Publications by authors named "Ahmad Adeli"

25 Publications

  • Page 1 of 1

Optimization of culture conditions for high-level expression of soluble and active tumor necrosis factor-α in E. coli.

Protein Expr Purif 2021 Mar 5;179:105805. Epub 2020 Dec 5.

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Anti-TNF inhibitors exert their therapeutic effect by inhibition of the excessive amounts of TNF-α within the body. Recombinant TNF-α should be produced in a soluble refolded form to investigate the effectiveness and efficiency of anti-TNF-α compounds. In this research, the designed cassette was subcloned in the pET28a expression vector and expressed in E. coli BL21 (DE3). The identity of the protein was confirmed through SDS-PAGE and Western blotting. After optimizing expression conditions, protein purification was performed using native Ni-NTA affinity chromatography. The biological activity of the soluble recombinant TNF-α was investigated using MTT assay. Also, the affinity of an anti-TNF-α agent, Altebrel, was investigated against the expressed protein through ELISA. Optimization of TNF-α expression conditions represented that the highest expression could be achieved at 37 °C using 0.5 mM IPTG 6 h post-induction. The recombinant protein represented an inhibitory effect on the L929 murine fibroblast cell line and was successfully detected by Altebrel in ELISA. Binding kinetics were also studied using Cimzia as an anti-TNF-α molecule and 7.2 EM was calculated as the equilibrium dissociation constant value (K). The significant expression level of the recombinant protein in the soluble form, its high purity, and assessment of its biological activity showed that the expressed protein could be used in tests of ELISA and MTT to assess the activity of anti-TNF-α agents.
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http://dx.doi.org/10.1016/j.pep.2020.105805DOI Listing
March 2021

Enhancement of Expression Level of Modified t-PA (TNKase) in Leishmania tarentolae by Induction System

Iran Biomed J 2019 07 17;23(4):272-9. Epub 2018 Sep 17.

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Background: The expression of bio-therapeutic proteins in mammalian cells, such as CHO, attains high homogeneity related to post-translational modifications. Although CHO remains the most popular cell line for bestselling biotherapeutic proteins on the market, there are still drawbacks such as expensive culture media, long time line, and high drug cost. Recently, researches on a novel Leishmania protozoan system have confirmed that this low-level eukaryote could represent a competitive alternative to the mammalian cell lines.

Methods: The full length of coding sequence of modified tPA TNKase (tenecteplase) was synthesized and cloned into an inducible expression vector of L. tarentolae T7-TR cells.

Results: The expression of the construct was driven by a Tet-inducible promoter. A Leishmania secretory signal sequence was also added to the expression cassette to facilitate the release of the recombinant protein into the medium. The secretory recombinant protein was analyzed and confirmed by SDS-PAGE and Western blot analyses. The expression level of TNKase in this novel system of L. tarentolae was 810 IU/mL after induction, which means that the percentage of expression increases two times compared to previous models in L. tarentolae. The TNKase activity was comparable with Activase.

Conclusion: Our results suggested that expressed TNK (modified tPA) is functionally compatible with Activase regarding their effect on fibrinolysis. Given the post-translational modification similarities between mammalian and L. tarentolae, it is speculated that this system is capable of producing complex proteins such as tPA similar to mammalian system, with easier manipulation and non-expensive method.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6462294PMC
July 2019

Development of Genetically Modified Chinese Hamster Ovary Host Cells for the Enhancement of Recombinant Tissue Plasminogen Activator Expression.

Malays J Med Sci 2016 Mar;23(2):6-13

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, 1316943551, Iran.

Background: Chinese hamster ovary (CHO) cells are the most commonly used host system for the expression of high quality recombinant proteins. However, the development of stable, high-yielding CHO cell lines is a major bottleneck in the industrial manufacturing of therapeutic proteins. Therefore, different strategies such as the generation of more efficient expression vectors and establishment of genetically engineered host cells have been employed to increase the efficiency of cell line development. In order to examine the possibility of generating improved CHO host cells, cell line engineering approaches were developed based on ceramide transfer protein (CERT), and X-box binding protein 1s (XBP1s).

Methods: CHO cells were transfected with CERT S132A, a mutant variant of CERT which is resistant to phosphorylation, or XBP1s expression plasmids, and then stable cell pools were generated. Transient expression of t-PA was examined in engineered cell pools in comparison to un-modified CHO host cells.

Results: Overexpression of CERT S132A led to the enhancement of recombinant tissue plasminogen activator (t-PA) expression in transient expression by 50%. On the other hand, it was observed that the ectopic expression of the XBP1s, did not improve the t-PA expression level.

Conclusion: The results obtained in this study indicate successful development of the improved CHO host cells through CERT S132A overexpression.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4976708PMC
March 2016

Enhanced truncated-t-PA (CT-b) expression in high-cell-density fed-batch cultures of Pichia pastoris through optimization of a mixed feeding strategy by response surface methodology.

