amir savardashtaki

Brucellosis is one of the most common shared diseases between humans and animals worldwide, causing various economic damages in the livestock industry and posing a threat to human health. Nowadays, the production of multi-epitope antigens, due to increased sensitivity and specificity in diagnostic efficiency, and reduced costs compared to the production of individual antigens is very useful in serological detection methods, especially ELISA. Therefore, this study aimed to bioinformatically design a multi-epitope antigen for the diagnosis of human brucellosis. Many studies demonstrate the effectiveness of Brucella outer membrane proteins to diagnose this disease. Therefore, to design a multi-epitopic antigen, Omp16, Omp25, and BP26 antigens were used. After identifying the epitopes with high antigenic properties from each antigen, the epitopes were connected using an appropriate linker (EAAAK). Subsequently, the physicochemical and antigenic properties were investigated. Following this, the three-dimensional structure (3D) was predicted, and the predicted structure was validated. After converting the amino acid sequence of the multi-epitopic antigen to a nucleic acid sequence, sequence optimization was performed for the successful expression of the protein in Escherichia coli (E. coli). The results from bioinformatics investigations show that the designed multi-epitopic antigen is stable and possesses solubility. Additionally, the optimization results indicate the successful expression of this antigen in a prokaryotic host. Therefore, based on the obtained results, the antigenic protein designed can be used with sensitivity and high specificity for the diagnosis of human brucellosis.

Human chorionic gonadotropin (hCG) is a polypeptide hormone synthesized during pregnancy and is also upregulated in some pathologic conditions such as certain tumors. Its measurement is essential for diagnosing pregnancy and malignancies. Despite numerous attempts to introduce an accurate method capable of detecting hCG levels, several limitations are found in previous techniques. This study aimed to address the limitations of current hCG assay methods by designing an electrochemical biosensor based on voltammetry for the rapid, selective, inexpensive, and sensitive measurement of hCG levels.

A carbon paste electrode was prepared and functionalized by para‑aminobenzoic acid. The primary anti‑β‑hCG monoclonal antibody was immobilized on the electrode surface by activating the carboxyl groups with 1‑ethyl‑3‑(3‑dimethylaminopropyl) carbodiimide and N‑hydroxysuccinimide solutions. The study also involved optimizing parameters such as the time for primary antibody fixation, the time for hCG attachment, and the pH of the hydrogen peroxide solution to maximize the biosensor response. Different concentrations of hCG hormone were prepared and loaded on the electrode surface, the secondary antibody labeled with HRP enzyme was applied, thionine in phosphate‑buffered saline solution was placed on the electrode surface, and the differential pulse electrical signal was recorded.

The linear range ranged from 5 to 100 mIU/ml, and the limit of detection was calculated as 0.11 mIU. The relative standard deviation was 3% and 2% for five repeated measurements of commercial standard samples with concentrations of 2 and 20 mIU/mL, respectively. The percent recovery was obtained from 98.3% to 101.5%.

The sensor represents a promising advancement in hCG level measurement, offering a potential solution to overcome the existing limitations in current diagnostic strategies. Simple and inexpensive design, detecting hCG in its important clinical range during early pregnancy, and successful measurement of hCG in real serum samples are the advantages of this sensor.

Context: 

Human papillomavirus (HPV) infections contribute to the cause of 15% - 20% of all human cancers. This review aims to examine and provide updated information on various aspects, with a particular focus on topics that are of interest to dermatologists. 

Evidence Acquisition:

 Using published studies, the pathogenicity of HPV was investigated. Subsequently, the existing vaccines were explored, followed by a review of the loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) method.

For HPV detection, the polymerase chain reaction (PCR), self-sustained sequence replication (3SR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA) were used. These methods can have good detection but still have problems. In comparison with nested PCR, the detection of HPV16 and HPV18 using LAMP-LFD has higher sensitivity.

Loop-mediated isothermal amplification-lateral flow dipstick is a simple and rapid method for the specific and sensitive detection of HPV. Thus, along with the previous HPV16 and HPV18 diagnostic tools, LAMP-LFDmight be useful in field studies or local hospitals.

