犬瘟热病毒扬州分离株融合蛋白F和附着蛋白H基因的序列分析

根据Genbank犬瘟热病毒(CDV)毒株序列分别设计融合蛋白F和附着蛋白H全基因的2对引物,以2个CDV扬州分离株(CDV-YZ0101、CDV-YZ0102)为模板RT-PCR扩增出H基因和F基因2个片段,将其克隆进pGEM-Teasy载体,并进行序列分析.结果表明H基因的开放阅读框架(ORF)为1 824 bp,2种分离株间核苷酸和编码氨基酸同源性达98%左右,与中国长春分离株、美国、日本分离株的同源性分别为96%、93%～95%和90%～91%.氨基酸分析显示扬州分离株与其他分离株的H蛋白有8～9个可能的天冬酰胺糖基化位点,而OP-CDV疫苗株只有4个类似位点.扬州分离株与长春分离株同属一个系,与美国、日本分离毒株以及疫苗株相比,均存在着明显的差异.F基因ORF为1 989 bp,不同毒株间的核苷酸及编码氨基酸差异主要表现在信号肽区域,而编码的F0前体蛋白表现出较高同源性(97%～99%),且所有的13个半胱氨酸残基、4个可能的天冬氨酰糖基化位点和2个疏水区域均完全一致.因此CDV F与H蛋白基因相比有很高的同源性,证明中国分离株与国外参考株有差异.

SEQUENCE ANALYSIS OF F GENE AND H GENE OF YANGZHOU ISOLATES

Using RNA extracted from the Yangzhou isolates (CDV-YZ0101, CDV-YZ0102) as template, the F protein gene and the H protein gene were amplified with RT-PCR. The PCR products were cloned into pGEM-Teasy vector routinely, and the positive recombinants of two genes obtained were then sequenced and analyzed. Sequence analysis showed the H gene of two Yangzhou isolates contained one 1 824 bp large open reading frame, and the two Yangzhou isolates had 98% identity with each other, 96%, 93%-95% with Changchun isolates, American and Japan isolates, but only 90%-91% identity with the vaccine strains (Ondersteroop strain, Convac strain). Amino acid alignment showed that there were 8-9 potential asparagines glycosylation site in the protein of the Chinese isolates and other isolates , while there were only four in OP-CDV. Phylogenesis study suggested that our isolate and Changchun isolate could be clarified to the same lineage, which was different from American and Japanese isolates, and there were evident difference between field isolates and vaccine strains. The ORF in the F gene f two Yangzhou isolates was 1 989 bp, the major difference among various CDV strains was the long signal peptide domain while the immature protein (FO) exhibited high identity, and all of the 13 serine residues, four potential asparagine glycosylation sites and two hydrophobic regions supposed to affect the fusion function were completely identical. These data supports the view that F protein is more conservative than H protein in CDV, and with repect to the H gene, there are differences between Chinese isolates and other country isolates.