Cloning and expression ofSpodoptera lituranucleopolyhedrovirusgp41gene inEscherichia coliand preparation of antibody

2005 ◽  
Vol 2 (2) ◽  
pp. 113-117
Author(s):  
Pan Li-Jing ◽  
Li Zhao-Fei ◽  
Yin Chong ◽  
Lv Lei ◽  
Pang Yi
Keyword(s):  
Fusion Protein ◽  
Prokaryotic Expression ◽  
Recombinant Plasmid ◽  
Nitrilotriacetic Acid ◽  
Cloning And Expression ◽  
Reading Frame ◽  
Pcr Product ◽  
Prokaryotic Expression Vector ◽  
Polymerase Chain

AbstractGP41, a major glycoprotein, identified in the occlusion-derived virions (ODV) of baculoviruses, is required for the egress of nucleocapsids from the nucleus in the pathway of budded virion (BV) synthesis. Using the polymerase chain reaction (PCR), the open reading frame (ORF) ofSpodoptera lituranucleopolyhedrovirus (SpltMNPV)gp41gene was obtained from SpltMNPV genomic DNA. The PCR product was cloned into pMD18-T vector to get the recombinant plasmid (pT-gp41). Thegp41gene was recombinedin vitrowith prokaryotic expression vector pQE30 and transformed intoEscherichia coliM15 [pREP4]. The M15 [pREP4] strain, containinggp41recombinant plasmid, expressed a 37.9 kDa 6×His-tag fusion protein after induction with 1 mmol/l isopropylthio-β-d-galactoside (IPTG). The fusion protein was purified with a nickel-nitrilotriacetic acid (Ni–NTA) resin column and used as the immunogen to raise GP41-specific antibody. Western blotting analysis indicated that the antibody was suitable to be used for further analysis of GP41 protein.

Molecular cloning and characterization of a glutathione S-transferase in the tropical liver fluke, Fasciola gigantica

2009 ◽  
Vol 84 (1) ◽  
pp. 55-60 ◽  
Author(s):  
A. Jedeppa ◽  
O.K. Raina ◽  
S. Samanta ◽  
G. Nagar ◽  
N. Kumar ◽  
...  
Keyword(s):  
Prokaryotic Expression ◽  
Liver Fluke ◽  
Nitrilotriacetic Acid ◽  
Fasciola Gigantica ◽  
Glutathione S Transferase ◽  
Chain Reaction ◽  
Prokaryotic Expression Vector ◽  
Polymerase Chain ◽  
Dot Elisa

AbstractGlutathione S-transferase from an Indian isolate of Fasciola gigantica of buffalo origin was isolated and characterized. Total RNA was transcribed to cDNA by reverse transcription and an amplicon of 657 bp glutathione S-transferase gene was obtained by polymerase chain reaction (PCR). The present isolate showed 99.1% sequence homology with the published sequence of the F. giganticaGST gene of cattle origin, with six nucleotide changes causing an overall change of four amino acids. Glutathione S-transferase protein was expressed in Escherichia coli using a prokaryotic expression vector pPROEXHTb. The recombinant protein was purified under non-denaturing and denaturing conditions by nickel nitrilotriacetic acid (Ni-NTA) affinity chromatography. Recombinant GST protein detected F. gigantica infection in naturally infected buffaloes by dot-ELISA.

scholarly journals Prokaryotic Expression, Purification and Identification of Human PDE9A Protein

2018 ◽  
Vol 10 (1) ◽  
pp. 22
Author(s):  
Huan Rui ◽  
Liqun Wang
Keyword(s):  
Prokaryotic Expression ◽  
Expression Vector ◽  
Cyclic Guanosine Monophosphate ◽  
Culture Conditions ◽  
Guanosine Monophosphate ◽  
Reading Frame ◽  
E Coli ◽  
Prokaryotic Expression Vector ◽  
Solid Foundation ◽  
Polymerase Chain