CRISPR-Cas9, enable precise DNA manipulation via RNA-guided breaks. KHDC3L and PEG3 genes are vital; CRISPR-Cas9 studies their roles in reproduction, development, and gene regulation. Focusing on KHDC3L's impact on PEG3 promoter in HCT116 cells, insights into gene functions and disease mechanisms emerge, informing potential therapies. This study aims to design sgRNAs for the KHDC3L gene using CRISPR tools involved ranking, off-target evaluation, and cloning. HCT116 cells were cultured, synchronized, and transfected with sgRNAs using lipofectamine. Successful transfections were confirmed by fluorescence microscopy. Clonal expansion followed, with DNA extracted and genotyped using PCR and Sanger sequencing. Bisulfite conversion analyzed DNA methylation, employing restriction enzymes for CpG site analysis. Statistical significance (p≤0.05) was assessed using SPSS software. The neighboring regions exhibited significant genomic changes. The designed sgRNAs were cloned into the PX458 plasmid, directing Cas9 to create double-strand breaks (DSBs) in KHDC3L exon 3. Transfected cells showed around 65% efficiency. Gap-PCR confirmed knock-out in 3 out of 17 clones. COBRA analysis revealed allele-specific CpG island methylation in PEG3, indicating the impact of KHDC3L knock-out on PEG3 promoter methylation and expression. The study demonstrates increased PEG3 promoter methylation upon KHDC3L deletion, indicating its role in modulation. Knockout correlates with reduced cell proliferation and colony formation, suggesting KHDC3L's role in promoting cell growth. The gene's relevance in PEG3 regulation and potential therapeutic implications are underscored, though further mechanistic insights are warranted.

Corona virus disease-19 (COVID-19) is an evolving global disease which has burst into 2019. SARS-CoV-2 infects human cells through recognition of and binding to angiotensin converting enzyme-2 (ACE2) through the spike (S) glycoprotein. Spike is an immunogenic protein that can elicit immune responses. Multi-epitope vaccines are novel and efficient class of vaccines which are designed by linking the B and T cells. These epitopes stimulate both humoral and cellular immunity.

Based on bioinformatics online tools, appropriate epitopes of S protein were selected, linked together via suitable linkers, a TLR4 binding adjuvant was added, and a multi-epitope construct was constructed. The 3D model of the construct was predicted, refined, and validated. The antigenicity, allergenicity, solubility, and physico-chemical properties of vaccine were checked. The B cell conformational epitopes and IFN-γ inducing parts were detected. The adjuvant and TLR4 binding were evaluated by docking and protein-protein complex stability was assessed by elastic-mode analysis. The coding sequence of the vaccine construct was optimized and sub-cloned in expression vector through an in silico approach. Finally, the structure, energy, and stability of vaccine coding mRNA were evaluated.

Ten continuous B cell epitopes, 9 T helper epitopes, and 8 CTL epitopes were chosen. The results showed that multi-epitope vaccine is a stable and soluble protein which can stimulate humoral and cellular immunity. Besides, the vaccine could stimulate immunity without inducing allergenicity in human body.

Finally, the vaccine can bind the TLR4 appropriately and can be expressed by a recombinant vector. The designed multi-epitope vaccine against COVID-19 could be considered as a suitable candidate for experimental studies.

The Coronavirus disease 2019 (COVID-19) pandemic has raised concerns regarding the application of assisted reproductive technology (ART) in the world. Many ART programs have been delayed or continued with new precautions due to the ambiguity about vertical transmission and pregnancy safety. Regarding the possible risks of SARS-CoV-2 infection on ART and the resultant embryos, this study aimed to investigate the presence of SARS-CoV-2 in follicular fluid, granulosa cells, and oocytes of COVID-19-infected women undergoing ART.

COVID-19-positive polymerase chain reaction tests were reported for five women undergoing ART cycles on the day of oocyte retrieval. SARS-CoV-2 tests were performed on oocytes, granulosa cells, and follicular fluid obtained from these COVID-19-infected women.

SARS-CoV-2 RNA was detected only in one follicular fluid sample; however, other follicular fluid samples, granulosa cells, and oocytes were negative regarding viral RNA.

Given the unknown effects of COVID-19 on human reproduction and ART, strict precautions should be taken during the COVID-19 pandemic.

Serological diagnosis of Strongyloides stercoralis (S. stercoralis) is frequently challenging because of cross-reactivity with other parasitic nematodes. Therefore, it is necessary to introduce novel serological tests with high performance to properly diagnose this neglected parasitic infection. The purpose of the current study was to design a multi-epitope construct for the diagnosis of S. stercoralis.

