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Background and Objectives:HBV and HTLV-I are life threatening infectious agents in patients who receive blood and blood products. Although serological methods have been proved to be useful, detection of these viruses has remained a challengingissue due to the many obstacles. By the advent of Nucleic Acid Testing methods, especially in multiplex format, more precise detection is possible.The objective of this study was to develop a reliable, rapid and cost- effective method tosimultaneously detect HBV and HTLV-I. Materials and Methods: We have developed a multiplex Real time-PCR assay for simultaneous detection of HBV and HTLV-I. Primer sets were designed for highly conserved regions of genome of each virus. Using these primers and standard plasmids, we determined the limit of detection, clinical and analytical specificity and sensitivity of the assay. Monoplex and multiplex Real-time PCRs were performed. Results: Analytical sensitivity was considered to be 1000 and 100 copies/ml for HBV and HTLV-I, respectively. High concentration of one virus had no adverse effect on detection of t low concentrations of the other one. By analyzing 30 samples, clinical sensitivity of the assay was determined to be 87% and 96% for HBV and HTLV-I, respectively. Using different viral and human genome samples, the specificity of the assay was verified to be 100%. Conclusions:We have developed a reliable, rapid and cost effective method tosimultaneously detect HBV and HTLV-I.Our results indicatedthe high capability of this simple and rapid method for detecting these viruses in clinical samples.

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Background: L-lysine is essential amino acid for human and animal nutrition. L-lysine is useful as medicament, chemical agent, food material (food industry) and feed additive (animal food). The industrial production of lysine has become an economically important industrial process. Several hundred thousands tones of L-lysine are produced annually worldwide, almost exclusively using Corynebacterium glutamicum. Study methods: To amplify LysA gene from C.glutamicum, two primers with NheI and HindIII restriction sites were designed. PCR was performed and PCR product was ligated with pTZ57R/T. Recombinant plasmid sequence was determined. LysA with sticky end was ligated with digested pET28a vector and ligation mixture was transformed in E.coli BL21(DE3). The recombinant plasmid was isolated with enzymatic digestion and sequencing. Results: LysA gene, a fragment with 1.3 kb, was cloned. PCR products and enzymatic digestion of extracted vectors with HindIII and NheI, sequencing and SDS-PAGE confirmed the authenticity of cloning. Recombinant bacterial colonies were investigated and confirmed by two methods (PCR and enzymatic digestion). Conclusion: In this study for the first time, the expression rate of Meso- diaminopimelate decarboxylase enzyme (EC 4.1.1.20) in this expression vector was investigated and was increased significantly.

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Aims: Nowadays, treatment of bacterial infections is one of the most important challenges in the world. Medicinal plants offer a great hope to overcome these needs because of their chemical diversity and their significant role in the drug development. The aim of this study was to evaluate the in vitro antibacterial activity of the thyme (Thymus vulgaris) essential oil against Mycobacterium tuberculosis.
Materials and Methods: In this experimental study, thyme herb plants were collected and thyme essential oil was extracted. The Minimum Inhibitory Concentration (MICs) tests were performed to determine the antimicrobial activity of Thymus plant against the first (Isoniazid, Rifampicin, Ethambutol) and second (Cycloserine, Streptomycin, Kanamycin) drug antibiotics of mycobacterium. Data were analyzed by SPSS 21 software, using one-way ANOVA test.
Findings: The MICs for Isoniazid, Ethambutol, Streptomycin and Cycloserine were less than 10µg/ml and the MIC values for Rifampicin and Kanamycin were 40µg/ml. The limits of minimal inhibitory concentration of essential oil was between 0.5-40µg/ml (p<0.05).
Conclusion: Thyme essential oil has antibacterial activity against Mycobacterium tuberclusis.