Bioprocess Biosyst Eng 2016 Apr 13;39(4):565-73. Epub 2016 Jan 13.

Department of Industrial Biotechnology, National Institute of Genetic Engineering and Biotechnology, PO Box 14965-161, Tehran, Iran.

Recently, Pichia pastoris has been the focal point of interest as an expression system for production of many recombinant proteins. The study and optimization of feeding strategy are of major importance to achieve maximum volumetric productivity in fed-batch cultivations. Among different feeding strategies used in P. pastoris fed-batch cultures, those trying to maintain a constant specific growth rate have usually resulted in superior productivities. The objective of the present study was to investigate and optimize the co-feeding of glycerol and methanol to attain maximum expression of t-PA in P. pastoris fed-batch cultures with constant specific growth rate. The experiments were designed by response surface methodology, considering the specific feeding rates of methanol and glycerol as independent variables. In each experiment, glycerol and methanol were fed according to a predetermined equation to maintain a constant specific growth rate. It was found that with glycerol feeding for higher specific growth rates, the inhibitory properties of glycerol are more pronounced, while the best expression level was achieved when the ratio of µ set glycerol to that of methanol was around 1.67. In all specific growth rates tested, almost a similar ratio of the specific glycerol feeding rate to that of methanol led to the maximum protein production and activity. The statistical model predicted the optimal operating conditions for µ set glycerol and that of methanol to be 0.05 and 0.03 h(-1), respectively. Applying the optimum strategy, maximum of 52 g/L biomass, 300 mg/L t-PA and 340,000 IU/mL enzyme activity were obtained.
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http://dx.doi.org/10.1007/s00449-016-1538-4DOI Listing
April 2016

Design of a novel chimeric tissue plasminogen activator with favorable Vampire bat plasminogen activator properties.

Enzyme Microb Technol 2014 Dec 22;67:82-6. Epub 2014 Sep 22.

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran. Electronic address:

Fibrinolytic agents are widely used in treatment of the thromboembolic disorders. The new generations like recombinant tissue plasminogen activator (t-PA, alteplase) are not showing promising results in clinical practice in spite of displaying specific binding to fibrin in vitro. Vampire bat plasminogen activator (b-PA) is a plasminogen activator with higher fibrin affinity and specificity in comparison to t-PA resulting in reduced probability of hemorrhage. b-PA is also resistant to plasminogen activator inhibitor-1 (PAI-1) showing higher half-life compared to other variants of t-PA. However, its non-human origin was a driving force to design a human t-PA with favorable properties of b-PA. In the present study, we designed a chimeric t-PA with desirable b-PA properties and this new molecule was called as CT-b. The construct was prepared through kringle 2 domain removal and replacement of t-PA finger domain with b-PA one. In addition, the KHRR sequence at the initial part of protease domain was replaced by four alanine residues. The novel construct was integrated in Pichia pastoris genome by electroporation. Catalytic activity was investigated in the presence and absence of fibrin. The purified protein was analyzed by western blot. Fibrin binding and PAI resistance assays were also conducted. The activity of the recombinant protein in the presence of fibrin was 1560 times more than its activity in the absence of fibrin, showing its higher specificity to fibrin. The fibrin binding of CT-b was 1.2 fold more than t-PA. In addition, it was inhibited by PAI enzyme 44% less than t-PA. Although the presented data demonstrate a promising in vitro activity, more in vivo studies are needed to confirm the therapeutic advantage of this novel plasminogen activator.
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http://dx.doi.org/10.1016/j.enzmictec.2014.09.005DOI Listing
December 2014

Expression of a novel chimeric-truncated tPA in Pichia pastoris with improved biochemical properties.

Mol Biotechnol 2014 Dec;56(12):1143-50

Biotechnology Research Center, Pasteur Institute of Iran (IPI), No. 69, Pasteur Avenue, Tehran, 1316943551, Iran.

Thrombolytic therapy by plasminogen activators (PAs) has been a main goal in the treatment of acute myocardial infarction. Despite improved outcomes of currently available thrombolytic therapies, all these agents have different drawbacks that may result in less than optimal outcomes. In order to make tissue plasminogen activator (tPA) more potent, while being more resistant to plasminogen activator inhibitor-1 (PAI-1) and having a higher affinity to fibrin, a new chimeric-truncated form of tPA (CT tPA) was designed and expressed in Pichia pastoris. This novel variant consists of a finger domain of Desmoteplase, an epidermal growth factor (EGF) domain, a kringle 1 (K1) domain, a kringle 2 (K2) domain, in which the lysine binding site (LBS) was deleted, and a protease domain, where the four amino acids lysine 296, arginine 298, arginine 299, and arginine 304 were substituted by aspartic acid. The chimera CT tPA showed 14-fold increase in its activity in the presence of fibrin compared to the absence of fibrin. Furthermore, CT tPA showed about 10-fold more potency than commercially available full-length tPA (Actylase(®)) and provided 1.2-fold greater affinity to fibrin. A residual activity of only 68 % was observed after incubation of Actylase(®) with PAI-1, however, 91 % activity remained for CT tPA. These promising findings suggest that the novel CT tPA variant might be an acceptable PA with superior characteristics and properties.
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http://dx.doi.org/10.1007/s12033-014-9794-5DOI Listing
December 2014

Engineering the cellular protein secretory pathway for enhancement of recombinant tissue plasminogen activator expression in Chinese hamster ovary cells: effects of CERT and XBP1s genes.

