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1.  Evaluation of cellular responses for a chimeric HBsAg-HCV core DNA vaccine in BALB/c mice 
Background:
Fusion of Hepatitis B virus surface antigen (HBsAg) to a DNA construct might be considered as a strategy to enhance cellular and cytotoxic T-lymphocytes (CTL) responses of a Hepatitis C Virus core protein (HCVcp)-based DNA vaccine comparable to that of adjuvanted protein (subunit) immunization.
Materials and Methods:
pCHCORE vector harboring coding sequence of HBsAg and HCVcp (amino acids 2-120) in tandem within the pCDNA3.1 backbone was constructed. The corresponding recombinant HCVcp was also expressed and purified in Escherichia coli. Mice were immunized either by adjuvanted HCVcp (pluronic acid + protein) or by pCHCORE vector primed/protein boosted immunization regimen. The cellular immune responses (proliferation, In vivo CTL assay and IFN-γ/IL-4 ELISpot) against a strong and dominant H2-d restricted, CD8+-epitopic peptide (C39) (core 39-48; RRGPRLGVRA) of HCVcp were compared in immunized animals.
Result:
Proper expression of the fused protein by pCHCORE in transiently transfected HEK 293T cells and in the expected size (around 50 kDa) was confirmed by western blotting. The immunization results indicated that the pCHCORE shifted the immune responses pathway to Th1 by enhancing the IFN-γ cytokine level much higher than protein immunization while the proliferative and CTL responses were comparable (or slightly in favor of DNA immunization).
Conclusion:
Fusion of HBsAg to HCVcp in the context of a DNA vaccine modality could augment Th1-oriented cellular and CTL responses toward a protective epitope, comparable to that of HCVcp (subunit HCV vaccine) immunization.
doi:10.4103/2277-9175.148296
PMCID: PMC4300588  PMID: 25625119
Cytotoxic T-lymphocyte response; Deoxyribonucleic acid vaccine; ELISpot; Hepatitis C virus; HCV core protein; HBsAg
2.  A novel combined method for cost–benefit production of DNA ladders 
Background:
Molecular deoxyribonucleic acid markers are one of the most important tools in molecular biology labs. The size of DNA molecule is determined by comparing them with known bands of markers during gel electrophoresis. In this study, we have suggested an efficient strategy to produce molecular weight markers in an industrial scale.
Materials and Methods:
A combination of two previously known methods, restriction enzyme digestion and polymerase chain reaction (PCR), was used. The enzymatic digestion process was based on designing and constructing plasmids which equaled in size with the bands of ladder and produce the DNA fragment by plasmid linearization through digestion. In the PCR method, the DNA fragments with length 102 bp lesser than the related bands in DNA ladder are amplified by PCR and cloned in pTZ57T/A cloning vector. Then, PCRs with forward and reverse 100-bp primers on the resulting plasmids amplify the ladder fragments. F100 and R100 primers bind to the backbone of pTZ57R (without insert) and amplify a 100-bp PCR product. PCR on the plasmid with insert amplifies DNA fragment with 102+ insert length bp size.
Results:
Upon application of this strategy, 2000-10,000 bp DNA fragments were produced by enzymatic digestion of plasmids of the same size. Moreover, 100-1500 bp fragments were produced during PCR using only a set of forward and reverse (100 bp) primers.
Conclusion:
The highest advantage of this cost–benefit approach is to produce different types of molecular weight markers by using an effective and short protocol.
doi:10.4103/2277-9175.148298
PMCID: PMC4300601  PMID: 25625121
DNA ladder; DNA marker; molecular weight
3.  Expression and Purification of Functional Human Vascular Endothelial Growth Factor-A121; the Most Important Angiogenesis Factor 
Advanced Pharmaceutical Bulletin  2014;4(4):323-328.
Purpose: Angiogenesis or formation of new blood vessels is an essential process for tumor growth, invasion and metastasis. Vascular Endothelial Growth Factor (VEGF) and its receptors play an important role in angiogenesis-dependent tumors. VEGF-A is the most important factor in angiogenesis process. Human VEGF-A gene consists of eight exons that undergoes alternative exon splicing and produce five different proteins consisting of 121, 145, 165, 189 and 206 amino acids (named VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206).
