Infectious bronchitis virus was isolated from swollen proventriculi of clinically ill chicken. The suspected virus samples (2/97, 3/97, 1/98) were adapted in SPF chicken embryos for virus isolation and identification....Infectious bronchitis virus was isolated from swollen proventriculi of clinically ill chicken. The suspected virus samples (2/97, 3/97, 1/98) were adapted in SPF chicken embryos for virus isolation and identification. All the virus isolates were able to agglutinate chicken erythrocytes after treatment with trypsin, and interfer with the reproduction of Newcastle disease virus in chicken embryos, and have low antigenic relat-edness values with reference positive IBV. The isolates 2/97, 3/97, 1/98 RNAs extracted from the allantoic fluid of inoculated embryonated eggs were converted to cDNA by reverse transcription with 3'-primer of S1 gene of (IBV). Polymerase chain reaction (PCR) was performed with two primers which span the S1 gene. Amplified product of 1. 93 kb was subjected to EcoR I and BamH I digestion and the fragments obtained were the same as expected size. The PCR product was ligated to pBlueScript-SK ( + ) vector, and its nucleotide sequence was determined by the dideoxy-mediated chain termination method. Nucleotide sequence analysis showed 73. 6 - 99. 7% homology between the isolated IBV and the IBV strains in GenBank. The homology of amino acid was 71. 4 - 99.4%.展开更多
[Objective] To study the prokaryotic expression and antigenicity identification of S1 gene from avian infectious bronchitis virus （IBV）. [Method] The S1 gene was cloned into a pMD18-T vector to yield a recombinant p...[Objective] To study the prokaryotic expression and antigenicity identification of S1 gene from avian infectious bronchitis virus （IBV）. [Method] The S1 gene was cloned into a pMD18-T vector to yield a recombinant plasmids pMD18-T-IBV-S1. Then S1 gene was inserted into the multiple cloning site of a prokaryotic expression vector pET-32a （ ＋ ）. The recombinant plasmid was transformed into E. coil BL21. The recombinant protein was induced by IPTG and measured by SDS-PAGE and western-blotting. [Result] The S1 gene was successfully expressed in E. coil BL21, the fusion proteins were about 66.0 kDa in a form of inclusion body. Western-blotting test showed that the recombinant proteins could be identified by IBV polyclonal antibody. [ Conclusion] The recombinant proteins of S1 gene have the antigenicity, which lays a good foundation for further research on new generation vaccine of IBV.展开更多
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
文摘Infectious bronchitis virus was isolated from swollen proventriculi of clinically ill chicken. The suspected virus samples (2/97, 3/97, 1/98) were adapted in SPF chicken embryos for virus isolation and identification. All the virus isolates were able to agglutinate chicken erythrocytes after treatment with trypsin, and interfer with the reproduction of Newcastle disease virus in chicken embryos, and have low antigenic relat-edness values with reference positive IBV. The isolates 2/97, 3/97, 1/98 RNAs extracted from the allantoic fluid of inoculated embryonated eggs were converted to cDNA by reverse transcription with 3'-primer of S1 gene of (IBV). Polymerase chain reaction (PCR) was performed with two primers which span the S1 gene. Amplified product of 1. 93 kb was subjected to EcoR I and BamH I digestion and the fragments obtained were the same as expected size. The PCR product was ligated to pBlueScript-SK ( + ) vector, and its nucleotide sequence was determined by the dideoxy-mediated chain termination method. Nucleotide sequence analysis showed 73. 6 - 99. 7% homology between the isolated IBV and the IBV strains in GenBank. The homology of amino acid was 71. 4 - 99.4%.
基金supported by Science and Technology Star Project of Beijing (2005B35)
文摘[Objective] To study the prokaryotic expression and antigenicity identification of S1 gene from avian infectious bronchitis virus （IBV）. [Method] The S1 gene was cloned into a pMD18-T vector to yield a recombinant plasmids pMD18-T-IBV-S1. Then S1 gene was inserted into the multiple cloning site of a prokaryotic expression vector pET-32a （ ＋ ）. The recombinant plasmid was transformed into E. coil BL21. The recombinant protein was induced by IPTG and measured by SDS-PAGE and western-blotting. [Result] The S1 gene was successfully expressed in E. coil BL21, the fusion proteins were about 66.0 kDa in a form of inclusion body. Western-blotting test showed that the recombinant proteins could be identified by IBV polyclonal antibody. [ Conclusion] The recombinant proteins of S1 gene have the antigenicity, which lays a good foundation for further research on new generation vaccine of IBV.
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.
