| 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RT-PCR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8 ku和28 ku。【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。 |
| 关键词: TGEV 河南分离株（HN-2012） ORF3a和ORF3b基因 293T细胞 真核表达 |
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| 基金项目: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) |
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| Genetic variation and eukaryotic expression of ORF3a and ORF3b from porcine TGEV HN-2012 strain |
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CAO Beibei,LAN Peiying,HAN Li,et al
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| Abstract: |
| 【Objective】The genetic variation of ORF3a and ORF3b genes of porcine transmissible gastroenteritis 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 amplify 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 analyze 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 relationship with other strains in China.This study also achieved the expression of ORF3a and ORF3b in 293T cells. |
| Key words: TGEV isolates in Henan （HN 2012） ORF3a and ORF3b gene 293T cells eukaryotic expression |