J Microbiol Biotechnol 2013 Aug;23(8):1116-22

Biotechnology Research Center, Pasteur Institute of Iran, Tehran 1316943551, Iran.

Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERTS132A- based secretion engineering could be an effective strategy for enhancing recombinant t- PA production in CHO cells.
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http://dx.doi.org/10.4014/jmb.1302.02035DOI Listing
August 2013

Immune responses against a new HIV-1 p24-gp41/pCAGGS-IL-12 DNA vaccine in Balb/c mice.

Iran J Immunol 2012 Jun;9(2):86-97

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran, e-mail:

Background: Development of an effective vaccine is highly needed in order to restrict the AIDS pandemic. DNA vaccines initiate both arms of immunity without the potential of causing disease. HIV-1 p24 and gp41 (gag and env) proteins play important roles in viral pathogenesis and are effective candidates for immune induction and vaccine design.

Objective: In this study, new DNA vaccine candidates constructed from HIV-1 fused p24-gp41 or gp41 alone were evaluated in Balb/c mice for induction of cellular and humoral immune responses.

Methods: Recombinant plasmids, pcDNA3.1/Hygro expression vector containing immunogenic sequences of fused p24-gp41 or gp41alone were produced. Dendrosome used as a system for carrying vectors in laboratory animals, and an IL-12 containing vector (pCAGGS-IL-12) was co-immunized with the p24-gp41 vector as a genetic adjuvant. Induction of effective immune responses against the designed vectors as DNA vaccine candidates in Balb/c mice was evaluated. Levels of total antibodies, IgG isotypes (IgG2a and IgG1); IFN-γ and IL-4 were measured by ELISA. MTT assay was used to evaluate lymphoproliferation.

Results: The results confirmed that the immunogenic epitopes of both p24 and gp41 genes are highly effective inducers of immune responses, and administration of fused p24-gp41 alone or along with IL-12 resulted in further enhancement of immune responses. Group 4 that received fused fragments (p24-gp41) along with an IL-12 expressing vector demonstrated a significantly higher Stimulation Index (SI) and IFN-γ production (p<0.0001) with a significant increase in IgG2a/IgG1 ratio, indicating the stimulation of CMI towards Th1. Although gp41 containing vector (group 6) also showed significant increases in both proliferation and IFN-γ production, the responses were persistently lower than that of p24-gp41 containing vectors. Total antibody production was highest in group 6 as expected.

Conclusion: Dendrosome proved to be an efficient carrier of recombinant plasmids constructed in this study. Further studies are necessary to evaluate these constructs as HIV vaccine candidates.
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http://dx.doi.org/IJIv9i2A2DOI Listing
June 2012

Combined TGE-SGE expression of novel PAI-1-resistant t-PA in CHO DG44 cells using orbitally shaking disposable bioreactors.

J Microbiol Biotechnol 2011 Dec;21(12):1299-305

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

An important modification of thrombolytic agents is resistance to plasminogen activator inhibitor-1 (PAI-1). In previous studies, a new truncated PAI-1-resistant variant was developed based on deletion of the first three domains in t-PA and the substitution of KHRR 128-131 amino acids with AAAA in the truncated t-PA. The novel variant expressed in a static culture system of Chinese Hamster Ovary (CHO) DG44 cells exhibited a higher resistance to PAI-1 when compared with the full-length commercial drug; Actylase. In the present study, the truncatedmutant protein was expressed in CHO DG44 cells in 50 ml orbital shaking bioreactors. The final yield of the truncatedmutant in the culture was 752 IU/ml, representing a 63% increase compared with the static culture system. Therefore, these results suggest that using the combined features of a transient and stable expression system is feasible for the production of novel recombinant proteins in the quantities needed for preclinical studies.
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http://dx.doi.org/10.4014/jmb.1106.05060DOI Listing
December 2011

A novel variant of t-PA resistant to plasminogen activator inhibitor-1; expression in CHO cells based on in silico experiments.

BMB Rep 2011 Jan;44(1):34-9

Biotechnology Research center, Pasteur Institute of Iran, Tehran, Iran.

