Co-Expression Of IBV-S1 Gene And The Chicken Interleukin-18 Gene In Fowlpox Virus Vector

To study the molecular epidemiological background of infectious bronchitis virus isolated in HeNan and to develop efficient new type vaccine,from HeNan RuZhou we isolated a field strain,(IBV-HN05),from flocks which broke out IB.But because of different genotypes,IB outbreaks frequently in China in vaccined flocks.It is suggested that fowl pox virus as a vector is safty and efficient.We have obtained recombinant viruses rFPV-S1 and rFPV-S1-ChIL-18,it paved the way for the study of immune dose.One pair of primers was designed and synthesized according to chicken infectious bronchitis virus S1 gene sequences in the GenBank. S1 gene of the infectious bronchitis virus ( IBV) Henan isolate (HN05) was amplified by using reverse transcription-polymerase chain reaction (RT-PCR). The purified PCR product was cloned into pGEM-T Easy vector and then was sequenced. The results show that S1 gene sequence of HN05 strain consisted of 1739 bp,containg S1 gene and partial S2 gene. Sequence comparison and analysis Show S1 genes of HN05 and other strains of IBV published previously in the GenBank, shared 99. 8% homology with JAAS, and78.1%-84.2% homology with that of other strains of IBV. Evolution analysis indicated that the HN05 isolate had near relation to JAAS and far relation to other strains of IBV.In order to develop recombinant fowlpox viruses(rFPV) expressing IBV S1,the S1 cDNA was cloned into BamHI site of pSY538 plasmid which contained fowlpox virus early/late promoter LP2EP2, then LacZ which contained fowlpox virus late promoter P11 was cloned into SfiI site of plasmid pSY681.The fragment of S1 was subcloned into NotI site of pSY681 plasmid containing LacZ gene. After construction of transfer vectors,it was transfected on the chicken embryo fibroblasts cell (CEF) pre-infected with S-FPV-017. Then,by selection of blue plaques on the CEF overlaid with agar containing X-gal ,the rFPV-S1 recombinants were obtained,and identified by restriction enzyme analysis and PCR. The results indicated that recombinant fowlpox viruses contained S1 gene and had the stable genetic properties. The expression of S1 by rFPV-S1 was detected in the recombinant virus-infected CEF by indirect immunofluorescence using antibody against IBV. Development of recombinant fowlpox virus paved the way for future study of IBV live vaccine.In order to develop recombinant fowlpox viruses(rFPV) coexpressing chicken IL-18 andIBV S1,chicken IL-18 was cloned into transfer plasmid containing S1 gene. After construction of transfer vectors,it was transfected on the chicken embryo fibroblasts cell (CEF) pre-infected with S-FPV-017. Then,by selection of blue plaques on the CEF overlaid with agar containing X-gal, the rFPV-S1-ChIL-18 recombinants were obtained,and identified by restriction enzyme analysis and PCR. The results indicated that recombinant fowlpox viruses contained ChIL-18and S1 gene and had the stable genetic properties. The expression of S1 by rFPV-S1 -ChIL-18 was detected in the recombinant virus-infected CEF by indirect immunofluorescence using antibody against IBV. The expression of chicken IL-18 by the rFPV-S1-ChIL-18 was detected in the recombinant virus-infected CEF fluid by MTT. The virus titration showed the recombinant rFPV-S1-ChIL-18 has the activity against vesicular stomatitis virus at CEF. Development of recombinant fowlpox virus(rFPV-S1-ChIL-18) paved the way for future study of biological function of expressed product and development of recombinant fowlpox viruses(rFPV) coexpressing chicken IL-18 and protective antigen gene.To summarize,IBV S1gene was obtained, recombinant fowlpox viruses rFPV-S1 was obtained successfully and the expression of S1 was detected in the recombinant virus-infected CEF, recombinant fowlpox viruses rFPV-S1-ChIL-18 was obtained successfully and can enhance the cellular immune responses and antivius. Development of recombinant fowlpox virus(rFPV-S1-ChIL-18) paved the way for the adjuvant effect of chicken IL-18 and recombinant fowlpox viruses(rFPV) coexpressing chicken IL-18 and protective antigen gene.