| Tuberculosis (TB) is a social and economic problem around the world, mostly in developing countries, especially in the South-East Asia, African and Western Pacific regions. With estimate of the World Health Organization (WHO), one-third of the world’s population has latent TB, over nine million people is infected with TB, and annual incidence of about three million TB cases is reported to be responsible for more adult deaths every year than any other single infectious disease. Vaccine is the best way for prevention TB and disease control. While not highly effective, Mycobacterium bovis Bacille Calmette-Guerin (BCG) is the only vaccine against TB currently available, and it has been used widely. New effective vaccines are urgently needed to replace BCG. Early, rapid, and accurate diagnosis method is essential for prevention of TB. There is no clinical diagnosis method is specific and sensitive. Immunologic examination has the inherent advantage than other laboratory methods. The strategy based on fusion antigen proteins from Mycobacterium tuberculosis is one of the main directions for developing novel subunit TB vaccines, diagnosis methods and reagents. The latest researchs show that Ag85B and ESAT-6proteins can be used as the desirable target antigens for developing TB vaccines and clinical diagnosis reagents. WbbL is the unique extracellular protein of Mycobacterium tuberculosis, and its sequence and structure are conserved, specific. The potential antigen epitopes exist in WbbL protein and may be a specific molecular marker of TB infection. Transgenic plants as bioreactors provide a new way for medical production of oral TB vaccine and diagnosis reagent.We cloned rfbpB, esxA and wbbLl genes, and obtained rfbpB-esxA, rfbpB-esxA-rwbbL fusion antigen genes in this research, then expressed them in E. coli and tobacco leaves by means of prokaryotic and plant transient expression mode mediated by pET-30a, potato virus X pgR107and pBI121vector, gained specific antigen proteins, evaluated immunogenicity, protection effect in vitro and in vivo, elucidated clinical diagnosis, vaccine application value and immune response mechanism of various antigens. The main research results are as follows: 1) The wbbLl gene encodes a protein named rhamnosyl transferase, composes of301amino acids. It contains no signal peptide and transmembrane helices, locates outside of membrane. Secondary structure analysis revealed that the protein contained a-helix (46.8%), extended strand (14.6%) and random coil (38.6%). It possesses one glycosyl transferase domain, six potential antigenic epitopes. Both sequences and structures are conservative and especial either in gene or in protein. Rhamnosyl transferase might be a desirable molecular target for immunological tests, vaccine against tuberculosis, anti-tuberculosis drugs. There is no change for immunogenicity of Ag85B and ESAT-6proteins due to adhibiting of hydrophobic linker (Gly4Ser)3.2) The fbpB, esxA and rwbbLl genes were amplified from genome of Mycobacterium tuberculosis H37Rv by PCR. The rfbpB-esxA, rfbpB-esxA-rwbbLl fusing genes ligated by (Gly4Ser)3linker were gained by means of Splicing by Overlapping Extension PCR (SOE-PCR). The genes were cloned into prokaryotic expression vector pET-30a. The recombinant plasmids were identified by PCR, enzyme digestion and sequencing, and showed vectors were constructed successfully. We induced proteins expression in E. coli BL21(DE3) using IPTG as inducer. SDS-PAGE analysis showed that Ag85B, ESAT-6, WbbL, Ag85B-ESAT-6and Ag85B-ESAT-6-WbbL proteins were expressed in E. coli, but mostly in the form of inclusion bodies. The soluble protein fractions were gained through the optimization of induction conditions, ESAT-6, WbbL and Ag85B-ESAT-6-WbbL protein in especial. Purified proteins were obtained using the method of affinity chromatography. Western blotting identification showed that the proteins and His-tag formed fusion proteins, and expression was correct.3) The structures of genes were reconstructed by application of PCR. We achieved single genes and fusing genes for plant expression, then constructed into pgR107and pBI121vector. The recombinant plasmids were identified by PCR, enzyme digestion and sequencing, and showed vectors were constructed successfully. The infected fluids infected tobacco leaves by leaf injection with OD6000.8～1.0. The maximum time stage of extraneous proteins expressed in tobacco leaves was determined by detection of green fluorescent protein and GUS. The highest expression of PVX carrier and pBI121series vector is in the seventh day, second days after infection, respectively. SDS-PAGE analysis of total leaf protein extraction showed that Ag85B, ESAT-6, WbbL and Ag85B-ESAT-6proteins were expressed in tobacco. Western blotting identification showed that expression of the proteins was correct.4) The ELIS A method was established using the purified proteins expressed in E. coli and tobacco as antigens, serum of TB patients as first antibody. We evaluated diagnosis value of various proteins by means of above-mentioned ELIS A method. The results showed the specificity is better (more than90%) and the sensitivity is different. The sensitivity of Ag85B-ESAT-6-WbbL protein was maximal in all, and fusion protein was better than single protein. Since the entire expression of WbbL protein in TB infection, it may be more suitable for diagnosis in mid-term and later period. The experimental results of immunization of mice by prime-boost vaccination strategy showed the proteins had good immunogenicity and could inspire Thl cell immune response. The immunological effect of proteins expressed in E. coli and tobacco was almost consistent. Compared with NS control group, serum IgG, IFN-γ and IL-2levels were significantly higher with statistical difference (P<0.05).This study will provide new ways, new materials and theoretical foundation for development of new TB vaccines shared by animals and humans and clinical diagnosis reagents, and has obvious clinical significance on TB prevention and treatment. |
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