scholarly journals Cloning and Expression of Ama r 1, as a Novel Allergen of Amaranthus retroflexus Pollen

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Payam Morakabati ◽  
Mohammad-Ali Assarehzadegan ◽  
Gholam Reza Khosravi ◽  
Bahareh Akbari ◽  
Fatemeh Dousti
Keyword(s):  
Specific Ige ◽  
Cross Reactivity ◽  
Amaranthus Retroflexus ◽  
Protein Family ◽  
Cloning And Expression ◽  
Binding Potential ◽  
Amino Acid Residues ◽  
Serum Samples ◽  
Reading Frame ◽  
The Family

Sensitisation to Amaranthus retroflexus pollen is very common in tropical and subtropical countries. In this study we aimed to produce a recombinant allergenic Ole e 1-like protein from the pollen of this weed. To predict cross-reactivity of this allergen (Ama r 1) with other members of the Ole e 1-like protein family, the nucleotide sequence homology of the Ama r 1 was investigated. The expression of Ama r 1 in Escherichia coli was performed by using a pET-21b(+) vector. The IgE-binding potential of recombinant Ama r 1 (rAma r 1) was evaluated by immunodetection and inhibition assays using 26 patients’ sera sensitised to A. retroflexus pollen. The coding sequence of the Ama r 1 cDNA indicated an open reading frame of 507 bp encoding for 168 amino acid residues which belonged to the Ole e 1-like protein family. Of the 26 serum samples, 10 (38.46%) had significant specific IgE levels for rAma r 1. Immunodetection and inhibition assays revealed that the purified rAma r 1 might be the same as that in the crude extract. Ama r 1, the second allergen from the A. retroflexus pollen, was identified as a member of the family of Ole e 1-like protein.

Cloning and Expression of a Novel Protease with Fibrinolytic Activity from Arenicola cristata

2014 ◽  
Vol 998-999 ◽  
pp. 210-213
Author(s):  
Chun Ling Zhao ◽  
Wen Jing Yu ◽  
Ji Yu Ju
Keyword(s):  
Amino Acid ◽  
Recombinant Protein ◽  
Active Form ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Reading Frame ◽  
Arenicola Cristata ◽  
Fibrin Plate ◽  
Serine Protease Family

cDNA of a novel protease, designated as AFEI, was cloned from digestive tract of Arenicola cristata by RACE. The cDNA of AFEIcomprised 897bp and an open reading frame that encoded polypeptides of 264 amino acid residues. AFEIshowed similarity to serine protease family and contained the conserved catalytic amino acid residues. The gene encoding the active form of AFEIwas expressed in E.coli and the purified recombinant protein could dissolve an artificial fibrin plate with plasminogen, which indicated the recombinant protein might be a plasminogen activator for thrombosis therapy.

scholarly journals Identification, Cloning, and Expression of the CAMP-Like Factor Autotransporter Gene (cfa) of Bartonella henselae

2005 ◽  
Vol 73 (7) ◽  
pp. 4205-4213 ◽  
Author(s):  
Christine M. Litwin ◽  
Joel M. Johnson
Keyword(s):  
Amino Acid ◽  
Bartonella Henselae ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cloning And Expression ◽  
Cosmid Library ◽  
Group B Streptococci ◽  
Reading Frame ◽  
Calculated Molecular Weight ◽  
The Family

ABSTRACT The CAMP reaction was first described by Christie et al. (R. Christie, N. E. Atkins, and E. Munch-Petersen, Aust. J. Exp. Biol. 22:197-200, 1944) as the synergistic lysis of sheep red blood cells by Staphylococcus aureus sphingomyelinase and CAMP factor (cohemolysin), a secreted protein from group B streptococci. We observed a CAMP-like reaction when Bartonella henselae was grown in close proximity to S. aureus on 5% sheep blood agar. This study describes the cloning, sequencing, and characterization of a CAMP-like factor autotransporter gene (cfa) from B. henselae. A cosmid library of B. henselae ATCC 49793 was constructed using SuperCos1 in Escherichia coli XL1-Blue MR. Cosmids were screened for the CAMP reaction, and a quantitative cohemolysis microtiter assay was developed using purified sphingomyelinase. Cosmid clones with the strongest cohemolytic reaction had similar restriction enzyme patterns. A DNA fragment that expressed the cohemolysin determinant was subcloned in a 7,200-bp StuI-BamHI fragment which contained a 6,024-bp open reading frame. The deduced amino acid sequence showed homology to the family of autotransporters. The autotransporters are a group of proteins that mediate their own export through the outer membrane. They contain an N-terminal passenger region, the α-domain, and a C-terminal transporter region, the β-domain. The α-domain contained four, nearly identical 42-amino-acid repeats and showed homology to the family of RTX (repeat in toxin) hemolysins. The concentrated supernatant of the recombinant strain expressed a protein with a molecular mass of 180 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis consistent with the calculated molecular weight of the secreted α-domain. In conclusion, we have characterized a novel secreted cohemolysin autotransporter protein of B. henselae.