For the purpose of this study, first, highly antigenic segments and potential immunodominant epitopes of S. stercoralis were identified from two antigenic proteins, and then all of the selected parts were linked by an appropriate linker. Next, the physico-chemical features of the designed construct were analyzed. Then, tertiary structures of the construct were built and evaluated to find out the best one. Lastly, the amino acid sequence was reverse-translated and optimized for over-expression in Escherchia coli (E. coli).

The bioinformatic evaluation indicated that the designed protein construct could be hydrophilic, thermostable, and acidic and the estimated half-life was more than 10 hr in E. coli.

According to the results of the study, the designed construct could be used as an efficient antigen in the ELISA system for serological diagnosis of human strongyloidiasis.

The most prevalent cancer in women over the world is breast cancer. Immunotherapy is a promising method to effectively treat cancer patients. Among various immunotherapy methods, tumor antigens stimulate the immune system to eradicate cancer cells. Preferentially expressed antigen in melanoma (PRAME) is mainly overexpressed in breast cancer cells, and has no expression in normal tissues. FliCΔD2D3, as truncated flagellin (FliC), is an effective toll-like receptor 5 (TLR5) agonist with lower inflammatory responses. The objective of the present study was to utilize bioinformatics methods to design a chimeric protein against breast cancer.

The physicochemical properties, solubility, and secondary structures of PRAME+FliCΔD2D3 were predicted using the tools ProtParam, Protein-sol, and GOR IV, respectively. The 3D structure of the chimeric protein was built using I-TASSER and refined with GalaxyRefine, RAMPAGE, and PROCHECK. ANTIGENpro and VaxiJen were used to evaluate protein antigenicity, and allergenicity was checked using AlgPred and Allergen FP. Major histocompatibility complex )MHC( and cytotoxic T-lymphocytes )CTL( binding peptides were predicted using HLApred and CTLpred. Finally, B-cell continuous and discontinuous epitopes were predicted using ABCpred and ElliPro, respectively.

The stability and solubility of PRAME+FliCΔD2D3 were analyzed, and its secondary and tertiary structures were predicted. The results showed that the derived peptides could bind to MHCs and CTLs. The designed chimeric protein possessed both linear and conformational epitopes with a high binding affinity to B-cell epitopes.

PRAME+FliCΔD2D3 is a stable and soluble chimeric protein that can stimulate humoral and cellular immunity. The obtained results can be utilized for the development of an experimental vaccine against breast cancer.

Recombinant AgB8/1 as the most evaluated antigen for serological diagnosis of Cystic Echinococcosis (CE) can provide early and accurate diagnosis for proper management and treatment of the disease. Thus, the secretory production of this recombinant protein is the main goal and the application of signal peptides at the N terminus of the desired protein can help to achieve this goal. The present study applied few bioinformatics tools to evaluate several signal peptides to offer the best candidate for extracellular production of AgB8/1 of Echinococcus granulosus in Escherichia coli. The sequences related to signal peptides were obtained from “Signal Peptide Website” and were checked by “UniProt”. In addition, UniProt was employed to retrieve the sequence of AgB8/1. Then, the probable signal peptide sequences and their cleavage site locations were determined by SignalP 4.1 followed by evaluation of their physicochemical features, using ProtParam. The solubility of the target recombinant proteins was accessed by SOLpro. Finally, PRED-TAT and ProtCompB were implemented to predict protein secretion pathways and final destinations. Among the 39 candidate signal peptides, ENTC2_STAAU and ENTC1_STAAU are the best ones which are stable and soluble in connection with AgB8/1 and can secrete target protein through Sec pathway. The signal peptides recommended in this investigation are valuable for rational designing of secretory stable and soluble AgB8/1. Such information is useful for future experimental production of the mentioned antigen.

Breast cancer is one of the most prevalent cancers among women. Common cancer treatment methods are not effective enough, and there is a need for a more efficient treatment procedure. Cancer vaccine is a novel immunotherapy method that stimulates humoral and/or cellular immunity against cancer. Placenta-specific protein 1 (PLAC1) is a cancer/testis antigen, prevalent in breast cancer and rarely found in normal tissues. FliC, as a bacterial adjuvant, when fused to PLAC1 can elicit humoral and cellular responses. Therefore, PLAC1-fliC is a chimeric protein, which can be considered a suitable candidate against breast cancer.