Methods: In this study, VEGF121 gene synthesized and cloned into the pET-26b plasmid. The recombinant plasmid was transferred into appropriate expression strain of BL-21. Expression of VEGF121 induced by IPTG (Isopropyl β-D-1-thiogalactopyranoside) and confirmed by SDS-PAGE and Western-Blotting. Recombinant VEGF121 was purified by nickel affinity chromatography. HUVECs (Human Umbilical Vein Endothelia Cells) cells were isolated from umbilical vein and the effect of VEGF121 on tube formation of endothelial cells was investigated.
Results: SDS-PAGE and Western-Blotting results verified the purification of VEGF121. The final yield of recombinant protein was about 5mg per liter. Endothelial cell tube formation assay results showed that VEGF121 leads to tube formation of endothelial cell on matrix and induces angiogenesis in vitro.
Conclusion: Recombinant VEGF121 is important factor in tube formation of endothelial cell, so it could be used in different cancer researches and angiogenesis assay.
doi:10.5681/apb.2014.047
PMCID: PMC4137420  PMID: 25436186
VEGF121; Angiogenesis; Endothelial tube formation assay
4.  Monitoring and Comparison of Antibiotic Resistant Bacteria and Their Resistance Genes in Municipal and Hospital Wastewaters 
Background:
Human exposure to antibiotic resistant bacteria (ARB) is a public health concern which could occur in a number of ways. Wastewaters seem to play an important role in the dissemination of bacteria and antibiotic resistant genes (ARGs) in our environment. The aim of this study was to evaluate the occurrence of three groups of ARB and their resistance genes in hospital and municipal wastewaters (MWs) as possible sources.
Methods:
A total of 66 samples were collected from raw MWs and hospital wastewaters (HWs) and final effluents of related wastewater treatment plants (WWTPs). Samples were analyzed for the detection of three groups of ARB including gentamicin (GM), chloramphenicol (CHL) and ceftazidime resistant bacteria and their ARGs (aac (3)-1, cmlA1 and ctx-m-32, respectively).
Results:
The mean concentration of GM, CHL and ceftazidime resistant bacteria in raw wastewater samples was 1.24 × 107, 3.29 × 107 and 5.54 × 107 colony forming unit/100 ml, respectively. There is a variation in prevalence of different groups of ARB in MWs and HWs. All WWTPs decreased the concentration of ARB. However, high concentration of ARB was found in the final effluent of WWTPs. Similar to ARB, different groups of ARGs were found frequently in both MWs and HWs. All genes also detected with a relative high frequency in effluent samples of MWs WWTPs.
Conclusions:
Discharge of final effluent from conventional WWTPs is a potential route for dissemination of ARB and ARGs into the natural environment and poses a hazard to environmental and public health.
PMCID: PMC4124567  PMID: 25105001
Antibiotic resistance genes; antibiotic resistant bacteria; ceftazidim; chloramphenicol; gentamicin
5.  A preliminary step of a novel strategy in suicide gene therapy with lentiviral vector 
Background:
One of the challenges in lentiviral vector–based suicide gene therapy by toxin or apoptosis-inducing genes is death of packaging cells. Therefore, the process of production of these lentiviral particles would be stopped in this step. We proposed that insertion of a reverse promoter between R and U5 regions of 5′ long terminal repeat (LTR) in transfer plasmid could be considered as a solution for this problem. But it is not known, whether the insertion of RΔU3 sequence between the promoter and target gene in proviral genome during the life-cycle of lentivirus may interfere whit gene expression in target cells.
Materials and Methods:
These following methods were performed in this study: insertion of RΔU3 sequence in pEGFP-N1 plasmid, evaluation of the expression of eGFP gene after calcium phosphate co-precipitation transfection of pCMV-RΔU3-GFP construction in 293T cells, and quantitative assay of eGFP gene by flow cytometry technique.
Results:
Our results from flow cytometry technique analysis showed that there was no significant difference between the expression of eGFP gene in transfected cells with pEGFP-N1 and pCMV-RΔU3-GFP plasmids (P > 0.05).