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 Identification of Allergenic Component Tyr p 8 from Tyrophagus putrescentiae and Cross-Reactivity with Der p 8

2013 ◽  
Vol 20 (4) ◽  
pp. 506-512 ◽  
Author(s):  
En-Chih Liao ◽  
Yi-Hsueh Lin ◽  
Chih-Liang Chiu ◽  
Ting-Chu Lin ◽  
Jaw-Ji Tsai
Keyword(s):  
Sequence Homology ◽  
Positive Response ◽  
Specific Binding ◽  
Major Allergen ◽  
Specific Ige ◽  
Cross Reactivity ◽  
House Dust Mites ◽  
Tyrophagus Putrescentiae ◽  
Serum Samples ◽  
Content Type

ABSTRACTGroup 8 mite allergens exhibit sequence homology to glutathioneS-transferases (GSTs), such as that fromDermatophagoides pteronyssinus(Der p 8). GSTs have been identified as important allergens in studies of allergens from house dust mites, cockroaches, and fungi. Our objective was to purify the native group 8 allergen fromTyrophagus putrescentiae(nTyr p 8) and generate recombinant Tyr p 8 (rTyr p 8) for immunological characterization. The allergenicity was determined by antibody recognition, IgE inhibition, and triggering of the basophil-sensitized release of histamine, usingT. putrescentiaehypersensitivity sera. The results showed that the mRNA transcript of nTyr p 8 is 657 bp long, contains 218 amino acids with a molecular mass of 26 kDa, and exhibits 83% sequence homology to Der p 8. Serum samples from the allergic patients with an IgE-positive response toT. putrescentiaewere analyzed to determine their IgE response to rTyr p 8. The results showed that the sera of 48 subjects (45.3%) had specific IgE against rTyr p 8. However, sera of only 19 subjects (17.9%) had specific IgE against rTyr p 8 afterD. pteronyssinusabsorption. Histamine release was observed fromT. putrescentiae-allergic subjects in the presence of rTyr p 8. Both the nTyr p 8 andT. putrescentiaecrude extract had been demonstrated to possess GST enzymatic activity. Although the specific binding of serum IgE to rTyr p 8 was only 17.9%, which indicates that rTyr p 8 was not a major allergen, the positive response to rTyr p 8 was due to the cross-reactivity with Der p 8. The group 8 mite allergen might be of use in the design of a suitable allergen for diagnosis and for the development of novel immunotherapies.

scholarly journals Analysis of IgM, IgA, and IgG Isotype Antibodies Directed Against SARS‑CoV‑2 Spike Glycoprotein and ORF8 in the Course of COVID‑19.

2020 ◽  
Author(s):  
Denise Meinberger ◽  
Manuel Koch ◽  
Annika Roth ◽  
Gabriele Hermes ◽  
Jannik Stemler ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Immunoblot Analysis ◽  
Cross Reactivity ◽  
Spike Glycoprotein ◽  
Serum Samples ◽  
Reading Frame ◽  
Reliable Assessment ◽  
Insight Into ◽  
Inflammation Parameters

Abstract Immunoassays are a standard diagnostic tool assessing immunity in severe acute respiratory syndrome coronavirus type 2 (SARS‑CoV‑2) infection. However, immunoassays do not provide information about contaminating antigens or cross‑reactions and might exhibit inaccurately high sensitivity and low specificity. We aimed to gain insight into the serological immune response of SARS‑CoV‑2 patients by immunoblot analysis.We analyzed serum immunoglobulins IgM, ‑A, and ‑G directed against SARS‑CoV‑2 proteins by immunoblot analysis from 12 infected patients. We determined IgG isotype antibodies by commercially available ELISA and assessed the clinical parameters of inflammation status and kidney and liver injury.We found evidence for antibody cross‑reactivity, which calls into question a reliable assessment of serum samples tested negative for anti‑SARS‑CoV‑2 antibodies by immunoassays. Nevertheless, for the detection of IgG anti‑SARS‑CoV‑2 antibodies, our data suggest that the use of the spike glycoprotein in immunoassays should be sufficient to identify positive patients. Using a combination of the spike glycoprotein and the open reading frame 8 protein could prove to be the best for the detection of patients positive for anti‑SARS‑CoV‑2 IgM antibodies. We found that the antibody response alone is not decisive for the course of the disease, but inflammation parameters are promising indicators.

