传染性喉气管炎病毒河南株gB基因的克隆、序列分析及其真核表达载体的构建

Gene cloning,sequence analysis and eukaryotic expression vector construction of gB gene of chicken infectious laryngotracheitis virus Henna isolate

【目的】构建传染性喉气管炎病毒（ILTV）的真核表达载体。【方法】从传染性喉气管炎病毒河南株（ILTV-XY）感染的鸡胚绒毛尿囊膜中提取病毒DNA,进行PCR扩增,PCR产物进行T-A克隆与测序,获得了鸡IL-TV-XYgB全基因序列;对ILTV-XY株gB基因与GenBank中读取的5株ILTVgB基因核苷酸序列及推导的氨基酸序列进行了比较分析;并将该基因片段克隆到真核表达载体pcDNA3.1中,对构建的重组质粒pcDNA3.1/gB进行酶切、测序鉴定。【结果】序列测定表明,gB全基因核苷酸长度为2 629 bp,编码873个氨基酸,推导氨基酸序列有8个潜在的N-糖基化位点,有12个与二硫键形成有关的半胱氨酸。与GenBank中读取的澳大利亚SA2疫苗株、辽宁疫苗株、烟台株、美国632强毒株、英国Throne强毒株gB基因进行比较,核苷酸序列同源性为99%以上,与ILTV-SA2株gB基因比较发现,ILTV-XY株gB基因核苷酸序列在第89位均缺失1个碱基G,而在第102位又都插入1个碱基A,从而引起该毒株gB基因推导的氨基酸序列中第28-32位5个氨基酸的移码突变。构建的重组质粒pcD-NA3.1/gB经酶切、测序鉴定,证实含有目的片段,且连接、构建正确。【结论】成功构建了ILTV的真核表达质粒pcDNA3.1/gB,为ILTV核酸疫苗的研究奠定了基础。

【Objective】 The research was done to construct eukaryotic expression vector of ILTV.【Method】 One pair of primers was designed and synthesized according to the chicken infectious laryngotracheitis virus（ILTV） gB gene nucleotide sequence（M64927）published in GenBank.gB gene of ILTV henan isolate（ILTV-XY） was amplified by PCR from viral DNA extracted from chicken chorioallantoic membrane infected by ILTV-XY.The purified PCR product was inserted into pGEM-T Easy vector,transforming competent cell JM109.By identification of blue-white spot screening,plasmid PCR and enzyme digestion,positive clones were sequenced.gB gene of ILTV-XY strain was compared with gB gene nucleotide sequence and deduced amino acid sequence of 5 ILTV strains published in GenBank.Then,the gB gene was subcloned into the eukaryotic expression vector pcDNA3.1 to construct recombinant plasmid pcDNA3.1/gB,recombinant plasmid pcDNA3.1/gB was identified for PCR and enzyme digestion.【Result】 The sequencing results indicate gB gene nucleotide sequence of LTV-XY strain is 2 629 bp in length,which includes one open-reading frame（2 622 bp）,encoding 873 amino acid residues,eight potential N-glycosalation sites and twelve cysteines in deduced amino acid sequence.Comparison of the gB gene nucleotide sequence with that of ILTV 632 strain,SA2 strain,Throne strain,yantai strain,liaoning strain,published previously in GenBank,revealed over 99.0% similarity.When compared with ILTV SA2 strain,ILTV-XY strain had one base deletion of G at 89 nt and one base insertion of A at 102 nt within gB gene resulting in five amino acid frame shift mutation.Recombinant plasmid was confirmed that the sequence of recombinant plasmid pcDNA3.1/gB in the reading frame and the ligation part was correct by identification of plasmid PCR,enzyme digestion and sequencing.【Conclusion】 The result showed that the recombinant plasmid pcDNA3.1/gB was constructed correctly,which paved the way of DNA vaccine.