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Aims: Vibrio cholerae is one of the intestinal gram-negative bacteria, causing cholera disease in developing countries; the two serogroups of O1 and O139 are the main causes of diarrhea. The bacteria resistance pattern to antibiotics varies in different countries. The aim of this study was to determine the resistance pattern of the isolates to representative antibiotics.
Materials & Methods: A total of 20 V. cholerae clinical strains were isolated from patients with cholera in Sistan and Baluchestan province of Iran during 2012-2013 outbreaks. After being identified by biochemical and molecular techniques, antibiotic susceptibility testing was performed for 6 antibiotics according to CLSI standards. Then, minimum inhibitory concentration (MIC) was also determined for tetracycline and erythromycin, using E-Test method.
Findings: All of the isolates were EL Tor biotype, O1 serogroup, and Inaba serotype. All of isolates were resistant to erythromycin and nalidixic acid, and 50% were resistant to tetracycline, while no resistance was observed against to ciprofloxacin, gentamicin, and ampicillin.
Conclusion: The sensitivity of all clinical isolates to antibiotics mentioned suggests that these antibiotics can likely be used in cholera disease treatment.

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 Aims
 Urinary tract infection (UTI) is one of the most common infections worldwide. The aim of this study was to investigate the association between ESBLs genes and quinolone resistance in Uropathogenic Escherichia coli isolated from patients with urinary tract infection .
Materials & Methods
A total of 150 E. coli isolates were collected from patients with urinary tract infection referring to Firouzgar Hospital in Tehran, Iran. Antimicrobial susceptibility of isolates were determined by disk diffusion method. Double-disk diffusion test was performed for phenotypic identification of extended-spectrum β-lactamase- (ESBL) producing isolates. PCR was used for the detection of ESBL-encoding genes in addition to quinolone (qnr) resistance genes.

Findings

 There was a high resistance rate to most of the studied antimicrobial agents. Phenotypically, 75% of the isolates produced an ESBL enzyme and were resistant to different antimicrobial classes. In overall, 83% of the isolates carried ESBL genes, especially bla_TEM and bla_CTX-M  . 75% were positive for the quinolone resistance genes including qnrA , qnrB ,qnrS and qepA. These results indicate the association between the presence of various ESBLs genes and quinolone resistance in uropathogenic E. coli.

Conclusion

Resistance patterns show the increased incidence of antibacterial resistance in E. coli. Results of the current study indicate the high prevalence of ESBL-producing isolates and quinolone resistance genes. Simultaneous presence of genes responsible for antibacterial resistance has made the treatment of UTI more challenging than ever before.

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Background: With increasing infectious diseases as well as antimicrobial resistance in pathogens to existing drugs, researchers are now seeking for new drug candidates to be used as alternatives or complementary therapies. Maca is commonly used in traditional medication as herbal medicine.
Materials & Methods: In this research, the antibacterial and antifungal activities of maca powder and ethanolic extract were evaluated against Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC27853, Escherichia coli ATCC25922, Enterococcus faecalis ATCC29212, and Candida albicans ATCC10231 using Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), and disc diffusion methods.
Results: The obtained results showed that there was no significant difference between the MIC and MBC of maca powder and extract against the reference and clinical strains. Also, no strain showed zone of inhibition at 30, 40, 50, and 60 µl of reference concentration.
Conclusion: According to the results obtained in this study, maca powder and extract had a poor inhibitory effect on bacterial and fungal growth.

 

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Aims: Hepatitis B Virus (HBV) has infected more than million hundreds of people worldwide. Hence, a high rate of morbidity and mortality caused by liver-related diseases is due to HBV infection. However, a strong and effective treatment should be based on an accurate and correct diagnostic method. Hence, the present research provided a multidimensional study comparing and analyzing patients’ molecular and serological tests results.
Materials & Methods: In this research, the HBV DNA molecular tests results were studied by examining patients’ gender, age, and HBsAg strip results.
Findings: Among the female patients (29 persons) studied in this research, 55.1% were positive for HBV DNA and HBsAg strip tests, and 17.3% were negative for both tests. Also, among the male patients  (44 persons), 65.9% were positive, and 6.8% were negative for both tests.
Conclusion: The present study results shed light on the correlation between the HBV DNA and HBsAg tests. Also, the significance of HBV DNA tests was highlighted for particular diagnostic purposes and for the differentiation and interpretation of the pathophysiological conditions of patients with hepatitis B.