ProtParam was used to evaluate the physicochemical properties of PLAC1-fliC. Second structures were determined using the GOR V server. PLAC1-fliC 3D structure was modeled by Phyre2, and it was refined using GalaxyWEB. The refined model was submitted to RAMPAGE, PROCHECK, and ProSA-web for validation. Antigenicity and allergenicity of the construct were predicted by ANTIGENpro, VaxiJen, AllergenFP, and SDAP databases. Then MHC-I- and MHC-II-binding epitopes of PLAC1-fliC were forecasted by NetMHC 4.0 and NetMHCII 2.3 Servers. Finally, Ellipro and CTLpred were employed to predict B-cell and cytotoxic T lymphocyte epitopes.

The construct was evaluated as a stable fusion protein, which could be antigenic and could stimulate B and T cells against breast cancer.

PLAC1-fliC, as a cancer vaccine candidate, might be suitable and specific for breast cancer, which could evoke humoral and cellular immunity against this type of tumor.

Breast cancer is the most prevalent type of cancer among the female population, and about 15% to 20% of patients with breast cancer is human epidermal growth factor receptor 2 (HER2)-positive. The current cancer treatment methods such as surgery, radiation, and chemotherapy are not sufficiently effective in decreasing mortality rates; however, immunotherapy is a novel approach in the treatment of cancer that is more efficient and less harmful to the body. Anti-cancer immunotoxins are chimeric molecules containing two parts, namely the immuno part, which is an antibody or a binding segment of antibody, and toxin part, which is a killer toxin molecule.

In this study, we sought to design a novel immunotoxin, including the anti-HER2 receptor, trastuzumab, derived from a single-chain variable fragment (scFv) with a connection to the functional part of Campylobacter jejuni cytolethal distending toxin (Cj-CdtB).

The chimeric protein’s physicochemical properties, solubility, and secondary structure were analyzed, using ProtParam, PROSO II, and GORV, respectively. A three-dimensional (3D) model was built, using I-TASSER and refined, using GalaxyRefine. The model’s structure was evaluated before and after refinement, using PROCHECK and RAMPAGE. The AlgPred server was employed to predict immunotoxin allergenicity, and mRNA stability was evaluated by RNAfold. Finally, the immunotoxin and HER2 were docked, using ZDOCK.

Analysis showed that the chimeric protein could be a stable and soluble protein and the secondary structure of its parts would not change and the protein had a robust 3D structure that might have a stable mRNA structure and could bind to HER2 receptor.

The designed immunotoxin was a stable and soluble protein with the ability to bind to HER2 receptors, making it an appropriate immunotoxin candidate for breast cancer treatment. The results of the current work could be useful for future experimental studies.

Methicillin-resistant Staphylococcus aureus (MRSA) is a challenging infectious agent worldwide. The ever growing antibiotic resistance has made the researchers to look for new anti-staphylococcal agents. Autolysins are staphylococcal enzymes that lyse bacterial cell wall for cell division. Autolysins can be used as novel enzybiotics (enzymes have antibiotic effects) for staphylococcal infections. LytU is a newly explored autolysin. SH3b is a potent cell wall binding domain that can be fused to lytic enzymes to increase their activity. The aim of this study was to design a novel and efficient fusion enzybiotic that could lyse staphylococcal cell wall peptidoglycan by disrupting the bacteria. LytU-SH3b fusion construct was synthesized and LytU was amplified through the construct, using overhang PCR. The fusion and native forms that had his-tag were synthesized by recombinant technology in Escherichia coli BL21 (DE3) strain and purified utilizing Ni-NTA agarose beads. LytU and LytU-SH3b activity and potency were assessed using plate lysis assay, turbidity reduction assay and minimal inhibitory concentration (MIC) tests. All these tests showed that LytU-SH3b has more activity and potency than LytU. LytU-SH3b has MIC 421 fold lesser than LytU. Finally, LytU-SH3b is a novel and efficient recombinant enzybiotic that can lyse MRSA as an alternative to chemical small molecule antibiotics.

