鸡传染性支气管炎病毒N基因片段的RT-PCR扩增及限制性内切酶分析

The RT-PCR amplification and restriction endonuclease analysis of IBV N gene fragment

根据鸡传染性支气管炎病毒 (IBV)的已报道基因序列设计了 1对可扩增 N基因片段的引物 ,并得到了预期的 438bp扩增产物 ;将目的条带纯化并回收后 ,克隆到 PMD18- T质粒载体中 ,对插入片段进行序列测定 ,并与已发表的 IBV N基因序列进行了比较 ,证实扩增和克隆的产物是正确的 ;根据扩增的 N基因的序列 ,选择了 Bst X 酶对 IBV进行分析 ,发现 M41和澳大利亚 T株 Bst X 酶切图谱存在明显的差异 ,从而建立了一种新的鉴定 IBV病毒的方法。

We designed a pair of primers to amplify the N gene fragment of IBV M41 and T strain based on the reported gene sequences.The anticipated 438 bp products were acquired.After purification and recovery,the products were recombined into PMD18 T vector and sequenced.The sequence were compared with the reported N gene sequences and testified that the RT PCR products were correct.According to the sequences,we found some restriction endonuclease sites.At last,we selected Bst XⅠ enzyme to analyze the two IBV strains and found the maps of the two strains were significant different.So we can use this method (RFLP) to identify and distinguish IBV M41 and T strain.