Resistance to PAI-1 is a factor which confers clinical benefits in thrombolytic therapy. The only US FDA approved PAI-1 resistant drug is Tenecteplase®. Deletion variants of t-PA have the advantage of fewer disulfide bonds in addition to higher plasma half lives. A new variant was developed by deletion of the first three domains in t-PA in addition to substitution of KHRR 128-131 amino acids with AAAA in truncated t-PA. The specific activity of this new variant, 570 IU/μg, was found to be similar to those found in full length t-PA (Alteplase®), 580 IU/μg. A 65% and 85% residual activity after inhibition by rPAI-1 was observed for full length and truncated-mutant form, respectively. This new variant as the first PAI-1 resistant truncated t-PA may offer more advantages in clinical conditions in which high PAI-1 levels makes the thrombolytic system prone to re-occlusion.
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http://dx.doi.org/10.5483/BMBRep.2011.44.1.34DOI Listing
January 2011

Increased expression of recombinant human tissue plasminogen activator in Leishmania tarentolae.

Biotechnol J 2010 Nov;5(11):1198-206

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

Recombinant tissue plasminogen activator (rt-PA) is one of the most important thrombolytic agents for treating cardiovascular obstructions such as stroke. Glycoprotein rt-PA is a serine protease, consisting of 527 amino acids of which 35 are cysteine residues. A variety of recombinant protein expression systems have been developed for heterologous gene expression in prokaryotic and eukaryotic hosts. In recent years, Leishmania tarentolae has been considered because of its safety aspects and special attributes in expression of complex proteins. In this study, two expression cassettes, each one including two copies of t-PA cDNA, were used for integration into the L. tarentolae genome by electroporation. Transformed clones were selected in the presence of appropriate antibiotics. Expression of active rt-PA was confirmed by Western blot and Zymography tests. Real-time PCR analysis was applied to investigate the presence of multiple t-PA gene copies in the parasite genome. Correlation of t-PA gene dosage and production rate was confirmed with real-time PCR. It was shown that the expression level of rt-PA in L. tarentolae is at least 480 IU/mL of culture media. This concentration of rt-PA is seven times higher than what was reported in previous studies in L. tarentolae and some other eukaryotic systems.
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http://dx.doi.org/10.1002/biot.201000233DOI Listing
November 2010

Expression of a novel chimeric truncated t-PA in CHO cells based on in silico experiments.

J Biomed Biotechnol 2010 22;2010:108159. Epub 2010 Sep 22.

Biotechnology Research Center, Pasteur Institute of Iran (IPI), no. 69, Pasteur Avenue, Tehran 1316943551, Iran.

Tissue plasminogen activator (t-PA) is one of the fibrin-specific serine proteases that play a crucial role in the fibrinolytic system. The rapid clearance of the drug from the circulation, caused by its active uptake in the liver, has lead to complicated clinical applications. Different forms of plasminogen activators have been developed to treat thrombotic disease. Deletion of the first three domains of t-PA by gene manipulation techniques has shown a significant increase in its plasma half life. In order to compensate the disadvantage of higher bleeding risk, a novel chimeric truncated form of t-PA with 394 amino acids and more fibrin affinity compared to the truncated form was designed to be expressed in Chinese Hamster Ovarian (CHO) cells. The recombinant chimeric plasminogen activator consists of kringle 2 and serine protease (K2S) domains of t-PA, namely GHRP-SYQ-K2S. The level of expression was found to be 752 IU/ml with 566,917 IU/mg specific activity, based on amidolytic activity. The fibrin binding of this novel chimeric truncated t-PA was 86% of the full length t-PA at a fibrinogen concentration of 0.2 mg/ml. This could be a promising approach with more desirable pharmacodynamic properties compared to existing commercial forms.
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http://dx.doi.org/10.1155/2010/108159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2946600PMC
January 2011

Human Tissue Plasminogen Activator Expression in Escherichia coli using Cytoplasmic and Periplasmic Cumulative Power.

Avicenna J Med Biotechnol 2010 Jul;2(3):131-6

Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran ; Iranian Center for Breast Cancer (ICBC), ACECR, Tehran, Iran.

Tissue plasminogen activator (tPA) is a serine protease, which is composed of five distinct structural domains with 17 disulfide bonds, representing a model of high-disulfide proteins in human body. One of the most important limitations for high yield heterologous protein production in Escherichia coli (E. coli) is the expression of complex proteins with multiple disulfide bridges. In this study the combination of two distinct strategies, manipulated cytoplasm and native periplasm, was applied to produce the functional full length tPA enzyme in E. coli. Using a PelB signal peptide sequence at 5' site of tPA gene, the expression cassette was prepared and subsequently was transformed into a strain with manipulated oxidizing cytoplasm. Then the induction was made to express the protein of interest. The SDS-PAGE analysis and gelatin hydrolysis confirmed the successful expression of functional tPA. The results of this study showed that complex proteins can be produced in E. coli using the cumulative power of both cytoplasm and periplasm.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3558155PMC
July 2010

Cloning and expression of functional full-length human tissue plasminogen activator in Pichia pastoris.

