isolation rt pcr and gene sequencing

... presented in this paper expands upon those earlier studies to pro- Methods Cloning of N and NSs genes PCR was used to amplify the open reading frame of N and NSs from the pCAGGS N and NSs vectors respectively ... study and the molecular cloning AKM performed the cloning, gene expression, purification, immunoassays and drafted the manuscript STN conceived of the study and participated in its design and coordination ... is important for vaccine acceptance given regulations restricting movement and export of infected animals in the affected areas In addition to indirectly reducing human morbidity and mortality...

Ngày tải lên: 12/08/2014, 04:21

... investigated, especially using the endogenous conotoxins as substrates In this article, we report gene cloning, recombinant expression and enzymatic activity analyses of a novel Conus marmoreus PDI (cPDI) ... using various substrates and compared with those of hPDI The recombinant cPDI and hPDI shared similar reductase, oxidase and isomerase activities PDI exhibits both chaperone and antichaperone activities ... Cl, pH 7.5, mM EDTA) The filled and open circles denote native and linear tx3a, respectively (B) Refolding carried out in buffer B (buffer A plus mM GSH and mM GSSG) and in buffer C (buffer B plus...

Ngày tải lên: 07/03/2014, 05:20

... hVb2.1hCb2 and hVb1hCb2 (genes kindly provided by U Utz and R P Sekaly, University of Montreal, Canada), were cloned between the NdeI and EcoRI restriction sites of the pET17b expression vector and ... pyogenes superantigen (SSA) The ssa-1 gene was PCR amplified from Streptococcus pyogenes DNA (ATCC 51500 strain) or clinical isolates of S pyogenes with 5¢ and 3¢ terminal oligonucleotides specific ... SMEZ1 and 2), three have one Cys (SEI, SEK and SPEC), two have three Cys (SEG and SPEA) and only SSA has more than that, five Cys As discussed later, the fact that SSA has two Cys residues (Cys26 and...

Ngày tải lên: 07/03/2014, 16:20

... patterns, RT- PCR analyses were carried Ó FEBS 2003 4676 K Asawatreratanakul et al (Eur J Biochem 270) Fig Overexpression of HRT1 and HRT2 in E coli and purification of HRT2 The pETHRT1 and pETHRT2 were ... resulting plasmids designated pJRHRT1 and pJRHRT2, contained HRT1 and HRT2 respectively The SNH23-7D yeast strain was transformed with plasmid pJRHRT1 and pJRHRT2 according to the protocol of ... amplified by RT- PCR with the latex poly(A)+ RNA as the template and then sequenced Finally, two cDNAs were obtained and designated HRT1 and HRT2, respectively DNA sequencing analysis Materials and methods...

Ngày tải lên: 07/03/2014, 21:20

... ST1 and SULT1 ST2 in cultured zebrafish liver cells and whole zebrafish To examine the presence of mRNA encoding zebrafish SULT1 ST1 or SULT1 ST2, RT- PCR was used As shown in Fig 7A, a discrete PCR ... sample (lane 3) and the whole zebrafish sample (lane 4) The authenticity of the PCR products corresponding to SULT1 ST1 and cDNAs was confirmed by nested PCR using the primary PCR products as templates ... zebrafish SULT1 ST1 and ST2 mRNAs and (B) Western blot analysis of zebrafish SULT1 ST1 protein (A) Detection of zebrafish SULT1 ST1 and ST2 mRNAs in cultured zebrafish cells (lanes and 3) and whole zebrafish...

Ngày tải lên: 08/03/2014, 02:20

... subtilis and B circulans should belong to subgroup Ib Expression and purification of AATB3 and its mutants To produce recombinant AATB3 and the three mutant proteins, the aatB3 gene and its mutants ... AAT and PLP, the Asp232 and Lys270 residues (corresponding to Asp222 and Lys258 in E coli AAT) were replaced with Asn and His using site-directed mutagenesis to obtain the mutants D232N and K270H, ... residues and is encoded by the aatB3 gene We found the typical promoter and terminator regions upstream and downstream, respectively, of this new gene To examine explicitly the phylogenetic relationship...

Ngày tải lên: 14/03/2014, 23:20

... peptides, the putative TPS gene from M tuberculosis was identified In this report, we describe the cloning and expression of the M tuberculosis tps gene in E coli, and the production of active ... 20 amino acids starting at position 397 of the M tuberculosis TPS, and another sequence of about 16 amino acids starting at position 421 of the M tuberculosis TPS Isolation and purification of ... has also been found in bacteria and involves three genes (treZ, treX and treY) that encode enzymes that convert sugars from glycogen into trehalose [42] These genes have been found in Sulfolobus...

