Development Of A RT-LAMP Method For Detection Of Infectious Bronchitis Virus And The Screening Of T Cell Epitopes From S1Protein

Infectious bronchitis (IB) is one of the most main disease of the poultry industry.It is an acute, highly contagious respiratory disease caused by infectious bronchitis virus (IBV), a member of Coronaviridae. It is difficult to completely detection and control IBV because the genetic changes of the virus occur far too frequently to develop and provide vaccines of the corresponding serotype. In order to explore the IBV multi phenotype generic detection methods and vaccines, we carried out the following studies:1. To develop a rapid, sensitive and specific approach the detection of infectious bronchitis virus(IBV), reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay with four pairs of primers designed according to the conserved sequences of IBV N gene was established in the present study. With optimized reaction condition, the positive results of RT-LAMP assay were judged through visible green. Subsequently, the specificity was evaluated in detecting IBV, NDV, AIV and MG of different strains. The viral RNA was amplified with Bst DNA polymerase within30min. The sensitivity was10times higher than that of conventional RT-PCR. RT-LAMP assay was specific and simple to operate with no need for special equipments. The approach established here provided a promising method for detection of IBV.2. The aim of the study was to provide a candidate target for screening of T cell epitopes from IBV. The constructed M and SI gene of IBV was subcloned into eukaryotic expression vectors pVAX1, respectively.The positive recombinant plasmids were transfected into Hela cells and detected by Western-blot and IFA. The results showed that the recombinant plasmids were constructed successfully and the transient expression of both genes was confirmed by respective antibodies. After that several SPF chickens were immuned with the recombinant plasmids and respective proteins, ELISA test and vaccination-challenge test were used to analyze the immune response of different groups. It is showed that compared with the PBS group, the immuned with IBV S1and M proteins respectively can induce certain level of antibodies and the immuned with IBV pVAX-S1can provide a higher level of protection than other groups against virulent IBV challenge(p<0.05). 3. To confirm and characterize T-cell epitope of SI protein in IBV different strains.SPF chickens were immunized with DNA vaccine which constructed in last chapter. According to a designed twenty-one single peptides and used to test for IFN-γ production from splenocytes of immunized chickens by an enzyme-linked immuno sorbent (IFN-γ ELIspot) assay. After three rounds of screening, individual peptide9、10、16and17could elicit high expression of IFN-y(p<0.01), which could possibly be T-cell epitope of S1protein in IBV different strains. This study provided important basis to the discovery of new type immune-enhancing vaccine against IBV.