| Objective Bacterial outer membrane vesicles(OMV)contain a variety of pattern recognition molecules that activate the body’s natural immunity,and are mostly used as carriers and vaccines.Diphtheria toxin mutant protein(CRM197)is mostly used in conjugate vaccine vectors because of its non-cytotoxicity.The host bacteria for CRM197 protein expression is mainly Diphtheria bacillus.However,the fermentation conditions of Diphtheria bacillus are extremely harsh and the fermentation cost is relatively high.Therefore,this topic In order to explore the soluble expression of CRM197 protein and whether OMV has an adjuvant effect,CRM197 protein was used as a vaccine to study the adjuvant activity of OMV.Methods(1)Preparation and optimization of recombinant protein CRM197: The recombinant plasmid CRM197 and molecular chaperone PG-KJE8 were co-expressed in a prokaryotic expression vector,and the OD600 value of induction conditions,induction temperature,induction time,concentration of inducer IPTG,etc.Carry out the corresponding optimization,subject the protein induced by various conditions to SDS-PAGE electrophoresis,detect the expression of the target protein,and select the best induction conditions for the target protein.The induced protein was purified by nickel column affinity chromatography,washed and eluted with imidazole buffer of different concentrations to remove contaminated proteins,and detected by Western Blotting technology.(2)Preparation and identification of OMV: using strains expressing CRM197 recombinant protein,after 16 hours of liquid culture,the culture supernatant was collected and centrifuged in a floor-standing refrigerated high-speed centrifuge,the precipitate was discarded,and the supernatant was used in a 100 KD ultrafiltration tube Concentrate,centrifuge with an ultra-high-speed centrifuge after concentration,discard the supernatant,resuspend the pellet in sterile PBS and centrifuge to prepare OMV,and observe the shape,quantity,and size of the prepared OMV with a transmission electron microscope;then use nanoparticles Diameter detector measures its size.(3)Animal immunization and vaccine adjuvant activity determination.The purified CRM197 recombinant protein and the prepared OMV are immunized to mice for 6 weeks.After the immunization,the antibodies in the serum are detected by ELISA and Western Blotting,and the qRT-PCR technology is used.Detect the changes of immune activity-related genes in the spleen,and finally use the HE staining method to observe the morphology of each tissue to detect the safety of the vaccine.Results(1)In the process of protein synthesis,molecular chaperones can recognize the partially folded conformation of a stable polypeptide chain,thereby promoting the folding and assembly of new peptide chains.By adding molecular chaperones for co-expression,it can promote the soluble expression of the target protein,and continue to improve Induction conditions such as OD600 value,induction temperature,induction time,concentration of inducer IPTG,etc.,successfully found the best conditions for inducing the target protein.(2)After continuous experimentation,the optimal concentration of purification was found,that is,p H6.5 250 mmol/L imidazole buffer is the optimal elution concentration.According to the analysis of Image J software,the purification rate was 48.93%.(3)Through observation under transmission electron microscope,it is found that the prepared OMV has a lipid bilayer structure with a relatively uniform size.The average particle size of OMV is found on the nano particle size detector at 250 nm,which is in line with the size of OMV.Western Blotting technology identified that the prepared OMV was expressed at 58 KD.(4)Use the ELISA kit to detect the antibody titer in the serum.When the titer is the same,the antibody concentration in the serum of the CRM197+OMV high-dose group is higher than the CRM197+OMV low-dose group and the CRM197 single group.The results show that OMV is in common During the immunization process,the immunogenicity of the CRM197 protein is enhanced,and at the same time the cellular immune response is stimulated.,;Through Western Blotting technology,it was found that under the condition of the same amount of sample,the purified protein concentration was detected to be 8.4 μg/μl,the protein was set to 25μg,and each well was loaded with 3 μl.The CRM197+OMV high-dose group The expression level was the highest,followed by the CRM197+OMV low-dose group,and finally the CRM197 group alone.The test results were consistent with the ELISA results;through qRT-PCR technology,it was found that compared with the control group,the IL4 in the CRM197 alone group(p<0.01)The mRNA level of IL10(p<0.01)was significantly reduced,while the mRNA level of IL2(p<0.01)was significantly increased;compared with the CRM197 alone group,the level of IL4 in the CRM197+OMV high-dose group(p<0.01),The mRNA level of IL10(p<0.01)was significantly reduced,while the mRNA level of IL2(p<0.01)was significantly increased.Both IL4 and IL10 are immunosuppressive factors,and their expression decreases during the immunization process;IL2 can promote the production of effector cells Th1 and Th2.Therefore,during the immunization process,the expression of IL2 increases.The qRT-PCR test results are consistent with expectations.The results are consistent.(5)HE staining experiment and various biochemical indicators of serology were used to detect the safety of the vaccine.It was found that the tissue morphology of the experimental group and the control group were consistent with no pathological changes,indicating that under the adjuvant of OMV,CRM197 The immune effect of the test group has been significantly improved.From the serum biochemical indicators of alanine aminotransferase(ALT),aspartate aminotransferase(AST),urea(UREA),creatinine(CREA),etc.,it can be seen that the experimental group compared with the control group,There is no statistical significance,and there is little difference between the two,indicating that the vaccine prepared this time is safe and non-toxic.Conclusion After the addition of molecular chaperones,the soluble expression of the target protein was successfully promoted;a uniform and stable OMV was successfully prepared by using sucrose to extract OMV;after 6 weeks of immunizing mice with the recombinant protein CRM197 and OMV,it was found that the CRM197+OMV high-dose group The antibody titer in the serum was the highest,and the test results were consistent with the Western Blot results,indicating that OMV played an adjuvant role in the immunization process;HE staining showed that there was no significant change in the morphology of the same tissue among the groups;from serological biochemistry The indicators of alanine aminotransferase(ALT),aspartate aminotransferase(AST),urea(UREA),creatinine(CREA),etc.can be seen that compared with the control group,the experimental group is not statistically significant,and the difference between the two is not significant,indicating that the The prepared vaccine is safer. |
PDF Full Text Request