牛病毒性腹泻病毒基因1型全基因组的测序分析

以我国新疆地区分离的1株牛源牛病毒性腹泻病毒基因1型毒株为研究对象,参照GenBank中的牛病毒性腹泻病毒1型基因组全序列,根据保守序列设计6对重叠引物,通过RT-PCR扩增出该毒株不同的cDNA片段,分别克隆到pMD19-T载体,转化受体菌JM109,挑取阳性克隆进行PCR、酶切鉴定及测序分析,为了解该毒株的遗传变异及基因结构与生物功能的关系,为开发牛病毒性腹泻病毒新型疫苗奠定基础。测序结果表明,该病毒全基因序列核苷酸为12 302nt(GenBank登录号:JN704144),该病毒基因组具有一个11 694nt开放阅读框编码的多聚蛋白,5′-UTR长382nt,3′-UTR长224nt,BVDV1-3156的全基因组序列分析发现该毒株为BVDV1b亚型。Blast比对分析显示,该毒株与VEDEVAC株的相似性最高,为90.9%。该毒株与BVDV1型其他毒株的全基因组序列具有较大的差异。

Sequencing and analysis of complete genome of bovine viral diarrhea virus genotype 1

In the present study,the BVDV1-3156 virus was used as material to sequence the complete genome of bovine viral diarrhea virus to provide direct information about the genomic structure and its possible relationships to the biological functions,and to provide aid for the development of new BVDV vaccines.Six pairs of primers were designed according to the sequence of BVDV1 virus prototype strain Oregon C24V.The cDNA fragments of BVDV1-3156 strain were amplified from infected MDBK cells using RT-PCR.Positive clones were screened by using PCR method with the products checked by enzyme digestion.Sequence analysis showed that the complete genome of BVDV1-3156 was composed of 12 302 nucleotides in length(GenBank accession No.JN704144),which included three regions:a 382 nt 5′-untranslated region(UTR),a 11 694 nt single large open reading frame encoding a polyprotein,and a 224 nt 3′-UTR.Phylogenetic analysis showed that the BVDV1-3156 strain was classified as BVDV type 1b.Blast revealed that the strain shared the highest similarity with VEDEVAC(90.9%) and low similarity to other strains of BVDV1.