IBRV内蒙古分离株gD基因的分子克隆与序列分析

CLONING AND SEQUENCE ANALYSIS OF THE gD GENE OF IBRV FROM INNER MONGOLIA

根据已发表的牛传染性鼻气管炎病毒(GenBank NC_001847)gD基因序列设计1对特异性引物,采用PCR方法扩增出内蒙古分离株gD基因的cDNA,重组到PMD19-T载体中,并进行克隆及序列测定。经限制性内切酶谱分析和序列分析,证明所克隆的cDNA为gD基因。该基因片段长1 559bp,包含1个由1 251bp组成的完整开放阅读框(ORF),共编码417个氨基酸。序列比较和系统进化树分析结果显示:内蒙古分离株gD基因与GenBank上已发表的其它分离株相比,核苷酸同源性在99.1%～99.9%之间,其推导的氨基酸同源性在90..6%～99.8%之间,在系统进化树中内蒙分离株与AJ004801株亲缘关系较近,属于同1个分支。生物信息学分析表明,该基因编码的蛋白具有亲水性,有很强的抗原性,有3个潜在糖基化位点,可能存在2个蛋白激酶C磷酸化位点,存在4个酪蛋白激酶Ⅱ磷酸化位点。

According to the published gD gene sequence of IBRV in GenBank（NC_001847）,one specific pair of primer was designed.The gD gene cDNA of Inner Mongolia strain was amplified by PCR and then cloned into PMD19-T plasmids and then sequenced.The results of restriction endonuclease pattern analysis of recombinant plasmid and DNA sequencing demonstrated that the cDNA is belong to the gD gene.The cDNA were 1 559bps long and contain a open reading frame（ORF） of 1 251 bases which encoding 417 amino acid.The comparison of the sequences and phylogenetic analysis showed that the homology of gD gene of Inner Mongolia strain compared with others in GenBank were 99.1% to 99.9% in nucleotide,90.6% to 99.8% of the deduced amino acid sequence.Phylogenetic tree revealed that the gD gene and the AJ004801 strain which were consistently located in the same lineages.Bioinformatics analysis of this sequence showed that the protein of the gD gene was hydrophilic and highly antigenic,includes three potential N-glycosylation site,two potential protein kinase C phosphorylation site,four potential casein kinase Ⅱphosphorylation site.