猪繁殖与呼吸障碍综合征病毒河南09分离株ORF5基因的克隆及结构分析

参照GenBank上登录的猪繁殖与呼吸障碍综合征病毒(PRRSV)美洲型AF494042株ORF5基因序列,设计1对特异性引物,应用RT-PCR扩增出PRRSV河南09分离株(HN-09)的ORF5基因,经与pGEM-T Easy载体连接后,筛选出阳性重组质粒进行序列测定,并利用DNASTAR软件对序列进行分析.结果表...

Cloning and structure analysis of ORF5 gene of porcine reproductiveand respiratory syndrome virus of HN-09 strain

A pair of the special primers were designed and synthesized according to the ORF5 sequence of porcine reproductive and respiratory syndrome virus (PRRSV) AF494042 strain published in GenBank. The ORF5 gene of field virulent strain that was collected from Henan province was amplified by RT-PCR and cloned into pGEM-T Easy vector. The nucleotide sequence was determined by sequencing and compared with other ORF5 genes in GenBank, and the structure of ORF5 gene was analyzed too. The results showed that the amplified fragment length was 721 bp. Compared with the corresponding region with other PRRSV strains, the results showed that the nucleotide sequence homology was 93.5% with AF494042 straine. There have high homologies when compared with Shanxi and Shandong isolates, from 97.8% to 99.2 %; and the nucleotide sequence homologies were from 93.4% to 98.7% when compared with Hebei, Anhui and Heilongjiang isolates; while there has the lowest homology when compared with the Henan 2004 isolates, just 88.7%. The further analysis of the field strain was done by the phylogenetic tree. In all of the PRRSV isolates, the HN-09 sequenced strain has closer relation with Shanxi and Shandong isolates, but it showed distant relationship with the Henan isolates in 2004. The composition of amino acid and percentage of secondary structure of GP5 protein were compared analyse between HN-09 and AF494042 isolate using the ANTHEPROT software. The results showed that the composition of amino acid and secondary structure of HN-09 isolate have some difference when compared with AF494042 isolate, but the distribution of secondary structure unit did not change too much.