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Introduction: Brucellosis is one of the most important zoonotic diseases that causes miscarriage and infertility in animals and causes human fever. The use of the common SS9 strain of Brucella abortus has several side effects for livestock. Brucella P39 protein is one of the plasma peripheral space proteins that is considered as one of the important immunogenic indicators. With the production of the new protein combination of P39, more studies can be done on the ability of this protein to stimulate immune responses against Brucella. Therefore, in this research, the production and purification of this protein in Escherichia coli bacteria has been done as a new compound.
method: In this experimental study, using the polymerase chain reaction, the P39 gene was propagated by the bacterium Brucella abortus. After purifying the P39 gene, it was cloned into plasmid carriers pSK+ and pGEX4T1. Therefore, pSK+-P39 and pGEX4T1-P39 structures were prepared. To produce the recombinant protein P39, the plasmid structure pGEX4T1-P39 first entered the Escherichia coli bacterium BL21. The protein was then produced by IPTG by induction of pGEX4T1-P39 plasmid. The resulting protein was purified using the orderly purification protein glutathione S-transferase. The amount of purified protein was measured using the Brad Ford method.
Results: The nucleotide sequence of the gene propagated by the cloned PCR in the plasmid carrier  pSK+ was exactly the same as the P39 gene of Brucella abortus. Production of P39 protein was performed by induction of pGEX4T1-P39 plasmid. The purified protein content was 200 micrograms per milliliter.
Conclusion: The production of the new protein P39 compound Brucella Abortus, which is unstable in the cytoplasm of the Escherichia coli bacterium, is possible using carriers with additive proteins such as pGEX4T1 in the host of Escherichia coli strain BL21.

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Introduction: In many studies, immunogenicity of Brucella proteins such as P39 in animals is investigated. In this study, we evaluated antigenicity of recombinant P39 from Brucella abortus in patients with Brucellosis. Materials and Methods: In this experimental study, at first recombinant P39 was produced in Escherichia coli. Sera reactivity of six infected individuals against the recombinant P39 protein was analysed by Western Blot. Results: Data indicated that P39 protein from Brucella abortus was recognized by patients, sera antibodies. Conclusion: Our data showed that recombinant P39 protein can be detected as an antigen by sera in infected human. Therefore, recombinant P39 have same epitopes with natural form of this antigen.

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Introduction: Streptolysin O (SLO) is an antigenic protein that is secreted by Streptococcus pyogenes. Streptococcal infections are diagnosed with anti streptolysin O. At present, streptolysin O is produced by vectors that have fusion protein. In this study streptolysin O without fusion protein vectors is produced. Materials and Methods: In this experimental study, Streptolysin O gene was amplified by Polymerase chain reaction (PCR) method and subcloned to prokaryotic expression vector pET28a. Escherichia coli BL21-DE3-plySs were transformed with pET28a-SLO and gene expression was induced by IPTG. Then it was purified by Ni-NTA kit. The concentration of SLO was assayed by Bradford method. To confirm recombinant SLO Western Blot was used. Results: The sequencing result was confirmed by Sanger method and was the same as SLO gene. Escherichia coli BL21 (DE3) pLysS was transformed with pET28a-SLO and gene expression was induced by IPTG. The expressed protein was purified by affinity chromatography with Ni-NTA resin. The concentration of purified protein was 100µg/ml. The integrity of product was confirmed by Western Blot analysis using a mouse anti streptolysin O. Conclusion: Data showed that recombinant SLO protein can be produced by pET28a in Escherichia coli. This protein maintains its antigenic effect very well. Therefore, recombinant SLO has same epitopes with natural form of this antigen.