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Objectives: Bordetella pertussis is a Gram-negative coccobacillus bacterium which is the causative agent of whooping cough. In recent years, the number of whooping cough cases has been rised. This bacterium has important virulence factors such as fimbriae and pertactin. In this study, polymorphism of Serotype 2 and 3 fimbriae genes and 2 Regions of pertactin gene were surveyed.
Materials & Methods: Totally, 20 B. pertussis clinical isolates were tested. DNA was extracted using the kit. Serotypes 2 and 3 fimbriae genes and pertactin Region 1 and 2 were identified using PCR method; finally, 13 samples were randomly sequenced.
Findings: No mutation was observed in the pertactin Region 2. In relation to the region1 of pertactin, 77% and 23% of the strains had prn2 and prn1 alleles, respectively. In relation to fim2 gene, 70% and 30% of the strains had fim2-2 and fim2-1 alleles, respectively. Also, in relation to fim3 gene, 70% and 30% of the strains carried fim3B and fim3A alleles, respectively.
Conclusion: In general, the present study results were similar to those of the previous studies conducted in Iran, but there were some differences in fim2 gene polymorphism so that the dominant allele changed from fim2-1 to fim2-2. Considering the fact that vaccine strains of Bp134 and Bp509 carry fim3A allele, which is different from the dominant circulating allele (fim3B), it is suggested that strains more similar to the dominant circulating strains should be used in designing vaccines.

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Background: Aureobasidin A is known as a cyclic depsipeptide antibiotic with toxic effects against yeasts such as Candida spp at low concentration. Combination therapy is used as a conventional treatment for fungal infections, especially drug-resistant cases. The current study aimed to investigate the combined effects of fluconazole and Aureobasidin A on fluconazole-resistant C. albicans isolates using broth microdilution method.
Materials & Methods: Antifungal activity of Aureobasidin A (AbA) compared to fluconazole against C. albicans ATCC 76615 strain was determined using the standardized broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI, document M27-Ed4) guidelines. The checkerboard method was used to test the combined effects of Aureobasidin A and fluconazole. The synergy, indifference, and antagonism were defined based on the fractional inhibitory concentration values below 0.5, 0.5-4, and more than 4 μg/mL, respectively.
Findings: MIC50 and MIC90 evaluations of Aureobasidin A and fluconazole were done at concentrations of 0.25-2 and 32-64 μg/mL against C. glabrata isolates, respectively. The synergy between fluconazole and Aureobasidin A was observed against Candida isolate. A reduced MIC was demonstrated against C. albicans isolate when fluconazole was combined with Aureobasidin A at 4 to 0.12 μg/mL concentrations.
Conclusion: The present study findings revealed that Aureobasidin A combined with fluconazole exhibited potent inhibitory effects against fluconazole-resistant C. albicans isolates. Further studies is recommended to investigate the synergistic effects of Aureobasidin A and other antifungal drugs.

 

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Purpose: Evaluation of humoral and cellular immune responses of naturally infected dogs against type I (rCPB) (Recombinant cycsteine proteinase B), and II (rCPA) (Recombinant cycsteine proteinase A) recombinant cysteine proteinases and C-terminal extension (CTE) of Leishmania infantum (L. infantum). Materials and Methods: In this study, fourteen infected dogs (7 with symptoms, 7 asymptomatics) from an endemic area and three uninfected dogs from a nonendemic region were selected and their humoral and cellular responses against type I and II recombinant cysteine proteinases, C-terminal extension (CTE) and F/T of Leishmania infantum were evaluated using the ELISA and lymphocyte proliferation assay, respectively. The level of specific IgG isotypes (IgG1 and IgG2) and lymphocyte proliferative response against rCPA, rCPB, CTE and Freezed/Thawed lysate (F/T) of L. infantum were examined. Results and Discussion: The results showed that in both of the symptomatic and asymptomatic dogs there is a high lymphoproliferative response to F/T antigens and moderate responses were observed when rCPs (Recombinant cycsteine proteinase) (rCPA and rCPB) and CTE were used. The level of antibody (total IgG, IgG1 and IgG2) recognition toward rCPA was low in the both groups of the dogs. In contrast, the CTE stimulates similarly as the CPB both of the humoral and cellular responses of all the infected animals and the level of total IgG and IgG2 isotypes against these antigens compared to the IgG1 was higher in the asymptomatic dogs. Since, the CTE is the terminal fragment of the CPB, it seems that the immunogenicity of the CPB is dependent on the CTE. Conclusion: The results of our investigation indicates that the CPB and CTE stimulate both humoral and cellular immune responses of L. infantum infected dogs, wherase the CPA is a weaker immunogen.

