IBDV超强毒HZ株VP2基因的克隆和真核表达质粒的构建

Cloning and sequence analysis of the VP2 gene in HZ strain of very virulent infectious burial disease virus and construction of its expression plasmid

利用反转录 -聚合酶链反应 (RT- PCR) ,扩增克隆传染性法氏囊病病毒超强毒 (vv IBDV) HZ株 VP2基因 ,并对 VP2基因进行全序列测定、序列分析和聚类分析 ,同时将VP2基因与真核表达载体 pc DNA3相连接。结果克隆到 VP2基因全序列长 1 356bp,分析表明 HZ株与欧洲超强毒株 UK661高度相似 ,同时将 VP2基因正向插入 pc DNA3的 CMV启动子下游 ,得到了 VP2基因的真核表达质粒。为

According to the published sequence of IBDV strain 52/70,a pair of primers that could amplify the cDNA of protective antigen VP2 gene was designed and synthesized.By RT PCR,a single DNA fragment of about 1.5 kb was obtained from HZ strain of IBDV.Then the VP2 cDNA was cloned into PUC119 at SmaI site.The nucleotide sequence of the expected VP2 gene was determined by Sangers DNA sequencing method,and then the amino acid sequence was deduced.Both the nucleotide sequence and amino acid sequence were compared with five published sequence of VP2 gene of IBDV strain.It was shown that HZ strain was mostly closely related to the very virulent strain UK661 but different from other serotype I strains.The IBDV VP2 gene was recovered from plasmid HZ PUC119 by digestion with XbaI.Then the VP2 gene was subcloned into the downstream of CMV promoter of the same treated expression vector pcDNA3.The recombinant plasmid HZ pcDNA3 containing IBDV VP2 gene was identified by restriction digestions and PCR amplification.The expression plasmid can be a potentially valuable gene vaccine against IBDV.