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

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-T Easy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mRNA,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.

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

One pair of primers was designed according to the nucleotide sequence of chicken infectious la-ryngotracheitis 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 DH5a 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 cell s and gB protein expression in CEF cells was detected by the indirect immunofluorescence test.