传染性喉气管炎病毒河南株gB基因的克隆与序列分析

根据传染性喉气管炎病毒(ILTV)gB基因的核苷酸序列,设计、合成1对引物,应用PCR扩增鸡ILTV河南株(ILTV-CG)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-CG株gB基因,全长为2 629 bp,包含一个完整的开放阅读框,共编码873个氨基酸的多肽,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.序列分析表明,ILTV-CG株gB基因与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基因核苷酸序列同源性为99%以上,与ILTV-SA2株gB基因比较发现,ILTV-CG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.

Cloning and Sequence Analysis of gB Gene of Chicken Infectious Laryngotracheitis Virus Henan Isolate

One pair of primers was designed and synthesized based on the nucleotide sequence(M64927)of chicken infectious laryngotracheitis virus(ILTV) gB gene pulished in the GenBank.gB gene of ILTV Henan isolate(ILTV-CG) was amplified by PCR.The purified PCR product was inserted into pGEM-T Easy vector,and then transformed competent cell JM109.By idenfication of blue-white colony screening,plasmid PCR and enzyme digestion,positive clones were sequenced.The sequencing results indicated that gB gene nucleotide sequence of ILTV-CG strain was 2 629 bp in length,which included one openreading frame(2 622 bp,encoding 873 amino acid residues,with eight potential N-glycosalation sites and twelve cysteines in deduced amino acid sequence.Comparison of the gB gene nucleotide sequence with that of the ILTV 632 strain,SA2 strain,Throne strain,Yantai strain and Liaoning strain published previously in GenBank,revealed similarity of more than 99%.When compared with ILTV-SA2 strain,ILTV-CG strain had one base deletion of G at 89 nt and one base insertion of A at 102 nt within gB gene,resulting five amino acid frame shift mutation.This study provided foundation for the development of recombinant vaccine against ILTV.