Appl Biochem Biotechnol 2010 Nov 9;162(7):2037-48. Epub 2010 May 9.

Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran.

Human tissue plasminogen activator (t-PA) plays a pivotal role in the treatment of acute myocardial infarction, ischemic stroke, and deep vein thrombosis. It has the benefit of generating no adverse effects such as fibrinogen depletion, systemic hemorrhage, and immunologic reactions. Human t-PA is a serine-protease enzyme containing 527 amino acid residues in five structural domains. The correct folding of t-PA requires the correct pairing of 17 disulfide bridges in the molecule. A gene encoding full-length human t-PA was cloned into pPICZαA expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from Saccharomyces cerevisiae and flush with the kex2 cleavage site to express the protein with a native N terminus. The methylotrophic yeast, Pichia pastoris GS115 strain, was transformed with this cassette, and methanol utilizing (mut+) transformants were selected for production and secretion of human t-PA into culture media. SDS-PAGE and Western blot analysis showed the expressed bands of t-PA protein. Zymography test indicated suitable folding and proper function of the expressed recombinant human t-PA in conversion of plasminogen to plasmin and gelatin lysis. Amidolytic activity test showed the amidolytic activity of 1,650 IU/ml. The results of this study concluded that P. pastoris methylotrophic yeast can be a suitable alternative for mammalian and prokaryotic expression systems to produce t-PA.
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http://dx.doi.org/10.1007/s12010-010-8979-zDOI Listing
November 2010

Molecular cloning of cDNA of mammalian and chicken II gonadotropin-releasing hormones (mGnRHs and cGnRH-II) in the beluga (Huso huso) and the disruptive effect of methylmercury on gene expression.

Fish Physiol Biochem 2010 Sep 11;36(3):803-817. Epub 2009 Oct 11.

Department of Fisheries, Faculty of Marine Natural Resources, Khorramshahr University of Marine Science and Technology, Khorramshahr, Khouzestan, Iran.

Two gonadotropin-releasing hormone (GnRH) isoforms were identified in the beluga (Huso huso) brain by cDNA sequencing: prepro-mammalian GnRH (mGnRH) and prepro-chicken GnRH-II (cGnRH-II). The nucleotide sequences of the beluga mGnRH and cGnRH-II precursors are 273 and 258 base pairs (bp) long, encoding peptides of 91 and 86 amino acids, respectively. To investigate the effect of methylmercury (MeHg) on GnRH gene expression, animals were fed with four diets containing increasing levels of MeHg (0 mg kg(-1) [control]; 0.76 mg kg(-1) [low]; 7.8 mg kg(-1) [medium]; 16.22 mg kg(-1) [high]) for 32 days. The effects of MeHg on brain GnRH mRNA levels were evaluated by real-time PCR. A significant decrease in brain mGnRH and cGnRH-II mRNA levels were detected in fish receiving high dietary MeHg dose compared to controls on day 11 (P < 0.05). On day 18 and 32, all treatment groups had significantly lower brain mGnRH and cGnRH-II mRNA levels compared to the control group (P < 0.05). These findings demonstrate a disruptive role of MeHg on the level of brain mGnRH and cGnRH-II mRNAs in immature beluga.
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http://dx.doi.org/10.1007/s10695-009-9356-0DOI Listing
September 2010

Cloning and expression of Leishmania major superoxide dismutase B1: A potential target antigen for serodiagnosis of Leishmaniasis.

Iran J Immunol 2009 Sep;6(3):130-40

Department of Immunology, Shiraz University of Medical Sciences, Iran.

Background: Leishmaniasis- a neglected public health problem- is a group of diseases affecting an estimated 12 million people worldwide.

Objective: In the present study, recombinant Leishmania major superoxide dismutase B1 (rLmSODB1) has been utilized as a potential antigen for the serodiagnosis of human cutaneous (CL) and visceral leishmaniasis (VL) in the endemic regions of southern part of Iran. Additionally, the sensitivity and specificity of ELISA-based serodiagnosis using rLmSODB1 and the soluble Leishmania antigen (SLA) were compared.

Methods: For the first time, rLmSODB1 has been cloned successfully and used for ELISA-based serodiagnosis. Sera from 30 CL and 24 VL cases were included in this study. Additional studies were also done for the evaluation of cross-reactivity using sera from 41 endemic controls including normal endemic donors (n=20), systemic lupus erythematosus patients (n=5), rheumatoid arthritis patients (n=5), and patients with tuberculosis (n=11).

