Acording to Avian Infectious Brochitis virus( IBV) Beaudette strain S1 gene sequence, a pair of primers were designed and synthesized. With the primers , IBV Guangdong isolation strain GD05 S1 gene was successively am...Acording to Avian Infectious Brochitis virus( IBV) Beaudette strain S1 gene sequence, a pair of primers were designed and synthesized. With the primers , IBV Guangdong isolation strain GD05 S1 gene was successively amplified by RT-PCR.PCR product was digested with BstY I,\%Hae\% III and \%Pst\% I respectively, the result showed RFLP pattern of was the same as that of M41 S1 gene. IBV GD05 strain was thought as Mass serotype primaryly. IBV GD05 S1 gene was cloned into pGEM-T vector and sequenced, its sequence was consisted of 1611 base pairs. By comparison , the nucleotide sequence was 97.14% identical to that of IBV M41. IBV GD05 S1 gene was subcloned into expression vector pET21d. SDS-PAGE experiment showed that it expressed in \%E.coli.\%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Acording to Avian Infectious Brochitis virus( IBV) Beaudette strain S1 gene sequence, a pair of primers were designed and synthesized. With the primers , IBV Guangdong isolation strain GD05 S1 gene was successively amplified by RT-PCR.PCR product was digested with BstY I,\%Hae\% III and \%Pst\% I respectively, the result showed RFLP pattern of was the same as that of M41 S1 gene. IBV GD05 strain was thought as Mass serotype primaryly. IBV GD05 S1 gene was cloned into pGEM-T vector and sequenced, its sequence was consisted of 1611 base pairs. By comparison , the nucleotide sequence was 97.14% identical to that of IBV M41. IBV GD05 S1 gene was subcloned into expression vector pET21d. SDS-PAGE experiment showed that it expressed in \%E.coli.\%
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.