Identification and characterization of phenol hydroxylase from phenol-degrading Candida tropicalis strain JH8

2014 ◽  
Vol 60 (9) ◽  
pp. 585-591 ◽  
Author(s):  
Yan Long ◽  
Sheng Yang ◽  
Zhixiong Xie ◽  
Li Cheng
Keyword(s):  
Amino Acid ◽  
Candida Tropicalis ◽  
Amino Acid Identity ◽  
Phenol Hydroxylase ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Native Molecular Mass ◽  
Its Sequence Analysis

The gene phhY encoding phenol hydroxylase from Candida tropicalis JH8 was cloned, sequenced, and expressed in Escherichia coli. The gene phhY contained an open reading frame of 2130 bp encoding a polypeptide of 709 amino acid residues. From its sequence analysis, it is a member of a family of flavin-containing aromatic hydroxylases and shares 41% amino acid identity with phenol hydroxylase from Trichosporon cutaneum. The recombinant phenol hydroxylase exists as a homotetramer structure with a native molecular mass of 320 kDa. Recombinant phenol hydroxylase was insensitive to pH treatment; its optimum pH was at 7.6. The optimum temperature for the enzyme was 30 °C, and its activity was rapidly lost at temperatures above 60 °C. Under the optimal conditions with phenol as substrate, the Km and Vmax of recombinant phenol hydroxylase were 0.21 mmol·L–1 and 0.077 μmol·L–1·min−1, respectively. This is the first paper presenting the cloning and expression in E. coli of the phenol hydroxylase gene from C. tropicalis and the characterization of the recombinant phenol hydroxylase.

scholarly journals Analysis of IgM, IgA, and IgG isotype antibodies Directed against SARS-CoV-2 spike glycoprotein and ORF8 in the course of COVID-19

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Denise Meinberger ◽  
Manuel Koch ◽  
Annika Roth ◽  
Gabriele Hermes ◽  
Jannik Stemler ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Immunoblot Analysis ◽  
Cross Reactivity ◽  
Clinical Parameters ◽  
Spike Glycoprotein ◽  
Reliability Of Results ◽  
Serum Samples ◽  
Reading Frame ◽  
Insight Into

AbstractImmunoassays are a standard diagnostic tool that assesses immunity in severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection. However, immunoassays do not provide information about contaminating antigens or cross-reactions and might exhibit inaccurately high sensitivity and low specificity. We aimed to gain insight into the serological immune response of SARS-CoV-2 patients by immunoblot analysis. We analyzed serum immunoglobulins IgM, -A, and -G directed against SARS-CoV-2 proteins by immunoblot analysis from 12 infected patients. We determined IgG isotype antibodies by commercially available ELISA and assessed the clinical parameters of inflammation status and kidney and liver injury. Unexpectedly, we found no correlation between the presence of antibodies and the future course of the disease. However, attention should be paid to the parameters CRP, IL-6, and LDH. We found evidence of antibody cross-reactivity, which questions the reliability of results for serum samples that tested negative for anti-SARS-CoV-2 antibodies when assessed by immunoassays. Nevertheless, for the detection of IgG anti-SARS-CoV-2 antibodies, our data suggest that the use of the spike glycoprotein in immunoassays should be sufficient to identify positive patients. Using a combination of the spike glycoprotein and the open reading frame 8 protein could prove to be the best way of detecting anti-SARS-CoV-2 IgM antibodies.

scholarly journals Analysis of IgM, IgA, and IgG Isotype Antibodies Directed Against SARS‑CoV‑2 Spike Glycoprotein and ORF8 in the Course of COVID‑19.

2020 ◽  
Author(s):  
Denise Meinberger ◽  
Manuel Koch ◽  
Annika Roth ◽  
Gabriele Hermes ◽  
Jannik Stemler ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Immunoblot Analysis ◽  
Cross Reactivity ◽  
Spike Glycoprotein ◽  
Serum Samples ◽  
Reading Frame ◽  
Reliable Assessment ◽  
Insight Into ◽  
Inflammation Parameters