Cerebral aggregation of beta amyloid plaques (Aβ) and neurofibrillary tangles is responsible for the onset of Alzheimer’s disease (AD), and PDE9A inhibition rescues Aβ-induced deficits in synaptic plasticity and cognition. This study was aimed to express active PDE9A protein for subsequent inhibitor screening. The PDE9A gene was cloned from human cDNA by real-time polymerase chain reaction, and then the gene sequence and its amino acid sequence were analyzed on Lasergene. An inducible expression vector was constructed by enzyme digestion-seamless cloning and transformed into Escherichia coli BL21 (DE3) for PDE9A expression with isopropyl β-D-1-thiogalactopyranoside (IPTG) as an inducer. The recombinant protein was purified by Ni-NTA affinity chromatography and its activity was determined by a phosphodiesterase assay kit. It was found the open reading frame of PED9A was 1035 bp long, the deduced protein was composed of 345 amino acids, and its predicted isoelectric point was about 4.84. The E. coli vector ST6-PDE9A successfully expressed the recombinant PDE9A protein in the supernatant of bacterial lysate. The optimal culture conditions were that the bacterium ST6-PDE9A was grown first in a lysogeny broth at 37 ºC to an OD600 of 0.6-0.8 and then at 16 ºC for 40 h with the addition of 1 M IPTG. Activity test showed PDE9A significantly hydrolyzed the substrate cyclic guanosine monophosphate. In conclusion, we constructed a prokaryotic expression vector and expressed active proteins, laying a solid foundation for screening PDE9 inhibitors.

PROKARYOTIC EXPRESSION OF RECOMBINANT MOUSE OP-1 AND ITS OSTEOGENESIS IN VITRO

2002 ◽  
Vol 06 (01) ◽  
pp. 9-15
Author(s):  
Zhenhua Lu ◽  
T. Sam Lindholm
Keyword(s):  
Recombinant Protein ◽  
Prokaryotic Expression ◽  
Development Research ◽  
Activity Assay ◽  
Rt Pcr ◽  
Iptg Induction ◽  
Reading Frame ◽  
Prokaryotic Expression Vector ◽  
Osteogenesis In Vitro

OP-1 is a main member of the BMP family. It plays diverse and significant roles in growth and differentiation. RT-PCR amplified the open reading frame of mouse OP-1 from cDNAs synthesis from CD-1 mouse embryo. By coding the gene of mOP-1 was inserted into pTrcHis 2B, the prokaryotic expression vector through IPTG induction, and the recombinant protein was isolated and purified with Ni-NTA resin. In an ALP activity assay, rmOP-1 has shown osteogenesis activity in vitro. It would offer a fast and inexpensive means of the productions of recombinant protein and an easy tool for growth and development research on rmOP-1.

Automated closed-vessel system for in vitro diagnostics based on polymerase chain reaction

1993 ◽  
Vol 39 (9) ◽  
pp. 1927-1933 ◽  
Author(s):  
J B Findlay ◽  
S M Atwood ◽  
L Bergmeyer ◽  
J Chemelli ◽  
K Christy ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Pcr Amplification ◽  
Automated System ◽  
Closed Vessel ◽  
Chain Reaction ◽  
Panel Testing ◽  
Pcr Product ◽  
Pcr Products ◽  
Polymerase Chain

Abstract An automated system for polymerase chain reaction (PCR) amplification and detection combats false-positive results caused by "PCR product carryover." The system uses a single vessel for both PCR amplification and the subsequent detection of PCR products, eliminating the need to handle PCR products in an open environment and risk product carryover. The sample and PCR reagents are introduced into one compartment within the vessel, and amplification occurs as they are thermally cycled. Other compartments contain the reagents for detection of PCR products. Pressure from a roller provides for sequential delivery of the contents of the compartments to a detection area. The PCR products are biotinylated at their 5' ends during amplification through the use of biotinylated primers. After delivery to the detection area, they are specifically captured by hybridization with immobilized oligonucleotide probes. Subsequent reaction with streptavidin-horseradish peroxidase conjugate forms a complex that catalyzes dye formation from dye precursor. Wash steps minimize nonspecific background. This format is amenable to multiplexing, permitting internal controls, speciation of bacteria, typing of viruses, and panel testing. An HIV assay performed with this system demonstrated 100% sensitivity and 95% specificity for 64 patients' samples relative to a conventional PCR assay based on 32P solution hybridization. Similarly, an automated closed-vessel assay of cytomegalovirus exhibited 97.5% sensitivity and 100% specificity.

