Molecular cloning and expression of the full-length tropomyosin gene from Trichinella spiralis

2003 ◽  
Vol 77 (1) ◽  
pp. 57-63 ◽  
Author(s):  
T. Nakada ◽  
I. Nagano ◽  
Z. Wu ◽  
Y. Takahashi
Keyword(s):  
Trichinella Spiralis ◽  
Expression System ◽  
Muscle Layer ◽  
Full Length ◽  
Cloning And Expression ◽  
Western Blots ◽  
Reading Frame ◽  
Muscle Larvae ◽  
Taxonomical Position ◽  
Good Agreement

AbstractA clone, designated as TsTM, was selected from the cDNA library of newborn larvae (NBL) of Trichinella spiralis through immunoscreening against infected sera. The clone contained a cDNA transcript of 855 bp in length with a single open reading frame, which encoded 285-amino acids (33 kDa in the estimated molecular weight). A sequence analysis revealed that the clone TsTM encoded the full-length of tropomyosin gene. The phylogenetic analysis of the tropomyosin gene was in good agreement with the classical taxonomical position of T. spiralis. The fusion proteins encoded by the clone TsTM were produced in an Escherichia coli expression system and affinity purified, and the antibody was raised against the protein for the following studies. The antibody against the fusion protein positively bound to the hypodermal muscle layer in immunolocalization analysis, and the 35 kDa band in crude extracts of muscle larvae but not in excretory and secretory (ES) products on Western blots. The antigenicity of the clone TsTM was recognized by host mice but exhibited little species specificity.

scholarly journals A Novel Selenite- and Tellurite-Inducible Gene inEscherichia coli

2000 ◽  
Vol 66 (11) ◽  
pp. 4972-4978 ◽  
Author(s):  
Julie Guzzo ◽  
Michael S. Dubow
Keyword(s):  
Expression System ◽  
Dot Blot ◽  
Protein Motif ◽  
Reading Frame ◽  
Sodium Tellurite ◽  
Dot Blot Analysis ◽  
Toxic Trace Element ◽  
Good Agreement ◽  
Inducible Gene

ABSTRACT Selenium is both an essential and a toxic trace element, and the range of concentrations between the two is extremely narrow. Although tellurium is not essential and is only rarely found in the environment, it is considered to be extremely toxic. Several hypotheses have been proposed to account for the toxic effects of selenite and tellurite. However, these potential mechanisms have yet to be fully substantiated. Through screening of an Escherichia coli luxABtranscriptional gene fusion library, we identified a clone whose luminescence increased in the presence of increasing concentrations of sodium selenite or sodium tellurite. Cloning and sequencing of theluxAB junction revealed that the fusion had occurred in a previously uncharacterized open reading frame, termed o393or yhfC, which we have now designated gutS, for gene up-regulated by tellurite and selenite. Transcription fromgutS in the presence of selenite or tellurite was confirmed by RNA dot blot analysis. In vivo expression of the GutS polypeptide, using the pET expression system, revealed a polypeptide of approximately 43 kDa, in good agreement with its predicted molecular mass. Although the function of GutS remains to be elucidated, homology searches as well as protein motif and secondary-structure analyses have provided clues which may implicate GutS in transport in response to selenite and tellurite.

Epitope Mapping of a Monoclonal Antibody against VWF Which Diminishes the Proteolytic Cleavage of VWF by ADAMTS13 Under Flow.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2133-2133
Author(s):  
Jingyu Zhang ◽  
Zhenni Ma ◽  
Ningzheng Dong ◽  
Jian Su ◽  
Anyou Wang ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Proteolytic Activity ◽  
Recombinant Proteins ◽  
Expression System ◽  
Guanidine Hydrochloride ◽  
Proteolytic Cleavage ◽  
Terminal Region ◽  
Full Length ◽  
Western Blots ◽  
A2 Domain