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Introduction: Staphylococcus Aureus is one of the most important pathogenes in human. Regarding the ability of this bacterium in nasal colonization, carriers can increase the incidence of many nosocomial infections. This study was designed to compare the efficacy of two antimicrobial regimens (topical nasal Mupirocin and oral Ciprofloxacin) in treatment of Staphylococcus Aureus carriers and its recurrence. Materials and Methods: This study is a triple blind clinical trial that was performed on 366 cases of Vali-e-asr hospital personnel. Nasal cultures were prepared from all these cases. Among them, 45 cases were carriers which were divided in two groups (A and B). Group A were treated by single dose of oral Ciprofloxacin (1500 mg) and Vit A+D ointment as placebo for 5 days (twice a day) and group B were treated by single dose of oral placebo and nasal Mupirocin ointmdent for 5 days (twice a day). After this period, nasal cultures were repeated in the two groups to evaluate the efficacy of treatment. Also after 5 weeks the last cultures were performed in order to determine the prevalence of reinfection. Results were analyzed using Chi-square test. Results: Results showed that, 12.9% of Vali-e-asr hospital personnels were nasal carriers of Staphylococcus Aureus. Also the efficacy of topical nasal Mupirocin regimen (89.5%) was significantly higher than single dose of oral Ciprofloxacin regimen (55%) (p=0.019). But there was no significant difference between prevalence of reinfection in topical (13.3%) and oral regiment (20%). Conclusion: According to the present data, it seems that topical Mupirocin therapy is more effective than single dose of oral Ciprofloxacin in treating Staphylococcus Aureus carriers. However there is no significant difference in the prevalence of reinfection between the two methods.

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Background: In molecular diagnosis of microbial agent, purification of chromosome is very important step. In this study, after cell destruction, DNA replication was done by increasing the denaturation time, without DNA purification. Methods and Materials: In this experimental study eight different dilution of E.coli (8/100, 4/100, 2/100, 1/100, 1/200, 1/400, 1/800 and 1/1600) solution were madce in D.W, Bacteria were separated by filtration. Polymerase chain reaction method was used to propagate 162 rRNA gene by design primers without DNA Purification. In order to confirme sensitivity of PCR, contamination of 15 different sources of Arak well water wafer was compared by MPN method. For confirmed sensitivity of PCR, 15 sources of water in Arak were examined and compared with MPN method. Results: Present of bacteria in diution sou tion were confirmed by culture. Polymerale Chain reaction (PCR) data were shown this method is able to recognize bacteria in above dilutions after filtration. This study showed high sensitivity of PCR method in compare to MPN method. Conclusion: Results were shown without stages of extraction of DNA, PCR were done without losing chromosome. Therefore false negative results were decrease and avoided from difficult phases.

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Background: In pyoderma infections, the density of pus is related to desoxiribo-nucleoproteins. The use of streptodornase (DNase) in combination with streptokinase can help dissolve purulent secretions of infections which results in healing the wound through the discharge of pus from the necrotic tissue. The aim of this study was to produce recombinant streptodornase from group A strain of Streptococcus pyogenes which is highly efficient in terms of active streptodornase production using expression vector. Materials and Methods: In this applied-fundamental study, genomic DNA of streptodornase gene (sd) was extracted by phenol-chloroform. Then by using specific primers of streptodornase gene, it was amplified through PCR. The resulting streptodornase gene was cloned in pGEX4T1-sd transformer for expression and the pGEX4T1-sd plasmid was transferred to the sd. E.coli BL21. Protein production was done by induction via IPTG and optimization of the conditions. The recombinant protein was purified using the glutathione sepharose 4B kit. Results: The nucleotide sequence of PCR and group A streptodornase Streptococcus was totally the same. The production of the streptodornase recombinant protein was done by inducing pGEX4T1-sd plasmid via Isopropyl β-D-1-thiogalactopyranoside. Protein purification was done through affinity-chromatography by using glutathione sepharose 4B. The recombinant protein was reacted with anti-streptodornase mouse serum through Western-Blot method. Conclusion: Recombinant streptodornase can be produced by pGEX4T1 in E. coli. The recombinant protein maintains its antigenic property desirably. Noticing the domestic need in Iran, low rate of production, and pathogenesis of streptococci, production of this recombinant product is feasible.

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Background: Hyaluronidase A is an antigenic protein that is secreted by Streptococcus pyogenes. Nowadays, streptococcal infections are diagnosed by tracking down anti-hyaluronidase A antibodies. In this study, the attempt was made to generate recombinant hyaluronidase A in E. coli. Materials and Methods: In this experimental study, through designing specific primers and polymerase chain reaction (PCR), hyaluronidase A gene was amplified and after purification, it was sub-cloned in plasmid expression vector pET32a. Then pET32a-hylA was transferred to E. coli BL21-DE3-plySs. Protein generation induced by IPTG. The recombinant protein was purified by Ni-NTA kit and its concentration was assayed by Bradford method. Western-Blot analysis was run for verifying the recombinant hyaluronidase A. Results: The nucleotide sequencing of the gene amplified by PCR was the same as hyaluronidase A gene from Streptococcus pyogenes. Production of the recombinant hyaluronidase A via induction by pET32a-hylA plasmid was done through IPTG. The expressed protein was purified by affinity chromatography by Ni-NTA resin. The concentration of purified protein was 500µg/ml. analysis using a mouse anti-hyaluronidase A serum was reacted with the generated protein using Western-Blot analysis. Conclusion: Recombinant HylA protein can be generated in E.coli and the resulting protein maintains its antigenic properties desirably.

