Preparation Of ASFV P72 Antibody And Its Expression In Lactobacillus Casei

African swine fever(ASF)is a kind of animal disease which is legally reported by the Office International des pizooties(OIE)and is also a kind of widespread animal disease in China in the past two years.Its pathogen is African swine fever virus(ASFV).ASFV is the only member of family Asfarviridae,genus Asfivirus and it is the only DNA arbovirus virus.The natural host of African swine fever virus are wild pigs and ticks.And the virus mainly infects domestic pigs,wild pigs and soft ticks.The symptoms produce in domestic pigs by ASFV infection are internal bleeding,high fever and respiratory disorders.The mortality rate can reach up to 100%.Because ASFV has a long survival time,a wide range of transmission routes,and no effective vaccines to prevent and control disease,the epidemic can only be controlled by mass killing the diseased and infected pigs and burying them in situ.At present,it caused serious economic losses to the global pig industry.Therefore,the development of African swine fever vaccine with good efficacy and high safety is urgent need.As one of the probiotics in human and animal gut,respiratory tract and urogenital system,Lactobacillus has long been proved safe in the field of food and fermentation.Lactobacillus can improve the immunity of human and animals nonspecifically,especially the function of mucosal immunity.Lactobacillus can also be used as the expression vector of exogenous protein to induce specific mucosal and systemic immune responses.ASFV has a genome of 170-190 kb,151-167 open reading frames which encode 235 proteins.P72 protein is the main structural protein,which is located on the surface of virus capsid and has four major epitope exposure regions(ER).It is the main protective antigen of African swine fever virus.In this laboratory,four ER region DNA molecules were synthesized by chemical synthesis,which expressed the corresponding proteins in E.coli prokaryotic expression system,immunized mice to produce specific antibodies,and provided a new idea for the design of ASFV vaccine.Lactobacillus casei is a food safety strain,which has significant advantages in promoting intestinal balance and nonspecific immunity.The expression of P72 protein ER region by Lactobacillus casei combine the prebiotic effect of Lactobacillus casei with the antigenicity of ER region,providing a new idea for the design and development of ASFV vaccine.In this study,the E.coli prokaryotic expression system was used to successfully express the P72 epitope sP72,and it was purified by Ni-NTA beads.The highest concentration was determined to be 1.33 ?g / ?L.The Freund's adjuvant was used to emulsify the sP72 protein.After immunizing the mice three times,the eyeballs were removed to obtain anti-sP72 serum.After ELISA titer determination,the highest titer was up to 1: 64000.Thus,an effective serum was obtained for the later Western Blotting detection of Lactobacillus casei expressing sP72 protein.This study also identified the strain type of Lactobacillus casei,explored for the heat shock temperature,ice bath time of chemical transformation and various preparation methods of competent cell,and tested the optimal procedure for electrical conversion.The final conclusion is that the chemical transformation method can not effectively introduce foreign genes into Lactobacillus casei,and the foreign plasmid can only rely on the electric transformation method to enter Lactobacillus casei.In this experiment,the prokaryotic expression system of L.casei was used to express the sP72 protein.Western Blotting results showed that it could specifically bind to sP72 mouse antiserum,indicating that L.casei successfully expressed sP72 protein.In summary,this study successfully obtained anti-sP72 mouse serum,summarized the best method for L.casei competent preparation and the most suitable conditions for transformation,and successfully made L.casei express sP72 protein.