犬副流感病毒分子克隆的构建及全基因组序列分析

Construction and Genome Sequence Analysis of Molecular Clone of Canine Parainfluenza Virus

为了解犬副流感病毒（CPIV）的基因组结构与遗传进化特征，应用RT—PCR分段扩增HRB—V毒株的5个片段，f1、f2、f3以及5′-末端和3′-末端，克隆到pHW2000栽体中并进行测序。经序列拼接获得全基因组序列，与OenBank登录的副流感病毒代表毒株进行对比分析。基因序列分析表明，HRB-V株基因组全长15246nt，含5′-末端、3′-末端和7个ORF具有副流感病毒基因组结构的典型特征；将结构蛋白F、HN以及全基因序列与参考毒株进行比较分析并做遗传进化树，结果表明，HRB—V与CPI一和CPI＋属同一分支，亲缘性相近。核苷酸以及氨基酸序列同源性分析结果表明，HRB-V与各病毒株的F基因的核苷酸同源性在96．5％～98．8％之间，氨基酸同源性为94．2％～98％；HN基因的核苷酸同源性在98．1％～99．5％之间，氨基酸同源性为97％～99．3％。以上试验证明副流感病毒人、猴、猪和犬sV5分离株基因差异较小，具有高度同源性。研究副流感病毒分子克隆的结构，为研究犬副流感病毒反向遗传操作技术平台奠定基础。

In order tO understand the molecular evolution of canine parainfluenza virus （CPIV）, the full- length molecular clone of HRB-V strain was constructed by ligation of overlapping RT-PCR fragments. The five fragments 5′ UTR, 3′UTR, fl, f2, f3, were digested with specific restriction enzymes and cloned into expression plasmid pHW2000. Then the complete genome was sequenced and compared with sequences of PIV5 reference strains in GenBank. The isolate genome was 15 246 nucleotides in length including 5′UTR,70RFS and 3′ UTR. Fusion （F） gene,haemagglutinin （HN） gene and complete genome analysis demonstrated that HRB-V is closely related with reference strains CPI-/CPI＋ The nucleotide sequence homologies of F and HN genes with the reference strains were between 96.5%-98.8% and 98.1%-99.5%, and amino acid sequence homologies were between 94.2%-98% and 97%-99.3%, respectively. Above all suggested the virus strain of HRB-V had little differences and close genetic relationships with other PIV5 isolates. The construction of molecular clone laid the base for further development CPIV reverse genetics system and mutation analysis.