Ngày tải lên: 17/03/2014, 10:20

... smegmatis treS gene and expressed it as active enzyme in E coli In this report, we describe the purification of TreS from M smegmatis, as well as the isolation of active recombinant TreS, and its enzymatic ... peptides allowed the gene for TreS to be identified in the genome and its cloning and expression in E coli properties as the purified 68-kDa protein from M smegmatis On the other hand, active TreS, ... carbohydrate analyzer to separate and quantify maltose and trehalose Trehalose formation could also be measured using a specific trehalase to convert trehalose to glucose, and then determining the amount...

Ngày tải lên: 23/03/2014, 13:20

... sole carbon source and it is thought to be an important organism in the degradation of biomass Six T fusca cellulase genes and a xylanase gene have been cloned, expressed and characterized [8–11] ... xyloglucanase and investigate its properties and its possible role in the degradation of plant biomass Materials and methods Protein production and purification Clone KPR17269, containing the gene for ... into BL21 gold (DE3) (Stratagene) and the correct strain (pMC1, D1212) was identified, as described above The portion of each plasmid created by PCR was sequenced and no unwanted mutations were...

Ngày tải lên: 23/03/2014, 21:20

... lane 0, protein standards (C) Protein bands after affinity chromatography and renaturing process Lanes and 2, protein bands after separation by affinity column; lanes and 4, protein bands after renaturing ... the gene cloning and characterization of three avian cathelicidin orthologs, namely Cc-CATH precursors from Coturnix coturnix, is reported, and the relationship between quail cathelicidins and ... 5¢-UTR and a highly conserved domain encoding the signal peptide of fowlicidins, and coupled with CDS III ⁄ 3¢ PCR primer The half nested PCR conditions consisted of two parts The first part comprised:...

Ngày tải lên: 28/03/2014, 23:20

... DNA sequencing Gene 28, 351– 359 25 Bibb, M.J., Findlay, P.R & Johnson, M.W (1984) The relationship between base composition and codon usage in bacterial genes and its use in the simple and reliable ... for min, and 72 °C for min) and an additional extension step at 72 °C for 10 The PCR product (about 1.1 kb) was purified by agarose gel electrophoresis and then subcloned into the NdeI and HindIII ... overnight in Luria–Bertani medium supplemented with ampicillin (100 lgÆmL)1), and then 40 mL Luria–Bertani medium was inoculated with 40 lL of the above fresh overnight culture and incubated at 37...

Ngày tải lên: 31/03/2014, 08:20

... cells using Western blotting, and analyzed the DPV gE gene transcription in DPV-infected cells using the real time PCR and RT- PCR Results Cloning of DPV gE gene and the correct recombinant plasmid ... quantitative PCR with SYBR Green I and reverse transcription -PCR (RT- PCR) , the PCR samples amplified were detected by 1.0% agarose gel electrophoresis (Fig 6A) As shown in Fig 6B, the gE gene was ... Page of 11 tion of the DPV gE gene using RT- PCR and real-time quantitative PCR DPV gE earliest transcripts were detected at h post-infection by RT- PCR, and markedly increased at 36 h post-infection...

Ngày tải lên: 12/08/2014, 04:20

... were based on the phylogenetic analysis of a few housekeeping genes, virulence factors genes (cagA and vacA alleles) Apart from the housekeeping genes and the virulence factor genes, transposable ... and Hook-Nikanne et al (1998) reported four types of insertion sequences in H pylori isolates in the last few years These insertion sequences are IS605, IS606, IS607 and IS608 The function, genetic ... (or Ks/Ka > 1), gene is under negative selection and conserved; if Ka/Ks > (or Ks/Ka < 1), gene is under positive selection and diverged (Hurst, 2002) Calculation of Ks and Ka of genes has been...

Ngày tải lên: 12/09/2015, 10:19

... competent cells and transformation 50 2.5.1.8 Selection and DNA sequencing 51 2.5.2 Other subclonings 52 2.5.2.1 Generation of pJG4-5-OSH6cc construct 52 2.5.2.2 Generation of YEplac-OSH7-GFP and YCplac-OSH7-GFP ... al., 1998), worms (C elegans Sequencing Consortium, 1998), and fungi (Jiang et al., 1994; Schmalix and Bandlow, 1994; Fang et al., 1996; Daum et al., 1999; Hull and Johnson, 1999) These proteins ... pathway Furthermore, ESCRT-I (endosome sorting complex required for transport) was showed to act in the recognition of ubiquitinated cargoes at the endosome and 35 initiate transport of these...