Cystic echinococcosis (CE) is a helminthic disease caused by the larval form of Echinococcus granulosus. In the present study, the B8/2 subunit of antigen B (AgB) of E. granulosus was expressed in E. coli host and then applied in a diagnostic ELISA set up. The DNA sequence of AgB8/2 subunit from E. granulosus was extracted from the GenBank and codon-optimized. The target sequence was cloned in an expression vector (pGEX-4T-1). The produced antigen was used in an ELISA system, and its performance for the diagnosis of human hydatid cyst was evaluated, using sera from CE and non-CE patients, along with the sera from healthy subjects. Moreover, the diagnostic value of the recombinant protein was compared with native AgB, as well as with a commercial kit. Antibodies to hydatid cyst were detected in 27 out of 30 patients corresponding to a sensitivity of 90% (95% CI: 73-98%). Cross-reaction with sera of non-CE subjects was seen in two cases resulted in a specificity of 93.5% (95% CI: 82-98%) for the test. A sensitivity of 87% and specificity of 90% were found for the native form of the antigen, while the ELISA commercial kit had a sensitivity of 97% and specificity of 95%. Our data show that rEgAgB8/2 is an appropriate source of antigen for the serological diagnosis of human hydatid cyst. Co-expression of the rEgAgB/2 along with other subunits of AgB may enhance the performances of these antigens for the serodiagnosis of human CE.

An elevated cholesterol level might lead to cardiovascular disease (CVD). Statins block the cholesterol synthesis pathway in the liver. Atorvastatin is the most widespread statin worldwide and, its chemical synthesis requires toxic catalysts, resulting in environmental pollution. Hence, enzymatic synthesis of atorvastatin is desirable. This process could be done by Lactobacillus kefir alcohol dehydrogenase (LKADH). Therefore, recombinant enzyme secretion by Escherichia coli using signal peptides (SPs) might result in easy production and purification. To achieve this objective, we used some online bioinformatics web servers to evaluate the suitable SPs for translocation of LKADH into extracellular spaces. “Signal Peptide Website” and “UniProt” were utilized to retrieve the SPs and LKADH sequences. “SignalP 4.1” was used to determine SPs and their cleavage site location and the results were rechecked by “Philius”. Physicochemical features of SPs were evaluated by “ProtParam”, then solubility of their fusion with LKADH was assessed by “Protein-sol”. Finally, secretion pathway and sub-cellular localization of the selected stable and soluble LKADH fusions were predicted by “PRED-TAT” and “ProtCompB”. Amongst the 41 evaluated SPs, only LPTA_ECOLI, SUBF_BACSU, CHIS_BACSU, SACB_BACAM, CDGT_BACST and AMY_BACLI could translocate LKADH out of cytoplasm. The six selected SPs in the result section were suitable to design a soluble secretory LKADH that accelerate its scale-up production and might be useful in future experimental researches.

Saffron (Crocus sativus) is one of the indigenous plants of Iran. Recently its possible effects have been reported on lipid abnormalities and oxidative stress. The aim of this study was to investigate the effects of saffron consumption on lipid profiles, especially the reduction of blood cholesterol, and lipid peroxidation in male hamsters under a high-fat diet. Twenty-six hamster rats were categorized in control group, high-fat diet group (HFD), and high-fat diet group treated with an aqueous saffron extract (HFD+S). The HFD and HFD+S groups were subjected to high-fat diet for 30 days. During last 10 days of this course, the HFD+S group received 100 mg/kg/day saffron through gavage. Saffron administration along with a high fat diet significantly decreased serum levels of total cholesterol, low molecular weight lipoprotein (LDL) cholesterol, malondialdehyde (MDA), and some hepatic enzymes. These results support the possible effects of saffron consumption in the prevention and treatment of cardiovascular disease.

Cardiac Radiofrequency (RF) ablation is used for treating some types of heart rhythm problems. The number of RF ablation procedures is increasing rapidly due to lower complication risks than surgery and high success rates. Due to higher patient exposure to X-ray radiation in different cardiac ablation procedures, public concerns are increasing regarding the detrimental effects of ionizing radiation, including skin injury, genetic effects, and malignancy.
This study aimed to determine patient absorption doses during Electrophysiological Study (EPS) and RF ablation of different cardiac arrhythmias in an electrophysiology laboratory unit with a flat panel detector.
Patients and This cross-sectional study was performed on 223 patients who underwent cardiac EPS and RF ablation. All procedures were executed on a single panel angiography unit with floor mounted C-arm. Dose Area Product (DAP), Entrance Skin Dose (ESD), and Fluoroscopy Time (FT) were recorded in all different procedures. Also, Total FT (TFT), total DAP, and total ESD were analyzed in the 223 procedures separately. Pearson’s correlation test was used to estimate the relationships between FT and DAP, FT and ESD, and ESD and DAP.
In this study, 56.1% of the patients were female. The mean age of male and female patients was 43.9 and 47.7 years, respectively. Medians of TFT, total ESD, and total DAP were 7.4 min, 165 mGy, and 19.2 Gycm2, respectively. Total ESD was strongly correlated to DAP (r = 0.945, P For prevention of deterministic and stochastic effects of radiation exposure, such as skin damage and cancer, operators should attempt to reduce patients’ radiation exposure as low as reasonably achievable. In the current study, none of the patients’ ESDs exceeded the threshold dose. The maximum ESD and DAP values were attributed to AF ablation procedures. Significant correlations between DAP and FT as well as between ESD and FT and the strong correlation between DAP and ESD showed that ESD could be reduced by reducing FT and DAP.

