Recombinant Expression Of RHDV VP60 Protein In Pasteurella Mutant

Rabbit Pasteurellosis and Rabbit hemorrhagic disease(RHD),characterized by its high morbidity and mortality,have caused serious damage to the rabbit farming industry and threatened the endangered species of wild rabbits.Vaccination is the main strategy for defending these two infectious diseases.Currently,the most widely used vaccination in the market is inactivated pasteurella vaccine and tissue inactivated vaccine from RHDV infected rabbit liver.There are many drawbacks to this type of vaccine,such as low immunity and low cross protection rate,short immunization duration and low security.Many researchers have turned their attention to the research and development of the new vaccine for Rabbit Pasteurellosis and Rabbit hemorrhagic disease.Among them,using gene editing method to develop genetic engineering vaccine has a broad prospect.Na Ago/ g DNA gene editing system,has some advantages including high nucleic efficiency of acid cutting,low frequency of off-target effect and easy to design and operate,is extremely valuable to research.In the previous study in our lab,the feasibility of genetic manipulation of Na Ago/ g DNA system in pasteurella was successfully verified.The study used g DNA/ Ng Ago gene editing system and homologous recombination principle to construct several gene deletion strains of pasteurella successfully.They are ?lyi-GXPM??opa-GXPM??hyae-GXPM??pilia-GXPM ??fhgb-GXPM.It was also found that Ng Ago proteins can still target the gene for cutting in the absence of g DNA.Maybe the homologous arm sequences play the role in guiding cutting.This study shows that Ng Ago protein can greatly increase the probability of homologous recombination in the existence of homologous arms,and the effective knockout of target gene is achieved.RHDV capsid protein VP60 is the main structural protein of the virus.The expressed recombinant VP60 protein has the characteristics of spontaneous assemble into virus like granules(VLPs).This VLPs has antigenic properties similar to RHDV,and can produce an effective immune response.Based on previous research,this study use Ng Ago gene editing system and homologous recombination principle and choose Pasteurella C51-17 strain as the carrier.The Lyi gene in the genome of the C51-17 strain was replaced by VP60 gene of a strong RHDV strain,and a recombinant strain of Pasteurella c51-17 was constructed with a VP60 gene inserted.Then,Kana promotor has been found to promote the expression of recombinant VP60 protein in that Pasteurella mutant strain.But the efficiency of its expression needs to be improved and its immunogenicity need to be further confirmed.