间核苷酸的同源性为92.6%～100%,ORF3b基因与其他毒株间核苷酸的同源性为98.6%～99.7%。Western blot[结果]表明,ORF3a蛋白和ORF3b蛋白的分子质量约为8ku和28ku。[结论] TGEV HN-2012株的ORF3a基因与CH/JLY2/08、CH/HLJH/08株等亲缘关系较近,ORF3b基因与TS株、Miller M6株等亲缘关系较近,与我国其他毒株关系较远;成功实现了ORF3a和ORF3b蛋白在293T细胞中的表达。[Objective] The genetic variation of ORF3a and ORF3b genes of porcine transmissible gas troenteritis virus （TGEV） HN-2012 was investigated. [Method] A pair of specific primers were designed and synthesized according to TGEV ORF3a and ORF3b sequences in GenBank. RT-PCR was used to ampli- fy ORF3a and ORF3b from HN-2012 strain. Homology analysis was conducted on gene sequences of HN 2012 and other TGEV strains from various sources in NCBI. Phylogenetic tree was also constructed to ana- lyze genetic variation. Then ORF3a and ORF3b were cloned into eukaryotic expression vectors to construct pCAGGS-ORF3a-flag vector and pCAGGS-ORF3b-flag vector, which were transfected into 293T cells to express. Western blot analysis was conducted using flag to tag antibody. [Result] The homology of ORF3a gene between TGEV HN-2012 strain and other strains was 92. 6%- 100%, and that of ORF3b was 98.6%-99.7%. Western blot indicated that molecular weights of ORF3a protein and ORF3b protein were 8 ku and 28 ku,respectively. [Conclusion] The ORF3a gene of TGEV HN-2012 strain was close to CH/ HLJH/08, CH/JLY2/08, while ORF3b gene was close to TS strain and Miller M6 strain. They had distant rela- tionship with other strains in China. This study also achieved the expression of ORF3a and ORF3b in 293T cells.

关 键 词：TGEV河南分离株(HN-2012) ORF3a和ORF3b基因 293T细胞 真核表达 

分 类 号：S852.659.6[农业科学—基础兽医学]