Showing 14 results for Recombinant
Hamid Abtahi, Ali Hatef Salmanian, Sima Rafati, Ghorban Behzadian Nejad,
Volume 7, Issue 1 (3-2004)
Abstract
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.
Mohsen Khaki, Mostafa Ghavamian,
Volume 8, Issue 4 (12-2005)
Abstract
Introduction: Hepatitis B is a disseminated liver inflammation from HBV, that causes diseases and a large number of deaths. Regarding the fact that some of the vaccinated people are non responder (NR), evaluation of immunity in vaccinated ones and identification of NR especially in high risk group is necessary.
Materials and Methods: In this descriptive study blood samples of all medical students of Borujerd Azad university at the age of 18-25 and vaccinated personnel of Borujerd Shariaty hospital were tested for Anti.HBS-Ab level by ELISA method with Radim kit (cat.KHB31). Results were analyzed according to the number of received vaccines, the duration of vaccination and demographic criteria using descriptive statistics.
Results: About 90% of samples had protective immunity and 10% were NR. 8% of immune group had more than 1000, 17.2 % between 500-1000 and 74.8 % between 10-500 miu/ml of Ab titer. About 75% of immune samples had received two vaccines. In NR group 53% had received three vaccines and 47% had two. 4% of samples were immune with the duration less than one month after vaccination which 85 % of them had two vaccines.
Conclusion: Herd immunity was 90% which is accordant to most studies. In some studies with different results the effective criteria were not differentiated. So regarding these differences, vaccinated people are recommended to evaluate their HBS.Ab level.
Dr Hamid Abtahi, Dr Ali Hatef Salmanian, Dr Sima Rafati,
Volume 9, Issue 1 (3-2006)
Abstract
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.
Raziyeh Khalesi, Jafar Salimian, Shahram Nazarian, Zahra Ehsaei , Ali Asghar Rahimi, Nafiseh Amini, Seyed Mohammad Moazzeni,
Volume 15, Issue 1 (4-2012)
Abstract
Background: Enterotoxigenic Escherichia coli bacterium is the most important bacterial agent causing diarrhea. Specific virulence factors, such as enterotoxins and colonization factors, distinguish ETEC from other classes of diarrheagenic E.coli. In this study, heat-labile toxin was purified which could be utilized for anti-toxin assay in GM1 gangelioside receptor-ELISA based method and for identification of ETEC producing toxin.
Materials and Methods: In this experimental study, bacterial strain producing heat-labile toxin was first cultivated for production and purification of toxin. Then supernatant soluble proteins were precipitated with ammonium sulfate and purified using biochemical methods. Finally, purified protein was dialyzed against Tris 0.02 mM pH 8 and analyzed on gel electrophoresis. GM1 gangelioside receptor-ELISA based method was used for detection and assessment of the purified toxin. Through this method, the effect of anti-recombinant heat-labile toxin B subunit neutralization on heat-labile toxin was investigated.
Results: Toxin purification was revealed by the presence of 12 and 28 KD protein bands. This study demonstrated that anti-recombinant heat-labile toxin B subunit antibody can detect the purified toxin and can inhibit its binding to GM1 receptor up to 80%.
Conclusion: Purification of heat-labile toxin and gangelioside receptor-ELISA assay can be used for accurate detection and epidemiological study of clinical isolates.
Sarah Najafi, Farida Behzadian , Fatemeh Fotuhi, Jalil Fallah Mehrabadi,
Volume 15, Issue 5 (10-2012)
Abstract
Background: In recent years Influenza viruses have caused widely spread moderate to severe infection in all around the world and there is no Influenza vaccine which can protect people only with one dose injection till now. Therefore , producing a universal vaccine based on virus like particle (VLP) could be ideal. In this study one of the molecular structures was considered for VLP based Influenza vaccine. Materials and Methods: In this experimental study, the human influenza virus (A /New Caledonia 20/1999/ (H1N1)) was propagated in MDCK cell culture. Viral RNA was extracted using RNX-plus solution. Complementary DNA synthesis was carried out using uni-12 primer and random hexamer as specific and general primers, respectively. Neuraminidase open reading frame (1413-bp) was amplified by PCR and cloned into pBlue-script SK. Neuraminidase coding frame sub cloned into pFastBac11 plasmid through SalI/XhoI sites. After verification of cloned Neuraminidase by restriction analysis, it was subjected to automated sequencing bi-directionally. The recombinant pFastBac Neuraminidase vector was transformed to E.coli DH10Bac cells which harbor bacmid DNA and helper plasmid to create Neuraminidase recombinant bacmid. Results: Neuraminidase recombinant bacmid was created by homologous recombination between pFastBacNA and bacmid and was verified by PCR using Neuraminidase specific and M13 universal primers. Conclusion: Recombinant baculovirus expressing Neuraminidase gene can be also used with other individual recombinant baculoviruses expressing HA and M1 genes in production of influenza VLPs or proteins resulting from this structure could be purified in specific insects for vaccine research studies.
