牛病毒性腹泻病毒P20和P14基因的克隆及序列分析

参考GenBank中BVDV Oregon C24V株的基因组序列设计两对引物,利用套式PCR方法扩增P20基因,扩增出预期525 bp的目的片段.扩增产物克隆至pMD18-T载体,经酶切鉴定获得阳性重组质粒并对其进行测序.测序结果与参考序列Oregon C24V比较,二者的核苷酸同源性仅为80.95%,推导氨基酸同源性为87.50%.测序结果经NCBI上的Blast(Http:/www.ncbi.nlm.nih.gov/BLAST/)同源性比较,克隆得到的基因与Osloss株同源性最高,核苷酸同源性为93.65%,推导氨基酸同源性为95.83%.根据P20的测序结果,参照GenBank中BVDV Osloss株设计一对引物,扩增P14基因,经同源性比较,扩增的P14基因与Osloss株核苷酸同源性为94.77%,推导氨基酸同源性为95.10%,通过系统发生分析,推测P20基因和P14基因与Osloss株在进化上比较接近.

Cloning and sequence analysis of the P20 and P14 gene of bovine viral diarrhea virus

According to the sequence data of BVDV Oregon C24V strain published by GenBank,two set of primers were designed and used to amplify P20 gene by the method of nested polymerase chain reaction(PCR).A specific 525 bp DNA product was amplified,which was cloned into pMD18_T vector,and the positive recombinant clone was identified by restriction enzyme digestion.The recombinant plasmid was sequenced and compared with P20 gene of Oregon C24V strain.The nucleotide homololgy was 80.95 %,and the deduced amino acid homology was 87.50 %.Compared with blast on line (http://www.ncbi.nlm.nih.gov/bLAST/),the P20 gene of Osloss_like strain exhibits the highest homology with Osloss strain,they shares 93.65 % nucleotide sequence identity and 95.83 % amino acid identity.According to the sequencing result of P20 gene,one set of primers were designed for amplifying P14 gene on base of BVDV Osloss strain published by Genbank.The P14 gene of Osloss_like strain shares 94.77 % nucleotide sequence identity and 95.10 % deduced amino acid identity with Osloss strain.It suggests that P20 and P1 gene was phylogeneticly closer to BVDV Osloss strain after phylogenetic analysis.