鸡传染性支气管炎病毒H株纤突蛋白S1基因的克隆及其在毕赤酵母中表达

Cloning of Spike Protein S1 Gene of Avian Infectious Bronchitis Virus H Strain and Its Expression in Pichia pastoris

鸡传染性支气管炎病毒(IBV)河南分离株H,经SPF鸡胚增殖,差速离心纯化病毒,SDS-蛋白酶K法抽提病毒RNA。参照IBVBeaudette株纤突蛋白S1基因序列设计并合成引物,以其进行RT-PCR,成功地扩增出IBVH株S1基因。扩增产物经BstYⅠ、HaeⅢ和PstⅠ酶切分析,结果表明,IBVH株S1基因的RFLP图谱与M41株S1基因的完全一致,初步断定IBVH株为Mass血清型。将IBVH株S1基因克隆于pGEM-T载体中进行序列分析,结果表明该基因全长为1611bp(从ATG到S前体蛋白裂解位点),与标准株M41和Beaudette的S1基因序列相比较,同源率分别达到97.39%和97.27%。将IBVH株S1基因亚克隆到pPICZ-A表达载体,转化毕赤酵母中,SDS-PAGE实验证实了IBVH株S1基因在毕赤酵母中得以表达,进一步用鸡抗IBV血清做Westernblot检测,证实了表达产物的抗原特异性。

Avian ?infectious ?bronchitis ?virus (IBV) H ?strain ?isolated ? from ? Guangdong ?Province ?was ?grown ?in SPF chicken embryos and purified by differential centrifugation and its RNA was extracted by SDS-proteinase K method.According to the spike glycoprotein S1 gene sequence of IBV Beaudette strain,a pair of primers were designed and synthesized.With the primers,IBV H S1 gene was successively amplified by RT-PCR.The PCR product was digested wi簦? Bst Y Ⅰ,Hae  Ⅲ and  Pst  Ⅰ separately.The result showed that the RFLP pattern of IBV H S1 gene was the same as that of M41 S1 gene.IBV H strain was considered as Mass serotype.IBV H S1 gene was cloned into pGEM-T vector and sequenced,the result showed it consisted of 1611 base pairs(from ATG to the possible cleavage site of spike glycoprotein).By comparison,the nucleotide sequence of IBV H S1 gene was 97.39% and 97.27% identical to those of IBV M41 and Beaudette respectively.IBV H S1 gene was subcloned into expression vector pPICZ-A,SDS-PAGE experiment showed it was expressed in  Pichia pastoris.The expressed product had antigen specificity by determination of Western blot with avian anti-IBV serum.