Construction Of Meq Gene Deletion Strain Of MDV Vaccine RMDV-LTR Strain By CRISPR/Cas9 Gene Editing System And Conditional Exploration Of Knock-in Platform

Marek’s disease(MD)is a widespread and highly contagious chicken lymphoproliferative disease caused by Marek’s disease virus(MDV).MDV mainly causes chicken T-cell lymphoma,accompanied by edema and extensive damage to the nervous system,eventually leading to paralysis and death of chickens,causing huge economic losses to the poultry industry in china and the world.Although the vaccination of MD vaccine has achieved temporary success,with the continuously increasing of MDV virulence,the prevention and treatment of the disease is still very difficult.Meq gene is the most important oncogene of MDV,which can be continuously expressed during latent and lytic infection and directly induce tumorigenesis.The deletion of Meq gene does not affect the replication of MDV.so the recombinant MDV vaccine with the deletion of Meq gene and insertion of foreign genes targeting Meq gene has a good development prospect.CRISPR/Cas9 gene editing technology,as a rapidly developing gene editing method in recent years,has been successfully used to edit large DNA virus genome,providing new ideas for the research of MDV functional genes and the construction of recombinant vaccines.By comparing the immunization of a live MDV vaccine(rMDVLTR strain)incorporating the long terminal repeat(LTR)of avian reticuloendotheliosis virus(REV)with the parental strain(CVI988/Rispens),it is found that the rMDV-LTR strain has good immune protection efficiency,and is a safe and effective vaccine,and can be used as a vaccine carrier strain.By constructing the prokaryotic expression plasmid p ET28(a)-Meq expressing MDV Meq protein,BALB/C mice were immunized with the induced product,and polyclonal antibody serum was obtained which could be used for IFA detection,providing a detection method for the study of MDV Meq gene function.Using CRISPR/Cas9 gene editing technology,the rMDV-LTR strain was used as the research model,constructed knockout plasmids targeting MDV Meq gene,and verified that px-sg RNA11 and px-sg RNA12 plasmids had good cutting effect;single plaques were continuously selected for subculture,and finally a MDV Meq gene deletion strain was obtained;the MDV gene deletion strain has been successfully edited,and has similar plaque morphology and replication ability with the parent virus.This experiment demonstrates the effectiveness and high efficiency of CRISPR/Cas9 technology in viral gene editing,and provides a simple and effective method for accelerating the study of MDV gene function.In order to rapidly develop MDV gene recombinant vaccine with high safety and good immune effect,the Knock-in platform with the m Cherry expression cassette inserted at the cut of MDV Meq gene was explored by using CRISPR/Cas9 homologous recombination repair method.By constructing and continuously optimizing the Donor plasmid,puromycin resistance and m Cherry reporter gene can be used as means to improve the screening efficiency of positive infected cells.However,the cells still contained parental virus and recombinant virus,indicating that it is still difficult to screen and purify the recombinant virus constructed at Meq gene,and further optimization and improvement are still needed.This study can provide a reference for the rapid development of multivalent recombinant MDV vaccine containing protective antigen genes of avian pathogens based on CRISPR/Cas9 technology.In conclusion,the results show that the immune protection efficiency of the MDV live vaccine rMDV-LTR strain integrating REV-LTR gene is higher than that of the parental CVI988/Rispens strain,indicating that the recombinant rMDV-LTR strain has a good immune effect,and was selected as the carrier for subsequent experiments.The polyclonal antibody for detecting MDV Meq protein was successfully prepared with good specificity;MDV Meq gene deletion strain rMDV-LTR-（？）Meq was successfully obtained by CRISPR/Cas9 gene recombination technology,which has similar erosion morphology and replication ability with the parental virus;and the conditions of MDV Knock-in platform were explored and finally the dual screening conditions of fluorescent gene and puromycin drug were determined to improve the screening efficiency of positive infected cells to a certain extent.The screening efficiency of the Knock-in platform was successfully constructed,which is important for the rapid development of new multivalent recombinant vaccines expressing protective antigen genes against other avian pathogens using CRISPR/Cas9 technology in the future.