Résumé
Objective To construct five types of recombinant modified vaccinia virus Ankara (MVA) carrying genes encoding antigen 85A (Ag85A), early secretory antigenic target (ESAT-6) or IL-2 and to investigate the immunogenicity of these recombinant MVA in mice. Methods The genes encoding Ag85A and ESAT-6 were amplified by PCR from Mycobacterium tuberculosis H37Rv genomic DNA. The am-plified DNA fragments were sub-cloned into vector p18. The recombinant plasmids were introduced respec-tively into MVA by homologous recombination. The five types of recombinant MVA clone were selected in the MPA selection medium and their expressions of TB antigens were confirmed by Western blot. The recombi-nant MVA were used to immunize BALB/c mice. Ag85A-ESAT-6 fusion protein induced proliferation of spleno-lymphecytes from immunized mice was detected by MTT test. The concentration of IFN-γ in superna-tant of spleno-lymphocytes cultures was also analyzed by ELISA. The delayed type hypersensitivity (DTH) response to tuberculosis antigens was measured after injection of tuberculin purified protein derivative (PPD) in hind footpads of immunized mice. Results The five types of recombinant MVA were successfully con-structed by confirming their expression of TB antigen with Western blot. After co-cultured with the fusion protein Ag85A-ESAT-6 in vitro, the spleno-lymphocytes from recombinant MVA immunized mice showed sig-nificant proliferation activity (P<0.01) and increased IFN-γ secretion (P<0.05) while the same cells showed no response when co-cultured with saline. The spleno-lymphocytes from recombinant MVA immu-nized mice also demenstrated significant proliferation activity (P<0.01) and increased IFN-γ secretion (P<0.01) under Ag85A-ESAT-6 inducement, compared with the spleno-lymphocytes from mice immunized with wild type MVA or saline. The significant DTH response to PPD was detected in recombinant MVA-immunized mice (P<0.05). Conclusion The five types of recombinant MVA carrying genes of TB anti-gens were constructed. The constructed recombinant MVA could induce specific cellular immunity against tu-berculosis antigen in mice.
Résumé
In this study, we produced a biphasic absorbable calcined bone (CB) of beta-TCP/HAP, and evaluated its function as a carrier of bone marrow derived mesenchymal stem cells(BMSCs) in BMP-2 gene medicine. Biphasic CB was manufactured and its surface was coated by collagen. X-ray diffraction analysis, scanning electron microscopy and biomechanical measurement were performed on the product. Heterotopic bone induction of product as carrier of BMP-2 gene transferred BMSCs and its biodegradability were tested in nude mice and goats. X-ray diffraction analysis showed biphasic patterns of HAP and beta-TCP. Scanning electron microscopy showed the porosity were similar to those of the cancellous bone, and the adhesion of cells on the CB surface were better after surface-coating with collagen. It had certain biomechanical strength and appropriate biodegradability. Biphasic CB loaded with Adv-hBMP-2 transduced BMSCs could induce much bony callus at the subcutaneous site of nude mice and the tibial bone defects of goats. The results showed biphasic CB is a superior carrier of cells in BMP-2 gene medicine.