Conclusion:
In this step of our strategy, we demonstrated that modification of orientation and location of promoter may overcome some issues in lentiviral suicide gene therapy, especially when toxin or apoptosis-inducing genes are used.
doi:10.4103/2277-9175.124634
PMCID: PMC3928841  PMID: 24592361
Apoptosis-inducing genes; CMV promoter; gene therapy; GFP; lentiviral vectors; RΔU3 sequence
6.  Immunization of Mice by BCG Formulated HCV Core Protein Elicited Higher Th1-Oriented Responses Compared to Pluronic-F127 Copolymer 
Hepatitis Monthly  2013;13(10):e14178.
Background
A supreme vaccine for Hepatitis C virus (HCV) infection should elicit strong Th1-oriented cellular responses. In the absence of a Th1-specific adjuvant, immunizations by protein antigens generally induce Th2-type and weak cellular responses.
Objectives
To evaluate the adjuvant effect of BCG in comparison with nonionic copolymer-Pluronic F127 (F127) as a classic adjuvant in the formulation of HCV core protein (HCVcp) as a candidate vaccine for induction of Th1 immune responses.
Materials and Methods
Expression of N-terminally His-Tagged HCVcp (1-122) by pIVEX2.4a-core vector harboring the corresponding gene under the control of arabinose-inducible (araBAD) promoter was achieved in BL21-AI strain of E.coli and purified through application of nitrilotriacetic acid (Ni-NTA) chromatography. Mice were immunized subcutaneously (s.c.) in base of the tail with 100 μl of immunogen (F127+HCVcp or BCG+HCVcp; 5 μgHCVcp/mouse/dose) or control formulations (PBS, BCG, F127) at weeks 0, 3, 6. Total and subtypes of IgG, as well as cellular immune responses (Proliferation, In vivo CTL and IFN-γ/IL-4 ELISpot assays against a strong and dominant H2-d restricted, CD8+-epitopic peptide, core 39-48; RRGPRLGVRA of HCVcp) were compared in each group of immunized animals.
Results
Expression and purification of core protein around the expected size (21 kDa) was confirmed by Western blotting. The HCVcp + BCG vaccinated mice showed significantly higher lymphocyte proliferation and IFN-γ production but lower levels of cell lysis (45% versus 62% in CTL assay) than the HCVcp+F127 immunized animals. “Besides, total anti-core IgG and IgG1 levels were significantly higher in HCVcp + F127 immunized mice as compared to HCVcp + BCG vaccinated animals, indicating relatively higher efficacy of F127 for the stimulation of humoral and Th2-oriented immune responses”.
Conclusions
Results showed that HCVcp + BCG induced a moderate CTL and mixed Th1/Th2 immune responses with higher levels of cell proliferation and IFN-γ secretion, indicating that BCG may have a better outcome when formulated in HCVcp-based subunit vaccines.
doi:10.5812/hepatmon.14178
PMCID: PMC3842517  PMID: 24348641
HCV; Adjuvant; Bacillus Calmette-Guerin (BCG); PluronicF127
7.  Recombinant expression and purification of human placental growth factor 1 and specific camel heavy chain polyclonal antibody preparation 
Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family. Unlike VEGF, PlGF is dispensable for normal cell development as well as playing various roles in pathological angiogenesis which occurs in tissue ischemia, inflammation, and malignancy. The PlGF-1 has been considered as a potential candidate for the diagnosis and targeting of pathological angiogenesis. Camelidae serum contains an important fraction of functional antibodies, called heavy-chain antibodies (HcAbs) that are naturally devoid of light chains. Camelid HcAbs recognize their cognate antigens by a single variable-domain, referred to as VHH or Nanobody.
Here, we describe the expression and purification of recombinant human PlGF-1 (rhPlGF-1). This protein was subsequently used for the preparation of camel heavy chain polyclonal antibody against rhPlGF-1.
