Construction Of The Chimeric Subunit Vaccine Of Mycobacterium Tuberculosis Using The Norovirus P Domain As The Vector

Tuberculosis is the most serious global infectious disease caused by Mycobacterium tuberculosis.It is estimated that one third of the world’s population is infected with Mycobacterium tuberculosis in the incubation period that can change to active TB at any time.Vaccination against TB remains the most effective way to improve public health in both industrialized and developing countries.BCG(Bacille Calmette Guerin)is the only currently available TB vaccine,yet it is largely ineffective in preventing adult TB and does not prevent LTBI(Latent Tuberculosis Infection).Therefore,we urgently need an effective TB vaccine to prevent TB.The main purpose of this paper is to enhance the immunogenicity of the main antigenic protein CFP10 of Mycobacterium tuberculosis by using the norovirus P particle as the antigen presenting vector,to construct a subunit vaccine expressing the CFP10 antigen,and to conduct a preliminary immunological evaluation on it,so as to provide data and theoretical basis for the development of a new anti-tuberculosis vaccine.The research mainly includes the following three parts.In the first part,the prokaryotic expression plasmids containing CFP10 and P domain were constructed by gene recombination technology,and the spatial structure of chimeric proteins was predicted.In the second part,the expression of recombinant protein was induced by IPTG,After the chimeric protein was purified by affinity purification,the GST tag was removed to obtain the target protein.In the third part,mice were immunized with the target protein,and the immunogenicity of the target protein was preliminarily evaluated by ELISA and flow cytometry.Through the above studies,we obtained the following results:1)The recombinant plasmid containing the main antigenic protein CFP10 and P domain of Mycobacterium tuberculosis was successfully constructed and the spatial structure of the protein was predicted.2)The purified chimeric protein can spontaneously form nanoparticles with a diameter of about 20nm.3)ELISA was used to detect the antibody obtained after 8 weeks in immunized mice.The antibody titer of PBS and P particle protein in immunized mice was lower and could be considered negative.The serum of mice immunized with P particle and CFP10 chimeric protein had antibody titers of about1:13440,1:7680 and 1:163200 for three antigenic peptides CFP1011-25,CFP1027-39 and CFP1071-85,respectively.The serum of mice immunized with CFP10 protein had antibody titers of about 1:1600,1:1600 and 1:1280 for three antigenic peptides CFP1011-25,CFP1027-39 and CFP1071-85.4)After immunizing mice with PBS,P particles,P particle-CFP10 and CFP10,flow analysis of splenic cells and cultured bone marrow cells revealed that the proportion of central memory T cells in the four immunized groups was 1.39%,2.21%,2.12%and 2.84%,respectively,and the proportion of effector memory T cells was 6.89%,6.93%,6.77%and 5.81%,respectively.The antigen specific T cells of splenic cells accounted for 0.15%,0.09%,0.11%and 0.10%of lymphocytes,respectively.The proportion of macrophages induced by immunized mice in bone marrow cells was 1.82%,4.11%,0.38%and 3.17%,respectively.In this study,we obtained a chimeric nanoparticle containing CFP10and P domain,and proved that P particles as a carrier can enhance the immunogenicity of the presented antigen CFP10.The chimeric nanoparticle was used to immunize mice,which produced high-potency antibodies against the TB antigen peptide and memory T cells against the antigen.In summary,this study has laid a foundation for the future research and development of tuberculosis subunit vaccine,and also provided new ideas for other subunit vaccines.