Chun Fang Wang

Molecular Differentiation of Nontuberculous Mycobacterium Isolated from Different Animals

To investigate the occurrence and species diversity of mycobacteria in different animals, lymphonodi mandibulares and lymphonodi mesenterici samples were collected from slaughter house of swine and cattle. Mycobacteria in each lymphonodi sample were isolated by decontamination using cetylpyridinium chloride (CPC) and cultivation on Lowenstein-Jensen medium and Middlebrook 7H9 agar, and then identified by nacterial smear, sequencing of 16SrDNA and the 65-kDa heat-shock protein gene (hsp65 gene). The most frequently isolated species was Mycobacterium fortuitum. The result demonstrated that animals are an important environmental source of mycobacteria and the combined application of 16SrDNA and hsp65 sequencing was more reliable to accurately identify mycobacteria present in animals.

Isolated Strains of Nontuberculous Mycobacterium Interfere with Immune Responses Associated with Mycobacterium Bovis BCG Vaccination

Prior exposure of a vaccine to certain species of environmental mycobacteria can prime the immune system against common mycobacterial antigens, which can in turn reduce the subsequent efficacy of live attenuated mycobacterial vaccines (such as Mycobacterium bovis BCG), in both human and livestock vaccination programs. In this study, five strains of nontubeculous mycobacterium, all isolated from lymphonodi mandibulares and lymphonodi mesenterici samples of swine and cattle, were investigated to determine their growth characteristics and effects on the immune system in murine models. Markedly, different effects on the immune system were observed. The different results may be linked to the inherent growth characteristics of the five strains, The implications of these findings for BCG vaccination protocols are discussed.

Prokaryotic Expression and Identification on the ag85a and mpb70 Fusion Gene of Mycobacterium bovis

Based on splicing by overlapping extension (SOE) polymerase chain reaction (PCR) ,the ag85a and mpb70 were amplified and the fusion gene ag85a-mpb70 were cloned into pMD18-T vector, and then we got the recombinant plasmid pMD-85a-70. pMD-85a-70 and pET28a (+) were digested by BamHI and EcoRI double enzymes. The purified ag85a-mpb70 fusion gene was subcloned into the expression vector pET28a (+),and the prokaryotic expression vector pET-85a-70 was constructed. Plasmid containing pET-85a-70 was transformed into competence Escherichia coli BL21(DE3).The bacterium was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 49 kDa fusion protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that Ag85A-MPB70 was of antigenic activity of Mycobacterium bovis. These results could serve as a basis for further studies on the usefulness of the fusion gene and its expression product in the development of novel vaccine against bovine tuberculosis.

Prokaryotic Expression and Identication on the 20kDa Protein Gene of Mycobacterium paratuberculosis

The gene encoding 20kDa protein gene from Mycobacterium paratuberculosis C-2, chromosomal DNA was amplified by using polymerase chain reaction (PCR), the PCR product was approximately 520bp DNA segment. The PCR product was cloned into pGEM-T vector and the cloning plasmid pGEM-T-20 was constructed successfully. The purified 20kDa protein gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression plasmid pET28a-20 was constructed. Plasmid containing pET28a-20 was transformed into competence Escherichia coli BL21 (DE3). The bacterium was induced by isopropyl-β-D- thiogalactopyranoside (IPTG) and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 23kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that the protein was of antigenic reactivity of M. paratuberculosis. The results were expected to lay foundation for further studies on the subunit vaccine and DNA vaccine of 20kDa protein gene in their prevention against bovine paratuberculosis.

Fusion, Expression and Identification on the mpb51 and mpb63 Genes of Mycobacterium bovis

The DNA fragments of mpb51 and mpb63 were fused by splicing by overlapping extension(SOE) polymerase chain reaction(PCR),and the fusion gene mpb51-63 were cloned into pMD18-T vector, and then we got the recombinant plasmid pMD-51-63. pMD-51-63 and pET28a(+) were digested by BamHI and EcoRI double enzymes. The purified mpb51-63 fusion gene was subcloned into the expression vector pET28a(+),and the prokaryotic expression vector pET-51-63 was constructed. Plasmid containing pET-51-63 was transformed into competence Escherichia coli BL21(DE3).The bacterium was induced by isopropyl-β-D-thiogalactopyranoside(IPTG)and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE),approximately 43ku exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that the protein was of antigenic activity of Mycobacterium bovis. These results could serve as a basis for further studies on the usefulness of the fusion gene and its expression product in the development of subunit vaccine, DNA vaccine and diagnostic reagents against bovine tuberculosis.

