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

2014 ◽  
Vol 884-885 ◽  
pp. 498-502
Author(s):  
Xin Liu ◽  
Chun Fang Wang ◽  
Hong Xia Ma ◽  
Yun Hang Gao ◽  
Jia Ning Guan ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Prokaryotic Expression ◽  
Expression Vector ◽  
Fusion Gene ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Antigenic Activity ◽  
Expression Product ◽  
Sds Page ◽  
Prokaryotic Expression Vector

Based on splicing by overlapping extension (SOE) polymerase chain reaction (PCR) ,theag85aandmpb70were 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 byBamHI andEcoRI 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 competenceEscherichia coliBL21(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 ofMycobacterium 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.

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

2013 ◽  
Vol 791-793 ◽  
pp. 212-215
Author(s):  
Jia Ning Guan ◽  
Chun Fang Wang ◽  
Jia Ming Lin ◽  
Hong Xia Ma ◽  
Shuang Hou ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Prokaryotic Expression ◽  
Expression Vector ◽  
Fusion Gene ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Antigenic Activity ◽  
Sds Page ◽  
Prokaryotic Expression Vector ◽  
Single Antigen

Based on the (Gly4Ser)3 linker, thempb83andmpb63gene were fused for raising the antigenicity of single antigen. The DNA fragments ofmpb83andmpb63were 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 byBamH I andEcoR 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 ofMycobacterium 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.

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

2013 ◽  
Vol 319 ◽  
pp. 140-145
Author(s):  
Yun Hang Gao ◽  
Chun Fang Wang ◽  
Xiu Yun Jiang ◽  
Hong Xia Ma ◽  
Da Ming Gao ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Expression Vector ◽  
Fusion Gene ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Antigenic Activity ◽  
Expression Product ◽  
Sds Page ◽  
Prokaryotic Expression Vector ◽  
Polymerase Chain

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.

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

2013 ◽  
Vol 421 ◽  
pp. 354-358
Author(s):  
Jia Ming Lin ◽  
Chun Fang Wang ◽  
Jia Ning Guan ◽  
Hong Xia Ma ◽  
Shuang Hou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Bovis ◽  
Expression Vector ◽  
Fusion Gene ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Antigenic Activity ◽  
Sds Page ◽  
Prokaryotic Expression Vector ◽  
Single Antigen

Based on the (Gly4Ser)3 linker, theesat-6andcfp-10gene were fused for raising the antigenicity of single antigen. The DNA fragments ofesat-6andcfp-10were 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 byBamHI andEcoRI 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 competenceEscherichia coliBL21(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 ofMycobacterium 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.

Expression and Identification on the MPB83-MPB70 Fusion Gene of Mycobacterium bovis in Escherichia coli

2014 ◽  
Vol 997 ◽  
pp. 210-214
Author(s):  
Yun Hang Gao ◽  
Yan Liu ◽  
Ying Nan Zhang ◽  
Yan Yu ◽  
Chun Lei Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Bovis ◽  
Expression Vector ◽  
Fusion Gene ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Antigenic Activity ◽  
Sds Page ◽  
Prokaryotic Expression Vector ◽  
Single Antigen

For raising the antigenicity ofMycobacterium bovissingle antigen, fusion protein of two genes was acquired. The DNA fragments ofmpb83andmpb70were 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 byBamH I andEcoR 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 competenceEscherichia coliBL21 (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 ofM.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.

Construction of Prokaryotic Expression Vector Containing Flocculation Gene FLO5 and Expression of FLO5 in E. coli

2013 ◽  
Vol 310 ◽  
pp. 157-161 ◽  
Author(s):  
Jie Xue ◽  
Wen Qiang Wei ◽  
Dong Yan Zhang ◽  
Yong Li Li ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Prokaryotic Expression ◽  
Genomic Dna ◽  
Expression Vector ◽  
Gene Fragment ◽  
Form Expression ◽  
Protein Fragments ◽  
E Coli ◽  
Sds Page ◽  
Prokaryotic Expression Vector