scholarly journals Properties of human testis-specific lactate dehydrogenase expressed from Escherichia coli

1991 ◽  
Vol 273 (3) ◽  
pp. 587-592 ◽  
Author(s):  
K M LeVan ◽  
E Goldberg
Keyword(s):  
Escherichia Coli ◽  
Lactate Dehydrogenase ◽  
Prokaryotic Expression ◽  
Specific Activity ◽  
Human Testis ◽  
Reading Frame ◽  
E Coli ◽  
Heat Stable ◽  
Prokaryotic Expression Vector ◽  
Tac Promoter

The cDNA encoding the C4 isoenzyme of lactate dehydrogenase (LDH-C4) was engineered for expression in Escherichia coli. The Ldh-c open reading frame was constructed as a cassette for production of the native protein. The modified Ldh-c cDNA was subcloned into the prokaryotic expression vector pKK223-3. Transformed E. coli cells were grown to mid-exponential phase, and induced with isopropyl beta-D-thiogalactopyranoside for positive regulation of the tac promoter. Induced cells expressed the 35 kDa subunit, which spontaneously formed the enzymically active 140 kDa tetramer. Human LDH-C4 was purified over 200-fold from litre cultures of cells by AMP and oxamate affinity chromatography to a specific activity of 106 units/mg. The enzyme was inhibited by pyruvate concentrations above 0.3 mM, had a Km for pyruvate of 0.03 mM, a turnover number (nmol of NADH oxidized/mol of LDH-C4 per min at 25 degrees C) of 14,000 and was heat-stable.

scholarly journals Ochratoxigenic fungi and Ochratoxin A determination in dried grapes marketed in Tunisia

2020 ◽  
Vol 70 (1) ◽  
Author(s):  
Samir Chebil ◽  
Wafa Rjiba-Bahri ◽  
Souheib Oueslati ◽  
Hanen Ben Ismail ◽  
Anis Ben-Amar ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Ochratoxin A ◽  
High Performance ◽  
Its Region ◽  
Production Test ◽  
Potential Health ◽  
Dried Grapes ◽  
Pcr Product ◽  
Polymerase Chain

Abstract Purpose With the present work, we aimed to assess the occurrence of ochratoxigenic fungi and Ochratoxin A (OTA) in dried grapes from Tunisia. Methods Dried grapes samples (n = 90) were investigated for the presence of ochratoxigenic fungi, which were further characterized at the species level through amplification of the internal transcribed spacer (ITS) region and polymerase chain reaction (PCR) product sequencing. Fungal isolates were tested for their ochratoxigenic potential by high-performance liquid chromatography with fluorescence detection (HPLC-FLD), as well as dried grapes samples after an immunoaffinity column (IAC) clean-up procedure. Results Black Aspergilli isolates were the dominant genre among the filamentous fungi found in dried grapes samples and were the only OTA-producing fungi encountered. Aspergillus niger aggregate were the most frequently found isolates reaching 70%, 80%, and 85% in dried grapes samples from regions of Kelibia, Sfax, and Rafraf, respectively, while covered 100% of the relevant mycobiota found in imported samples. Aspergillus carbonarius isolates were found only in Sfax’s and Kelibia’s samples, while uniseriate Aspergilli were found between 7 and 20% in dried grapes from Kelibia, Sfax, and the imported samples. The in vitro OTA production test showed that 88.9% of OTA-producing isolates belonged to A. carbonarius with OTA levels varying from 0.06 to 1.32 μg/g of Czapek Yeast Agar (CYA). The remaining OTA-producing fungi (11.1 %) belonged to A. niger aggregate group having a maximum OTA potential of 2.88 μg/g CYA, and no uniseriate Aspergilli isolate was able to produce OTA. All dried grapes samples were free of OTA presence. Conclusion According to the present study’s findings, no OTA contamination was recorded in the investigated samples from Tunisian market. Nevertheless, the presence of strong OTA producers A. carbonarius in samples originated from the two out of three studied Tunisian regions, as well the high incidences of Aspergillus niger aggregate group with an attested potential for OTA production in all samples, necessitates further research on Tunisian dried grapes. Additionally, a continuous analysis of staple food of the Mediterranean diet is imperative to insure the best quality for the consumers and prevent potential health problems.