Spleen is an unusual location for hydatid cyst. Here we report a case of primary splenic hydatid cyst in a 41-yr-old Iranian woman from Yasuj, southwest of Iran. The patient had been admitted to Shahid Beheshti Hospital because of abdominal pain. Abdominal sonography revealed a hypoechoic lesion of 150 X 130 mm in the spleen, suggestive of hydatid cyst. Splenectomy was performed for the patient and surgical interventions revealed a hydatid cyst occupying most of splenic parenchyma. She was discharged on the 5 day of her operation. Postoperative diagnosis and confirmation of hydatid cyst was done by histopathological, molecular and serological approaches. Histopathological evaluation revealed the classical laminated layer of hydatid cyst. DNA was extracted from a part of cyst and PCR amplified. Sequencing and analysis of PCR product revealed that the isolate has the most similarity with G1 strain of Echinococcus granulosus. Patient’s serum was positive for IgG anti-hydatid cyst antibodies, using antigen-B ELISA.

Cystic echinococcosis (CE), as a chronic parasitic disease, is a major health problem in many countries. The performance of the currently available serodiagnostic tests for the diagnosis of CE is unsatisfactory.
The current study aimed at sub-cloning a gene, encoding the B8/1 subunit of antigen B (AgB) from Echinococcus granulosus, using gene optimization for the immunodiagnosis of human CE.
The coding sequence for AgB8/1 subunit of Echinococcus granulosus
was selected from GenBank and was gene-optimized. The sequence was synthesized
and inserted into pGEX-4T-1 vector. Purification was performed with GST tag affinity
column. Diagnostic performance of the produced recombinant antigen, native antigen B
and a commercial ELISA kit were further evaluated in an ELISA system, using a panel
of sera from CE patients and controls.
SDS-PAGE demonstrated that the protein of interest had a high expression level and purity after GST tag affinity purification. Western blotting verified the immunoreactivity of the produced recombinant antigen with the sera of CE patients. In an ELISA system, the sensitivity and specificity (for human CE diagnosis) of the recombinant antigen, native antigen B and commercial kit were respectively 93% and 92%, 87% and 90% and 97% and 95%.
The produced recombinant antigen showed a high diagnostic value which can be recommended for serodiagnosis of CE in Iran and other CE-endemic areas. Utilizing the combination of other subunits of AgB8 would improve the performance value of the introduced ELISA system.

Detection of antibodies against HCV is the initial step in chronic HCV diagnosis. HCV NS4B is among the most immunogenic HCV antigens and it has been widely used in commercial Enzyme Immunoassays (EIA) for HCV diagnosis. Furthermore, since NS4B is a key protein in the virus replication, it is an alternative target for antiviral therapy. Hence, rapid, high yield, and economical production of recombinant HCV NS4B is an obligation for producing HCV diagnostic kits and developing anti-HCV drugs. In the current study, we aimed to develop a new method for high-level expression and purification of NS4B coding region. Viral RNA was purified from the serum of an HCV positive patient and NS4B coding region was amplified using nested RT-PCR. PCR products were cloned into pET102/D-TOPO expression vector and transformed into E. Coli BL21. Induction was performed by adding 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) to the culture medium. Immunogenicity of the purified recombinant proteins was evaluated by immunoblotting and indirect enzyme-linked immunosorbent assay (ELISA). The accuracy of the construct was confirmed by colony PCR and sequencing. SDS-PAGE analysis showed successful expression of the recombinant protein. ELISA and western blotting confirmed the immunoreactivity of the recombinant NS4B. The directional TOPO cloning provides an efficient and easy method for cloning and expression of recombinant HCV NS4B. The directional TOPO cloning should be evaluated for production of other viral proteins.