Results: Analysis indicated that rLmSODB1 was recognized by 62.5% and 13.3% of sera from patients with VL and CL, showing a sensitivity of 72.7% and 53.6%, respectively. However 95.8% of VL and 30% of CL sera reacted with SLA, revealing sensitivities of 96% and 58.8%, respectively. Additionally, from 41 sera collected either from healthy subjects or patients affected with other diseases, 97.5% were negative with SLA or rLmSODB1 (specificity 97.6%).

Conclusion: These results show that rLmSODB1 almost does not react with sera from patients with tuberculosis and autoimmune diseases and may be considered as a candidate antigen for the specific immunodiagnosis of visceral leishmaniasis.
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http://dx.doi.org/IJIv6i3A3DOI Listing
September 2009

Prevalence of viral hepatitis and molecular analysis of HBV among voluntary blood donors in west Iran.

New Microbiol 2009 Apr;32(2):193-8

Biotechnology Research Centre, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran.

To determine the prevalence of viral hepatitis infection and hepatitis B virus (HBV) molecular characterization, 11,200 blood donors from citizens of Shahrekord (a city located in west of Iran) were investigated. Results showed HBsAg-positive in 1.78% of persons (n=200), anti-HDV-positive in 3% of HBsAg-positive cases (n=6) and anti-HCV-positive in 0.67% of donors (n=76). HBV phylogenetic analysis disclosed HBV genotype D, sub-genotype D1, and subtype ayw2. Amino acid mapping of the HBV pol region revealed various HBV drug-resistance mutations though donors had no antiviral therapy. In conclusion, this study demonstrated notable viral hepatitis seroprevalence rate in blood donors in west of Iran.
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April 2009

Clinical, virologic and phylogenetic features of hepatitis B infection in Iranian patients.

World J Gastroenterol 2008 Sep;14(35):5448-53

Department of Genetics, Faculty of Basic Sciences, Tarbiat Modares University, Tehran, Iran.

Aim: To characterize the clinical, serologic and virologic features of hepatitis B virus (HBV) infection in Iranian patients with different stages of liver disease.

Methods: Sixty two patients comprising of 12 inactive carriers, 30 chronic hepatitis patients, 13 patients with liver cirrhosis and 7 patients with hepatocellular carcinoma (HCC) were enrolled in the study. The HBV S, C and basal core promoter (BCP) regions were amplified and sequenced, and the clinical, serologic, phylogenetic and virologic characteristics were investigated.

Results: The study group consisted of 16 HBeAg-positive and 46 HBeAg-negative patients. Anti-HBe-positive patients were older and had higher levels of ALT, ASL and bilirubin compared to HBeAg-positive patients. Phylogenetic analysis revealed that all patients were infected with genotype D (mostly ayw2). The G1896A precore (PC) mutant was detected in 58.1% patients. HBeAg-negative patients showed a higher rate of PC mutant compared to HBeAg-positive patients (c2 = 9.682, P = 0.003). The majority of patients with HCC were HBeAg-negative and were infected with PC mutant variants. There was no significant difference in the occurrence of BCP mutation between the two groups, while the rate of BCP plus PC mutants was higher in HBeAg-negative patients (c2 = 4.308, P = 0.04). In the HBV S region, the genetic variability was low, and the marked substitution was P120T/S, with a rate of 9.7% (n = 6).

Conclusion: In conclusion, HBV/D is the predominant genotype in Iran, and the nucleotide variability in the BCP and PC regions may play a role in HBV disease outcome in HBeAg-negative patients.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2744168PMC
http://dx.doi.org/10.3748/wjg.14.5448DOI Listing
September 2008

Expression of human tissue plasminogen activator in the trypanosomatid protozoan Leishmania tarentolae.

Biotechnol Appl Biochem 2007 Sep;48(Pt 1):55-61

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

A variety of recombinant protein expression systems have been developed for heterologous genes in both prokaryotic and eukaryotic systems such as bacteria, yeast, mammals, insects, transgenic animals and transgenic plants. Also, it has been reported that Leishmania tarentolae, a trypanosomatid protozoan parasite of the white-spotted wall gecko (Tarentola annularis), has the capability of expressing heterologous genes. Trypanosomatidae are rich in glycoproteins, which can account for more than 10% of total protein. The oligosaccharide structures of their glycoproteins are similar to those of mammals with N-linked galactose, and sialic acid residues. For a variety of reasons, including the glycosylation patterns and the secondary structures of some of these proteins, synthesis in eukaryotic system is highly preferable. In addition, formation of native disulfide bonds in complex eukaryotic proteins is tremendously important. In the present study, we tried to express the tPA (tissue plasminogen activator) gene in L. tarentolae. This protein is a thrombolytic agent with 527 amino acid residues. tPA possesses serine-protease activity, with 35 cysteine residues that participate in the formation of 17 disulfide bonds. We have used an expression cassette, including the alpha intergenic regions of Leishmania major and two sites at the 3'- and 5'-ends, for homologous recombination in L. tarentolae, in addition to antibiotic-resistant genes. Southern-blot analysis showed that the human tPA gene had been inserted into the genome of the parasite. The expression of the tPA at the mRNA and protein levels was confirmed. It was shown that the expressed tPA in this system was 70 i.u. (international units)/ml of culture media, which is much higher than levels reported previously in other systems.
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http://dx.doi.org/10.1042/BA20060217DOI Listing
September 2007

Molecular analysis and phylogenetic characterization of HIV in Iran.

