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.

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 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.

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.

Expression of β-agarase I DagA in prokaryotic cell and its activity identification

2009 ◽  
Vol 6 (2) ◽  
pp. 153-159
Author(s):  
Zhou Yan-Sheng ◽  
Wang Bao-Li ◽  
Qu Dong
Keyword(s):  
Molecular Chaperones ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bacterial Protein ◽  
Gene Encoding ◽  
Sds Page ◽  
Optimum Ph ◽  
Pseudoalteromonas Atlantica ◽  
Polymerase Chain ◽  
Ph Range

AbstractThe DagA gene and DagA(▽), which is a DagA gene encoding sequence without signal peptide, were cloned from genome DNA ofPseudoalteromonas atlantica19262 by polymerase chain reaction (PCR). After ligation with pET21 vector, DagA and DagA(▽) were respectively expressed inEscherichia coliER2566 using molecular chaperones DsbC and FkpA. A strain of ER2566-pET21a-DagA(▽)-DsbC was screened as a highly effective expressing system in the form of an inclusion body that had the target protein with up to 60% total bacterial protein. DagA protein was renatured and purified by dissolving it in 8 mol/l of urea, using Ni-NTA resin affinity chromatography and refolding using the urea gradient method. DagA with a molecular weight of ~30.8 kDa was identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and had the ability to digest agarose. In a pH range of 4.8–6.8, DagA maintained a bioactivity greater than 60%, with 5.8 being the optimum pH, and it exhibited activity at temperatures from 37°C to 60°C, with 55°C being the optimum temperature.

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

2013 ◽  
Vol 749 ◽  
pp. 177-181
Author(s):  
Yan Ru Zheng ◽  
Chun Fang Wang ◽  
Yun Hang Gao ◽  
Jia Ning Guan ◽  
Jia Ming Lin ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Mycobacterium Bovis ◽  
Recombinant Plasmid ◽  
Expression Vector ◽  
Fusion Gene ◽  
Eukaryotic Expression ◽  
Chain Reaction ◽  
Eukaryotic Expression Vector ◽  
Polymerase Chain ◽  
I Gene

Based on the splicing by overlapping extension (SOE) polymerase chain reaction (PCR),fusion gene mpb83-70 andil-15were 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 enzymesHindIII andEcoRI, 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.

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

2013 ◽  
Vol 421 ◽  
pp. 308-312
Author(s):  
Shuang Hou ◽  
Chun Fang Wang ◽  
Yan Ru Zheng ◽  
Jia Ning Guan ◽  
Jia Ming Lin ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Mycobacterium Bovis ◽  
Recombinant Plasmid ◽  
Expression Vector ◽  
Fusion Gene ◽  
Eukaryotic Expression ◽  
Chain Reaction ◽  
Eukaryotic Expression Vector ◽  
Polymerase Chain

Based on the polymerase chain reaction (PCR),ag85aandmpb70Fusion gene ofMycobacterium boviswere ligated and cloned into pMD18-T, then recombinant plasmid pMD-85a-70 was constructed. pMD-85a-70 and pVAX1-BMS were digested by double enzymesHindIII andEcoRI, 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.

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).

Identifikasi Daging Tikus Pada Produk Baso Dengan Metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

2018 ◽  
Vol 26 (2) ◽  
pp. 058
Author(s):  
Anna P. Roswiem ◽  
Triayu Septiani
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Sds Page

<em>Bahan<strong> </strong>baku untuk membuat baso adalah daging hewan, pada umumnya dari daging sapi, ayam, ikan dan babi. Di beberapa daerah di Indonesia terjadi kasus baso tikus. Tujuan penelitian ini adalah menguji ada tidaknya kandungan daging tikus pada produk baso yang dijual di pasar Cempaka Putih-Kecamatan Kramat Jakarta Pusat dan di pedagang baso atau mie baso di sekitar kampus Universitas YARSI Jakarta. Daging adalah protein salah satu metode untuk mengidentifikasi protein adalah metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE).<strong> </strong>Hasil penelitian menunjukkan bahwa dari 6 sampel baso terindikasi ada 2 sampel baso dengan nomor 1 dan 5 yang dibuat dari campuran daging sapi dan tikus; ada 1 sampel baso dengan nomor 6 yang terbuat dari daging tikus; dan 2 sampel baso dengan nomor 2 dan 3 yang terbuat dari campuran sapi  dan babi, dan hanya 1 sampel baso dengan nomor sampel 4 yang benar-benar terbuat dari daging sapi.</em>

Majra Honey Abrogated the Normal and Cancer Cells Proliferation Inhibition by Juniperus procera Extract and Extract/Honey Generated AgNPs

2020 ◽  
Vol 20 (8) ◽  
pp. 970-981
Author(s):  
Hamed A. Ghramh ◽  
Essam H. Ibrahim ◽  
Mona Kilnay
Keyword(s):  
Anticancer Activity ◽  
Cancer Cells ◽  
Biological Activities ◽  
Sugar Content ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bioactive Molecules ◽  
Sds Page ◽  
Splenic Cells ◽  
Juniperus Procera

Background: Juniperus procera and Majra honey are well-known as a folk medicine in many countries. Objectives: This work aimed to study the immunomodulatory effects after mixing Majra honey, J. procera water leaves extract and silver Nanoparticles (AgNPs) on immune or cancer cells. Methods: Juniperus procera water leaves extract and 20% Majra honey were prepared. Both the extract and honey were used separately to synthesize AgNPs. AgNPs were characterized using UV/Vis spectrophotometry and electron microscopy. Bioactive molecules in honey and the extract were explored using Fourier Transform Infrared (FT-IR) spectroscopy. Protein profile of honey was explored using Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) and honey sugar content was determined using High- Performance Liquid Chromatography (HPLC). Biological activities of honey and the extract were tested. Results: The results demonstrated the ability of the extract/honey to produce AgNPs in a spherical shape. The extract/honey contained many functional groups. SDS-PAGE of Majra honey showed many protein bands. HPLC revealed honey is of good quality and no external additives are added to it. The extract and extract+ AgNPs inhibited the growth of normal rat splenic cells while honey stimulated it. The extract+honey turned stimulatory to the splenic cells’ growth and significantly diminished the inhibitory potential of the extract containing AgNPs. Both the extract and honey have antimicrobial activities, this potential increased in the presence of AgNPs. Honey and Honey+AgNPs inhibited HepG2 cancer cell proliferation while Hela cell growth inhibited only with honey+AgNPs. Conclusion: Both honey and the extract have antibacterial and immunomodulatory potentials as well as the power to produce AgNPs. Majra honey alone showed anticancer activity against HepGe2 cells, but not against Hela cells, and when contained AgNPs had anticancer activity on both cell lines. Mixing of Majra honey with J. procera extract showed characterized immunomodulatory potentials that can be described as immunostimulant.

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