Abstract Abstract 2133 Poster Board II-110 Introduction: In our former study, we have found that SZ-34, a monoclonal antibody to Von Willebrand factor (VWF), can inhibit the proteolysis of VWF by ADAMTS13 under shear stress. But the precise epitope of this antibody (SZ-34) on VWF is not clear for it is generated by immunizing mouse with native full-length VWF purified from pooled human normal plasmas. Thus, the objective of this study is to map the epitope of SZ-34 and to explore the effect of VWF structrue on the proteolytic activity by ADAMTS13. Materials and Methods: Firstly we constructed and expressed a series of recombinant proteins of different domains or polypeptide fragments of human VWF in prokaryotic cell expression system, including A1A2A3, D′D3, A1, A2, A3, A1A2, A2A3 and five sub-fragments of A2 domain. Then native VWF and these recombinant proteins or polypeptide fragments were subjected to polyacrylamide gel electrophoresis (PAGE) and analyzed by Western blots with SZ-34. Results: Different recombinant proteins of VWF were successfully expressed and purified. Results of Western blot showed that SZ-34 could bind specifically some recombinant proteins, such as full-length VWF, A1A2A3, A2 and GST-D1459D1596 in which the last was a fusion protein of a sub-fragment of A2 domain with GST. But SZ-34 couldn't bind to others, including A1, A3, D′D3, GST-D1459E1554, GST-E1554D1596, GST-D1596R1668 (VWF73) and GST- E1554R1668. In addition, the reacting activity of SZ-34 with native VWF was significantly stronger than with unfolded VWF, such as heat-treated or 1.5M guanidine hydrochloride-treated VWF. Conclusions: The epitope of SZ-34 is located within N-terminal region fore-VWF73 inside VWF-A2 domain. Besides, SZ-34 maybe is a conformation-specific monoclonal antibody. Combining with our former findings that SZ-34 inhibits the proteolytic cleavage of VWF by ADAMTS13, we can conclude that N-terminal region fore-VWF73 inside VWF-A2 domain also regulates the proteolytic activity of VWF by ADAMTS13, although VWF73 is considered as the minimal substrate for ADAMTS13. Disclosures: No relevant conflicts of interest to declare.

A panel of antigens of muscle larvae of Trichinella spiralis and T. pseudospiralis as revealed by two-dimensional Western blot and immunoelectron microscopy

Parasitology ◽  
1999 ◽  
Vol 118 (6) ◽  
pp. 615-622 ◽  
Author(s):  
Z. WU ◽  
I. NAGANO ◽  
Y. TAKAHASHI
Keyword(s):  
Western Blot ◽  
Trichinella Spiralis ◽  
Cross Reactivity ◽  
Microscopical Study ◽  
Detectable Level ◽  
Antigenic Peptides ◽  
Two Dimensional ◽  
Western Blots ◽  
Muscle Larvae ◽  
Isoelectric Points

This study characterized antigens of Trichinella spiralis and T. pseudospiralis muscle larvae recognized by mice infected with the worms. Two-dimensional (2-D) Western blot analysis revealed some profile of antigenic peptides including: (1) molecular weight (MW); (2) isoelectric points (pI), (3) reactivity to well-defined monoclonal antibodies (mAb) and (4) cross-reactivity between the 2 species. Antigenic peptides of T. spiralis consisted of about 100 spots. The MW ranged from 22 to 80 kDa, and pI ranged from 4 to 7. The mAb against TSL-1 stained most of the T. spiralis excretory–secretory (E–S) peptides migrating at 40, 45 and 50 kDa, and the mAb against TSL-4 stained non-E–S peptides. Antigenic peptides of T. pseudospiralis consisted of about 20 to 30 peptide spots. The MW ranged from 25 to 80 kDa, and pI ranged from 4 to 7. The mAb against TSL-1 stained most of the T. pseudospiralis E–S peptides migrating at 35 and 45 kDa, and the mAb against TSL-4 stained non-E–S peptides. Two-dimensional Western blots showed that the E–S products of T. spiralis and T. pseudospiralis were highly cross-reactive with each other. The non-E–S peptides were, however, not recognized by T. pseudospiralis-infected sera but were recognized by T. spiralis-infected sera. An immunoelectron microscopical study showed the similar result that stichocyte granules and cuticle surface (known to contain E–S antigen) had cross-reactive antigens between the two species. T. pseudospiralis-infected sera stained very weakly the cuticle inner layers and haemolymph (known to contain non-E–S antigen). This evidence implies that mice infected with T. pseudospiralis do not evoke antibodies against non-E–S antigen at the detectable level.

scholarly journals Cloning and expression of a prostaglandin E receptor EP3 subtype from human erythroleukaemia cells

1994 ◽  
Vol 298 (2) ◽  
pp. 263-267 ◽  
Author(s):  
S P Kunapuli ◽  
G Fen Mao ◽  
M Bastepe ◽  
L Y Liu-Chen ◽  
S Li ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Human Kidney ◽  
High Specificity ◽  
Full Length ◽  
Cloning And Expression ◽  
Prostaglandin E ◽  
Hybridization Probe ◽  
Reading Frame ◽  
Prostaglandin Receptor ◽  
Ep3 Receptor