FLO5 has been identified as a dominant flocculation gene. The goal of this study is to clone the FLO5 gene from Saccharomyces cerevisiae and express it in E. coli. In this study, the FLO5 gene amplified by PCR from S. cerevisiae was cloned into prokaryotic expression vector pET-28a to form expression vector pET28a-FLO5, finally, transferred into E.coli BL21. Methods: FLO5 gene was amplified by PCR from genomic DNA extracted from Saccharomyces cerevisiae. The amplified FLO5 gene fragment was then recombined with clone vector pMD18-T to form clone vector pMD18-T-FLO5 amplified in E.coli JM109. After confirmed with sequencing, FLO5 fragment cut out from pMD18-T-FLO5 by enzyme EcoRI and NotI was recombined into expression vector pET-28a to form vector pET28a-FLO5. Vector pET28a-FLO5 was then transferred into E. coli BL21 and protein FLO5 was expressed in E. coli BL21 by the induction with IPTG. Expressed protein fragments separated by SDS-PAGE showed a band with the size of protein FLO5 suggesting the expression of gene FLO5. with the expected This study will lay the foundation for further research in studying flocculating effect of exogenous protein expressed by genetic engineering and making new flocculating agent through recombinant engineering.

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

2013 ◽  
Vol 319 ◽  
pp. 146-150
Author(s):  
Yun Hang Gao ◽  
Chun Fang Wang ◽  
Xiu Yun Jiang ◽  
Hong Xia Ma ◽  
Fan Li Zeng ◽  
...  
Keyword(s):  
Prokaryotic Expression ◽  
Protein Gene ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mycobacterium Paratuberculosis ◽  
Chromosomal Dna ◽  
Gene Encoding ◽  
Pcr Product ◽  
Sds Page ◽  
Antigenic Reactivity

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.

Cloning, Expression and Characterization of β-Glucosidase from L. Delbrueckii Subsp. Delbrueckii in Escherichia coli

2011 ◽  
Vol 301-303 ◽  
pp. 347-351
Author(s):  
Xiu Hong Zhao ◽  
Jie Zeng ◽  
Hai Yan Gao ◽  
Chang Biao Li ◽  
Chang Jiang Liu
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Prokaryotic Expression ◽  
Expression Vector ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gene Encoding ◽  
Prokaryotic Expression Vector ◽  
Strain Bl21

Gene encoding β-glucosidase was amplified through PCR by using the genome DNA extracted from L .delbrueckii subsp. delbrueckii as a template. The gene encoding β-glucosidase was inserted into a prokaryotic expression vector pET-28a(+) and expressed in E.coli strain BL21(DE3). The gene encoding β-glucosidase was of 1380bp. The nucleotide sequence of the gene encoding β-glucosidase from L. delbrueckii subsp. delbrueckii showed as high as 97.9% homology comparing with that from L. delbrueckii subsp. bulgaricus indicating that the gene encoding β-glucosidase is highly conservative. The enzyme activity was about 34U/mg and the molecular weight of β-glucosidase is about 51 kDa analyzed by SDS-polyacrylamide gel electrophoresis.

Expression of a Modified Cry1Ie Gene in E. coli and in Transgenic Tobacco Confers Resistance to Corn Borer

2004 ◽  
Vol 36 (4) ◽  
pp. 309-313 ◽  
Author(s):  
Yun-Jun Liu ◽  
Fu-Ping Song ◽  
Kang-Lai He ◽  
Yuan Yuan ◽  
Xiao-Xia Zhang ◽  
...  
Keyword(s):  
Transgenic Tobacco ◽  
Prokaryotic Expression ◽  
Expression Vector ◽  
Wild Type ◽  
Corn Borer ◽  
E Coli ◽  
Plant Expression Vector ◽  
Sds Page ◽  
Prokaryotic Expression Vector ◽  
Efficient Expression

Abstract The wild-type Cry1Ie gene from Bacillus thuringiensis was modified for its efficient expression in transgenic plants. Modified Cry1Ie gene (designated as Cry1Iem) was cloned into prokaryotic expression vector pET28b and its expression in E. coli was confirmed by SDS-PAGE analysis. Bioassays using crude expression products in E. coli revealed that Cry1Iem protein had a similar toxicity to corn borer as wild-type Cry1Ie. Cry1Iem gene was then inserted downstream of the maize ubiquitin-1 promoter in plant expression vector p3301. Transgenic tobacco plants carrying Cry1Iem showed insecticidal activity against corn borer.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

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