Construction Of Recombinant Marek's Disease Virus Strain 814 Expressing The Green Fluorescent Protein

Marek's disease (MD) is a highly contagious malignant lymphomatous disease of chickens, causedby Marek's disease virus (MDV). MDV vaccine strain is a kind of favourable viral vector. using therecombinant multivalency vaccine constructed with it to induce immune, can attain the destination ofpreventing one or multiple diseases of Avian.MDV serotypeâ… (MDV-1) strain 814 with better immunogenicity is a natural low virulent strainisolated from China, and used as vaccine in longterm. We chose it as the viral vector to constructrecombinant virus is profit to the prevention and cure of MD and other Avian diseases. The recombinantvirus which was constructed with MDV serotypeâ… (MDV-1) strain 814 as the viral vetor, could beused for the prevention and cure of MD and other Avian diseases. Meanwhile, the recombinant viruscan be used to research the functions of MDV genes and the tumorigenesis mechanisms of MD.The green fluorescent protein (GFP) gene cassette, Newcastle disease virus(NDV) Fusion(F) genecassette and Avian Influenza virus(AIV)Hemagglutinin(HA) gene cassette were inserted into MDV-1strain 814 US10 gene, respectively, to get three kinds of recombiant transfer vector plasmid. Arecombinant MDV strain 814 expressing GFP was constructed instantaneously.According to the US1-US10-SORF3 genes sequences of the strain GA of MDV-1 published inGenBank, 2 pairs of primers were designed to amplify two fragments by PCR method. The upperprimer of the first fragment located at the central region of US1 gene and the lower primer located at theupstream of US10 gene, the upper primer of the second fragment located at the central region of US10gene and the lower primer located at the upstream of SORF3 gene, a restriction enzyme site wasintroduced into the 5' end of each primer. The two PCR products cloned into the plasmid pUC19 in turnto construct the recombinant MDV transfer vector plasmid named pUC-US10. The result of sequenceanalysis showed that the nucleotide sequence of MDV-1 strain 814 is highly homology with the samelocation of strain GA.Based on the sequence of commercial plasmid pEGFP-N1, a pair of primers was designed, arestriction enzyme site was introdunced into the 5' end of each primer. The GFP gene expressioncassette from the plasmid pEGFP-N1 was amplified by PCR method with the primers located at theupstream of promoter and the downstream of polyA. The PCR products including the CMV promoter,SV40 polyA and GFP open reading frame. The GFP gene expression cassette was inserted into the Sphlsite of the vector plasmid pUC-US10, the recombinant MDV transfer vector plasmid namedpUC-US10-GFP including the GFP gene expression cassette was obtained.Two pairs of primers specific for NDV F gene expression cassette and AIV HA gene expressioncassette corresponding to the plasmids pEGFP-F and pCIHA sequences were designed. PCR products were inserted into the Sphl site of pUC-US10, respectively, then the recombinant plasmids namedpUC-US10-F and pUC-US10-HA were obtained.The DNA of recombinant MDV transfer vector plasmid pUC-US10-GFP and MDV strain 814 totalDNA cotransfected secondary generation chick embryo fibroblast (CEF) with calcium phosphateprecipitation method, screened the plaques given off green fluorescence, gained the recombinant MDVstrain 814 expressing GFP. Identification the GFP cassette by PCR showed that GFP gene expressioncassette was inserted into the MDV-1 strain 814 genome DNA. After passaged 20 generations in CEF,the GFP gene expression cassette can be detected from the recombinant virus, showed that therecombinant virus generated stably in vitro. The detection result of the plaque number of therecombinant virus showed that the recombinant virus was familiar with the parental virus in the aspectof growth status and so on.The recombinant MDV strain 814 expressing GFP were successfully constructed, which is benefitfor research to MD vaccine and MDV gene function. We also constructed rencombinant plasmidscontained AIV HA gene and NDV F gene, which made a preparation for getting recombinant MDVstrain 814 expressing AIV HA protein or NDV F protein. AIV HA protein and NDV F protein all haveexcellent antigencity, they can induce humoral immunity efficiently, in that we can attain the destinationof prevent one disease or multiple diseases of Avian through constructed recombinant virus expressingthe proteins.