| Tuberculosis caused by Mycobacterium tuberculosis is a kind of chronic infectious diseases,which is a huge threat to human and animals.At present,BCG is still the only vaccine of tuberculosis worldwide.However,the protective efficacy of BCG declined constantly due to the different areas and years,the absence of some genes results in the decline.Therefore,the improvement methods of BCG are actively sought to improve its protection.Region of difference1(RD-1)of Mycobacterium tuberculosis is in the absence of all kinds of BCG compared with pathogenic Mycobacterium.The RD-1 region is consists of Rv3871-Rv3879 c,which encodes 9 proteins that play an important role in M.tuberculosis infection.With the completion of sequencing of Mycobacterium tuberculosis genome,research on Mycobacterium tuberculosis has entered molecular level,.Turning RD-1 gene recombinant vector into BCG for protein expression may provide a new idea for the improvement of tuberculosis vaccine.At first,the 6 p MV261-RD-1 vectors were obtained by molecular biology techniques and the recombinant plasmids were transformed into BCG by dot-puncturing to construct recombinant BCG.The animals were immunized with recombinant BCG and the samples were tested at the 6th week and the 12 th week after immunization respectively.The immune status of the mice and the analysis of their immune status were analyzed by animal immunization of splenic lymphocyte transformation,antibody level changes and cytokine IFN-? and NO content in the spleen lymphocyte culture supernatant.The experimental results are as follows:1.In this study,p MV261-Rv3872,p MV261-Rv3873,p MV261-Rv3874,p MV261-Rv3875,p MV261-Rv3876,p MV261-Rv3877 recombinant plasmids of RD-1 region were constructed successfully.2.Recombinant BCG named r BCG: ESAT-6,r BCG: Esp I,r BCG: Snm4 were successfully obtained by using electrotransformation technology.The growth curve of recombinant BCG did not change significantly compared with the BCG.The recombinant BCG can be used for far follow-up experiments.3.The weight of experimental animals were weighed every week after immunization.The weight of mice injected with BCG and recombinant BCG was not significantly changed compared with those have no significant change between experimental mice and mice injected with PBS.The recombinant BCG did not affect the growth of animals after injection.4.Antibody titer test results: After 6th week of immunization,Ig G levels of all the group were increased significantly compared with PBS group.After 12 th week of immunization,the titer of antibodies decreased.The Ig G levels in BCG group and r BCG: ESAT-6 group increased significantly compared with PBS group.5.The results of splenic lymphocyte transformation experiment showed that when stimulated with PPD,spleen lymphocytes proliferated to T cells in the 6 weeks after immunization,and r BCG: ESAT-6 could obviously stimulate spleen lymphocyte proliferation to T cells in the 6 weeks after the immunization.After 12 weeks of immunization,the SI of BCG and r BCG: ESAT-6 group after PPD stimulation was significant.Compared with PBS group and BCG group,r BCG: ESAT-6 group can maintain a higher level of cellular immunity.6.INF-? test results showed that: 6 weeks,12 weeks after immunization,the IFN-? in spleen cell supernatant of experimental group of mouse were significantly increased compared with PBS group.It is indicated that IFN-? secreted by cells is maintained at a high level of secretion,which is beneficial to the removal of pathogens.7.The results of NO assay showed that the expression of NO in spleen cell supernatant of every r BCG groups were significantly higher than that of PBS group when stimulated by PPD at 6 weeks after immunization.After 12 weeks of immunization,the expression of NO in BCG group and r BCG: ESAT-6 group was significantly higher than that in PBS group when stimulated by PPD.There was significant difference in NO production in the spleen cell supernatant of r BCG: Esp I and r BCG: Snm4 groups. |
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