传染性法氏囊病病毒疫苗株B87基因组A节段的克隆和序列分析

目的获得传染性法氏囊病病毒疫苗株B87 A节段全长cDNA,并对其进行序列分析。方法使用蛋白酶K、酚/氯仿抽提法提取病毒基因组,用LiCl分级沉淀方法纯化病毒基因组dsRNA,通过RT-PCR一步扩增获得A节段的全长cD-NA。将其克隆至pGEM-T载体上,测序,并用DNAStar软件进行序列分析。结果测序结果表明,克隆的B87株A节段全长为3 260 bp,与Gt株的同源性最高为99.5%,与其他毒株的核苷酸同源性介于95.2%~99.4%之间,与Ⅱ型毒株OH仅为84.0%。结论通过对20株IBDV编码氨基酸序列的分析、比较,推测A片段上7个独特的氨基酸位点可能与毒力相关。

Cloning and Sequencing of Genomic A Segment of Infectious Bursal Disease Virus Vaccine Strain B87

Objective To clone and sequence the full-length cDNA of genomic segment A of infectious bursal disease virus(IBDV) vaccine strain B87.Methods Extract the genomic RNA of IBDV by protease K digestion and phenol/chloroform extraction,then purify the genomic dsRNA by LiCl fractional precipitation for amplification of full-length cDNA of genomic segment A of IBDV strain B87 by one-step RT-PCR.Insert the amplified cDNA into pGEM-T vector,then sequence and analyze the result by using DNAStar software.Results Nucleotide sequencing proved that the homologies of cloned genomic A segment of IBDV vaccine strain B87,at a full-length of 3260 bp,were 99.5% and 95.2%~99.4% respectively to those of Gt and other strains,but was only 84.0% to that of strain OH of IBDV type Ⅱ.Conclusion The analysis of deduced amino acid sequences of 20 IBDV strains indicated that the 7 characteristic amino acid sites of A segment might be related to virulence.