一步法扩增IBDV上海株全长基因组cDNA

Amplifiction of Full-length cDNA of Infectious Bursal Disease Virus of Shanghai Strain

为了研究鸡传染性法氏囊病病毒 (IBDV)上海株的全部核苷酸序列 ,应用蛋白酶K消化、割胶纯化获得了高纯度的dsRNA ,应用随机引物合成了cDNA的第一链。参照基因库中相关IBDV毒株的基因序列 ,设计合成了两对引物 ,一步扩增出IBDV上海株全长基因组cDNA的A片段和B片段。为了验证获得的A片段和B片段的正确性 ,以扩增出的A片段为模板 ,成功扩增出IBDV的VP2 基因 ,并应用T载体进行了克隆和测序 ,测序结果显示 ,其与国内外数株IBDV毒株的同源性很高 ,表明本文建立的扩增IBDV全长基因组cDNA的一步法正确。

To determine the complete nucleotide sequence of infectious bursal disease virus of Shanghai strain,an efficient method was developed to amplify full_length cDNA of both the genomic A and B segment.dsRNA were extracted by proteinase K digestion based approach from purifed virions.The first strand synthesis using random primers was carried out,which catalyzed by Superscript Ⅱ RNase H_Reverse Transcriptase.The full_length cDNA of both A and B segment were amplified by using an optimized PCR.The VP 2 gene was generated and cloned using the amplified A segment as template.The sequence of VP 2 gene of IBDV of Shanghai strain was given in this paper.