[Objective]To design and express a recombinant protein rMKIBV incorporating confirmed antigenic epitopes of infectious bronchitis virus(IBV)as a vaccine to provide comprehensive protection.Additionally,it explores the...[Objective]To design and express a recombinant protein rMKIBV incorporating confirmed antigenic epitopes of infectious bronchitis virus(IBV)as a vaccine to provide comprehensive protection.Additionally,it explores the potential of polyclonal yolk antibodies(IgY)harvested from laying hens immunized with the rMKIBV vaccine in the prevention and control of IBV.[Methods]The antigenic epitope sequences of IBV,obtained from online databases,were compared with sequences of representative IBV strains from GenBank.Flexible peptides were designed to link all antigenic peptides.The constructed amino acid sequence was analyzed,reverse-translated,codon-optimized,and then inserted into the pET-28a(+)cloning vector.The recombinant vector was introduced into Escherichia coli for expression.The purified,desalted,and endotoxin-removed rMKIBV protein was used as a vaccine to immunize animals for investigation of its immunogenicity and ability to stimulate specific IgY production in laying hens.[Results]The retrieved IBV antigenic epitope sequences showed high similarity with the published N and S protein sequences of 22 representative IBV strains.The predicted isoelectric point and molecular weight of rMKIBV were 10.25 and 63.39 kDa,respectively.The secondary structure of rMKIBV included a high proportion of random coils,which suggested strong antigenicity.High-purity rMKIBV was obtained from E.coli transformed with the recombinant plasmid pET-28a-mkibv.This protein specifically bound to anti-His-tag antibodies,N protein antibodies,and S protein antibodies.The mice immunized with this protein showed increases in the spleen index(P<0.05),elevations in the levels of serum-specific IgG antibodies(P<0.01)and IFN-γ(P<0.05),and no significant change in the IL-2 level.Immunized laying hens successfully produced IgY in egg yolks,with specific IgY antibody levels significantly increasing.Moreover,the IgY antibody titer gradually rose after immunization,reaching the peak after about 50 days and then gradually declining to reach a stable level.[Conclusion]We successfully constructed and expressed the recombinant protein rMKIBV.The protein demonstrated good immunogenicity,stimulating specific antibody production in both mice and laying hens.Notably,the IgY extracted from the yolks of immunized laying hens offers a novel approach to IBV prevention and control.These findings hold significant scientific and practical value for the development of vaccines against IBV.展开更多
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
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[Objective]To design and express a recombinant protein rMKIBV incorporating confirmed antigenic epitopes of infectious bronchitis virus(IBV)as a vaccine to provide comprehensive protection.Additionally,it explores the potential of polyclonal yolk antibodies(IgY)harvested from laying hens immunized with the rMKIBV vaccine in the prevention and control of IBV.[Methods]The antigenic epitope sequences of IBV,obtained from online databases,were compared with sequences of representative IBV strains from GenBank.Flexible peptides were designed to link all antigenic peptides.The constructed amino acid sequence was analyzed,reverse-translated,codon-optimized,and then inserted into the pET-28a(+)cloning vector.The recombinant vector was introduced into Escherichia coli for expression.The purified,desalted,and endotoxin-removed rMKIBV protein was used as a vaccine to immunize animals for investigation of its immunogenicity and ability to stimulate specific IgY production in laying hens.[Results]The retrieved IBV antigenic epitope sequences showed high similarity with the published N and S protein sequences of 22 representative IBV strains.The predicted isoelectric point and molecular weight of rMKIBV were 10.25 and 63.39 kDa,respectively.The secondary structure of rMKIBV included a high proportion of random coils,which suggested strong antigenicity.High-purity rMKIBV was obtained from E.coli transformed with the recombinant plasmid pET-28a-mkibv.This protein specifically bound to anti-His-tag antibodies,N protein antibodies,and S protein antibodies.The mice immunized with this protein showed increases in the spleen index(P<0.05),elevations in the levels of serum-specific IgG antibodies(P<0.01)and IFN-γ(P<0.05),and no significant change in the IL-2 level.Immunized laying hens successfully produced IgY in egg yolks,with specific IgY antibody levels significantly increasing.Moreover,the IgY antibody titer gradually rose after immunization,reaching the peak after about 50 days and then gradually declining to reach a stable level.[Conclusion]We successfully constructed and expressed the recombinant protein rMKIBV.The protein demonstrated good immunogenicity,stimulating specific antibody production in both mice and laying hens.Notably,the IgY extracted from the yolks of immunized laying hens offers a novel approach to IBV prevention and control.These findings hold significant scientific and practical value for the development of vaccines against IBV.
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.
