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.

Expressing activator protein Ap36 inBacillus thuringiensisand the function of the recombined strain on disease resistance

2008 ◽  
Vol 5 (2) ◽  
pp. 121-126
Author(s):  
Peng Dong-Hai ◽  
Zhou Chen-Fei ◽  
Qiu De-Wen ◽  
Zhou Kang ◽  
Ruan Li-Fang ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Recombinant Strain ◽  
Phenylalanine Ammonia Lyase ◽  
Protein Gene ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Elicitor ◽  
Sds Page ◽  
Rot Disease ◽  
Derivative Strain

AbstractThe geneap36encoding a protein elicitor fromAlternariasp. was fused downstream of theslh(S-layer homology) motif ofBacillus thuringiensisS-layer protein genectc. The recombinant gene was then transferred intoB. thuringiensisplasmid-free derivative strain BMB171. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the SLH–Ap36 fusion protein was expressed inB. thuringiensisBMB171. After tomato (Lycopersicum esculentum) leaves were treated for 90 min with the recombinant strain cultured at 28°C for 24 h, the activity of peroxidase and the amount of proline of tomato leaves were increased to 57.14% and 131.59%, respectively, compared to the control, and after the tomato leaves were treated with the cultured recombinant strain for 4 days, the activity of phenylalanine ammonia lyase was also higher than that in the control. Furthermore, tubers of treated potato (Solanum tuberosum) plants showed higher resistance to rot disease caused byErwinia corotovoraSCG1 compared to the control treatments.

scholarly journals Comparison of the Proteosomes and Antigenicities of Secreted and Cellular Proteins Produced by Mycobacterium paratuberculosis

2006 ◽  
Vol 13 (10) ◽  
pp. 1155-1161 ◽  
Author(s):  
Donghee Cho ◽  
Michael T. Collins
Keyword(s):  
Solid Phase ◽  
Expression Profiles ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Infected Cattle ◽  
Mycobacterium Paratuberculosis ◽  
Serologic Tests ◽  
Sds Page

ABSTRACT The protein expression profiles and antigenicities of both culture filtrates (CF) and cellular extracts (CE) of Mycobacterium paratuberculosis were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), one-dimensional electrophoresis (1-DE) and 2-DE immunoblotting, and enzyme-linked immunosorbent assay (ELISA). The CF proteins were harvested from supernatants of stationary-phase liquid cultures and concentrated by size exclusion filtration. The CE proteins were extracted by mechanical disruption of cells using glass beads and a high-speed agitator. Analysis of SDS-PAGE gels showed that the majority of CF proteins had low molecular masses (<50 kDa), whereas CE protein mass ranged more evenly over a broader range up to 100 kDa. By 2-DE, CF proteins had a narrow array of pI values, with most being between pH 4.0 and 5.5; CE proteins spanned pI values from pH 4.0 to 7.0. The antigenicities of CF and CE proteins were first determined by 1-DE and 2-DE immunoblotting with serum from a cow naturally infected with M. paratuberculosis. The serum reacted strongly to more proteins in the CF than the CE. Sera from 444 infected and 412 uninfected cattle were tested by ELISA with CF and CE as solid-phase antigens. Receiver-operator characteristic curve analysis of the ELISA results showed a significantly greater area under the curve for CF compared to CE (P < 0.05). A high degree of variability in protein binding patterns was shown with 1-DE immunoblot analysis with 31 sera from M. paratuberculosis-infected cattle. Collectively, these results indicate that serologic tests for bovine paratuberculosis may be improved by using proteins derived from CF instead of CE. To maximize the diagnostic sensitivity of serologic tests, multiple proteins will be required. Even so, a CF ELISA may not be able to detect all M. paratuberculosis-infected cattle, in particular those in the early stages of infection that have yet to mount an antibody response.

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.