Hamid Kazemian, Mohammad Najafi-Mosleh, Hamid Abtahi,
Volume 15, Issue 7 (12-2012)
Abstract
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.
Hadi Ansarihadipour, Ali Bahadori Vatankhah, Saeed Ziraki, Mohamad Saiadi,
Volume 15, Issue 8 (1-2013)
Abstract
Background: The presence of oxidant agents yields higher levels of free radical reaction products in erythrocyte membrane proteins and serum proteins. The aim of this study is to investigate the oxidative modifications of recombinant human coagulation factor VIII (rHFVIII) by spectrophotometric analysis. Materials and Methods: In this experimental study, rHFVIII was incubated aerobically with vitamin C and ferro ions in metal catalyzed oxidation (MCO) system for 4 to 28 hr. Carbonyl assay was used as an index of protein oxidation. For this purpose, 2,4dinitrophenyl hydrazine (DNPH) was used. Reaction of this reagent with carbonyl groups produces dinitrophenylhydrazone derivatives that their concentration was estimated by spectrophotometry. Results: Carbonyl groups in rHFVIII changed in the presence of vitamin C and ferro ions. Dose-dependent effects of vitamin C showed a decrease in carbonyl groups of rHFVIII whileferro ions increased oxidation and carbonyl group formation in this protein. Conclusion: These findings indicate that changes in carbonyl groups in rHFVIII are related to the generation of reactive oxygen species. Also, antioxidant mechanisms are activated in a dose- and time-dependent manner.
Leila Hasanzadeh, Hamid Abtahi, Ehsanollah Ghaznavi-Rad , Safieh Soufian , Vahideh Farjadi ,
Volume 16, Issue 1 (4-2013)
Abstract
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
Vahideh Farjadi , Hamid Abtahi, Mohammad Reza Zolfaghari, Safieh Soufian, Leila Hasanzadeh,
Volume 16, Issue 7 (10-2013)
Abstract
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.
Roghayeh Rahimi, Mehdi Mahdavi, Massoumeh Ebtekar,
Volume 17, Issue 9 (12-2014)
Abstract
Background: Delivery of antigens directly to dendritic cells enhances the immune responses to the antigen and is an attractive approach for eliciting cellular immune responses against mutagenic pathogens like HIV virus. So the aim of this study is evaluation of immune responses elicited by delivered multi-epitopic HIV-1 tat/pol/gag/env recombinant protein to dendritic cells in sito using &alphaDEC-205 mAb.
Materials and Methods: In this study, recombinant protein expressed by pET23a-HIVtop4 plasmid including HIVtop4 sequence (Gag158-186, Pol150-190, ENV296-323, ENV577-610, Tat1-20 and Tat44-61) in BL21 E. coli cells was used as vaccine model. To exploiting dendritic cells, properties for immunization purposes, we conjugated this recombinant protein chemically to anti body against DEC-205 receptor on these cells. Balb/c mice immunized subcutaneously (s.c.) with conjugated multi-epitopic protein or un-conjugated one (as control) simultaneously with Poly I: C as dendritic cell maturation factor. Lymphocyte proliferation was measured by bromo di uridine assay, Cytotoxicity by Grenzyme B production activity, IL-4, IL-17, IFN- cytokines production and total antibody by direct and indirect ELISA methods in order.
Results: Immunization by anti DEC-205 conjugated peptide led to a significant increase in the proliferative responses of lymphocytes, production of Gr-B, IFN-&gamma, IL-4 and IL-17 cytokines and total antibody titer in comparison with the none targeted groups.
Conclusion: It is concluded that targeting of protein antigens to DEC-205+dendritic cells significantly enhances immune responses in compare to non-targeting strategies.
Bahador Behrouz, Nour Amirmozafari, Mohammad Mehdi Fizabadi, Nima Khoramabadi, Mahboobeh Bahroudi, Mehdi Mahdavi,
Volume 18, Issue 5 (8-2015)
Abstract
Background: Pathogenic Pseudomonas aeruginosa strains produce a polar flagellum that required for motility, adhesion, invasion and secretion of virulence factors. The aim of this study was to evaluate the effect of prevention and treatment with anti-recombinant type B flagellin antibody in a burned mouse model.
Materials and Methods: One hundred twenty six mice were divided into 16 groups injected with different regimen of anti-recombinant type B flagellin antibody. Infections were caused by sub-dermal injection of P. aeruginosa PAO1 and PAK strains at the burn site. Groups were monitored for mortality for one week. Additionally, functional activity of this antibody was assessed by motility inhibition and opsonophagocytosis assays.
Results: Immunotherapy with rabbit antisera substantially increased (85.71%) survival rate of mice challenged with a homologous strain PAO1 compared with the control PBS. The mortality rate in mice infected by the heterologous strain PAK was only 28.57%. This antibody increased phagocytosis killing of the homologous strain but it only had a slight effect on the heterologous strain.
Conclusion: Passive immunotherapy protected burned mice challenged with the homologous strain but showed lower efficacy against the heterologous strain.
