| Tuberculosis (TB) is a chronic respiratory infectious disease caused by the pathogen Afycobacterium tuberculosis (MTB). About one third of the world's population estimated by the World Health Organization (WHO) is infected with MTB and there are 10 million new cases and 3 million deaths annually. In recent years, such factors as increasing frequency of drug-resistance MTB, poverty, incidence of HTV-associated and increase of population movement, have aggravated further the opportunity of people worldwide face to the threat of TB. China is one of the twenty-two high-burden countries of TB, and at present there are 40 million people infected MTB, the number of contagious TB patients has got to 2 million. Prevention of TB is again becoming an important task. BCG is the most widely used vaccine against TB, but its efficacy is not good enough. Studies show that BCG can prevent and reduce the severity of TB in children, but shows little efficacy on the pulmonary TB of adults, its protective efficacy varies from 0 to 80%. The reasons for this change may result from the loss of protective antigens caused by the variation of BCG, the heredity of people, the difference of nutrition and the factors of environment. It should be very important to develop a more effective vaccine of TB.Studies have shown that the protective antigens of MTB are mostly in the short-term culture filtrate (ST-CF), which is composed of numerous moleculessecreted or released during the growth of MTB, including about 200 polypeptides. The ESAT-6 antigen is a dominant target for cell-mediated immunity in the early phase of TB, causing the memory T-cell proliferation and gamma interferon (IFN-7) production. DFN- can activate macrophage and enhance the effet of macrophage on the growth inhibition and killing of MTB. ESAT-6 protein is only in pathogenic MTB, but lack in strains of BCG.Several new types of TB vaccine, including subunit vaccines, live attenuated vaccines, DNA vaccines and recombinant BCG (rBCG), are currently investigated in experiments. rBCG is genetically engineered BCG by inserting foreign genes, which relis on BCG to multiply in host, express the foreign antigens and induce humoral and cell-mediated immune responses. So rBCG could potentially share the vehicle and adjuvant of BCG, different foreign fenes and living vaccine. It engenders strong and long-lived immune responses with only a single dose. Using developed molecular genetic tools and methods for mycobacteria have provided important means to develop new types of TB vaccine.In this study, we first inserted the gene encoding ESAT-6 protein of MTB into the vector pDE22 and constructed the Eco/f-Mycobacterium shuttle plasmid pDE22-ESAT-6, then transformed into BCG by electroporation and obtained the rBCG expressing secretory ESAT-6 protein. rBCG were grown in 7H9 medium for 2 week, and the culture supernatants were collected after heat induction. Sephadex G-50 gel chromatography was used to purify the low-molecular-mass. ESAT-6 protein was analyzed by Tricine-SDS-PAGE and identified by dot blotting. The result revealed that rBCG could express ESAT-6 secretively, and this was further confirmed by dot blotting. Inoculate BCG and rBCG in 7H9 medium respectively and culture them at 37 C. The growth curve was studied. The value of OD 600nm was detected. The result indicated that there was no significant difference in the growth rates between rBCG and BCG.BALB/c mice were immunized subcutaneously with approximately 106 CPU of BCG, rBCG or saline to compare the effect of immunization, after immunization,antibody levels of mice were evaluated by ELISA, the result showed that mice vaccinated with the two different mycobacterial strains could induce higher levels of antibody against culture filtrate proteins(CFP) of M. tuberculosis H37Rv. liters as high as 1:8000 were obtained with rBCG immunization, which is significantly higher than that induced by immunization with BCG (P < 0.05). The spleen lymphocytes of mice were isolated. Level of IFN-'y secreted by spleno... |
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