J Med Virol 2006 Jul;78(7):853-63

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

The rate of human immunodeficiency virus type 1 (HIV-1) infection in Iran has increased dramatically in the last few years. While the earliest cases were found in hemophiliacs, intravenous drug users are now fueling the outbreak. In this study, both the 122 clones of HIV-1 gag p17 and the 131 clones of env V1-V5 region were obtained from 61 HIV-1 seropositives belonging to these two groups in Iran. HIV-1 subtyping and phylogenetic analysis was done by heteroduplex mobility assays (HMA) and multiple clone sequencing. The result indicated all hemophiliacs are infected with HIV-1 subtype B and all intravenous drug users are infected with HIV-1 subtype A. Since intravenous drug abuse is the major transmission route in Iran, HIV-1 subtype A is likely to be the dominant viral subtype circulating in the country. The analysis of genetic distances showed subtype B viruses in Iran to be twice as heterogeneous as the subtype A viruses. In conclusion, this first molecular study of HIV-1 genotypes in Iran suggests two parallel outbreaks in distinct high-risk populations and may offer clues to the origin and spread of infection in Iran.
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http://dx.doi.org/10.1002/jmv.20634DOI Listing
July 2006

A serological screening assay of human immunodeficiency virus type 1 antibodies based on recombinant protein p24-gp41 as a fusion protein expressed in Escherichia coli.

J Biotechnol 2006 Sep 2;125(2):295-303. Epub 2006 May 2.

Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran.

The objective of this study was expression of a recombinant fusion protein p24-gp41 to gain a proper folding pattern of the proteins which could be recognized by specific antibodies against human immunodeficiency virus type 1 (HIV-1) for development of a reliable serodiagnostic kit. Serodiagnostic method using enzyme-linked immunosorbent assay (ELISA) with the expressed recombinant fusion protein p24-gp41 was carried out to test the sensitivity and specificity of the protein using human sera and various reference panels from Boston Biomedica Inc. (BBI). The level of the expression was determined to be 30% and the final recovery from fermentation and purification process was calculated as 80 mg/L with more than 98% purity. The developed ELISA assay was demonstrated to have 100 and 99.5% sensitivity and specificity, respectively, detecting anti-HIV-1 antibody using 900 positive and 10,000 negative human sera. The developed assay showed reliable results in comparison with other reference HIV ELISA kits using various BBI panels as well. In conclusion, the recombinant fusion protein p24-gp41 was expressed and used to develop a serodiagnostic kit for screening of the HIV-1 with high sensitivity (100%) and specificity (99.5%) which could be useful for screening large groups of blood donors.
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http://dx.doi.org/10.1016/j.jbiotec.2006.03.021DOI Listing
September 2006

Hepatitis B virus genotyping, core promoter, and precore/core mutations among Afghan patients infected with hepatitis B: a preliminary report.

J Med Virol 2006 Mar;78(3):358-64

Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran.

In spite of hepatitis B virus (HBV) vaccination, HBV infection remains an important public health problem worldwide. Although the HBV genotype distribution has been determined in some parts of South Central Asia, no survey has been conducted to determine the HBV genotype in Afghanistan. Twelve Afghan patients infected with HBV living in Afghanistan were enrolled in this study. Partial HBsAg and basic core promoter, precore, and core (BCP/preC/C) regions were amplified and subjected for direct sequencing. In parallel, precore G1896A mutation was also determined by an amplification-created restriction site method. Results revealed HBV genotype D (95% bootstrap value), sub-genotype D1 (98% bootstrap value), and subtype ayw2 in all Afghan isolates. Afghan isolates clustered in a separate branch in the D1 sub-genotype called D1', while supported by 82% bootstrap value. The percentage of intra-genotypic distance among Afghan isolates was 1.05% and inter-genotypic distance with the other genotype D was 2.87% and with other genotypes was 7.50%-11.1%. The wild-type, mixed infection, and precore mutant were found in six, two, and four HBV isolates, respectively. The A1762T/G1764A BCP dual mutation was found in one isolate. Three isolates presented single mutation in the BCP dual mutation region, whereas two showed a novel G1764T mutation. In conclusion, this preliminary study revealed HBV genotype D, sub-genotype D1, and subtype ayw2 of HBV among hepatitis B infected patients from Afghanistan. Further investigation should be carried out.
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http://dx.doi.org/10.1002/jmv.20547DOI Listing
March 2006

Complete genomic sequence and phylogenetic relatedness of hepatitis B virus isolates from Iran.

