Using Cre-loxP System To Self-excise Reportor Gene In Recombiant Fowlpox Virus Expressing Hemagglutinin Gene Of Subtype H5 Avian Influenza Virus

Green fluorescence protein and Lacz as a report gene are usually needed for selection and purification during the construction of recombiant virus. However, these recombniant viruses with reporter gene didn't accord with commercial biosafe criterion in genetic engineering vaccine. In this research, Cre-loxp systerm was used to self- excise reportor gene in recombiant fowlpox virus to make it express hemagglutinin gene of subtype H5 avian inluenza virus only. Finally the virus was gained. Its efficacy was testified by testing hemagglutinin inhibit.1. Construction of universal delivery vector for fowlpox virusThe recombinant arm gene was amplified by polymerase chain reaction (PCR) with extracted total DNA from fowlpox virus as template. The expression cassette including GFP gene controlled by FPV early and late promoter was cloned into F30 gene in fowlpox virus to gain the recombinant fowlpox virus expressing GFP. The GFP gene was self-excised from the recombinant fowlpox virus by homologous recombination mediated by Cre recombinant enzyme.The results proved the Cre-loxp system had advantage in removing the report gene of the recombinant fowlpox virus expressing target gene.2. Construction of recombinant fowlpox virus expressing hemagglutinin gene of subtype H5 avian influenza virusHemagglutinin gene of subtype H5 avian influenza virus was amplified by PCR to construct expression cassette containing FPV early and late promoter and SV40 polyA tail. Then vector was constructed by cloning hemagglutinin gene of subtype H5 and GFP gene into fowlpox virus corecombinant arm gene. The vector and LipofectamineTM 2000 were transfected into CEF cells preinfected FPV 282E4 strain virus. Recombinant fowlpox virus expressing the green fluorescence protein and hemagglutinin gene of subtype H5 was screened and purified many times in CEF cells. After...