鸡传染性喉气管炎病毒河南株gB基因真核表达载体的构建及表达

Construction and Expression of Eukaryotic Expression Vector of ILTV He'nan Strain gB Gene

根据已发表的传染性喉气管炎病毒(ILTV)gB基因序列,设计并合成1对引物,以河南分离株(ILTV-HN)接种10日龄鸡胚,从含痘斑的绒毛尿囊膜中提取基因组DNA扩增gB基因,将扩增片段克隆入pGEM-TEasy载体后,经蓝白斑筛选,菌液PCR和酶切鉴定为阳性的重组菌进行测序,结果表明克隆到ILTV-HN株gB基因,全长为2 629 bp,包含一个完整的开放阅读框。然后将其克隆至真核表达载体pcDNA3.1(+)中,构建真核表达载体pcDNA3.1-gB,转染鸡胚成纤维细胞(CEF),通过RT-PCR检测,转染细胞中含gB基因的mR-NA,间接免疫荧光检测,结果表明gB基因在CEF细胞中进行了瞬时表达。

One pair of primers was designed according to the nucleotide sequence of chicken infectious laryngotracheitis virus （ILTV） gB gene sequence published in GenBank. ILTV He＇ nan strain was inoculated into 10- day embryonated eggs,and gB gene was amplified by polymerase chain reaction （PCR） from genome DNA extracted from allantochorion contained variola. The purified PCR product was inserted into pGEM -T Easy vector, and then the competent cell DH5α was transformed. By identification of blue - white colony screening, plasmid PCR and enzyme digestion, positive clones were sequenced. The results showed that gB gene nucleotide sequence of ILTV - HN strain was 2 629 bp in length, which included one open - reading frame, gB gene was subcloned into eukaryotic expression vector pCDNA3.1 （ ＋ ）. After identification of PCR and restriction endonuclease, the positive recombinant plasmid pcDNA3.1 -gB was transfected into chicken embryo fibroblasts （CEF）. gB mRNA expression was found in CEF cells and gB protein expression in CEF ceils was detected by the indirect immunofluorescence test.