我国鸡传染性支气管炎病毒地方分离株S1基因的分离及初步鉴定

The Amplification of the S1 Gene of Five Chinese Avian Infectious Bronchitis Virus Isolates and Identification

根据IBVBeaudette株全基因组序列 ,借助基因分析软件自行设计合成了 3条引物 (you1+、you2 -、youM +) ,引物you1+和you2 -在S1基因两侧 ,跨幅为 16 11bp ,引物you2 -和youM +在S1基因的 3’端 ,跨幅为 5 35bp 用引物you1+和you2 -对 5个IBV地方分离株 (HN2、HN4、JX1、SC2和SC4)进行RT PCR ,均成功地扩增出预期大小的目的片段 用另 1对引物 (you2 -和youM +)对各毒株的RT PCR产物再进行PCR扩增 ,均得到一条约 5 30bp的目的片段 ;对RT PCR产物用限制性内切酶PstⅠ进行酶切分析 ,结果酶切产物中得到 2条条带 ,大小分别为 5 6 0和 10 5 0bp ,与GeneBank中 11株已发表的S1基因分析结果一致 初步鉴定结果显示 ,已经成功分离得到了IBV S1基因 .

According to the sequence of the genome of the Beaudette strain of IBV, and with a gene analysis program, three primers (you1+、you2- and youM+) were designed and synthesized. The primers you1+ and you2-were located respectively in the 5'most and the 3'most end of the S1 gene and approximately cover 1 611 bp and the primer youM+ lying in the middle of the S1 gene. RTPCR was used to amplify the S1 gene fragment of the five Chinese IBV isolates(HN2,HN4,JX1,SC2 and SC4) using the primers you1+ and you2-. The result indicated that the length of the RTPCR products was the same as expected. A sequence of 535 bp was amplified by PCR using the primers you2- and youM+ when every RTPCR product of the five IBV isolates was used for template. The RTPCR products were digested with the restriction enzyme PstⅠ and two fragments (560 bp and 1 050 bp) were seen, which were the same for the 11 published S1 sequences of IBV strains in Genebank. Therefore , all these results demonstrate that the S1 gene fragments of the five IBV isolates were isolated successfully.