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Backgrounds: Aspergillus fumigatus is a pathogen responsible for invasive aspergillosis and the main leading cause of death in immunosuppressed individuals. The present study aimed to evaluate the impact of eugenol-loaded chitosan nanoparticles on the expression of CYP51a and CYP51b, two well-known genes responsible for triazole drug resistance in A. fumigatus.
Materials & Methods: The minimum inhibitory concentration (MIC) of eugenol-loaded chitosan nanoparticles, chitosan, eugenol, and itraconazole was determined based on the Clinical and Laboratory Standards Institute M38-E3 method at concentrations of 4.6-2400, 11.7-12000, 2-2048, and 1-256 μg/mL, respectively. The expression of CYP51A and CYP51B was evaluated in A. fumigatus exposed to 0.5, 1, and 2× of MIC concentration of NPs and itraconazole using the real-time polymerase chain reaction.
Findings: The obtained results showed that eugenol-loaded chitosan nanoparticles sucessfully reduced A. fumigatus fungal growth at 300 μg/mL concentration. MIC of chitosan, eugenol, and itraconazole was measured to be 6000, 256, and 4 μg/mL, respectively. The results of real-time PCR also revealed that eugenol-loaded chitosan nanoparticles increased the expression of both CYP51A and CYP51B in a dose-dependent manner. The expression of fungal CYP51A and CYP51B at mRNA level was significantly increased 1.26, 1.93, and 3.1-fold as well as 1.2, 2.1, and 2.4-fold at concentrations of 150, 300, and 600 μg/mL, respectively (p<.05). However, it seems that the prepared nanoparticles had a lower impact on the expression of these genes compared to itraconazole.
Conclusion: Overall, these findings suggest that the treatment of A. fumigatus with eugenol-chitosan nanoparticles could increase the expression of the CYP51 gene, suggesting the anti-fungal property of these nanoparticles.

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Backgrounds: Allium cepa L. as a traditional medicine is a rich source of beneficial bioactive metabolites. In the present study, the effect of A. cepa ethanolic extract (EAC) was studied on Aspergillus fumigatus growth, ergosterol synthesis, gliotoxin production, and gliP gene expression.
Materials & Methods: The minimum inhibitory concentration (MIC) of EAC (125-4000 µg/mL) was determined against A. fumigatus isolates according to Clinical and Laboratory Standards Institute (CLSI) guidelines (M-38). Protease activity, gliotoxin production, cell membrane ergosterol content, ultrastructure, and gliP gene expression were evaluated in the fungus exposed to 0.5× MIC concentrations of EAC (1000 μg/mL) and fluconazole (FCZ: 64 μg/mL).
Findings: Ergosterol content was significantly reduced to 0.53 and 0.45 µg/mg in FCZ- and EAC-treated fungal cells, respectively (p< .001). The protease activity was significantly inhibited in both EAC- and FCZ-treated groups. The gliotoxin production was inhibited by 51.55 and 68.75% in the treated groups with FCZ and EAC, respectively. The expression of gliP in both EAC- and FCZ-treated A. fumigatus groups was significantly reduced by 0.40 and 0.53-fold, respectively (p< .05).
Conclusion: This study finding revealed that A. cepa ethanolic extract (EAC) effectively suppressed the growth and virulence factors of A. fumigatus, which could be attributed in part to its bioactive metabolites. Further studies are recommended to isolate and identify these metabolites as potential candidates for the development of antifungal drugs.