The recombinant expression plasmid pET-26b-hPlGF-1 was introduced into Escherichia coli BL21 cells to express the rhPlGF-1 protein. Purified rhPlGF-1 was used to immunize camel, the specific reactivity of HcAb was determined with ELISA and western blot. Western blot analysis indicated that the antiserum specifically reacted to the recombinant protein. The rhPlGF-1 protein and its antibody may be used for the development of detection assays needed for clinical research.
doi:10.1016/j.sjbs.2013.04.008
PMCID: PMC3937466  PMID: 24596498
PlGF; VHH; Polyclonal antibody; Angiogenesis; Heavy chain antibody
8.  Expression, purification, and characterization of a diabody against the most important angiogenesis cell receptor: Vascular endothelial growth factor receptor 2 
Antibodies and their derivative fragments have long been used as tools in a variety of applications, in fundamental research work, biotechnology, diagnosis, and therapy. Camels produce single heavy-chain antibodies (VHH) in addition to usual antibodies. These minimal-sized binders are very robust and bind the antigen with high affinity in a monomeric state. Vascular endothelial growth factor recepror-2 (VEGFR2) is an important tumor-associated receptor that blockade of its signaling can lead to the inhibition of neovascularization and tumor metastasis. Here, we describe the construction, expression, and purification VEGFR2-specific Diabody. Two variable fragments of a same camel anti-VEGFR2 antibody were linked together by the upper hinge segment of antibody to make a diabody. We showed the ability of diabody to recognition of VEGFR2 on the cell surface by FACS. Diabodies can be produced in the low-cost prokaryotic expression system, so they are suitable molecules for diagnostic and therapeutic issues.
doi:10.4103/2277-9175.100126
PMCID: PMC3544076  PMID: 23326765
Diabody; Nanobody; vascular endothelial growth factor recepror-2
9.  Thyroid Peroxidase Gene Mutation in Patients with Congenital Hypothyroidism in Isfahan, Iran 
Background. Thyroid peroxidase gene (TPO) mutations are one of the most common causes of thyroid dyshormonogenesis in patients with congenital hypothyroidism (CH). In this study, the prevalence of TPO gene mutations in patients with thyroid dyshormonogenesis in Isfahan was investigated. Methods. In this cross-sectional study, genomic DNA of 41 patients with permanent CH due to thyroid dyshormonogenesis was extracted using the salting out method. The 17 exonic regions of the TPO gene were amplified. SSCP technique was performed for scanning of the exonic regions of the TPO gene, except exon 8. DNA sequencing was performed for those with different migration patterns in SSCP by chain termination method. Exon 8 was sequenced directly in all patients. In 4 patients, all fragments were also sequenced. Results. One missense mutation c.2669G > A (NM_000547.5) at exon 15 (14th coding exon) in one patient in homozygous form and seven different single nucleotide polymorphisms (SNPs) in exons 1, 7, 8, 11, and 15 of TPO gene. Conclusion. The TPO gene mutations among CH patients with dyshormonogenesis in Isfahan were less frequent in comparison with other similar studies. It may be due to the presence of other unknown gene mutations which could not be detected by SSCP and sequencing methods.
doi:10.1155/2012/717283
PMCID: PMC3419406  PMID: 22919382
10.  Functional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen 
Iranian Biomedical Journal  2012;16(3):121-126.
Background: Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length transmembrane protein CD20. Methods: In this study, we expressed the extra membrane loop of hCD20 (exCD20) consisting of a non-glycosylated 47-amino acids region. The exCD20 coding sequence was amplified by PCR and cloned in pET32a(+) expression vector. The desired protein was expressed in fusion with thioredoxin and 6× His tag in E. coli Origami strain. ELISA and Western-blotting data were performed to indicate the functionality of this protein. Results: We have obtained the exCD20 recombinant protein which can be detected in ELISA and Western-blot experiments. This recombinant fusion protein was soluble and stable without aggregation and misfolding problems. Conclusion: The recombinant extra membrane loop of human CD20 protein in fusion with thioredoxin (exCD20) can be used in function assays and some applications such as ELISA, immuneblotting, affinity purification, immunization, screening, and development of anti-CD20 antibodies.
doi:10.6091/ibj.1082.2012
PMCID: PMC3629936  PMID: 23023212
E. Coli; CD20; Thioredoxin
11.  Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin 
Background:
Recombinant human erythropoietin (rhEPO) is considered to be one of the most pivotal pharmaceutical drugs in the market because of its clinical application in the treatment of anemia-associated disorders worldwide. However, like other therapeutic proteins, it does not have suitable pharmacokinetic properties for it to be administrated at least two to three times per week. Chemoselective cysteine PEGylation, employing molecular dynamics and graphics in in silico studies, can be considered to overcome such a problem.
