ABSTRACT: To study the function of Loa22 gene from virulent serovar Lai. L. interrogans by expressing its protein.
The recombinant plasmid with Loa22 was constructed and its expression was induced by IPTG. The expression product was identified by SDS-PAGE and Western Blotting and purified with GST-affinity chromatography. The purified protein was applied to mice macrophages ANA-1 cells purified by GST-affinity column and exerted to ANA-1 cells. The cytotoxicity of purified protein to ANA-1 was evaluated by detecting LDH activity in culture medium, XTT aborbtion and apoptosis ration.
The recombinant plasmid with Loa22 mature peptide was constructed successfully and the protein was purified subsequently. Significant higher level of LDH activity and apoptosis ratio and lower XTT absorption were observed by comparison of expression protein treated cells and those untreated.
Loa22 had a toxic effect on ANA-1 and the gene Loa22 might be a virulent-related gene of L. interrogans.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 06/2008; 39(3):351-4, 377.
ABSTRACT: Objective To construct recombinant Mycobacterium smegmatis expressing ESAT-6 of the human pathogen Mycobacterium tuberculosis.
ESAT-6 gene was amplified from M. tuberculosis genomic DNA and inserted into an E.coli-mycobacterium shuttle vector under the control of HSP60 promoter. The recombinant vector was transformed into M. smegmatis by electroporation. To assess the ability of recombinant M. smegmatis to activate macrophage, mouse macrophage ANA-1 was cocultured with recombinant M. smegmatis. The apoptosis of ANA-1 cells was detected by flow cytometry and iNOS mRNA expression of the cells was detected by reverse transcription-polymerase chain reaction (RT-PCR). The survival of M. smegmatis strains in ANA-1 cells was evaluated.
The recombinant vector was verified by restriction endonuclease digestion and DNA sequencing. ESAT-6 protein was expressed in M. smegmatis in response to heat shock and the molecular weight of the expression product was identical to the expected value. The growth curve of the new recombinant M. smegmatis was consistent with that of the wild-type strain, suggesting the absence of ESAT-6 protein toxicity against M. smegmatis. The recombinant M. smegmatis did not induce significant changes in mouse macrophage ANA-1 apoptosis. Coculture of the macrophages with recombinant M. smegmatis for 4 to 24 h could induce iNOS expression in the former, and the CFU of recombination M. smegmatis grown in ANA-1 cells was much less than that of the control bacteria.
The recombinant M. smegmatis expressing M. tuberculosis ESAT-6 gene possess immunogenicity, which provides experimental evidence for the development of novel M. smegmatis-based vaccine against tuberculosis.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 08/2006; 26(7):923-6.
ABSTRACT: To investigate the protein-protein interaction between ESAT-6 and CFP-10 of mycobacterium tuberculosis.
ESAT-6 gene and CFP-10 gene were amplified from mycobacterium tuberculosis genome DNA. The ESAT-6 gene was subcloned into pGBKT7 and the CFP-10 gene was subcloned into pGADT7-Rec. After being verified with restriction endonuclease digestion and DNA sequencing, the recombinant vectors were transformed into yeast cell AH109 by lithium acetate method.
The yeast cells co-transformed with pGBKT7-ESAT-6 and pGADT7-CFP-10 grew on SD/-Ade/-His/-Leu/-Trp plates, and beta-galactosidase activity assays showed positive results.
ESAT-6 and CFP-10 protein could interact with each other in yeast cells.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 06/2006; 37(3):349-52.