Preparation And Effect Evaluation Of Mycobacterium Tuberculosis Fusion Protein BfrB-GrpE

Tuberculosis is caused by Mycobacterium Tuberculosis（M.Tuberculosis）infection and is one of the most fatal infectious diseases caused by a single pathogen.Currently,Bacille Calmette-Guérin（BCG）is the only vaccine used for clinical prevention of tuberculosis,but its protection against tuberculosis in adults is not effective.A large number of studies have shown that the tuberculosis subunit vaccine can provide long-term immune protection and good safety,so it has a good prospect of application and development.When Mycobacterium tuberculosis infection occurs,there are multiple metabolic states in the host,such as stages of resuscitation,replication and dormancy,and different metabolic states can be converted to each other under certain conditions.Therefore,the new TB subunit vaccine should contain immunodominant antigens in different metabolic stages to form a multi-stage vaccine,which can effectively improve the anti-TB protection effect.Objective:In this study,an unlabeled fusion protein Bfr B-Grp E（referred to as BG）was constructed by combining the immunodominant antigen Bfr B in the replication and latent phase of Mycobacterium tuberculosis and the immunodominant antigen Grp E in the latent phase.The fusion protein BG with a purity of more than 95%was prepared and immunize C57BL/6 mice with DP（C）【DDA-Poly I:C-（cholesterol）】and Mn J（β）adjuvant respectively to evaluate the immunogenicity and protective effect of the tuberculosis subunit vaccine BG.Methods:1.By consulting a large number of literatures and software to predict antigen characteristics,the screened Mycobacterium tuberculosis immunodominant antigens Bfr B and Grp E gene fragments were optimized and sent to Beijing Huada Gene Company for synthesis and sequencing verification.The recombinant plasmid was transferred into E.coli BL21（DE3）-p Ly Ss to induce expression of the target gene protein,and the target protein was purified by two-step ammonium sulfate precipitation and Butyl HP hydrophobic chromatography.2.57BL/6 mice were immunized with Bfr B/DPC,Grp E/DPC,BG/DPC,MH/DPC and BG+MH/DPC vaccines for 3 times with 0,4,and 12-week immunization strategy,and control groups of BCG and PBS were set up at the same time.Splenic lymphocytes were isolated 12 weeks after the last immunization,and the levels of cytokines IL-2 and IFN-γwere detected by ELISA after being stimulated by a single antigen for 72 hours.At the same time,BG/DPC,MH/DPC,BG+MH/DPC and control group vaccine mice were challenged with the attenuated strain of Mycobacterium tuberculosis H37Ra via the tail vein,and the bacterial load in the lung and spleen of mice was detected after3 weeks.In addition,BG+MH/DPC,PBS and BCG vaccine groups were also aerosol challenged with Mycobacterium tuberculosis H37Rv strain,and the bacterial load in the lung and spleen of mice was detected after 6 weeks after challenge.3.C57BL/6 mice were immunized with BG/DP and BG/Mn J（β）vaccines for 3times with 0,2,4-week immunization strategy,and BCG and PBS groups were set up at the same time.Splenic lymphocytes were isolated 6 weeks after the last immunization,and the levels of cytokines IFN-γwere detected by ELISA after being stimulated by a single antigen for 68 hours.At the same time,the mice were challenged with attenuated strain of Mycobacterium tuberculosis H37Ra through tail vein,and the bacterial load in lung and spleen of mice was detected 3 weeks after challenge.Results:1.Construction and expression of fusion protein BG.The recombinant plasmid p ET30a（+）-Bfr B-Grp E was successfully constructed.A large amount of fusion protein BG was stably expressed as a soluble protein in E.coli BL21（DE3）-p Ly Ss.The protein was purified from the protein supernatant by ammonium sulfate precipitation and Buty1HP hydrophobic chromatography with a purity of more than 95%.2.Evaluation of immunogenicity and protective effect of subunit vaccines Bfr B/DPC,Grp E/DPC,BG/DPC and BG+MH/DPC.C57BL/6 mice were respectively immunized with subunit vaccine at 0,4 and 12 weeks,and the immunogenicity and protective effect were evaluated 12 weeks later.The results showed that compared with the BCG group,the levels of Bfr B-specific IFN-γand IL-2 secreted by splenocytes in the Bfr B/DPC and BG/DPC vaccine groups were increased（p<0.05）.The level of Grp E-specific IL-2 secreted by mouse splenocytes was significantly increased（p<0.0001）,and the level of Grp E-specific IFN-γsecreted by splenocytes of mice in the BG/DPC vaccine group was increased（p<0.05）.At the same time,the antibody titers against Bfr B and Grp E-specific antibodies Ig G,Ig G1 and Ig G2c in each vaccine group were significantly increased（p<0.0001）.Twelve weeks after the last immunization,the tail vein challenge of Mycobacterium tuberculosis H37Ra attenuated strain showed strong protective effect in the BG+MH/DPC vaccine group.Compared with the mice in the PBS group,the bacterial load in the lung decreased by 1.49 Log₁₀ CFU（p<0.0001）;the spleen decreased by 1.81 Log₁₀ CFU（p<0.0001）,and its protective effect against the H37Ra attenuated strain was comparable to the BCG group.While H37Rv strain aerosol challenge mice in BG+MH/DPC vaccine group,the protective effect of lung against H37Rv strain was comparable to BCG;the bacterial load in spleen was not significantly different from BCG（p=0.08）.3.Evaluation the immunogenicity and protective effect of fusion protein BG combined with DP and Mn J（β）adjuvant.The subunit vaccines BG/DP and BG/Mn J（β）were used to immunize C57BL/6 mice at 0,2,and 4 weeks.The immunogenicity and protective effect were evaluated 6 weeks later.The results showed that the levels of Bfr B and Grp E-specific IFN-γsecreted by spleen cells in the BG/DP vaccine group were significantly higher than those in BG/Mn J（β）group（p<0.05）.The 1g G1/Ig G2c ratio of serum antibody titers of mice showed that the BG/DP vaccine group preferred cellular immunity,while the BG/Mn J（β）vaccine group tended to humoral immune response,and the antibody titers against single antigen Bfr B and Grp E specific antibodies Ig G,Ig G1 and Ig G2c in each vaccine group were significantly higher than those in the BCG group（p<0.0001）.The tail vein challenge of the attenuated Mycobacterium tuberculosis H37Ra strain was performed 6 weeks after the last immunization.The results showed that compared with the control PBS group,the lung bacterial load of the mice in the BG/Mn J（β）group decreased by 1.32 Log₁₀ CFU（p<0.0001）;The bacterial load in the spleen was reduced by 2.00 Log₁₀ CFU（p<0.0001）,which indicated that its protective effect was comparable to that of the BCG group.Conclusion:A large number of fusion protein BG can be stably expressed in E.coli BL21（DE3）-p Ly Ss,and the target protein with purity more than 95%can be obtained by ammonium sulfate precipitation and hydrophobic column exchange chromatography.The fusion protein BG combined with DP adjuvant immunized mice showed good immunogenicity,while the protection effect against the attenuated strain H37Ra was poor.However,the protective effect of combined early fusion protein MH against Mycobacterium tuberculosis in the laboratory was stronger than that of the single fusion protein vaccine group.The protective effect of fusion protein BG combined with Mn J（β）adjuvant immunized mice was better than that of DP adjuvant adjuvant group.