| BACKGROUUND: According to the different profile of the cytokine secretion, the CD4+ T help lymphocyte can be divided into Th1 and Th2 subgroups, which can keep balance in the physiological condition, while the balance may be broken in some diseases. A lot of conditions can affect the differentiation of Thl/Th2, including antigens presented by APC. Besides, the allergy is now believed to be caused by the disorder of Thl/Th2 balance. It has already been demonstrated that Mycobacteria, such as Mycobacterium bovis , Mycobacterium tuberculosis (MTB) andMycobacterium vaccae (M.vaccae), can induce the Thl-like immune response and suppress the Th2-like immune response. As the main strain of Mycobacterium, BCG (Bacillus Calmette-Guerin) is regarded as the best and safest vaccine and therefore used worldwide. Thus, it has been suggested that the recombinant BCG (rBCG) containing foreign antigens can be used to regulate the Thl/Th2 balance and to prevent the occurrence of allergic diseases.AIM: (1)To construct the rBCG expressing foreign antigen Der p2 in the form of lipoprotein by using E.coli-BCG shuttle vector pOLYG. (2) To investigate the role of modulating immune response of mice with Der p2-rBCG vaccination.METHODS: The full length of Der p2 gene was constructed by ligating 11 fragments, which were synthesized individually. Then, the gene was cloned into the expression vector pProEX HTb, and the construct was transformed into DH5 a . After the gene was confirmed by sequencing, the recombinant expression was induced by IPTG and further checked by 15% SDS-PAGE and Western Blot. Next, the Der p2 gene was cloned into expressing plasmid pPICPK, and the construct was linealized by Bgl II and then transformed into Pichia pastoris strains SMD1168 by electroporation. The positive clone was obtained by G418 selective culture, and its antigen expression was confirmed by ELISA. At the same time, the gene fragment encoding 19kDa antigen (19-ss) was amplified by PCR from the Mycobacteria tubercullosis H37RV. After sequencing, the PCR product was cloned into the E.coli-BCG shuttle vector pOLYG, and then into pCW. The Der p2 gene was also cloned into pCW vector, and theDer p2-pCW-rBCG construct was induced into M.vaccae by electroporation. The expression was detected both molecular biologically and immunologically. Finally, two groups of seven 6 to 8 weeks old and two groups of seven newborn BALB/c mice were vaccined intraperitoneally with 100 μ L of 106CFU rBCG or BCG respectively. At the same time, the control group was injected with 100 μ L saline. Six weeks later, all animals were injected with Der p2 (20 μ g in 50 μ L). Further two weeks later, the concentrations of IL-4 and IFN-γ from both immunized mouse serum and splenocyte culture supernatant were determined by ELISA, and Th subgroups were determined by double flourescent staining and flow cytomety, so that the effects of rBCG on mouse immune response could be evaluated.RESULTS: It was confirmed by sequencing that the constructed Der p2 gene was identical withwhat had been designed, and the mutations did not change the amino acid sequence. By using the vector pPIC9K, Der p2 gene was integrated into the genome of Pichia pastor is successfully. The high effective expression was achieved by using G418 at the concentration of 6.0g . L-1. After 72 hours' methylol induction, the recombinant expression reached to its peak of up to 47% of total amount of bacteria protein. The results of both indirect ELISA and sandwich ELISA showed that the yeast expressed protein could be recognized very well by the polyclonal antibodies induced by natural antigen, therefore can be used as the Der p2 antigen. On the other hand, the 19ss gene was amplified and cloned into the vector pCW, which can successfully schlep and express the recombinant gene in a form of lipoprotein on the surface of M. vaccae. Finally, Der p2-rBCG was prepared, and its expression efficiency was confirmed by immunofluorescent staining and flow cytometry. The serum from both rBCG-...
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