Construction of recombinant modified vaccinia virus Ankara with Ag85A and ESAT-6 gene and examination of their immunogenicity in mice
- VernacularTitle:重组结核抗原痘苗病毒Ankara株的构建及其免疫原性研究
- Author:
Jueren LOU
;
Qun ZHANG
;
Lin ZHU
- Publication Type:Journal Article
- Keywords:
Tuberculosis;
Recombinant modified vaccinia vires Ankara;
Vaccine;
Immunogenicity
- From:
Chinese Journal of Microbiology and Immunology
2008;28(9):812-817
- CountryChina
- Language:Chinese
-
Abstract:
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.