Prostaglandins inhibit platelet activation by stimulating intracellular cyclic AMP formation. We have postulated that intracellular cyclic AMP levels in platelets are buffered by a distinct prostaglandin receptor that mediates inhibition of cyclic AMP formation. In order to provide evidence for the model, we have cloned the cDNA coding for a prostaglandin receptor EP3 subtype, which is coupled to inhibition of adenylate cyclase, from the megakaryocytic cell line human erythroleukaemia (HEL) cells. A PCR-generated hybridization probe, produced using primers based on the sequence of the mouse prostaglandin EP3 receptor published by Sugimoto, Namba, Honda, Hayashi, Negishi, Ichikawa and Narumiya [(1992) J. Biol. Chem. 267, 6463-6466], was used to screen a lambda gt11 HEL cell cDNA library. The composite full-length cDNA clone HEP3, generated from the two partial clones pHEP3-7 and pHEP3-5, is 1.6 kb long with an open reading frame coding for 390 amino acids. This clone is 83% identical to the alpha subtype of the mouse EP3 receptor. The full-length construct was transfected into COS-1 cells. The cloned receptor exhibited the properties of a prostaglandin EP3 subtype, inhibiting forskolin-stimulated cyclic AMP formation in response to prostaglandin E2 (PGE2) and binding PGE2 with high specificity and a Kd of 3.2 nM. Radiolabelled PGE2 could be displaced by prostaglandins in the order PGE2 = PGE1 > iloprost = PGD2. Northern blot analysis revealed that the receptor is also present in human kidney.

scholarly journals Characteristic band pattern in western blots for specific detection of anti-Trichinella spiralis antibodies in different host species

2014 ◽  
Vol 64 (1) ◽  
pp. 33-43 ◽  
Author(s):  
Nataša Ilić ◽  
Alisa Gruden-Movsesijan ◽  
Milena Živojinović ◽  
Ljiljana Sofronić-Milosavljević
Keyword(s):  
Host Species ◽  
Trichinella Spiralis ◽  
Standard Test ◽  
Antibody Detection ◽  
Reference Laboratory ◽  
Serum Samples ◽  
Western Blots ◽  
Muscle Larvae ◽  
Molecular Masses ◽  
Inhibition Studies

Abstract Western blot (Wb) is considered to be the gold standard test for Trichinella infection serology, since this method allows specific Trichinella antigens to be distinguished from cross-reactive antigens. This is not the case with widely used antibody assay techniques - indirect immunofluorescence and ELISA - which are sensitive, but subject to crossreactions that make the interpretation of weakly positive results difficult. Application of Trichinella spiralis muscle larvae excretory-secretory (ES) antigens for the specific antibody detection in ELISA resulted in improved specificity compared to that of crude worm extract that was previously in use, but since production of ES has not yet been standardized, differences among laboratories occur. For this reason, the Wb profile of serum samples from different T. spiralis infected host species: human, horse, swine and dog, was investigated in the Serbian National Reference Laboratory for Trichinellosis (NRLT). The common feature of the obtained Wb profiles was the appearance of a triad of bands with molecular masses (Mw) of 45, 49, and 53 kDa. The very same triad was recognized by a monoclonal antibody (mAb) 7C2C5 specific for an immunodominant epitope unique to the muscle larvae stage of all species in the genus Trichinella. Inhibition studies confirmed that mAb and anti-Trichinella antibodies from sera competed for the same parasite epitope. Based on the obtained results, the NRLT introduced the recognition of the above mention triad as the basis for specific anti-Trichinella antibodies detection in the sera of infected hosts.

Molecular cloning and characterization of a novel protein of Trichinella pseudospiralis excretory–secretory products

2002 ◽  
Vol 76 (2) ◽  
pp. 165-170 ◽  
Author(s):  
I. Nagano ◽  
Z. Wu ◽  
T. Nakada ◽  
T. Boonmars ◽  
Y. Takahashi
Keyword(s):  
Recombinant Protein ◽  
Expression System ◽  
Specific Expression ◽  
Reading Frame ◽  
Trichinella Pseudospiralis ◽  
Crude Extracts ◽  
Muscle Larvae ◽  
Secretory Products ◽  
Novel Protein

