Effect Of Vibrio Mimicus Targeted DNA Vaccine On The Activity And Antigen Presenting-related Gene Transcription Of Intestinal Macrophage

Vibrio mimicus?V.mimicus?is an intestinal pathogen which seriously endangering the development of aquaculture.Oral vaccination not only is easy to operate and not limited by fish size and culture scale,but also can induce intestinal mucosal immunity that plays an important role in the resistance to the pathogen infection in fish.Therefore,it is very necessary to develop a DNA vaccine that is safe,efficient,and can be used for oral immunization.The mechanism of oral or anal injection immunogenicity mainly depends on the intestinal mucosal immune system.Although there is a lack of lymphoid masses and dendritic cells that mammals possess in the intestinal mucosal immune system of teleost fishes,a large number of immune cells are distributed in the lamina propria in the middle and posterior intestine.Wherein,in addition to possessing killing function,intestinal macrophages are also antigen presenting cells?APCs?,which play a role as a bridge between innate immunity and acquired immunity.Therefore,the activity and function of intestinal macrophages in fish and the transcription level of genes associated with the antigen presentation pathway are important indicators for evaluating the immune response induced by oral or anal DNA injection,especially the level of intestinal mucosal immunity.At the same time,little research has been conducted on fish intestinal macrophage in vitro at home and abroad.In previous studies,we have constructed"naked"DNA vaccine of V.mimicus.on this basis,in this paper,the"naked"DNA vaccine was loaded into lyophilized DH5?-BGs to form targeted DNA vaccines.Then the activity and function of the intestinal macrophages of immunized grass carp were detected,and the transcription level of the genes associated with the antigen presenting pathway from intestinal macrophages also was detected.The main research work and results completed are summarized as follows:1)Optimization of lysis and loading conditions for E.coli DH5?ghosts.The OD_600nm00nm values of initial concentration were 0.3,0.4,0.5 and 0.6 recombinant Escherichia coli DH5??pBV220-LysisE?carried out 42?induced lysis,by DH5?strain lysis power curve,and the shape and cleavage efficiency junction structure,The optimal lysing condition for DH5?was determined as the DH5??pBV220-LysisE?broth with initial concentration OD_600nm00nm of 0.4,which was induced at 42°C for 2.5 h.A large number of freeze-dried DH5?strains were prepared under optimal lysis conditions.The effects of different ratios of freeze-dried bacterial ghosts,plasmids and membrane vesicles on the loadings were investigated.It was found the optimized ratio of the three was 5:3:1.2)Preparation,immunization and transcriptional expression of the target DNA vaccine of V.mimicus.Under optimal loading conditions,DH5?lyophilized ghosts were loaded eukaryotic expression plasmid of pcDNA3.1-Ovepis?"naked"DNA vaccine?and pcDNA3.1-Iclp-Ovepis?endogenous-targeted vaccine?to used to prepare the exogenous-targeted vaccine and double-targeted DNA vaccine.In three mimicus targeting DNA vaccine immunized carp anal injection,RT-PCR detection revealed that the vaccine gene was transcribed in the liver,head kidney,spleen,and intestine at 7,14 and 21days after immunization.3)Detection of the activity/function of macrophages in immunized grass carp and the antigen presenting-related gene.The grass carp intestine macrophages were isolated and cultured on days 7,14 and 21 after immunization.The oxygen respiratory burst activity,nitrogen respiratory burst activity and phagocytic energy of grass carp macrophages were detected by NBT reduction method,Griess method and neutral red method,respectively.Theresultsshowedthatthedetectionindexes of double-targeted DNA vaccine group of intestinal macrophages at each detection time point were superior to the two single-targeted DNA vaccine groups;on the 7th and 14th days after immunization,The detection indexes of intestinal macrophages in two single-targeted DNA vaccine groups were superior to those of the"naked"DNA vaccine group.In addition,the transcriptional levels of the antigen-presenting pathway-related genes?MHC-I?,MHC-II?,Iclp,STING?in intestinal macrophages were detected by real-time quantitative PCR,and the double-targeted DNA vaccine group of intestinal macrophages at each detection time point was found that the transcription levels of MHC-I?,MHC-II?,and Iclp genes were significantly higher than those of the single-targeted DNA vaccine group and the“naked”DNA vaccine group;Four DNA vaccines can significantly increase the transcription of STING gene in intestinal macrophages,but the difference between the groups at each time point is irregular.In conclusion,all three targeted DNA vaccines of V.mimicus can significantly enhance the activity/function and antigen presenting-related gene transcription of the macrophages from immunized grass carps,and the double-targeted DNA vaccine has the best enhancement effect.