PCR Detection of Mycobacterium bovis DNA in Cattle Bloods

For molecular biological detection of Mycobacterium tuberculosis, PCR methods with primers targeting different regions specific for mycobacterium tuberculosis complex are used worldwide. In this study,256 whole blood samples were detected using multiplex PCR.2 of all samples were found to be PCR positive. Homogenous comparison of Mycobacterium bovis specific genes with Mycobacterium bovis AF2122/97 all reached up to 99%.

Construction of Eukaryotic Expression Vector and Expression on Mycobacterium bovis ag85a and mpb70 Genes

Based on the polymerase chain reaction (PCR), ag85a and mpb70 Fusion gene of Mycobacterium bovis were ligated and cloned into pMD18-T, then recombinant plasmid pMD-85a-70 was constructed. pMD-85a-70 and pVAX1-BMS were digested by double enzymes HindIII and EcoRI, the purified ag85a-mpb70 was subcloned into pVAX1-BMS, then recombinant plasmid pVAX1-BMS-85a-70 was constructed and transient expressed in Marc145 cell. These results laid solid foundations for further studies on ag85a-mpb70.

Fusion Expression on the Esat-6 and cfp-10 Genes of Mycobacterium bovis in Escherichia coli

Based on the (Gly4Ser)3 linker, the esat-6 and cfp-10 gene were fused for raising the antigenicity of single antigen. The DNA fragments of esat-6 and cfp-10 were fused by splicing by overlapping extension (SOE) polymerase chain reaction (PCR),and the fusion gene esat-6-cfp-10 were cloned into pMD18-T vector, and then we got the recombinant plasmid pMD-esat-6-cfp-10. pMD-esat-6-cfp-10 and pET28a (+) were digested by BamHI and EcoRI double enzymes. The purified mpb esat-6-cfp-10 fusion gene was subcloned into the expression vector pET28a (+),and the prokaryotic expression vector pET-esat-6-cfp-10 was constructed. Plasmid containing pET-esat-6-cfp-10 was transformed into competence Escherichia coli BL21(DE3).The bacterium was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE),approximately 25 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that the protein was of antigenic activity of Mycobacterium bovis. These results could serve as a basis for further studies on the usefulness of the fusion gene and its expression product in the development of DNA vaccine; living carrier vaccine; subunit vaccine and diagnostic reagents against bovine tuberculosis.

Construction of Eukaryotic Expression Vector and Transient Expression on Mycobacterium bovis mpb83,mpb70 and Cattle il-15 Gene

Based on the splicing by overlapping extension (SOE) polymerase chain reaction (PCR),fusion gene mpb83-70 and il-15 were ligated and cloned into pMD18-T,then recombinant plasmid pMD-83-70-15 was constructed. pMD-83-70-15 and pVAX1-BMS were digested by double enzymes HindIII and EcoRI, the purified mpb83-70-il-15 was subcloned into pVAX1-BMS,then recombinant plasmid pVAX1-BMS-83-70-15 was constructed and transient expressed in Mark145 cell. These results laid solid foundations for further studies on mpb83-70-il-15.

Fusion and Prokaryotic Expression on the mpb83 and mpb63 Genes of Mycobacterium Bovis

Based on the (Gly4Ser)3 linker, the mpb83 and mpb63 gene were fused for raising the antigenicity of single antigen. The DNA fragments of mpb83 and mpb63 were fused by splicing by overlapping extension (SOE) polymerase chain reaction (PCR) ,and the fusion gene mpb83-63 were cloned into pMD18-T vector, and then we got the recombinant plasmid pMD-83-63. pMD-83-63 and pET28a (+) were digested by BamH I and EcoR I double enzymes. The purified mpb83-63 fusion gene was subcloned into the expression vector pET28a (+),and the prokaryotic expression vector pET-83-63 was constructed. Plasmid containing pET-83-63 was transformed into competence Escherichia coli BL21(DE3).The bacterium was induced by isopropyl-β-D-thioga-lactopyranoside (IPTG) and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE),approximately 38 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that the protein was of antigenic activity of Mycobacterium bovis. These results could serve as a basis for further studies on the usefulness of the fusion gene and its expression product in the development of DNA vaccine; living carrier vaccine; subunit vaccine and diagnostic reagents against bovine tuberculosis.