scholarly journals Cloning and expression of human liver dehydroepiandrosterone sulphotransferase

1993 ◽  
Vol 289 (1) ◽  
pp. 233-240 ◽  
Author(s):  
K A Comer ◽  
J L Falany ◽  
C N Falany
Keyword(s):  
Molecular Mass ◽  
Bile Acids ◽  
Blot Analysis ◽  
Human Liver ◽  
Molecular Size ◽  
Cloning And Expression ◽  
Reading Frame ◽  
Acid Protein ◽  
Liver Cdna Library

Dehydroepiandrosterone sulphotransferase (DHEA-ST) catalyses the 3′-phosphoadenosine 5′-phosphosulphate-dependent sulphation of a wide variety of steroids in human liver and adrenal tissue and is responsible for most, if not all, of the sulphation of bile acids in human liver. This report describes the isolation, characterization and expression of a cDNA which encodes human liver DHEA-ST. The DHEA-ST cDNA, designated DHEA-ST8, was isolated from a Uni-Zap XR human liver cDNA library and is composed of 1060 bp and contains an open reading frame encoding a 285-amino-acid protein with a molecular mass of approx. 33765 Da. Translation of DHEA-ST8 in vitro generated a protein identical in molecular size with that of DHEA-ST. Expression of DHEA-ST8 in COS-7 cells produces an active DHEA-ST protein which is capable of sulphating DHEA, has the same molecular mass as human liver DHEA-ST and is recognized by rabbit anti-(human liver DHEA-ST) antibodies. Northern-blot analysis of human liver RNA detects the presence of three different size transcripts; however, Southern-blot analysis of human DNA suggests that only one gene may be present in the genome. These results describe the cloning of a human ST which has an important role in the sulphation of steroids and bile acids in human liver and adrenals.

High Expression and Preliminary Purification of Human β-Defensin-2 Fusion Protein

2013 ◽  
Vol 781-784 ◽  
pp. 1076-1079
Author(s):  
Hong Tao Wei ◽  
Zhong Wen Lv ◽  
Xue Mei Han ◽  
Guo Li Zhang
Keyword(s):  
Fusion Protein ◽  
Metal Chelate ◽  
Genetically Engineered ◽  
High Expression ◽  
Soluble Form ◽  
Freezing And Thawing ◽  
Preliminary Purification ◽  
Prokaryotic Expression Vector ◽  
Genetically Engineered Bacteria

This study was undertaken to achieve high expression and preliminary purification of human β-defensin-2 fusion protein to lay a solid foundation for production of human β-defensin-2 using genetic engineering. A prokaryotic expression vector for human β-defensin-2 fusion protein was generated using in vitro gene synthesis before transformation into BL21 (l DE3) plysS TrX-B host bacteria. High expression of TrX-A-HBD-2 fusion protein was induced with IPTG in the bacteria exposed to various expression conditions. The fusion protein then underwent preliminary purification. The protein of interest was released from the genetically engineered bacteria after freezing and thawing. The expression of the target protein accounted for 16.12% of the total bacterial proteins. Fractional precipitation with saturated ammonium sulfate and metal chelate affinity chromatography yielded human β-defensin-2 peptide fusion protein, with a relative purity of 80.53%.Human β-defensin-2 fusion protein could be highly expressed in a soluble form, with a relatively high purity

scholarly journals Cloning and expression of a human choline/ethanolaminephosphotransferase: synthesis of phosphatidylcholine and phosphatidylethanolamine

