Optimization And Biosafety Evaluation Of Recombinant Marek's Disease Virus Expressing The VP2 Gene Of Infectious Bursal Disease Virus

Marek's disease(MD)and Infectious burial disease(IBD)are two highly contagious viral infectious diseases that cause severe economic losses to the world's poultry industry due to high morbidity and mortality.Vaccine immunization is the main means of preventing and controlling MD and IBD.However,the massive use of vaccines has led to the mutation of the virus and the emergence of more virulent strains,and traditional vaccines have been unable to control the development of the disease well.In recent years,genetic engineering vaccine has become an important direction of vaccine research and development,and recombinant live vector vaccine is one of the representative types of genetic engineering vaccines.Because the recombinant live vector vaccine uses transgenic technology,any transgenic microorganism with new target genes and expression products may bring new risks in the process of use.Therefore,for the purpose of protecting human and animal health and ecological environment safety,we must carry out biosafety assessment on transgenic microorganism products.In this study,the recombinant Marek's disease virus expressing the VP2 gene of Infectious Bursal Disease Virus was eliminated the BAC skeleton,and the recombinant virus with the eliminated BAC skeleton was purified by finite dilution method.The optimized recombinant virus was identified by PCR,and the results showed that the BAC skeleton of the recombinant virus was knocked out successfully.The recombinant virus was identified by PCR,Westernblot and IFA,and showed stable expression of the biologically active VP2 protein and good genetic stability.At the same time,the optimized recombinant virus was evaluated for transgenic biosafety.The results of safety tests on target and non-target animals showed that the mental state of test animals in each group was good,with normal development and no significant difference in body weight compared with the control group.The necropsy of the experimental animals showed that the tissues and organs developed normally without swelling or atrophy,and there was no significant difference compared with the control group.There were no obvious pathological changes in the histopathological tests.The results of cohabitation experiment showed that the recombinant virus had very low horizontal transmission ability and could not survive in the external environment.In conclusion,the recombinant virus belongs to GMO safety level I for animal transgenic microorganism and has good safety,which can be used for the development of double vaccine.