犬瘟热病毒野毒株融合蛋白主要功能区基因的变异研究

Variation of Fusion Protein Gene of Canine Distemper Virus Field Strains

根据犬瘟热病毒（ＣＤＶ）融合蛋白（Ｆ）基因的核苷酸序列，设计合成了１０条引物。用１对引物从延吉犬病料中扩增出了含Ｆ２区基因的３１４ｂｐ的片段，除两端引物序列外为２６５ｂｐ，编码８８个氨基酸。它与日本弱毒株（ＣＤＶ－Ｊ）、Ｏｎｄｅｒｓｔｅｐｏｏｒｔ弱毒株（ＣＤＶ－ＯＮ）、海豹瘟热病毒２型（ＰＤＶ２）、１型（ＰＤＶ１）在核苷酸和氨基酸水平上的同源性，分别为９８．１％和９５．５％、９６．２％和９３．２％、９４．３％和９３．２％、７７．４％和８７．５％。对５份来自不同地区的ＣＤＶ病料的融合区基因片段进行了扩增、克隆和序列分析，其中北京１号犬（ＣＤＶ－Ｂ１）、２号犬（ＣＤＶ－Ｂ２）、沈阳犬（ＣＤＶ－Ｓ）和长春犬（ＣＤＶ－Ｚ）的２８３ｂｐ片段完全相同，而与哈尔滨犬（ＣＤＶ－Ｈ）的该片段有３个核苷酸和２个氨基酸不同。在核苷酸和氨基酸水平，北京犬野毒株等与ＣＤＶ－Ｈ、ＣＤＶ－Ｊ、ＣＤＶ－ＯＮ、ＰＤＶ２、ＰＤＶ１的同源性分别为９８．９％和９７．９％、９７．９％和９５．８％、９６．５％和９７．９％、９３．３％和９８．９％、７７．０％和８４．２％。本研究揭示了ＣＤＶ的流行毒株和相关毒株在融合蛋白上的亲缘关系，进一步证实了ＰＤＶ２?

Ten oligonucleotide primers had been designed and synthesized whose sequences were based on the nucleotide sequences of the gene encoding the fusion protein (F) of canine distemper virus (CDV). A F2 gene fragment of a CDV field strain was successfully amplified from a dog′s liver in Yanji (CDV Y) by reverse transcriptase polymerase chain reaction (RT PCR). 265 nucleotides of the CDV Y F2 gene had been sequenced from the 314 bp fragment excluding the 2 primer sequences. At the nucleotide level, 265 bp fragment of CDV Y was 98.1%, 96.2%, 94.3% and 77.4% separately identical to Japanese modified live CDV strain (CDV J), CDV Onderstepoort strain (CDV ON), phocine distemper virus type 2(PDV2) and PDV1. 4, 6, 6 and 13 amino acid residues changed when the 88 deduced amino acid sequences were respectively compared with CDV J, CDV ON, PDV 2 and PDV 1. Gene fragments of fusion region in fusion protein gene of 5 CDV field strains were separately amplified, cloned and sequenced from 5 dogs that were respectively named Beijing 1 (CDV B1), Beijing 2(CDV B2), Shenyang 1 (CDV S), Changchun Zanggao(CDV Z) and Harbin 1(CDV H). There were 3 nucleotides and 2 amino acid residues changes between CDV H and other 4 CDV field strains. 283 bp of fusion region of CDV B F gene was separately 98.9%, 97.9%, 96.5%, 93.3% and 77% identical to CDV H, CDV J, CDV ON, PDV 2 and PDV 1. In addition, the percentage of identification of CDV B at the amino acids level to CDV H, CDV J, CDV ON, PDV 2 and PDV 1 were separately 97.9%, 95.8%, 97.9%, 98.9% and 84.2%. A 287 bp DNA fragment (ZP5 9) was amplified, cloned and sequenced in a 771 bp PCR production that amplified by primer 3 and primer 5 from CDV Z. The 21 bp at the 5′ end of the ZP5 9 fragment was identical to CDV primer 5 and 194 bp at the 3′ end of the fragment 99% identical to CDV ON. The other 72 bp fragment was identified as a component of cell gene by nucleic acid hybridization assay.