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.

scholarly journals Variation in Resistance of Mycobacterium paratuberculosis to Acid Environments as a Function of Culture Medium

2003 ◽  
Vol 69 (11) ◽  
pp. 6833-6840 ◽  
Author(s):  
Nackmoon Sung ◽  
Michael T. Collins
Keyword(s):  
Growth Rate ◽  
Culture Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acid Resistance ◽  
Culture Conditions ◽  
Mycobacterium Paratuberculosis ◽  
In Vitro Susceptibility ◽  
Sds Page

ABSTRACT Acid resistance of Mycobacterium paratuberculosis was examined as a function of growth conditions (i.e., in vitro growth medium and pH). M. paratuberculosis was cultured in either fatty acid-containing medium (7H9-OADC) or glycerol-containing medium (WR-GD or 7H9-GD) at two culture pHs (pHs 6.0 and 6.8). Organisms produced in these six medium and pH conditions were then tested for resistance to acetate buffer at pHs 3, 4, 5, and 6 at 20°C. A radiometric culture method (BACTEC) was used to quantify viable M. paratuberculosis cell data at various acid exposure times, and D values (decimal reduction times, or the times required to kill a 1-log10 concentration of bacteria) were determined. Soluble proteins of M. paratuberculosis grown under all six conditions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to identify proteins that may be associated with acid resistance or susceptibility. The culture medium affected growth rate and morphology: thin floating sheets of cells were observed in 7H9-OADC versus confluent, thick, waxy, and wrinkled pellicles in WR-GD. Culture medium pH affected growth rate (which was highest at pH 6.0), but it had little or no effect on D values for M. paratuberculosis at any test pH. When grown in 7H9-OADC, M. paratuberculosis was more acid resistant at all test pHs (higher D values) than when grown in WR-GD. Glycerol appeared to be the culture medium component most responsible for lower levels of M. paratuberculosis acid resistance. When glycerol was substituted for OADC in the 7H9 medium, D values were significantly lower than those of 7H9-OADC-grown M. paratuberculosis and were approximately the same as those for M. paratuberculosis grown in WR-GD medium. Comparison of the SDS-PAGE protein profiles for M. paratuberculosis cultures grown in 7H9-OADC, WR-GD, or 7H9-GD medium revealed that increased expression of 34.2- and 14.0-kDa proteins was associated with higher levels of acid resistance of M. paratuberculosis grown in 7H9-OADC medium and that 56.6- and 41.3-kDa proteins were associated with lower levels of acid resistance. This is the first report showing that in vitro culture conditions significantly affect growth characteristics, acid resistance, and protein expression of M. paratuberculosis, and the results emphasize the importance of culture conditions for in vitro susceptibility studies.

scholarly journals CS22, a Novel Human EnterotoxigenicEscherichia coli Adhesin, Is Related to CS15

2000 ◽  
Vol 68 (6) ◽  
pp. 3280-3285 ◽  
Author(s):  
Mariana Pichel ◽  
Norma Binsztein ◽  
Gloria Viboud
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Protein Band ◽  
Bacterial Surface ◽  
Pcr Product ◽  
Defective Mutant ◽  
Sds Page ◽  
Structural Subunit ◽  
Colonization Factors

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) expresses a broad spectrum of O:H antigens. Serogroup O20 is one of the most prevalent among the ETEC strains lacking any of the defined colonization factors (CFs), in Argentina. An O20:H− strain, ARG-3, adhered to Caco-2 cells and exhibited a thermoregulated 15.7-kDa protein band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An antiserum against this protein inhibited ARG-3 adhesion to Caco-2 cells and bound to very thin fibrilla-like structures on the bacterial surface. A 15.7-kDa protein-defective mutant failed to adhere to Caco-2 cells and lacked immunogold-labeled surface structures. The N-terminal amino acid sequence of the structural subunit showed 95% homology to that of CS15 of ETEC (former antigen 8786) and 65% homology with fimbria SEF14 of Salmonella enterica serovar Enteritidis. Nevertheless, the molecular size of ARG-3 adhesin was different from that of CS15, as revealed by SDS-PAGE and mass spectrometry. Both proteins are immunologically related, yet not identical, since an antiserum against the 15.7-kDa protein reacted solely with ARG-3 after absorption with bacteria bearing CS15. Moreover, only under low stringency conditions could DNA from strain ARG-3 be amplified by PCR using primers derived from thenfaA sequence of CS15. Thus, from the DNA sequence obtained from the ARG-3 PCR product, it could be deduced that the subunit protein differed in 30 residues from that of CS15. ARG-3 adhesin was found in 60% of the O20:H- CF-negative ETEC strains from Argentina; however, it appeared restricted to this serotype. We propose the designation CS22 for the herein identified nonfimbrial adhesin of human ETEC.

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.

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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