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Background: Streptokinase is one of the antigenic proteins secreted by streptococcus pyogenes. This protein has an important role in bacterial pathogenesis. The aim of this study was to produce recombinant forms of this enzyme so that the product would change in accordance with changes in the media. Materials and Methods: In this experimental study, we amplified the streptokinase gene by polymerase chain reaction (PCR) method. After extraction, it was sub-cloned to prokaryotic expression vector pET32a. pET32a-Ska was transferred to E.coli BL21-DE3-plySs strain. Protein production was induced by IPTG and optimization of culture media and OD of bacteria. The recombinant protein was extracted by Ni-NTA and its concentration was measured by Bradford assay. Western- Blot analysis was used to verify the recombinant protein. Results: The nucleotide sequence of the amplified gene was the same as streptokinase gene of the streptococcus pyogenes. The production of recombinant streptokinase by induction of plasmid pET32a-Ska was done by IPTG. The recombinant streptokinase had the same antigenic properties as natural streptokinase. The largest amount of recombinant protein was produced in bacteria concentrations with OD = 0.8. Also, the production of the recombinant protein was higher in media with no glucose. Conclusion: Changes in culture media can increase the production of recombinant proteins in host bacteria. The presence of nutrients, such as glucose, alone not only can not increase the amount of production but it might even decrease it

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Background: Brucella is a gram-negative intracellular bacterium. Since Brucella brings about health and socio-economic problems, its control is of primary importance. The common method of vaccination includes using live attenuated strains of this bacterium. This study was done to evaluate the immunogenicity of Brucella aburtus P39 gene in Balb/c mice. Materials and Methods: In this experimental study, P39 gene was amplified by polymerase chain reaction (PCR) method and after extraction, it was sub-cloned to eukaryotic expression vector pcDNA3. The intramuscular injection of the obtained plasmid to the Balb/c mice was done in three stages. After the last vaccination, immunologic tests, such as proliferative response in lymphocytes, IFN- assessment, IL-5, and determination of IgG2a and IgG1, were run. Results: The level of activation in splenic lymphocytes response was 3.6 and the measured IFN- was 3 ng/ml, whereas IL-5 was insignificant. IgG2a and IgG1 titers were 1.640 and 1.40, respectively. Conclusion: The findings of the immunological analysis show the appropriate immune response in Balb/c mice model after the injection of P39 gene containing plasmid. The immune system response was in Th1 form which decreased the number of bacteria in spleen. Therefore, P39 gene is of appropriate immunogenicity and DNA vaccination is efficient in the activation of cell immune response against this bacterium.

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Background: Enterococcus is known as an important pathogen in Iran like all around the world. The increasing use of vancomycin makes vancomycin-resistant enterococci (VRE) an important nosocomial pathogen. Vancomycin in combination with an aminoglycoside can provide effective treatment for severe enterococcus infections, while resistance to vancomycin antibiotic is increasing in enterococci. In this study, the pattern of antibiotic resistance and prevalence of vancomycin resistance enterococci have been explored. Materials and Methods: In this experimental study, after isolating and identifying 150 strains of enterococci from clinical specimens, the antibiotic resistance pattern of these strains to erythromycin, teicoplanin, vancomycin, ciprofloxacin, tetracycline, gentamicin, co-trimoxazole and linezolid was examined. The MIC test by using micro dilution broth method was performed for the vancomycin resistance enterococci specimens with the vancomycin and teicoplanin antibiotics. Results: Antibiotic susceptibility test showed 14% and 5.3% of the samples were resistant to vancomycin and teicoplanin respectively. Resistant to erythromycin, co-trimoxazole, ciprofloxacin, tetracycline, linezolid and gentamicin were 64, 40, 38.6, 6.6, 0, 38.76 percent respectively. Fourteen samples had high resistance to vancomycin which MIC were ≥ 256 µg/ml. Conclusion: Based on the results of present study, there are vancomycin-resistant enterococci in Arak as well as other parts of the world. The percentage of vancomycin resistance enterococci is high in Arak and appropriate treatment of infections caused by enterococcus is essential