1999 ◽  
Vol 339 (2) ◽  
pp. 291-298 ◽  
Author(s):  
Annette L. HENNEBERRY ◽  
Christopher R. McMASTER
Keyword(s):  
De Novo ◽  
Active Form ◽  
Fatty Acyl ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Kennedy Pathway ◽  
Membrane Spanning

Cholinephosphotransferase catalyses the final step in the synthesis of phosphatidylcholine (PtdCho) via the Kennedy pathway by the transfer of phosphocholine from CDP-choline to diacylglycerol. Ethanolaminephosphotransferase catalyses an analogous reaction with CDP-ethanolamine as the phosphobase donor for the synthesis of phosphatidylethanolamine (PtdEtn). Together these two enzyme activities determine both the site of synthesis and the fatty acyl composition of PtdCho and PtdEtn synthesized de novo. A human choline/ethanolaminephosphotransferase cDNA (hCEPT1) was cloned, expressed and characterized. Northern blot analysis revealed one hCEPT1 2.3 kb transcript that was ubiquitous and not enriched, with respect to actin, in any particular cell type. The open reading frame predicts a protein (hCEPT1p) of 416 amino acid residues with a molecular mass of 46550 Da containing seven membrane-spanning domains. A predicted amphipathic helix resides within the active site of the enzyme with the final two aspartic residues of the CDP-alcohol phosphotransferase motif, DG(X)2AR(X)8G(X)3D(X)3D, positioned within this helix. hCEPT1p was successfully expressed in a full-length, active form in Saccharomyces cerevisiae cells devoid of endogenous cholinephosphotransferase or ethanolaminephosphotransferase activities (HJ091, cpt1::LEU2 ept1-). In vitro, hCEPT1p displayed broad substrate specificity, utilizing both CDP-choline and CDP-ethanolamine as phosphobase donors to a broad range of diacylglycerols, resulting in the synthesis of both PtdCho and PtdEtn. In vivo, S. cerevisiae cells (HJ091, cpt1::LEU2 ept1-) expressing hCEPT1 efficiently incorporated both radiolabelled choline and ethanolamine into phospholipids, demonstrating that hCEPT1p has the ability to synthesize both choline- and ethanolamine- containing phospholipids in vitro and in vivo.

scholarly journals Other Natural Hosts of Potato virus T

Plant Disease ◽  
2000 ◽  
Vol 84 (7) ◽  
pp. 736-738 ◽  
Author(s):  
C. Lizárraga ◽  
M. Querci ◽  
M. Santa Cruz ◽  
I. Bartolini ◽  
L. F. Salazar
Keyword(s):  
Potato Virus ◽  
Germplasm Bank ◽  
Chain Reaction ◽  
Oxalis Tuberosa ◽  
Pcr Product ◽  
Ullucus Tuberosus ◽  
Natural Hosts ◽  
Polymerase Chain ◽  
Andean Tuber Crops

Potato virus T (PVT), a member of the genus Trichovirus, was isolated from leaves of naturally infected ulluco (Ullucus tuberosus), oca (Oxalis tuberosa), and mashua (Tropaeolum tuberosum). These Andean tuber crops are often grown in small plots in association with potato (Solanum tuberosum) in the Peruvian highlands. PVT isolates from ulluco, oca, mashua, and potato infected virus-free ulluco, oca, and potato genotypes by mechanical inoculation. The incidence of PVT in mashua, oca, and ulluco accessions from the International Potato Center (CIP) in vitro germplasm bank was less than 10%. A polymerase chain reaction (PCR) product of approximately 330 bp was obtained from each of the four isolates using primers designed from the published PVT sequence. Restriction enzyme digestions of the PCR product did not demonstrate variability.

Sign in / Sign up

Export Citation Format

Share Document