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Gao Y.H.,Jilin Agricultural University | Liu Y.,Changchun University of Science and Technology | Zhang Y.N.,Changchun University of Science and Technology | Yu Y.,Changchun University of Science and Technology | And 8 more authors.
Advanced Materials Research | Year: 2014

For raising the antigenicity of Mycobacterium bovis single antigen, fusion protein of two genes was acquired. The DNA fragments of mpb83 and mpb70 were fused by splicing by overlapping extension (SOE) polymerase chain reaction(PCR), and the fusion gene mpb83-mpb70 were cloned into pMD18-T vector, then we got the recombinant plasmid pMD-83-70. pMD-83-70 and pET28a(+) were digested by BamH I and EcoR I double enzymes. The purified pMD-83-70 fusion gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET-83-70 was constructed. Plasmid containing pET-83-70 was transformed into competence Escherichia coli BL21 (DE3). The bacterium was induced by isopropyl-β-D-thiogalactoside (IPTG) and the lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 41 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 M. 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 and DNA vaccine against bovine tuberculosis. © (2014) Trans Tech Publications, Switzerland. Source


Liu X.,Jilin Agricultural University | Wang C.F.,Jilin Agricultural University | Ma H.X.,Key Laboratory of Animal Production | Gao Y.H.,Key Laboratory of Animal Production | And 6 more authors.
Advanced Materials Research | Year: 2014

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. © (2014) Trans Tech Publications, Switzerland. Source


Lin J.M.,Jilin Agricultural University | Wang C.F.,Key Laboratory of Animal Production | Guan J.N.,Key Laboratory of Animal Production | Ma H.X.,Key Laboratory of Animal Production | And 5 more authors.
Applied Mechanics and Materials | Year: 2013

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. © (2013) Trans Tech Publications, Switzerland. Source


Wang C.F.,Jilin Agricultural University | Wang C.F.,Key Laboratory of Animal Production | Qi H.R.,Jilin Agricultural University | Jiang X.Y.,Jilin Agricultural University | And 3 more authors.
Advanced Materials Research | Year: 2014

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. © (2014) Trans Tech Publications, Switzerland. Source


Zhang S.,Jilin Agricultural University | Cao K.,Jilin Agricultural University | Liu D.,Jilin Agricultural University | Gaowa N.,Jilin Agricultural University | And 4 more authors.
International Journal of Food Engineering | Year: 2016

In this assay, rabbit anti-β-conglycinin polyclonal antibody (Pab) was used as primary antibody and goat anti-rabbit IgG-horseradish peroxidase (HRP) was used as secondary antibody. The optimum incubating time of blocking, HRP and TMB (3, 3'5, 5'-tetramethylbenzidine) detected by the response surface and orthogonal test was 58, 140, and 20 min, respectively. The practical working range for the determination of β-conglycinin was 20-320 ng ml-1. The recoveries of β-conglycinin in spiked soybean samples were between 95.8?% and 101.2?% with the relative standard deviation less than 6.9?% (intra-assay) and 8.2?% (inter-assay). The current method was used to analyze 80 different soybean obtained from different region of Jilin province and the detected results using the direct ELISA. Compared with the competitive ELISA, The results showed contents had no difference between two methods. The dcELISA assay provides a specific and sensitive method for detecting of soybean β-conglycinin in actual production. © 2016 by De Gruyter 2016. Source

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