Evaluation Of Efifcacy Of Recombinant Marek’s Disease Viruses Expressing Optimized Or Different Promoters Controlled F And HN Gene Of New Castle Disease Virus

Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a highly contagious viraldisease of poμltry, and its effects are most notable in domestic poμltry and brings about a lot of losses topoμltry industry. The development of recombinant virus provides new methods to prevent this disease.Marek’s Disease Virus (MDV) is a member of the Alphaherpesvirinae subfamily of Herpesviridae. Thegenome of MDV is about170kb and there are some nonessential regions in the genome. In this studywe construct various transfer-vecters with optimized/wild F gene under different promoter to gotdifferent recombinant viruses to select an economic and commercial recombinant vaccine.First, F gene was inserted into different eukaryotic expression vectors to construct differentcassettes. F gene of F48E9were inserted into pcDNA3.1(+) to get the cassette with gene under controlof CMV promotor; F gene was inserted into pYS2010to get the cassette with gene under control ofCMV enhance and beta chicken actor promoter; The optimized F and HN of F48E9were inserted intopcDNA3.1(+) respectively to get the cassettes with gene under control of CMV promotor.Then gpt selection marker and various cassettes were subcloned into pUAB orderly and thetransfer vectors were constructed: pUAB-gpt-wF(3.1)(+), pUAB-gpt-wF(3.1)(-),pUAB-gpt-wF(YS2010), pUAB-gpt-oF and pUAB-oF-oHN. These transfer vectors were co-transfectedwith MDV total DNA into CEF respectively to generate recombinant MDV. The recombinant MDVwith the foreign gene were obtained by gpt selection and identified by PCR. The expressions of foreigngene were verified by IFA, western blot test in infected CEF. All recombinant MDVs can both expressbiological NDV antigen.At last, all recombinant viruses were immunized into1-day-old chickens with8000PFU/chicken,to evaluate their immune protective effects. In NDV challenge immune protective assay, the protectiveindex of rMDV-wF(+), rMDV-wF(-), rMDV-wF(YS2010), rMDV-oF and rMDV-oF-oHN group were40.00%,42.11%,66.67%,55.00%and68.42%respectively, which indicated that protective index ofrecombinant MDVs is related to the construction of cassettes.In summary, we constructed various recombinant viruses and the expression of NDV genes wereevaluated in vitro and in vivo in this study, which proved optimization of gene and the CMV enhancerwith beta chicken actin promoter can improve the expression of gene and efficiency of recombinantvaccine dramatically.