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
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国家质检局科技计划项目(2009IK029)


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

    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。结果表明,扩增片段长度为721 bp,与预期目的片段大小一致,测序验证其为PRRSV的ORF5基因。序列分析表明,HN-09株与AF494042的同源性为93.5%,与山西、山东近年分离株的同源性为97.8%~99.2%,与河北、安徽、黑龙江等省分离株的同源性为93.4%~98.7%,但与2004年河南分离株的亲缘关系较远,其同源性为88.7%。用ANTHEPROT软件分析HN-09株和AF494042株编码蛋白质氨基酸的组成和二级结构的含量及分布,结果表明, HN-09株中碱基的突变,导致其氨基酸序列与美洲型有所不同,形成的二级结构略有差异,但二级结构元件的大致分布与美洲型相同。

    Abstract:

    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.

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杨春华,胡慧.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[J].湖南农业大学学报:自然科学版,2011,37(1):73-77.

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  • 收稿日期:2010-07-05
  • 最后修改日期:2010-10-26
  • 录用日期:2011-01-25
  • 在线发布日期: 2011-01-31
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