Somayeh Kadkhodayan, Shiva Irani, Seyed Mehdi Sadat, Fatemeh Fotouhi, Azam Bolhassani,
Volume 19, Issue 4 (7-2016)
Abstract
Background: Nef is one of the HIV-1 critical proteins, because it is essential for viral replication and AIDS disease progression and induction of immune response against it can partially inhibit viral infection. Moreover, a domain of the HIV-1 Trans-Activator of Transcription (Tat, 48-60 aa) could act as a cell penetrating peptide (CPP). In current study, cloning and expression of Tat-Nef fusion protein was performed in E. coli for the first time. The protein expression was confirmed by western blot analysis and was purified using reverse staining method.
Materials and Methods: In this experimental study, primarily, cloning of Tat-Nef fusion gene was done in pGEX6p2 expression vector. Then, the expression of Tat-Nef recombinat protein in E.coli BL21 (DE3) strain was performed by using IPTG inducer. The protein expression was confirmed by SDS-PAGE and western blotting using anti-Nef monoclonal antibody. Then, the recombinant fusion protein was purified from gel using reverse staining method.
Results: The results of PCR analysis and enzyme digestion showed a clear band of ~ 726 bp in agarose gel indicating the correct Tat-Nef fusion cloning in pGEX6p2 prokaryotic expression vector. In addition, a 54 kDa band of Tat-Nef on SDS-PAGE revealed Tat-Nef protein expression that western blot analysis using anti-Nef monoclonal antibody confirmed it.
Conclusion: The purified Tat-Nef recombinant fusion protein will be used as an antigen for protein vaccine design against HIV infection.
Pooneh Roghanian, Jafar Amani, Shoreh Zare, Zahra Nour Mohammadi,
Volume 22, Issue 1 (4-2019)
Abstract
Background and Aim: Enterotoxigenic Escherichia coli (ETEC) is one of the most common bacterial causes of diarrhea deaths among children and travelers in developing countries. The ETEC colonization factors, such as CFA/I and CS2 play an important role in the development of the disease. In this study, to produce the CFaE fusion recombinant protein, the tip subunits CFA/I(CfaE) and sub structural unit of CS2 (CotD) from ETEC, were used. Since mucosal immune responses to CFs can prevent disease, the aim of this study was to develop a chimeric antigen for developing the effective vaccine.
Materials and Methods: In order to amplify the cfae-cotd gene, a dual gene construct consisting of cfae and cotd, the PCR reaction was performed by designed primers. The propagated gene was cloned in the expression vector pET28a. Following the induction of a recombinant gene construct with IPTG, the recombinant protein was expressed and purified by Ni-NTA chromatography column and confirmed by western blotting by Anti-Histag.
Ethical Considerations: This study with research ethics code IR.IAU.SRB.REC.1397.066 has been approved by research ethics committee at Islamic Azad University, Science and Research Branch of Tehran, Iran.
Findings: Cloning accuracy was confirmed by PCR and enzyme digestion reaction. The presence of the band in the SDS-PAGE 10% gel in the 68 kDa region, the expression of the recombinant protein, and the presence of the band on the nitrocellulose paper in the Western blotting test confirmed the production of recombinant protein.
Conclusion: Optimization of codon and expression in heterologous hosts is a useful method for the production of recombinant proteins. The production of ETEC antigens as a candidate for vaccination against this bacterium is also prominent.
Mohammad Reza Soleyman, Mostafa Khalili, Alireza Soleyman Meiguni, Maryam Baazm,
Volume 22, Issue 5 (11-2019)
Abstract
Background and Aim recombinant DNA technique is a powerful and appropriate method for the production of protein biopolymers with specificity in amino acid sequence and spatial chemistry. Elastin-Like Polypeptide (ELP) is a biocompatible, biodegradable and non-immunological biopolymer used in various biotechnology studies. The ELP tag is a cheap, fast and non-chromatographic technique for purifying target proteins. In this study, pET expression vector was designed for the combination of ELP gene sequences and target recombinant protein in order to produce recombinant fusion protein with the ELP tag.
Methods & Materials MOD gene was transformed to E. coli-BL21 (DE3) cells after designing and synthesis among the XbaI and XhoI restriction sites in the pET-32a (+) vector of the clone. Then, colonies were isolated based on plasmid size and examined by cutting using restriction enzymes. The final recombinant colonies was verified using polymerase chain reaction method and DNA sequencing.
Ethical Considerations The Research Ethics Committee of Arak University of Medical Sciences approved all ethical considerations ofworking on laboratory animals (Code: 92-146-11).
Results Replacing the MOD sequence in the pET-32a vector (+) eliminated the components expressing the fusion tags (Thioredoxin, Histidine, and S-tag), the identification site of protease enzyme (tobacco etch virus), and multiple cloning site. In addition, it added specific restriction enzyme identification sequences of ELP gene and target gene. As a result, in the optimized pET-MODvector, 466 nucleotides reduced in size and the secondary structure was improved.
Conclusion Considering the improvement of spatial structure and reduction of pET-MOD vector size, as well as the possibility of the fusion of recombinant protein with the ELP tag, it is possible to use this vector for ELPyation of the target protein.
