| E.coli O157:H7 always brought blood diarrhea,anemia and hemolysis urine syndrome(HUS) in clinic infection.E.coli O157:H7 could release the cytotoxin during invading procession and the antibiotic could speed this cytotoxin release by the dissolution of cell wall,therefore the therapy of E.coli O157:H7 was conservative therapeutics.In order to prevent its occurrence,this study is carried in inspect of plant oral vaccine.The main invade path of E.coli O157:H7 is enteron,and the arising of effect mucous membrane immunization is the most emergent question in vaccine study.In the previous studies,the inject immunization was incapable to induce the efficient mucosal immune,and antigen without protection could not arrive the intestines effectively.Another question in oral vaccine study is the quantity of antigen arriving at the intestines is too low to raise the intestinal mucosal immunization.Aimed at those matters,plant vaccine could solve these questions of the immunization.During the protein expression in plant cell,plant cell wall acts as a capsule and provides protection to the recombinant protein,so recombinant protein in the protection of plant cell could pass the upper gastrointestinal,and release the antigen during the digestive in the intestine.Lettuce as a vegetable was planted widely,and it could be harvest in 50 days,so its cost is low as a plant candidate.Because lettuce could be eat in raw,and avoid antigen denaturing in the immunization,it has the unique advantages compared to tobacco,potato and rice.In this study,lettuce was used to express EspA and certified it might be the candidate immunogen in E.coli O157:H7 vaccine study.EspA was expressed in plant cells by the transient expression and stable expression for the first time.After EspA expression,BALB/C mice were immuned and specific antibodies were valued.The protections of the polyclonal antibodies of immuned mice were carried on HeLa cell monplays,and mice intestinal histopathologic slides were made after E.coli O157:H7 challenge.It was found the polyclonal antibody of EspA-immuned mice could protect the mice from E.coli O157:H7 infection.The first part:Mobile genetic elements decided the virulence of the pathogenic bacteria,and play an important role in the animal and plant incursion.TypeⅢsecretion systems (T3SS) are complex assemblies that require the function of more than 20 genes for their activity.The complex is composed of Esc/Sep proteins on which a filamentous structure of polymerized EspA is assembled.EspB and EspD are located at the distal end of the EspA filament,and it has been suggested that these proteins are involved in pore formation in the host cell membrane.Effector proteins such as Tir,EspB,EspG, EspF,and Map are translocated through this structure.Translocation of these molecules into host cells results in changes of cytoskeleton of the underlying epithelial cells.EspA was transient expressed in lettuce for the first time,and PBI121-espA, PBI121-ΩespA was introduced into Agrobacterium AGL1,EHA105,LBA4404, GV3101 and C58C1.Lettuces were cultured for 14 days and transfected by the Agrobacterium.After cultured for 3 days in 22℃,the concentration of EspA in lettuce extract was valued.The AGL1 group had the highest EspA concentration,and the lowest was LBA4404 group.By the detection of the PBI121-espA,PBI121-ΩespA group,the PBI121-ΩespA group had the higher EspA concentration.In this study,the transfection ability of Agrobacterium AGL1,EHA105,LBA4404,GV3101,C58C1 were valued and this is the first report of the transfection ability study in the lettuce. By the mice immune,BALB/C mice which were immunized with lettuce-derived EspA produced specific antibody titers with the highest IgA titer of 3,200 and IgG titer of 12,000.The second part:By E.coli adherence characterization,the EPEC and EHEC could be differentiated.HeLa cell was widely used in E.coli O157:H7 virulence study and the cytoskeleton aggregation was a characterization during E.coli O157:H7 invasion.In the study of polyclonal antibody protection,Fluorescence actin assay was frequently used. In this study,BALB/C mice were immuned with EspA expressed in Lactococcus. Lactis NZ9000(L.Lactis NZ9000) and lettuce,respectively.The polyclonal mice antisera were valued with the bacterial adherence assay,Fluorescent actin staining (FAS) and labelling of EspA filaments.In the following study,immuned mice polyclonal antibody blocked E.coli O157:H7-induced host cell actin re-arrangement and could label E.coli O157:H7 EspA filaments in vitro.But the polyclonal antibody could not inhibit E.coli O157:H7 adherence to HeLa cell in vitro.The polyclonal antibody could recognize the natural EspA on the surface of E.coli O157:H7 and the specific fluorescence were observed with the help of the HRP-marked goat anti mouse serum.In the previous studies,the prokaryotic expressed EspA could have the same protection effect in FAS,but this is the first report of the immune effect of EspA expressed in Lactococcus.Lactis NZ9000(L.Lactis NZ9000) and lettuce,respectively. Then it was approved that both of EspA expressed in Lactococcus.Lactis NZ9000(L. Lactis NZ9000) and lettuce had the same antigenic determinant,and could be the candidate antigen in the vaccine study of E.coli O157:H7.The third part:Cell culture is divided into cell culture,tissue culture and organic culture,and each of them has close relation with each other.Now,the study of tissue culture is widely applied in biology and medicine.The tissue culture condition could be controlled easily and the error is low,so it is easy to study the physical,chemical and biological factor in tissue culture.In this part,lettuce tissue culture was studied and the stable expression system was set.In the lettuce callus culture,0.2 mg-0.8 mg/l 6-BA could induce the lettuce callus,but callus could be browned in 6-BA 0.8 mg/l,NAA 0.12 mg/l;6-BA 0.3 mg/l, NAA 0.12 mg/l.At the same culture,callus grows well in 0.2,0.4 mg/l 6-BA,and it suggested the brown callus was not related with 6-BA.Adventive bud could differentiate into integrate plant in the 0.3 mg/l NAA MS culture medium.The rate of lettuce callus were 45.6%(AGL1),26.6%(C58C1),22.7% (EHA105),25%(GV3101),17.7%(LBA4404) in Kan antibiotic medium after agrobacterium transfection.The transfect ability was AGL1＞C58C1＞GV3101＞ EHA105＞LBA 4404.With the same method,the stable expression of EspA in lettuce callus was set,and the callus was generated for three times,and then was detected with PCR,ELISA and Western-blot.The genome of the lettuce callus was extracted with genome extract kit,and espA gene was present by the PCR detection.EspA protein in the callus was also detected by ELISA and Western-blot.The EspA in lettuce callus was 1.6μg/ml and the mice produced high IgG specific antiserum.The mice were attacked with E.coli O157:H7⊿stx-B and 50%mice survived after 5 days. The intestinal microvilli were intact and the fundus cells arrange wass tightness,but microvilli of the control group were broken off and the fundus cells arrange was in disorder.This study demonstrated the protection of the EspA plant vaccine in the histopathology.The fourth part:EHEC O157 produced Stx1 and Stx2 while the Stx2 is secreted into the medium and the Stx1 is expressed in cytoplasm.Both of the two toxin are composed of A subunit and five B subunits.The A subunit has the cytotoxin by the interaction with 28Sr RNA,and this is the base of the pathogenic mechanism of E.coli O157;the B subunit could bind to Gb3 and initiate the A subunit.According to the pathogenic mechanism of E.coli O157,the stx1-B gene was cloned from the pMD18-T-stx1 plasmid,and the expression vector pET15b-stx1B was constracted.The Stx1-B was purified by inclusion denaturing and renaturing. Meanwhile,espA gene and stx1-B fuse gene was cloned into pBI121,and the lettuce was transfected by Agrobacterium GV3101 harboring pBI121-espA-B.The mice were fed with transgenic plant,but the specific IgG was not detected in the mice serum. The possible reason might be the false folding of the fused protein and then caused the immune failure. |
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