| Vibrio mimicus is the pathogen that causes ascites disease in cultured fish,which seriously harmed the aquaculture industry.Numerous studies showed that Vibrio mimicus can produce a variety of virulence factors such as adhesins,enterotoxins and hemolysin,but its infection process and main target tissue were not clear.Bacterial labeled technique is to labele bacteria with useful markers,in order to distinguish the target bacteria,then locate and track it.Currently,the technology is widely used in bacterial translocation,dynamic process of bacterial infection,the interaction of bacteria with host,and other research fields.So this subject use the enhanced green fluorescent protein labeled Vibrio mimicus,through the green fluorescent signal trace the dynamic distribution of bacteria in infected Ctenopharyngodon idellus,determining the target tissue for adhesion,colonization,growth and reproduction,providing a scientific basis for prevent and control the fish ascites disease.Meanwhile,using pEGFP-N1 as the model plasmid,macrophage cell line RAW264.7 as the APC,to verify the feature of E.coli bacterial ghosts loading and targeting delivery,and layed the foundation for the development of Vibrio mimicus bacterial gene vaccine.This reasearch specific include the following respects:1 EGFP labeled Vibrio mimicus and its characteristic analysisEnhanced green fluorescent protein(EGFP)gene was cloned into prokaryotic expression vector p BAD24 to construct recombinant expression plasmid pBAD24-EGFP,and then the recombinant plasmid was transformed into Vibrio mimicus04-14 competent cells,to obtain labeled Vibrio mimicus.The labeled strain after L-arabinose induced,SDS-PAGE analysis showed that the molecular weight of about 27.0 ku EGFP protein was expressed;Fluorescence microscopy and flow cytometry found that EGFP-labeled Vibrio mimicus can emit significant green fluorescent signal.The morphological characteristics,growth characteristics,cell adhesion and pathogenicity of EGFP-labeled Vibrio mimicus almost have no change with the wild strain,and the labeled strain also have genetic stability,These results show that labeled Vibrio mimicus can be used for subsequent infection trace studies.2 The dynamic distribution of Vibrio mimicus in infected Ctenopharyngodon idellusUsing the EGFP-labeled Vibrio mimicus which concentration is 5 × 10~7 CFU / m L infected Ctenopharyngodon idellus for 5 h.Then transferred those infected fish into normal water for feeding.Three fish per group of experimental group and control groupwere random sacrificed at 4、6、8、10、12、24、48、72 and 85 h post infection,the gill,liver,spleen,head kidney,intestine,and muscle of fish were sampled separately and weighed,half of the weight of the tissue samples under fluorescence microscope to seen fluoresce;the other half of the weight of tissue samples for the detection of the number of bacteria.The green fluorescent signals appeared firstly in the intestine and gill at 4h after infection,and the number of bacteria reached up to 3.60 × 10~8 CFU / g and 2.36 × 10~6 CFU / g,respectively.The number of bacteria in the intestine and gill had no obvious changes from 4h to 10 h post infection,then declined gradually after 12 h,but the number of bacteria in the intestine than other organs until fish death.the green fluorescent signals in the head kidney,muscle,liver and spleen were found in 24 h post infection,and the number of bacteria increased at first but later decreased,48 h reached peak,being 8.75 ×10~4,9.58 × 10~4,4.50 × 10~4 CFU / g and 1.50 × 10~4,respectively.All lower than the number of bacteria in intestine.These results show that Vibrio mimicus is an intestinal pathogens,Intestinal mucosa is the primary target organ for adhesion,colonization,growth and reproduction.3 Feature verification of loading and targeting delivery for plasmid through E.coli bacterial ghostsRecombinant E.coli DH5α strain which carried temperature control lysis plasmid p BV220-E grow at 28 ℃ until logarithmic growth phase,then take the temperature rose to 42 ℃ quickly,induced the lysis gene E expression to lyse,obtained bacterial ghosts(BG).The BG maintaining the original shape and bacterial membrane structure seen under scanning electron microscope and transmission electron microscope,but cell surface have obvious transmembrane channel,substantially free of internal contents.After incubation with the plasmid pEGFP-N1 and BG together in different concentration proportions,added membrane vesicles closed overnight,and detected the loading efficiency by flow cytometry.Then,under the optimum loading conditions,BG with pEGFP-N1 transfected mouse macrophage cell line RAW264.7,and detected the phagocytic efficiency and protein expression by laser scanning confocal microscope and flow cytometry.The results showed that when BG and plasmid pEGFP-N1 concentration proportion was 5:2,the loading efficiency,phagocytic efficiency and protein expression efficiency was 90.13%,98.47% and 40.74%,respectively,which indicated-BG has favorable feature of loading and targeting delivery for plasmid.In summary,the intestinal mucosa of fish is the target tissue for adhesion,colonization,growth and reproduction of Vibrio mimicus.BG has favorable feature oflaoding and targeting delivery for plasmid,it might be used as targeted delivery vector for gene vaccine. |
PDF Full Text Request