Construction And Immunological Properties Of The Recombinant BCG Secreted Expressing The Protein Ag85B From Mycobacterium Tuberculosis

Tuberculosis(TB)is a c hronicr espiratory infectious disease caused by the pathogen Mycobacterium tuberculosis (MTB). TB is the only disease ever declared a global emergency by the WHO. About one third of the world's population estimated by the World Health Organization (WHO) is infected with MTB . Almost 8.8 million new cases of TB were reported in 2005, and 1.6 million deaths were attributed to the disease. Asia and Sub-Saharan Africa accounted for 7.4 million new cases of TB worldwide. TB continues to cause great mortality and suffering,especially in poor and less-developed countries. Its association with the HIV/AIDS pandemic forms a lethal combination. In addition, multidrug resistant (MDR) TB and the recently-described extensively drug resistant (XDR) TB– with further resistance to key second-line drugs and virtually incurable– severely complicate the management and control of the disease worldwide. Chinese number of TB patients ranks second in the world,and at present there are 40 million peoplein fected MTB,the number of contagious TB patientsh as got to 5million,The number of people who die from TB each year is 256 thousand people.BCG is currently the world's only application of the vaccine to prevent TB. But BCG different populations in different regions, there are differences. BCG can be effective in preventing children of the advantages of TB infection, but can not prevent adult tuberculosis, its preventive role ranges from 0-80%. BCG protect themselves because of the inadequacy, as well as the seriousness of the current TB epidemic, a new study tuberculosis vaccine for the prevention of TB has become a hot research at home and abroad.New TB vaccine, including subunit vaccines, DNA vaccines, viral vector vaccine, live attenuated vaccines such as BCG and recombinant BCG ,are currently investigated in experiments. Subunit vaccines and viral vector vaccine are mainly a class of immune boosting vaccine. Live attenuated vaccine and recombinant BCG vaccine are a kind of early vaccination. Recombinant BCG principle is foreign gene inserted into plasmid genomes or BCG certain areas so that the high expression, but does not affect the vaccine strain of survival and reproduction, this recombinant vaccine after vaccination, in addition to the original bacteria have protection , but also was inserted gene related to the protection of the disease. RBCG Set in a adjuvants and carriers, and a variety of exogenous gene integrated with the live vaccine, the first vaccination, will be a strong and lasting multiple specific immune. Therefore, the traditional BCG was reconstructed by the genetic engineering technology, the recombinant BCG is a new one of the major directions of tuberculosis vaccine.Studies have shown that the protect antigens of MTB are mostly in the short-term culture filtrate(ST-CF), which is composed of numerous molecules secreted or released during the growth of MTB,including about 200 polypeptides . The main ingredient is Ag85B complex. Ag85B protein is composed of 285 amino acid, which comprises 975 bp of the coding nucleotide, the former 120 bp of the coding nucleotide composed of 40 amino acid is signal peptide. It has Mycobacterium acid transfer enzyme activity, it is very easy to integrate people fibronectin, phagocytic cells can be mediated phagocytosis its. It can induce strong Th1 immune response, promoted Th1 cells proliferation and cytokine production.In this study ,we first extract BCG genome. Ag85B protein full length genes was acquired by using PCR method,it was cloned into the vector pSMT3 to construct Ecoli-Mycobacterium shuttle plasmid pSMT3-Ag85BFL,then transformed to BCG by electroporation and obtained the rBCG expressing secretory Ag85B protein. rBCG were cultured in sauton liquid medium, the culture supernatant was collected and concentrated supernatant Ag85B Protein was analyzed by SDS-PAGE electrophoresis and identified by western-blotting ,and Ag85B protein concentration was measured in the laboratory .The results showed that, the culture supernatant Ag85B rBCG content(14.78 ug/g) was much higher than the BCG culture supernatant Ag85B content(3.21 ug/g).Balb/c mice were immunized subcutaneously with approximately 5×106CFU of BCG and rBCG ,or saline and Ag85B protein control group to compare the efect of immunization, after immunization 12 weeks,different methods were used to detect the immune Mice specific humoral and cellular immunity levels.Antibody levels of mice were evaluated by ELLISA, the result showed that mice vaccinated with the two diferent mycobacterial strains and Ag85B protein could induce higher levels of antibody against Ag85B protein. Titer was 1∶12800 with Ag85B protein immunization ;Titers as high as 1∶3200 were obtained with rBCG immunization,which is significantly higher than that induced by immunization with BCG 1∶800(P < 0.05).The spleen lymphocytes of mice were isolate in vitro.Splenocytes proliferation of immunized mice in response to antigen and ConA ,restimulation was measured by MTT method..The results showed that the antigen peptide has not multiplied regarding control group's cell,and stimulation index is less than 1; The proliferation of rBCG has slight ,and stimulation index is greater than 1. ConA have the multiplication function regarding each group, regarding the rBCG stimulation index is 1.16. The change of CD4+ and CD8+ subset were examined by flow cytometry, the results of differentiation showed that rBCG stimulated the spleen lymphocyte subsets of CD4 + (61.5 percent) was significantly higher(42.8%) than control group(42.8%);But it stimulates the number of subsets CD8+(12.9%) ,it was not more obvious than control group (9.0 percent).CTL activity assay results show that recombinant BCG vaccine has 8% damaging effect to the specific target cell, but the BCG vaccine has 3% damaging effect; The ELLISPOT law examination IFN-γproduction level indicated that the recombinant BCG vaccine secretion IFN-γcell's integer is BCG vaccine secretion IFN-γcell's integer 1.3 times.In summary,the rBCG expressing the secertory Ag85B protein comparative with BCG may secrete antigen Ag85B massively;After the long time immunity's test result indicated that rBCG replies compared to the BCG stimulation strong humoral immunity, but in the cellular immunity response the specificity is not very strong. Therefore, the rBCG immunity protective function also waits for further studies.