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Background: Vibrio cholera is an important agent causing cholera in human. The expression of Flagellum and the movement of the bacterium are critical in the colonization and virulence of Vibrio cholera. FlaA gene is one the five genes encoding Flagellin which plays an important role in the activity and movement of the bacterium and its colonization which has a significant role in its immunogenicity. The aim of this study was to express and produce the recombinant FlaA protein in E.coli using Western blot method. Materials and Methods: In this experimental study, FlaA gene was proliferated by PCR method using the specific primers and cloned with BamHI and Xhol in pTz57R/T. Then it was proliferated and sequenced in DH5a vector of E.coli. The cloned FlaA gene was inserted into pGEX-4T-1 vector. The cloned vector was transformed to BL21-DE3 of E. coli and successfully expressed by induction of IPTG. The expressed protein was purified by GST affinity resin. For preparation of the primary antibody, the purified recombinant protein was injected to rats. Western blot assay method was used for determining the antigenicity of the recombinant FlaA. Results: Determination of gene sequencing showed that this gene has been proliferated properly and the antibody used in Western blot verified the production of the recombinant protein. Conclusion: The results of this study demonstrate that FlaA protein is immunogenic and can be evaluated in vaccine designing and as a diagnostic tool for detection of cholera infection.

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Background: Vibrio cholerae is a gram-negative bacterial pathogen that causes diarrheal disease cholera. One of the most pathogenic factors of Vibrio cholera is pili. Pili plays an important role in colonization and persistence of bacteria in small intestine. Materials and Methods: In this study, pili A (tcpA) gene was amplified by Polymerase chain reaction (PCR) method and sub-cloned into expression vectors such as pGEX4T-1. Escherichia coli competent cells were transformed by recombinant plasmids and the expression of protein with IPTG. The recombinant proteins were purified by affinity chromatography (GST) and immunoblot analysis was used for evaluation of new recombinant proteins antigenicity. The concentration of recombinant proteins was measured according to Bradford assay. Results: The results of this study indicated that recombinant proteins were expressed successfully in competent cell of E. coli, such as E. coli BL21 (DH3). The recombinant protein was purified by affinity chromatography (GST). The immunoreactivity pattern of anti-Tcp antibody with recombinant proteins of TcPA showed that the recombinant proteins had antigenic properties. Conclusion: Because these recombinant proteins are antigenic, these proteins may be considered as tentative candidates for designing cholera vaccine.

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Background: Helicobacter pylori is the most common bacteria causing chronic infections worldwide. An important virulence factor of H. pylori is a vacuolating cytotoxin (VacA) that induces the formation of acidic vacuoles in cytoplasm and damage to epithelial cells. The aim of this study was to examine the antigenic properties of the recombinant VacA of H. pylori in infected sera of mice and human.

Materials and Methods: In this experimental study, the highly antigenic region of VacA gene (1233 bp) was detected by bioinformatics methods, and it was amplified by PCR method and cloned into the pET32a expression vector. After expression and purification of the target protein, its antigenicity was studied by Western Blotting using human sera infected with H. pylori and sera from immunized mice infected with purified recombinant VacA.

Results: PCR and sequencing results showed that the target gene was correctly cloned into the recombinant vector. Antibodies used in Western Blotting indicated the production and expression of the recombinant protein (65kDa) with concentration of 2.1 mg/ml.

Conclusion: Recombinant VacA protein has antigenic and immunogenic properties thus, it is a proper candidate for designing H. pylori vaccine and diagnostic kits

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Background: Helicobacter pylori (H. pylori) is a gram negative bacilli that causes the stomach and duodenum diseases in human. An important virulence factor of H. pylori is a CagA gene that increases of colonization it in stomach epithelial cells and lead to inflammation and peptic ulcers. The aim of the present study was to production of recombinant protein containing highly antigenic region of CagA in E. coli.

Materials and Methods: In this experimental study, the antigenic region (1245 base pair) of CagA gene was detected by bioinformatics methods, proliferated by PCR method, digested by BamHI and XhoI restriction enzymes and cloned into pET32a plasmid and was expressed in the E. coli BL21 (DE3) pLysS with induced by IPTG. The expressed protein was purified with Ni-NTA kit and its antigenicity was studied by western blotting method.