AbstractA novel excretory–secretory (ES) protein of Trichinella pseudospiralis was produced. A cDNA library was constructed from mRNA of muscle larvae at 30 days post infection (p.i.) and immunoscreened with the antibody against ES products. A clone, designated Tp22-3, contained a cDNA transcript of 815 bp in length with a single open reading frame which encoded 244-amino acids (28407 Da in the estimated molecular mass). A database search revealed that no sequences had a homology to this predicted protein. The recombinant protein was produced in an Escherichia coli expression system. Stage specific expression of this protein was suggested from the following experiments. An antibody against the recombinant protein could stain proteins migrating at about 28 kDa (which is the expected size from the sequence) on Western blotting of crude extracts or ES products from 30 days p.i. muscle larvae, but failed to stain any proteins in crude extracts from newborn larvae or 15 days p.i. muscle larvae. The antibody reacted to the stichocytes of larvae at 30 days p.i., but did not react to 15 days p.i. muscle larvae. The production of an mRNA transcript for Tp22-3 gene was restricted largely to the 30 days p.i. muscle larvae and adult worms.

scholarly journals Isolation of the Astacin-like metalloprotease coding gene (astl) and assessment of its insecticidal activity against Spodoptera littoralis and Sitophilus oryzae

2019 ◽  
Author(s):  
Mervat R. Diab ◽  
Ibtissam H.A. Hussein ◽  
Mahmoud M. Ahmed ◽  
Ahmed Mohammed
Keyword(s):  
Insecticidal Activity ◽  
Spodoptera Littoralis ◽  
Catalytic Domain ◽  
Expression System ◽  
Sitophilus Oryzae ◽  
Full Length ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Spider Species ◽  
Yeast Expression System

AbstractAstacin- like metalloprotease (astl) is a multi-domain metallopeptidase that has protease activity against a number of organisms; including fish, frogs, birds and insects. In this present investigation, the full length of astl cDNA was cloned from spider species, Hasarius adansoni. Sequencing of the cloned astl cDNA proved that its full length including 802 bp with 714bp open reading frame encoding for 238 amino acids. The catalytic domain comprised of 489 nts was cloned and expressed by the yeast expression system Pichia pastoris and its insecticidal activity was determined against two species of agricultural insects Spodoptera littoralis (Lepidoptera:Noctuidae) and Sitophilus oryzae (Coleoptera:Curculionidae). Bioassay was performed using three concentrations (100,500 and 1000 ppm) for four days for S. littoralis and 14 days for S. oryzae. In addition, the astl was fused to the GNA snowdrop lectin in the same frame and expressed in P. pastoris. The synergistic effect of astl and GNA was examined on the S. littoralis larvae and S. oryzae adults. The mortality percentages of the fused protein (Ha-astl/GNA) “1000 ppm” after 4 days, were 78.6%± 4.16 and 71.66% ±3.51 for first and second spodpotera larval instars, respectively. While, lower mortality of the fused protein of the same concentration was observed on S. oryzae adults, 49.3±2.08 %.

Molecular Cloning and Expression of Rabbit Heparin Cofactor II: A Plasma Thrombin Inhibitor Highly Conserved between Species

1994 ◽  
Vol 71 (06) ◽  
pp. 778-782 ◽  
Author(s):  
William P Sheffield ◽  
Philip D Schuyler ◽  
Morris A Blajchman
Keyword(s):  
Amino Acids ◽  
Untranslated Region ◽  
Expression System ◽  
Open Reading Frame ◽  
Cloning And Expression ◽  
Thrombin Inhibitor ◽  
Cdna Clones ◽  
Mature Protein ◽  
Heparin Cofactor Ii ◽  
Reading Frame

SummaryHeparin cofactor II (HCII), a circulating plasma protein that inhibits thrombin, is a member of the serine proteinase (serpin) family of proteins. The extent to which HCII structure is conserved actross species lines was investigated, by obtaining cDNA clones encoding rabbit HCII. Overlapping clones corresponding to rabbit HCII were obtained by the combined use of hybridization screening of a rabbit liver cDNA library, and by rapid amplification of cDNA ends (RACE). The consensus sequence obtained spans 2178 nucleotides, and is comprised of a 5' untranslated region of 77 nucleotides, an open reading frame of 1440 nucleotides, and 3' untranslated region of 661 nucleotides that concludes with a poly A tract. The open reading frame is subdivided into a secretory signal sequence of 19 amino acids, and a mature protein of 461 amino acids. Within the region comprising the mature protein, 87% of the amino acid residues are identical to those seen in human HCII. Expression of an appropriately modified form of the rabbit HCII clone in an in vitro reticulocyte expression system yielded two major polypeptides, of 60 and 56 kD respectively, both of which were able to form SDS-stable complexes with human α-thrombin, in a reaction accelerated by dermatan sulphate. The remarkable degree of homology observed between rabbit HCII and its human conterpart, indicating a high degree of conservation of structure through evolution, suggests an important function of HCII in hemostatis.

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