Construction Of Recombinant Marek's Disease Virus Expressing Newcastle Disease Virus F Gene And Evaluation Of Its Immunoprotective Potency

Newcastle disease(ND)is a severe avian infectious disease caused by a virulent strain of Newcastle disease virus(NDV).The fatality rate of ND is extremely high,which causing huge losses to the global poultry industry economy and until now the most effective control method is vaccination.Although the traditional attenuated LaSota vaccine and the attenuated genetic VII virus independently developed in China are still the most used vaccines in China,the continuous outbreak of atypical Newcastle disease in recent years shows the insufficiency of existing vaccine.It demonstrates that the existing vaccines cannot resist the interference of maternal antibodies very well,and the high antibody level lasts for a short time,requiring multiple immunizations,which increasing economic costs and stress response and weakening the vaccine immune response.Therefore,it is urgent to develop a new vaccine for Newcastle disease to make up for the shortage of traditional vaccines.Recombinant live viral vector vaccine is a direction of new vaccines research,which can achieve the purpose of preventing multiple diseases with one injection and reduce stress response.In this study,we use Marek's disease virus as a live viral vector to construct a recombinant virus expressing F protein of Newcastle disease.It can effectively resist the interference of maternal antibodies and achieve a lifelong effect of one injection,which lays the foundation for the research of new Newcastle disease vaccine.1.Construction of recombinant Marek's disease virus expressing the eGFP geneIn this study,eGFP was used as the reporter gene and transferred into EL250 competent cells by electrical transformation to replace the Kan-SacB gene.Positive clones of successful recombination were obtained by double antibiotic screening and digestion of restriction enzyme,and then the BAC plasmid identified correct was transfected into CEF cells to rescue the recombinant virus.After 4-5 days transfection,CEF cells developed a typical MDV-infected cytopathy where appeared green fluorescence under the fluorescence microscope,which indicating that eGFP was successfully inserted into the MDV genome and expressed well.2.Construction of recombinant Marek's disease virus expressing the NDV-F geneIn this study,by inserting eGFP in the MDV genome,a recombinant virus expressing eGFP was successfully obtained,which verified the feasibility of inserting an exogenous gene at the gpt gene expression location.So the same method can be used to insert the F gene of Newcastle disease in the MDV genome.In this study,using the pcDNA 3.1-F plasmid constructed and preserved in our laboratory as a template,the F gene expression cassette with a 50 bp homologous recombinant arm was amplified and transfected into CEF cells to verify its expression in the cells.Subsequently,competent cells were prepared with pPoul vacc Kan-SacB EL250,and the F gene expression cassette was transferred into the competent cells via electrical transformation to replace the Kan-SacB gene by homologous recombination.The positive recombinant bacteria were screened by antibiotics,PCR identification and restriction enzyme digestion.Finally,we obtained three positive clones and further transfected into CEF cells to rescue the recombinant virus rCVI988-NDV-F.For 15 consecutive generations of viruses,PCR and IFA were used to identify the genetic stability of recombinant viruses and the expression of foreign genes every 5 generations.The results showed that recombinant viruses were stablely inherited in vitro and the expression of exogenous genes was stable.The growth characteristics determination results suggested that compared with the parental strain CVI988,the growth and replication ability of recombinant virus rCVI988-NDV-F in vitro was not affected.The insertion site was consistent with the expectation by sequenced.Therefore,we obtained a recombinant Marek's disease virus that can grow normally in CEF cells and stably express the NDV-F gene.3.Evaluation of immunoprotective efficacy of recombinant virus rC VI988-NDV-FIn this study,a recombinant Marek's disease virus rCVI988-NDV-F expressing the NDV-F gene was successfully constructed and its immunoprotective efficacy was further evaluated.Eighty 1-day-old SPF chicks were randomly divided into 4 groups of 20 in each group,two group was vaccinated with 5,000 PFU and 10,000 PFU recombinant viruses at 1 day age,respectively.In the 2nd,3rd and 4th weeks after immunization,serum samples from the immune group and negative control group were collected to detect the NDV-F specific antibody levels by commercial kit,and the results showed that 2 weeks after immunization,part of chickens produced antibodies gradually.Moreover,antibody levels increase over time and in week 4,more than 80%of the chicken produced antibodies,and the antibody levels reached the highest level of the three tests time.On the 28th day after immunization,the two immune group and positive control group were challenged with Newcastle disease virus(F48E8 strain)at 104ELD50.The mortality rate of positive control group achieved 100%,and the protection rate of the 5000 PFU and 10000 PFU immune groups was 90%and 100%,respectively.Taken together,the rCVI988-NDV-F has good immunoprotective efficacy against NDV in SPF chickens.