Results: Data showed the successful cloning and expression of the target gene. Recombinant CagA protein purified by Ni-NTA kit and dialysis with concentration of 1.5 mg/ml. In western blotting, the produced protein was interacted with infected human and mice sera.

Conclusion: Results indicated that recombinant CagA protein (65 KDa) maintains its antigenicity, so could be used for serological diagnosis of H. pylori diseases and production of vaccine.

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Background: Streptococcus pyogenes produce extracellular hyaluronidase enzyme which is directly associated with the spreading of the organism during infection. Hyaluronidase enzyme is able to break hyaluronic acid or interstitial cement. This enzyme might be used in cancer treatment.The objective of the present study was to clone and express the nucleotide sequence of this enzyme which is involved in hyaluronidase enzymatic activity.

Materials and Methods: The enzymatic region of hyaluronidase gene was detected by bioinformatics methods. The polymerase chain reaction method was used to amplify the region. The amplified product was cloned into the expression vector pET32a. E. coli BL21 (DE3) pLYsS was transformed with recombinant plasmids. Then gene expression was induced by IPTG. The expressed protein was purified successfully via affinity chromatography by NiNTA kit. The integrity of the product was confirmed by western-blot analysis.

Results: The nucleotide sequence of amplified gene was consistent with the streptocuccal hyaluronidase gene. The concentration of recombinant protein calculated to 500 mg purified protein per liter. The enzymatic region of recombinant protein from Streptococcus pyogenes was recognized by all five patient’s sera with Streptococcus infection.

Conclusion: In general, it is possible to produce the enzymatic regions of the Streptococcus pyogenes hyaluronidase in Escherichia coli. The antigenic property of the produced protein is well retained. Considering the product's domestic demand and also low efficiency of production and pathogenicity of Streptococcus species, it is possible to produce it as recombinant product.

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Background: Listeria monocytogenes is one of the most important causes of abortion and postpartum infection in newborns. Because of the importance of L . monocytogenes in the health of pregnant women and newborn babies, the aim of this study was to determine the prevalence of these bacteria in pregnant women and to compare the level of prevalence between women with a history of abortion and with no a history of abortion.

Materials and Methods: In this study, 540 samples of pregnant women were provided from Arak Taleghani hospital. The samples were cultured in enrichment media, then L .monocytogenesis was isolated in specific media.

Results: Of clinical samples, 14 cases had Listeria monocytogenes. Of these samples, 8 cases in women had a history of abortion, while women with no history of abortion were 6 Most cases of positive culture were related to the age of 25 to 34 years, including 7 cases, the lowest cases were 35 to 44 years old including 3 women and 4 women were between 17 and 24 years old.

Conclusion: The study showed that Listeria monocytogenes can cause infection in pregnant women. The use of Phenotypic methods and specific media can apparently isolate listeria monocytogenes from healthy pregnant women.

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Abstract

Background: Ureaplasma and M. genitalium species belong to a kind of bacteria that are sexually transmitted and are the possible cause of pelvic inflammatory disease and nongonococcal urethritis, and et al. The aim of this study was to determine the urea plasma and Mycoplasma genitalium species frequency in women with vaginal infection and various sexual partners who referred to women^, s health promotion and treatment center in Arak.

Materials and Methods: Endocervical swab samples from 110 women with vaginal infections referred to women^’s health promotion and treatment center in Arak, were prepared. Patients’ personal information and identities during reception process were registered. The samples were transferred to the laboratory in the transport environment and after DNA extraction, were evaluated according to Real-time PCR assay.

Results: Urea plasma and Mycoplasma genitalium bacteria existed in 96(87.27%) and 4(3.63%) of patients, respectively. Among them, 4 cases had both bacteria infections. The amount of isolation in young women between 30-39 years old was more than others.

Conclusion: The results show that the colonization of urea plasma species in adult women is 40-80% and in studied group is 87.27%. These results indicate that with due attention to the increasing number of sexual partners and the increase of sexual activity, the urea plasma colonization of women will increase. In view of the potential influence of mycoplasma species on side effects resulted from pregnancy infection of mothers and mortality, on-time diagnosis and treatment will be increasingly essential.

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