The recombinant baculovirus expressing S1 glycoprotein of nephropathogenic strain JS/95/03 of infectious bronchitis virus was generated by using the Bac-to-Bac baculovirus expression system.The BamH I-Sal Ⅰ fragment ...The recombinant baculovirus expressing S1 glycoprotein of nephropathogenic strain JS/95/03 of infectious bronchitis virus was generated by using the Bac-to-Bac baculovirus expression system.The BamH I-Sal Ⅰ fragment containing S1 gene from the recombinant plasmid pMDJS9503S1 was purified and cloned in frame into the baculovirus transposing vector pFASTBAC HTa under the polyhedrin gene promoter.The recombinant transposing plasmid pFASTJS9503S1 was screened and then transformed into Escherichia coli DH10BAC.The resulting recombinant bacmid rBacmidJS9503S1 was transfected into cells of the insect Spodoptera frugiperda(Sf9)and the recombinant baculoviruse rAcJS9503S1 was obtained.The lysates of cells infected with rAcJS9503S1 were analyzed by SDS-PAGE and the expressed product of S1 gene was detected by Western bloting and immunofluorescence assay(IFA).The results showed the recombinant baculovirus was fully capable of expressing S1 glycoprotein of JS/95/03.Maybe owing to the incomplete glycosylation in insect cells,the S1 gene product had a Mr of only 61000.In immunofluorescence test and Western blotting,the expressed product could react with polycolonal antibody against IBV M41 strain,indicating it possessed the antigenic properties specific for native S1 glycoprotein.展开更多
利用 Bac- to- Bac杆状病毒表达系统构建了 2株重组杆状病毒 r Ac JS95 0 3S1和 r Ac SD970 1S1,分别表达了 2株致病性不同的传染性支气管炎病毒的 S1基因。用感染重组杆状病毒的昆虫细胞裂解液对 2周龄的伊莎公雏进行免疫 ,根据鸡免疫...利用 Bac- to- Bac杆状病毒表达系统构建了 2株重组杆状病毒 r Ac JS95 0 3S1和 r Ac SD970 1S1,分别表达了 2株致病性不同的传染性支气管炎病毒的 S1基因。用感染重组杆状病毒的昆虫细胞裂解液对 2周龄的伊莎公雏进行免疫 ,根据鸡免疫后的抗体水平动态变化和攻毒后鸡肾脏和气管的保护力判定它们的免疫原性。结果显示 ,重组杆状病毒表达的 IBV S1蛋白可以诱导抗体产生和免疫保护反应 ,2毒株间也存在着一定程度的交叉免疫保护反应。展开更多
In this study, the genes encoding main structural proteins and 3′ untranslated region (UTR) of a commercial domestic vaccine strain H52 and a commercial foreign vaccine strain H52 of infectious bronchitis virus (IBV)...In this study, the genes encoding main structural proteins and 3′ untranslated region (UTR) of a commercial domestic vaccine strain H52 and a commercial foreign vaccine strain H52 of infectious bronchitis virus (IBV) were obtained by RT-PCR, then were cloned and sequenced respectively. The sequence analyses showed the genetic information of S1 gene, M gene, N gene and 3′UTR of domestic vaccine strain H52 was remarkably different from that of foreign H52 strain and that of published sequences of H52 strain. Compared with foreign H52 strain, this domestic H52 strain was genetically closer with M41 strain and was located in the same branch of phylogenetic tree based on the sequences of main structural protein genes. The results strongly suggested that it was necessary to build genetic information archives of seed stock of IBV vaccine strains.展开更多
文摘The recombinant baculovirus expressing S1 glycoprotein of nephropathogenic strain JS/95/03 of infectious bronchitis virus was generated by using the Bac-to-Bac baculovirus expression system.The BamH I-Sal Ⅰ fragment containing S1 gene from the recombinant plasmid pMDJS9503S1 was purified and cloned in frame into the baculovirus transposing vector pFASTBAC HTa under the polyhedrin gene promoter.The recombinant transposing plasmid pFASTJS9503S1 was screened and then transformed into Escherichia coli DH10BAC.The resulting recombinant bacmid rBacmidJS9503S1 was transfected into cells of the insect Spodoptera frugiperda(Sf9)and the recombinant baculoviruse rAcJS9503S1 was obtained.The lysates of cells infected with rAcJS9503S1 were analyzed by SDS-PAGE and the expressed product of S1 gene was detected by Western bloting and immunofluorescence assay(IFA).The results showed the recombinant baculovirus was fully capable of expressing S1 glycoprotein of JS/95/03.Maybe owing to the incomplete glycosylation in insect cells,the S1 gene product had a Mr of only 61000.In immunofluorescence test and Western blotting,the expressed product could react with polycolonal antibody against IBV M41 strain,indicating it possessed the antigenic properties specific for native S1 glycoprotein.
文摘In this study, the genes encoding main structural proteins and 3′ untranslated region (UTR) of a commercial domestic vaccine strain H52 and a commercial foreign vaccine strain H52 of infectious bronchitis virus (IBV) were obtained by RT-PCR, then were cloned and sequenced respectively. The sequence analyses showed the genetic information of S1 gene, M gene, N gene and 3′UTR of domestic vaccine strain H52 was remarkably different from that of foreign H52 strain and that of published sequences of H52 strain. Compared with foreign H52 strain, this domestic H52 strain was genetically closer with M41 strain and was located in the same branch of phylogenetic tree based on the sequences of main structural protein genes. The results strongly suggested that it was necessary to build genetic information archives of seed stock of IBV vaccine strains.