Ngày tải lên: 03/10/2015, 20:57

... further understanding of c-gliadin gene family structure Theor Appl Genet 103, 323–330 16 Hsia CC & Anderson OD (2001) Isolation and characterization of wheat x-gliadin genes Theor Appl Genet 103, ... c-gliadin genes and the general primary structure of c-gliadins Plant Sci 57, 141–150 15 Anderson OD & Hsia CC (2001) The wheat c-gliadin genes: characterization of ten new sequences and further ... genomic gene encoding x-5 gliadin using PCR method To amplify the coding region of the x-5 gliadin gene, PCR primers were designed at the position of the initiation and termination codons of the gene...

Ngày tải lên: 20/02/2014, 01:20

... carp IL-10 and b-actin genes were amplified using a range (21–30) of PCR cycles Following this procedure, an optimal number of PCR cycles (24 for IL-10 and 21 for bactin) was determined and subsequently ... and 57 °C (b-actin), and at 72 °C PCR products were electrophoresed on a 2.0% (w/v) agarose gel to enable detection of the specific bands Results Cloning of the IL-10 gene from carp By using PCR ... Expression analysis of IL-10 by RT- PCR RT- PCR was used to analyse expression of the IL-10 gene in normal (healthy) tissues of carp (Fig 5) Using the primers IL-10-Fw3 and IL-10-Rv3 (Table 1), a 284...

Ngày tải lên: 20/02/2014, 02:21

... LdEcR-R2 primers (52 °C) and with LdEcR-F3 and LdEcR-R3 primers (46 °C), respectively The first PCR with LdUSP-F1 and LdUSP-R1 primers and the second nested PCR with LdUSP-F2 and LdUSP-R1 primers ... cDNA cloning of LdEcR and LdUSP A 379-bp fragment was amplified by RT- PCR using degenerate primers, and its sequence was determined A deduced amino acid sequence of the PCR product was homologous ... corresponding part of EcRs of other insects Then we subsequently conducted 5¢-RACE and 3¢-RACE, and sequences of 1337-bp and 998-bp fragments were determined, respectively Combining these sequences of PCR...

Ngày tải lên: 07/03/2014, 21:20

... Discussion) Fig Puri®cation and properties of wild-type and mutant Rab31 and Rab32 expressed as GST±fusion proteins Samples of platelet particulate fraction protein and of puri®ed GST and GST±Rab proteins ... most strongly in placenta and brain and to a lesser extent in heart and lung, but no signal was detected from liver, skeletal muscle, kidney and pancreas HEL cells, and Ó FEBS 2002 to a lesser ... and at 72 °C, for a total of 21 cycles The PCR product was puri®ed using a QIAquickTM kit and cloned into the BamHI and XhoI sites of the pGEX-4T-1 expression vector The sequence of the insert...

Ngày tải lên: 08/03/2014, 16:20

... All PCR products were sizefractionated by agarose gel electrophoresis and the bands eluted, purified and subcloned in the PCRIITM TOPO vector containing the lac promoter and the b-galactosidase gene, ... primers N9 and N10, and by subsequent cloning and sequencing Based on this partial sequence, 5¢ RACE experiments and RT- PCR (using the primer pair Met and Coda) were performed as described in the ... Cloning and sequence analysis of full-length tryptase cDNAs A partial cDNA (690 bp) encoding a new bovine tryptase isoform (BLT) was obtained from lung mRNA by RT- PCR, using primers N9 and N10, and...

Ngày tải lên: 17/03/2014, 09:20

... vertebrates have been cloned and sequenced [6,7] It is now known that all cytosolic SULTs from vertebrates constitute a gene superfamily and, based on amino acid sequence homology, distinct gene ... SULT1 gene family [11,12], one belongs to the SULT2 gene family [13], and one appears to be independent of all known SULT gene families [14] We report here the molecular cloning, expression and ... 3,3¢,5¢-Triiodo-L-Thyronine (L -rT3 ) L-Thyroxine (L-T4) 17b-Estradiol Estrone 4-Androstene-3,17-dione Cholesterol Corticosterone Cortisone Dehydroepiandrosterone D-Dopa L-Dopa Dopamine Hydrocortisone 17a-Hydroxy...

Ngày tải lên: 23/03/2014, 15:20