 

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Backgrounds: Bacteriophage therapy could be an alternative strategy for the treatment of antibiotic-resistant bacteria. This study aimed to evaluate the antibacterial activities of isolated bacteriophages against methicillin-resistant Staphylococcus aureus (MRSA) isolates. 
Materials & Methods: A total of 16 clinical isolates of MRSA were collected from medical diagnostic laboratories in Tehran, Iran. A specific bacteriophage was isolated from hospital sewage using double-layer agar. Phage morphology was evaluated by transmission electron microscopy (TEM). Different bacteria were selected to determine the bacteriophage host range using spot test. Phage susceptibility to temperature and pH was evaluated by double-layer agar method.  In vitro assay was carried out on human epithelial type 2 (HEp-2) cells to investigate the effect of bacteriophage on the adhesion of MRSA to human epithelial cells. 
Findings: TEM suggested the Myoviridae family for the isolated phage. The effective titer of bacteriophages was 1.8×10⁷ PFU/mL. The isolated bacteriophage was stable at 4 ˚C and pH=8. The isolated bacteriophage was specific for all clinical isolates of MRSA and had no lytic activity against other pathogenic bacteria. In evaluating the binding and invasion of MRSA to the HEp-2 cell line, as expected, the lytic activity of specific bacteriophages was observed following inoculation.
Conclusion: The specificity and lytic activity of this phage on MRSA and MRSA-infected HEp-2 cell line emphasized that the isolated bacteriophage may serve as an effective prophylactic and alternative therapeutic agent in hospital settings.

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Background: Lipophilic yeasts of the genus Malassezia has been placed among the Basidiomycota phylum in the family of Cryptococcaceae. The genus Malassezia comprises yeasts with a natural habitat of the skin of many warm-blooded vertebrates, and human, but they are also associated with several skin diseases such as tinea versicolor, seborrehoeic dermatitis and even systemic infections. The genus Malassezia has recently been revised to include nine species by biochemical, morphological and molecular findings. Materials and methods: This study was aimed at the development of a DNA-based procedure applicable to rapid laboratory confirmation and identification of each Malassezia species. At first, conventional identification methods based on macroscopic, and microscopic features and physiological properties was performed. In the molecular findings a specific and unique restriction pattern was determined for each of the three currently recognized Malassezia species. Results: The primers ITS/4 produced a large amplificon (about 800 bp for M. furfur, and M. globosa) and the other amplificon is smaller (about 600 bp for M. sympodialis). For further species distinction with this amplicon, one restriction endonuclease proved tobe useful. Restriction patterns obtained by ECOR1 digestion of amplified products from the ITS region was distinguish. Conclusion: Application of polymerase chain reaction (PCR) technology for molecular diagnostics allows early and accurate identification of Malassezia Spp. The PCR-RFLP results were well correlated with those obtained from traditional identification procedures.

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Controlled delivery technology of protein/peptide drugs from biodegradable particles has emerged as one of the eminent areas to overcome problems related to macromolecules formulation. The goal of the present study was to develop protein-loaded micro-particles using biodegradable polymer, polycaprolactone (PCL) and hydrogel from beluga cartilage. Bovine serum albumin (BSA) was used as a model for protein/ peptide molecules such as GnRH. The double emulsion (W/O/W) technique was selected as one of the most appropriate methods for preparing a drug delivery system for soluble proteins in water. The first emulsion was prepared using ultrasonic and the mechanical agitator was used for achieving the second emulsion. The hydrogel prepared by enzymatic digestion was used in the first aquatic solution. At the present investigation, three groups were considered as the drug delivery system: G1; (PCL/hydrogel/BSA), G2; (PCL/BSA) and G3; (PCL/Alginate/BSA). Findings showed that the morphology of particles was spherical and non-conglomerated in all groups. The comparison of average particle size among groups was also indicated that the particles.

