传染性法氏囊病病毒疫苗B87株基因组B节段的克隆和序列分析

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

目的获得传染性法氏囊病病毒(IBDV)疫苗B87株B节段全长cDNA,并对其序列进行分析,为进一步在分子水平上研究病毒基因组的功能、抗原变异和毒力变异奠定基础。方法使用蛋白酶K和酚/氯仿抽提法提取病毒基因组RNA,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B节段的全长cDNA。将其克隆到pGEM-T载体上,然后测序,并用DNAStar软件进行序列分析。结果测序结果表明,克隆的B87株基因组B节段全长为2827bp,与超强毒参考株UK661和HK46的同源性分别为89.8%和89.3%,与强毒参考株Harbin-1和ZJ2000的同源性分别为90.2%和89.5%;与变异毒株GLS的同源性为97.8%;与弱毒参考株CEF94和Gt株的同源性均为99.8%,与P2株的同源性达100%。结论通过对9株IBDV编码氨基酸序列进行分析、比较,推测B节段上10个独特的氨基酸位点可能与毒力相关。

Objective To clone and sequence the full-length eDNA of genomic segment B of infectious bursal disease virus （IBDV） vaccine strain B87 and lay a foundation of study on the function as well as antigen and virulence variations of genome of IBDV strain B87 at a molecular level. 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 B of IBDV strain B87 by one-step RT-PCR. Insert the amplified eDNA into pGEM-T vector, then sequence and analyze the result by using DNAStar software. Results The eDNA of genomic segment B of IBDV, at a full length of 2 827 bp, was cloned, which showed homologies of 89. 80% and 89. 30% to high virulent reference strains UK661 and HK46,90. 2% and 89. 5% to virulent reference strains Harbin-1 and ZJ2000,97. 8% to variant strain GLS,both 99. 8% to avirulent reference strains CEF94 and Gt,and 100% to strain P2,respectively. Conclusion The analysis of deduced amino acid sequences of 9 IBDV strains indicated that the 10 characteristic amino acid sites of B segment might be related to virulence.