Methods:
A special kind of EPO analog was elicited based on a literature review, homology modeling, molecular dynamic simulation, and factors affecting the PEGylation reaction. Then, cDNA of the selected analog was generated by site-directed mutagenesis and subsequently cloned into the expression vector. The construct was transfected to Chinese hamster ovary/dhfr− cells, and highly expressed clones were selected via methotrexate amplification. Ion-immobilized affinity and size exclusion (SE) chromatography techniques were used to purify the expressed analog. Thereafter, chemoselective PEGylation was performed and a nanosize PEGylated EPO was obtained through dialysis. The in vitro biologic assay and in vivo pharmacokinetic parameters were studied. Finally, E31C analog Fourier transform infrared, analytical SE-high-performance liquid chromatography, zeta potential, and size before and after PEGylation were characterized.
Results:
The findings indicate that a novel nanosize EPO31-PEG has a five-fold longer terminal half-life in rats with similar biologic activity compared with unmodified rhEPO in proliferation cell assay. The results also show that EPO31-PEG size and charge versus unmodified protein was increased in a nanospectrum, and this may be one criterion of EPO biologic potency enhancement.
Discussion:
This kind of novel engineered nanosize PEGylated EPO has remarkable advantages over rhEPO.
doi:10.2147/IJN.S19081
PMCID: PMC3131188  PMID: 21753873
nanoPEGylated EPO; cysteine PEGylation; pharmacokinetic property
12.  Transient Expression Assay of Aγ-588 (A/G) Mutations in the K562 Cell Line 
Iranian Biomedical Journal  2011;15(1-2):15-21.
Background: In the previous study, we have shown that the presence of A allele at position -588 in Aγ-globin gene was highly frequent and closely associated with fetal hemoglobin elevation among β-thalassemia intermedia patients. Therefore, we decided to investigate whether this allele (A allele at -588) could result in an increase in Aγ-globin gene expression to ameliorate the severity of the disease in thalassemia patients. Methods: Three constructs containing µ locus control region, Aγ-globin and β-globin genes were designed and employed in the transient expression assay. The difference among constructs was in the promoter region of Aγ-globin gene (A and G alleles at -588). A construct with T to C base substitution at -175 of Aγ-globin, created by site-directed mutagenesis, was selected as positive control. The K562 cell line was transfected with the above constructs. Subsequently, the expression of Aγ-globin gene was determined by quantitative real-time reverse transcription-PCR. Results: There was not a significant increase in the expression of Aγ-globin gene in the construct containing A allele comparing the one with G allele at -588. Conclusions: -588 (A>G) mutation does not play a major role in regulation of Aγ-globin gene, suggesting that other factors may be involved.
PMCID: PMC3639736  PMID: 21725495
β-thalassemia; Aγ-globin; K562 cells
13.  High Expression of Methylotrophic Yeast-Derived Recombinant Human Erythropoietin in a pH-Controlled Batch System 
To accomplish the worldwide demand for recombinant human erythropoietin (rHuEpo) as a therapeutic, application of cost-efficient expression system of methylotrophic yeast Pichia pastoris (P. pastoris) rather than mammalian cells is indispensable. Herein, a report on high levels secreted-expression of Pichia-derived rHuEpo by batch fermentation in a pH stabilized format is presented. The full length cDNA of rHuEpo was inserted into pPICZαA vector under control of AOX1 promoter, downstream of the secretion-α-factor and electroporated into P. pastoris strain X33. The highest expression transformant was selected by screening among the colonies surviving high concentration of Zeocin (1.0 mg/ml), followed by comparative small scale expression analysis by ELISA. Stabilization of pH around 6.0 by adding phosphoric acid into the culture media during induction period, improved the yield of expression to 150 mg/l of the media. Single-step Nickel-affinity chromatography was employed for purification of rHuEpo-6xHis to 80% purity. Analyses by SDS-PAGE, Western blot and N-terminal protein sequencing confirmed the authenticity of the 33 kDa expressed rHuEpo with a native N-terminal indicating the proper cleavage of secretion-signal. Results of this study, further confirmed the possibility of employing methylotrophic yeast for scaled up production aims of rHuEpo as a cost-efficient expression system when provided evidence for higher expression yields through application of pH-controlled systems.
PMCID: PMC3558167  PMID: 23407145
Erythropoietin; Fermentation; Pichia pastoris; Yeasts

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