Study On Form Of DP Adjuvant And Preparation Of Mycobacterium Tuberculosis Fusion Protein LT29

Tuberculosis(TB),a chronic respiratory infectious disease caused by Mycobacterium tuberculosis(M.tuberculosis),is the most widespread,persistent and deadly disease in the world,and is one of the top 10 causes of death worldwide.At present,there is only one tuberculosis vaccine,BCG,in clinic.Although BCG can effectively prevent severe tuberculosis in children,it has a poor protective effect on adults.Therefore,it is urgent to develop new tuberculosis vaccine to prevent tuberculosis and protect uninfected and latent patients.Subunit vaccine is a new type of vaccine.Compared with inactivated whole bacteria vaccine and attenuated live vaccine,subunit vaccine selects components with effective immunogenicity and has fewer side effects.In our previous study,the predominantly expressed protein Rv2626c in the dormant period of M.tuberculosis and the dominant antigen ESAT6 in the growth period were combined into ESAT6-Rv2626c fusion protein(short for LT29).In this study,high-purity LT29 protein is prepared on a large scale through fermentation and protein purification technology.Protein subunit vaccines require adjuvants helping to induce a strong immune response,therefore it is important to select appropriate adjuvant.At present,only aluminum adjuvant is used in clinic in China.Aluminum adjuvant which promotes Th2 type humoral immune response,cannot cause Th1 type cellular immune response.Therefore,based on cationic liposome dimethyl dioctadecyl ammonium bromide(DDA)and toll-like receptor 3 agonist(Poly I:C),our laboratory constructed a novel adjuvant,DDA-Poly I:C adjuvant(short for DP)that can induce strong Th1 cellular immunity.However,it has poor stability and is prone to flocculation,so it cannot meet the requirement of clinical application.Therefore,this study aims to improve its stability and prepare suitable dosage forms,so as to lay a foundation for the application of DP adjuvant in clinic.Objective:(1)Using fermentation and purification techniques,to express and prepare fusion protein LT29 on a large scale.(2)Through high pressure homogenization technology,to prepare more stable DP adjuvant;To develop DP dosage form,especially powder needle dosage form.(3)To detect immunogenicity of subunit vaccine LT29-DP.Methods:1.The fusion protein ESAT6-Rv2626c(LT29)is expressed in E.coli BL21(DE3)and fermented in a ferment tank.The protein is purified by HiTrap DEAE FF ion exchange chromatographic column,HiTrap Butyl HP hydrophobic chromatographic column and HiLoad 16/600 Superdex 75 gel filtration chromatographic column on AKTA pure150 protein purification apparatus.2.The high-pressure homogenizer is used to homogenize the DP adjuvant,and the suitable pressure was screened to improve the stability of the vaccine adjuvant.In order to study the dosage form of freeze-drying,the freeze-drying protectants were investigated,and were used to freeze-dry and rehydrate the DP adjuvant.The particle size and Zeta potential of DP adjuvant prepared by various methods are tested.3.Immunogenicity of the subunit vaccine LT29-DP is detected in the mice model.Mice were immunized with PB,BCG,DP adjuvant,ESAT6-DP,Rv2626c-DP,LT29-DP,LT29-DP(homogenate processed)and LT29-DP(homogenate processed and freeze-dried)for three times at week 0,2 and 4,respectively.20 weeks after the last immunization,ELISA is to be used to detect the level of IFN-?secreted by splenic lymphocytes.Proliferation of lymphocytes was detected by EdU.The central memory T cells will be detected by flow cytometry.Results:1.The fusion protein LT29 was expressed in E.coli BL21(DE3)efficiently in the supernatant,especially in the ferment tank.Using the AKTA protein purification system,LT29 was gradually purified by HiTrap DEAE FF ion exchange chromatographic column,HiTrap Butyl HP hydrophobic chromatographic column and HiLoad 16/600 Superdex 75 gel filtration chromatographic column.At last,the purity of LT29 was higher than 95%.Compared with BCG,ESAT6 and Rv2626c,fusion protein LT29 can stimulate mice to produce strong cellular immune response.The lymphocytes of C57BL/6 mice injected with LT29 vaccine were more proliferative.The mice had longer immune memory.2.The DP adjuvant was homogenized at 200 bar by high pressure homogenization method.The particle size of DP adjuvant could be reduced from 542±17 nm to 428±25 nm,with uniform size.The Zeta potential increased from 23.48±2.93 mV to 45.19±1.02 mV,and the stability of DP adjuvant was significantly improved.The DP adjuvant was lyophilized with sucrose or trehalose as protective agents.When trehalose was used as freeze-drying protectant,the particle size increased to 664±28 nm after rehydration,and the Zeta potential decreased to 24.76±1.13 mV.The effect of freeze-drying and rehydration was feasible.However,when sucrose was used as protectant,the particle size increased too much,and the Zeta potential decreased too much.Using homogenizing and freeze-dried DP adjuvant combined with fusion protein to immunize C57BL/6 mice,the cellular immune response and immune memory were not as good as the untreated DP adjuvant.Conclusion:1.In this experiment,the conditions of fermentation and expression induction of fusion protein LT29 were explored.A three-step purification scheme of ion-exchange chromatography,hydrophobic chromatography and gel chromatography was developed to prepare fusion proteins with a purity higher than 95%.Immunoassay confirmed that fusion protein LT29 had better and longer immune protection effect than BCG,ESAT6 and Rv2626c.2.We improved homogeneity and stability of DP adjuvant through high pressure homogenization technology;Trehalose was selected as a lyophilized protective agent to prepare dry powder DP adjuvant.However,immunoassay showed that homogenate and freeze-dried DP adjuvant were less effective than untreated DP adjuvant.