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Objective: In this study, a SYBR Green real-time RT-PCR assay for quantification of HIV-1 viral RNA was developed. Materials and Methods: This assay was performed based on amplification of the pol region of HIV-1 and product analysis by an ABI 7500 system. We quantified HIV-1 viral load in 26 seropositive patients by this system and the data were subsequently compared with results obtained with a reference technique represented by COBAS AMPLICOR HIV-1 Monitor test. Results: The results demonstrated that this technique could detecte up to 500 HIV-1 RNA copies/ml of plasma. The linearity of this approach was conserved over a wide range of HIV-1 copy numbers (5×102-5×109). Since no positive signal was observed in seronegative volunteers, the specificity of the test was calculated as 100%. Comparison of the results with those obtained by the reference quantification method, revealed a significant correlation between the results (R2= 0.95). Conclusion: On the basis of the most recent recorded cases for HIV-1 infection and AIDS in Iran, the prevalence of this disease is rising rapidly and the situation has been called to be alarming by national health representatives. Determination of HIV-1 viral load in plasma has been considered as the most effective single prediction tool of clinical outcome. Indeed, the development and stabilization of HIV-1 RNA assays have given physicians a unique tool for monitoring HIV-1 patients treated with antiviral drugs. In this study, we have developed a SYBR-Green Real Time RT-PCR assay for quantitative analysis of HIV-1 in infected patients. Since a synthetic RNA standard was used in this assay, the upper limit of detection was detected to be higher than the standard test (5×10 9 versus 7.5×10 5). This can be important in patients with acute high viral load infections. Reproducibility was assessed by Intra assay and Inter assay analysis. Coefficient of variations Ct, in reproducibility tests for Intra assay and Inter assay variability were less than

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Objective: β-thalassemia is caused by absence or reduction of β-globin chain synthesis. One of the effective therapeutic methods for this disease can be gene therapy by viral vectors. The capacity of lentiviral vectors is approximately 8 kb, we designed a 6 kb construct containing mini LCR and β-globin gene instead of LCR region. The aim of this study is to make a recombinant lentiviruses containing miniLCR and β-globin gene for transfer to the target cells for gene therapy of β-thalassemia. Materials and Methods: HS2, HS3, HS4 segments (mini LCR) and β-globin gene with 5΄ and 3΄ UTR were amplified from the genomic DNA of a normal individual by PCR. Each segment was cloned in pTZ57R/T vector and then sub cloned first into the pBGGT vector and finally into the pLenti-Dest vector. Final transfer vector and the three helper packaging plasmids (Plp1, Plp2, Plp/VSVG) were cotransfected into 293T packaging cells using lipofectamine 2000. Harvested viruses were confirmed by RT-PCR on extracted RNA of these recombinant lentiviruses. Results: The titer of lentiviral stock determined in a K562 cell line and compared with COS-7 cell line. The titer in both cell lines was the same. Optimum MOI for COS-7 cell line was 5 and when polybrene was used transduction increased by 2 fold. The remaining transduced COS-7 colonies were expanded and DNA was extracted. By PCR, random integration of construct into the genome was evaluated. Conclusion: The produced lentiviruses can be an appropriate means for effective transfer of the designed construct into dividing and non-dividing cells such as hematopoetic stem cells for transplantation of beta thalassemia patients. Efficiency of transduction by leniviruses is more than the gene targeting technique. Also units of HS2, HS3 and HS4 regions in mini LCR and selection of larger HS3 unit may increase the expression of beta globin gene.

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Objective: The global HIV epidemic continues to expand and exceeding previous predictions. An effective vaccine represents the best hope to curtail the HIV epidemic. DNA vaccines induce humoral and cellular responses and mimic live vaccines without their pathogenic potential. The importance of CD8+ CTL responses in controlling HIV and SIV viremia has led to production of a series of vaccine candidates that effectively induce these responses. It is now widely believed that an HIV vaccine strategy must stimulate both a strong humoral (antibody) as well as cell-mediated (CTL) immune response.The p24 and gp41 play many important roles in host-virus interaction and pathogenesis. These proteins are considered as attractive vaccine candidate in which their immunogenecity and immunomodulatory effects have been confirmed. Materials and Methods: In this study, a construct, pcDNA3.1Hygro- (p24-gp41), was evaluated as a DNA vaccine candidate in Balb/C mice for generation of effective cellular immune responses. For immunizing, we used dendrosome, a novel family of vehicles for transfection and therapy. IFN-γ cytokine production and total antibody were detected by ELISA. Lymphoprolifration assay was performed by MTT test. Results: ELISA and MTT assays confirmed that the cited p24-gp41 fusion gene is able to enhance immune responses in mice. Conclusion: The construct that was used in this research can be a good candidate for DNA vaccine against HIV-1, if the future complementary tests demonstrate the same trends of immunogenic responses shown in this study.