Abstract Immunoassays are a standard diagnostic tool assessing immunity in severe acute respiratory syndrome coronavirus type 2 (SARS‑CoV‑2) infection. However, immunoassays do not provide information about contaminating antigens or cross‑reaction and might exhibit inaccurate high sensitivity and low specificity. We aimed to gain insight into the serological immune response of SARS‑CoV‑2 patients by immunoblot analysis. We analyzed serum immunoglobulins IgM, ‑A, and ‑G directed against SARS‑CoV‑2 proteins by immunoblot analysis from 12 infected patients. We determined IgG isotype antibodies by commercially available ELISA, and assessed clinical parameters of inflammation status, kidney, and liver injury. We found evidence for antibody cross‑reactivity, which calls into question a reliable assessment of serum samples tested negative for anti‑SARS‑CoV‑2 antibodies by immunoassays. Nevertheless, for the detection of IgG anti‑SARS‑CoV‑2 antibodies, our data suggest that the use of the spike glycoprotein in immunoassays should be sufficient to identify positive patients. Using a combination of the spike glycoprotein and the open reading frame 8 protein could prove to be the best for the detection of patients positive for anti‑SARS‑CoV‑2 IgM antibodies. We found that the antibody response alone is not decisive for the course of the disease but the inflammation parameters are promising indicators.

Enterovirus D-68 Molecular Virology, Epidemiology, and Treatment: an update and way forward

2020 ◽  
Vol 20 ◽  
Author(s):  
Mahmoud Ahmed Ebada ◽  
Notila Fayed ◽  
Souad Alkanj ◽  
Ahmed Wadaa Allah
Keyword(s):  
Current Knowledge ◽  
Rna Virus ◽  
Neurological Diseases ◽  
Cross Reactivity ◽  
Serological Tests ◽  
Molecular Virology ◽  
Acute Flaccid Myelitis ◽  
The Family ◽  
Pcr Products ◽  
Enterovirus D68

: Enterovirus D68 (EV-D68) is a single-stranded positive-sense RNA virus, and it is one of the family Picornaviridae. Except for EV-D68, the family Picornaviridae has been illustrated in literature. EV-D68 was first discovered and isolated in California, USA, in 1962. EV-D68 has resulted in respiratory disorders’ outbreaks among children worldwide, and it has been detected in cases of various neurological diseases such as acute flaccid myelitis (AFM). A recent study documented a higher number of EV-D68 cases associated with AFM in Europe in 2016 compared to the 2014 outbreak. EV-D68 is mainly diagnosed by quantitative PCR, and there is an affirmative strategy for EV-D68 detection by using pan-EV PCR on the untranslated region and/or the VP1 or VP2, followed by sequencing of the PCR products. Serological tests are limited due to cross-reactivity of the antigens between the different serotypes. Many antiviral drugs for EV-D68 have been evaluated, and showed promising results. In our review, we discuss the current knowledge about EV-D68 and its role in the development of AFM.

scholarly journals A Label and Probe-Free Zika Virus Immunosensor Prussian Blue@carbon Nanotube-Based for Amperometric Detection of the NS2B Protein

Biosensors ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 157
Author(s):  
Bárbara V. M. Silva ◽  
Marli T. Cordeiro ◽  
Marco A. B. Rodrigues ◽  
Ernesto T. A. Marques ◽  
Rosa F. Dutra
Keyword(s):  
Carbon Nanotube ◽  
Prussian Blue ◽  
Zika Virus ◽  
Point Of Care ◽  
Amperometric Detection ◽  
Cross Reactivity ◽  
Structural Protein ◽  
Serum Samples ◽  
Polypyrrole Composite ◽  
Congenital Disabilities

Zika virus (ZIKV) is a mosquito-borne infection, predominant in tropical and subtropical regions causing international concern due to the ZIKV disease having been associated with congenital disabilities, especially microcephaly and other congenital abnormalities in the fetus and newborns. Development of strategies that minimize the devastating impact by monitoring and preventing ZIKV transmission through sexual intercourse, especially in pregnant women, since no vaccine is yet available for the prevention or treatment, is critically important. ZIKV infection is generally asymptomatic and cross-reactivity with dengue virus (DENV) is a global concern. An innovative screen-printed electrode (SPE) was developed for amperometric detection of the non-structural protein (NS2B) of ZIKV by exploring the intrinsic redox catalytic activity of Prussian blue (PB), incorporated into a carbon nanotube–polypyrrole composite. Thus, this immunosensor has the advantage of electrochemical detection without adding any redox-probe solution (probe-less detection), allowing a point-of-care diagnosis. It was responsive to serum samples of only ZIKV positive patients and non-responsive to negative ZIKV patients, even if the sample was DENV positive, indicating a possible differential diagnosis between them by NS2B. All samples used here were confirmed by CDC protocols, and immunosensor responses were also checked in the supernatant of C6/36 and in Vero cell cultures infected with ZIKV.

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