传染性支气管炎病毒山东分离株S_1基因的克隆及序列比较分析

Cloning and Sequencing Analysis of S_1 Gene of the Isolated Strain of IBV in Shandong Province

参照Genbank中传染性支气管炎病毒 (IBC)M41纤突蛋白S1的基因序列 ,设计一对引物 ,对山东地区IBV分离株RNA进行反转录_聚合酶链反应 (RT_PCR) ,成功扩增出约 16 34bpS1基因片段。将扩增出的S1基因分别克隆到TEasy质粒载体中 ,获得重组质粒 ,提取质粒DNA ,利用Eco .RI酶切证实了重组质粒的特异性 ,并进行序列测序。克隆出的序列已被GeneBank收录 ,序列号为 :AY0 4 3313。将得到的序列与标准株M41进行核酸序列和氨基酸同源性比较分析 ,与M41的核苷酸同源率99 %。利用Omigo 2 .0软件对克隆出的山东IBV进行了理论酶切位点分析 ,在BstYI、AluI和BgⅢ位点与M41不同。结合血清学和分子生物学实验结果推测

A pair of primers of S1 gene of the infectious bronchitis virus strain M 41 were designed according to the Gene Bank.The IBV strain isolated from Shandong(A) was amplified by reverse transcription_polymerase chain reaction(RT_PCR).The amplified product was 1634bp,then get the recombinant plasmid by cloning the S1 gene into vector plasmid T Easy.Restriction endonuclease fragment length polymorphism analysis proved the speciality of the RT_PCR and the recombinant plasmid.Compared homology of the nucleic acid and amino acid sequence of the isolate with the IBV standard strain M 41 .The homologous rate of the nucleic acid sequence between the two stains was 99%.The homologous rate of the amino acid between the two strains was 97%.The sequence has been received by the Gene Bank(accession №AY043313).Analysis the sequence of the Shandong IBV with soft ware Omiga 2.0,we found that some difference between the isolate with M 41 in BstyI,AluI and BgⅢ.Those results combining serology test suggested that the isolate(A) was a variant strain of M 41 .