Molecular Epidemiology Of Guangxi Avian Infectious Bronchitis Viruses And The Immuneresponse Of Uniquitin Fusioned With Virual Main Antigen Genes

Infectious bronchitis is currently one of infectious diseases in chickens caused by infectious bronchitis virus(IBV).IBV cause the corresponding organization epithelial cell lesions and damage by violations chicken respiratory tract intestinal fallopian tubes and kidney,and serious damage when secondary pathogens like bacteria.IB is one of important causes of the biggest common respiratory syndrome in the chicken production.IBV has high potential of point mutation,gene insertion and recombination between different sources IBV genome happened easily,the result is that the biological characteristics such as serotype,genotype and pathogenicity are extremely easy to change,causing the clinical manifestations of IB were complicated and serotype was numerous.It also causes the failure of immune protection and control in poultry production with great difficulties due to poorer cross-protection between a number of IBV serotypes.The disease is still emerge frequently in Guangxi and could result in significant economic losses to the poultry though flocks were immunized with IB vaccines for several times.The purpose of this study was to help understand IB infection and epidemic circumstances of the poultry industry in Guangxi and prove up the distribution of major genotypes of IBV epidemic strains,transmission prevalent and damage study at the molecular level.The technology platform of fusion gene inoculated with ubiquitin and IBV DNA multivalent vaccines were constructed to improve the immune effection.The research main contents are as follows:1.IBV molecular epidemiological during 1985-2012 in Guangxi.We isolated sixty IB strains which were collected from major chicken-producing area in Guangxi during 1985-2012.The spike protein(S1),membrane protein(M),nucleoprotein(N)and 3' UTR gene sequences were analyzed and compared by RT-PCR.We also constructed nucleotide phylogenetic trees to analyze the evolutionary relationship between the sixty IBV Guangxi isolates and reference strains.The results showed that gene mutations,insertions or deletions were commonly found in these genes.Based on phylgenetic relationship,Guangxi isolated strains were divided into several groups and far away from vaccine strains used in China and abroad.This may be one of the main reasons why vaccine failure and genomic variation of Guangxi isolates occurred.With higher nucleotide identity in the same period and no differences or small differences in regional evolutionary relationship between S1?N and M genes,the viral genes is in a special evolution process.There were 7 different serotypes from the serotyping of sixty strains in Guangxi.The results demonstrated that there were different multiple IBV serotypes circulated in recent years,which maybe the main reason for the IB outbreak in Guangxi.The relationship between genotypes and serotypes is also discussed.2.Complete genome sequencing and bioinformatics analysis of avian infectious bronchitis viruses isolates GX-NN09032 and GX-C isolated in Guangxi in different period.According to molecular epidemiological studies,sequence of the complete genome GX-C and GX-NN09032 which were isolated in different period in Guangxi.Primer design used the whole IBV genome sequence as a reference which has released in GenBank.The 5'-terminal and 3'-terminal sequence of the two isolates genome was obtained with the commercial kit of 5' RACE and 3' RACE.The whole genome sequence of GX-C and GX-NN09032 were 27 701 bp and 27 684bp in length,respectively.The bioinformatics analysis of isolates GX-C and GX-NN09032 including analysis of insertion and deletion of nucleotide sequence,and homology analysis of whole genome sequence,construction of phylogenetic tree of whole genome nucleotide sequence and structure proteins segment amino acid sequence.The whole genome of GX-C and GX-NN09032 in the nucleotide sequence have the highest identity with the H120 and GX-YL5,respectively.Recombination phenomena between the isolates and reference strains was found by phylogenetic analysis in structural proteins.Obvious gene recombination and gene deletions in S and 3' UTR genes of GX-NN09032 were observed by further analysis.3.The immune response of infectious bronchitis virus main antigen gene with Ubiquitin gene.In this research,Ub?S1 and N gene of GX-YL5 were expressions and distributions,then these genes were inserted into a eukaryotic expression vector pVAXl,to construct these fusion expressing plasmids including pVAX1-N?pVAX1-Ub-linker-N?pVAX1-Ub-linker-N-S1 and pVAX1-N-S1.Furthermore,a flexible linker which could express the(G4S)3 polypeptide was used to link the Ub gene with N and S1 gene to construct fusion gene plasmid of eukaryotic expression vector.Eukaryotic expression vector were confirmed by restriction enzyme digestion and DNA sequencing.Target gene transcription and expression in Vero cell were detected by indirect immunofluorescence test and RT-PCR.The chicks immune indexes were detected in different treatment groups by Flow cytometry?qPCR and ELISA.Differences between the different plasmid immune protection effects were compared by toxicity attack test.Four plasmids were transcripted into target gene in cells that can be amplified specific N gene fragment by RT-PCR.The results of the transient transfection of these four fusion plasmids in vitro showed that the fused gene could express together in Vero cells.Therefore,it proved that the protein expressed by fusion gene could retain the bioactivities of proteins.The experimental chicken results showed that most of immune response parameters of four inoculated groups are higher than the control group,especially,the pVAX1-Ub-linker-N and pVAXl-Ub-linker-N-S1 groups which involved Ub.The details were as follows:the number of CD4+ and CD8+ T cell,the levels of IBV antibody in peripheral blood and IL-12?IFN-? mRNA expression in spleen of immune groups were all higher than those of control group(P<0.05).And the pVAXl-Ub-linker-N-S1 group was the most significant among those groups.Challenge study showed that immune protection ratio appear differences between immune protection ratio of different immune groups.Potection ratio is higher for which group has the high cellular immune response level.Conclusion:The results showed that IBV gene is in the process of genetic derivation and evolution,IB vaccine candidate strain can be determined with serotyping results.The evolution of IB is related to the epidemic status of IBV strain in the same period by analysing the complete Genome sequence of isolates from different period.A technology platform of IBV DNA multivalent vaccines of Ubiquitin fussion was performed by constructing eukaryotic expression plasmid of infectious bronchitis virus main antigen gene with Ubiquitin gene and studying immune response.Ultimately Provide the technical platform and theoretical basis for the development of efficiency and security of new vaccine.