J Med Virol 2005 Jul;76(3):318-26

Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran.

Hepatitis B virus (HBV) is one of the main etiological agents of acute and chronic liver disease that is still a major public health problem in the world. Numerous HBV isolates have grouped into eight genotypes, A to H, based on the complete genome sequence. To date, no study has been carried out on the complete HBV genome sequence in Iran. The objective of this study was to investigate the complete genome sequence organization and phylogenetic analysis of the five HBV strains, which obtained from Iranian chronic infected patients. Results showed that Iranian strains were closely related to each other, with 97-100% nucleotide similarity. Phylogenetic analysis based on the complete genome sequences and the precore/core gene sequences revealed that all strains were of genotype D, sub-genotype D1 with bootstrap value 100 and 99%, respectively. The S gene encoded Arg122, Pro127, and Lys160 corresponding to subtype ayw2. Iranian HBV isolates had closely related with Turkish HBV strains. All strains had a nucleotide length of 3,182 base pair (bp) except IR-P4 strain, with a 3,185 bp in length and with a unique Phe89 insertion in the X gene. The intragenotypic divergence of the complete genome sequence of Iranian strains was 1.8% and the intergenotypic in genotype D was 3.8% and with the other genotypes was 7.9-15.4%. In conclusion, this study revealed that the HBV genotype D, sub-genotype D1, subtype ayw2 dominates in the Iranian infected patients. A single Phe89 insertion in the X gene of the one Iranian strain with an unforeseen length of 3185 bp was identified.
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http://dx.doi.org/10.1002/jmv.20362DOI Listing
July 2005

A novel accurate amplification created restriction site method for determination of the wild type and the precore mutant hepatitis B virus variants.

J Virol Methods 2005 Jul 19;127(1):19-23. Epub 2005 Apr 19.

Biotechnology Department, Pasteur Institute of Iran, Pasteur Square, Tehran 13164, Iran.

The most commonly occurring hepatitis B virus (HBV) mutation is the G to A mutation at nucleotide 1896 in the precore region. The aim of this study was to develop a novel accurate amplification created restriction site (ACRS) method for determination of the TGG wild type and the TAG precore mutant HBV variants. Two conserved and consensus specific and diagnostic primers introducing BstXI and XagI cleavage sites were designed in order to determine the G1896 wild type and the A1896 precore mutant HBV variants in all HBV genotypes. The results of the ACRS method were compared with sequencing data. With the ACRS method, three different patterns could be distinguished for the wild type, the precore mutant and mixed infection HBV variants. The results of the ACRS method on 30 HBV isolates revealed the TAG precore mutant in 50% (15/30), the TGG wild type variant in 30% (9/30) and the mixed infection in 20% (6/30). The sequencing data of these samples were in agreement with the ACRS results. The ACRS method is a rapid and cost-effective technique for detecting both the TGG wild type and the TAG HBV precore mutant variants. It can be carried out for follow-up of G1896A precore mutant variant in hepatitis B virus infected subjects at routine molecular diagnostic laboratories.
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http://dx.doi.org/10.1016/j.jviromet.2005.02.011DOI Listing
July 2005

Genotype characterization and phylogenetic analysis of hepatitis B virus isolates from Iranian patients.

J Med Virol 2005 Feb;75(2):227-34

Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran.

Hepatitis B virus (HBV) is one of the major causative agents of acute and chronic liver disease worldwide and is believed to be responsible for a million deaths annually. Eight genotypes of HBV, A to H, have been described on the basis of similarity of the complete genomes sequence. Although, it is reported that the predominant HBV genotype in the Mediterranean area and the middle east is genotype D, there are no reports on HBV genotypes prevalent in Iran. In this study, the C and S regions of HBV from 26 chronic hepatitis B Iranian patients were amplified and sequenced. Phylogenetic analysis revealed that all Iranian HBV isolates sequences were classified into genotype D with bootstrap values of 100%, 73%, and 100% (1,000 replicates each) for S, C, and preS2 regions, respectively. The mean percent intra-distance of S and C regions were 0.8% and 2.3%, respectively. The mean percent inter-distance of S and C regions between Iranians and genotype D isolates were 1.7% and 3.0%, respectively, and the range of mean percent nucleotide distance of S and C regions between Iranians and the other reference isolates were 7.9%-17.5% and 4.8%-14.7%, respectively. Thirteen out of 23 HBV C region sequences showed nucleotide "A" at position 1896 (precore mutant) in C region. Nucleotide 1858 showed presence of "T" in all isolates. No insertion or deletion was found in both regions. SimPlot and BootScanning analyses did not show any recombination between Iranian isolates and other genotypes in both regions.
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http://dx.doi.org/10.1002/jmv.20261DOI Listing
February 2005