جستجوی مقالات مرتبط با کلیدواژه « کلونینگ » در نشریات گروه « زیست شناسی »

The wound healing process is a complex and dynamic process that involves many metabolic pathways. This process consists of three phases inflammation, cell proliferation, and tissue regeneration stages. Successful wound healing depends on careful regulation and coordination between the factors involved. Until recent years, the strategy of treating chronic wounds was limited to wound preparation, removal of necrotic tissue, and control of infection and inflammation, but recently the use of growth factors has been approved to accelerate the healing process and heal the wound. Human recombinant growth factor PDGF-BB is one of the first types of recombinant growth factors approved in treating diabetic wounds. Several studies have reported that PDGF is an important mediator in wound healing that helps to accelerate healing, improve inflammation, cell proliferation, angiogenesis, and tissue regeneration. In this study, the human PDGF-B gene sequence was inserted into the pET 21a (+) expression vector for cloning and then inserted under the T7 promoter for its expression in the E. coli shuffle host. Purification was done using a nickel agarose column and to check the activity of the purified protein, cell proliferation, migration, and interaction were checked. The results of this study showed that the dimer type of PDGF expressed and purified in the bacterial host, probably due to maintaining the correct folded structure, has both main activities, i.e., cell proliferation due to the active binding to the cell receptor, as well as the ability to bind to fibrinogen.

Programmed cell death is a vital cellular process that is highly conserved in evolution. Apoptosis, as a common mode of programmed cell death, is disturbed in the most human malignancies and leads the resistance of cancers to current treatment strategies. Caspase 9 is a key protein in mitochondrial apoptosis. Activated Caspase 9 leads to activation of Caspase 3/7, initiating a caspase cascade and killing cell. In this study, Caspase 9 gene was cloned into pcDNA3.1(+) and its expression and function evaluated in cell.

PCR amplification of Caspase 9 was performed by specific primers and ligated into pcDNA3.1(+) after double-digestion with KpnI and BamHI. After sequencing, pcDNA/Caspase 9 was transfected into SH-SY5Y cells and treated with doxorubicin. Caspase 9 function was determined by its effect on cell death level by trypan blue and PI staining, and Caspase 3 activity, and its expression in cells measured by western blotting.

Caspase 9 gene cloning was done and its expression in cell defined by western blot. Overexpression of Caspase 9 led to autoprocessing following homodimerization and induction of cell death and also increased cell sensitivity to doxorubicin treatment and declined cell viability.

The cloned Caspase 9 was functional in cell and enhanced apoptosis in the treated cells by doxorubicin through self-activation and subsequently amplification of Caspase 3 activation.

Recombinant antibody technology has revolutionized the production and use of these kinds of antibodies and removed some of the hybridoma technology limitations. Monoclonal antibodies have high potential as diagnostic and therapeutic agents, especially in targeting cancer cells, but their use has been limited due to their large size. The special structure of different regions of antibodies allows to divide them into efficient subunits, which can combine to form new molecules with efficient properties. Due to the reduced size of scFv molecules compared to the primary antibody, they have many advantage. The most important of which is the reduction of immune compare to complete mouse antibodies due to the lack of constant fragments. In this study, the variable regions of gene carcinoembryonic tumor marker monoclonal antibody were constructed as scFV and cloned into the phagemid vector. Then its specificity was investigated by the western blotting test. The scFV gene was successfully constructed. Then cloning and expression was performed. Finally, its specificity was confirmed by western blotting. The recombinant scFV antibody expressed in this study, due to size change, was able to specifically identify the carcinoembryonic tumor marker, and therefore it can be used in the diagnosis of some cancers. This recombinant antibody was specifically capable of carcinoembryonic tumor marker recognition. Therefore, it can be applied to some cancers diagnosis and monitoring. In addition, this method can be used to synthesize scFV-type recombinant antibodies against other antigens. So that they can be used in various diagnostic tests and treatment most of diseases.

 

Enterokinase is a gastrointestinal enzyme that acts as a protease with a specific sequence. Due to the ability of enterokinase to enzymatically digest the recombinant protein at its specific site (including the four amino acids Aspartic acid and one amino acid lysine), which is targeted at the protein, as a very useful tool for purification and separation Target protein from the non-target part is used in the pharmaceutical, food and industrial industries.

Independent variables used included OD (0.6, 1.2 and 1.8) at 600 nm, IPTG concentration (0.2, 0.5 and 0.8 mM) and The type of bacterial strain was (ShuffleT7, BL21, NICO21). The response surface methodology (RSM) in the form of a central composite scheme was used to predict independent variables on the production of enterokinase enzyme.

OD equal to 1.8 at 600 nm wavelength, IPTG concentration of 0.71 mM and type of ShuffleT7 bacterial strain, LB culture medium, 2.5 mM lactose concentration and 25 ° C induction temperature, for production Recombinant enterokinase was optimized.
Discussion and

Drug proteins play an important role in modern molecular medicine therapies. Enterokinase gene expression in the bacterial system facilitates the purification of low concentrations, so large-scale, highly purified recombinant proteins can be produced and optimized in the bacterial system.

With the advent of cloning technology and recombinant DNA, it became possible to produce drugs and vaccines that were not possible in the past. Recombinant protein occupies a large part of the biopharmaceutical industry, one of the most important of which is vaccination.

In this study, with the aim of obtaining a protein candidate for cholera vaccine, first cloning of a plasmid designed for the bacterial host E.coli DE3 Rossetagami was transformed, then expression was induced using IPTG and after protein expression was examined by SDS-PAGE gel electrophoresis, and then the protein was purified by nickel-sepharose (NI-NTA) chromatography column.

The results show that the purified protein has a molecular weight of 31 kDa and the amount of purified protein is 1 mg.

Based on previous studies as well as the results of this study, the resulting protein can be considered as a suitable candidate for the cholera vaccine.

Aim & Background:

 the genus Burkholderia (mallei and pseudomallei species) are classified as dangerous human pathogens. Diagnosing cultures for this bacterium is costly and time consuming and on the other hand it is very dangerous to work with and it is very difficult to access this bacterium. the porpose of this research Design and production of a genetically simulant construct for the detection of  B. pseudomaleii

initially, a simulant construct containing the reference and specific genes of the mallei and pseudomallei species was not observed in other species of the family, and then the operational efficiency of the designed construct was quantified using Real-Time Quantitative PCR. Quality (SYBER Green) and qualitative (using TaqMan probe) were evaluated. Finally, Real-Time PCR amplification products were analyzed by melting curve and electrophoresis.

the minimum and maximum sensitivity were 0.1 pg and 10 ng on the constructs designed in the present study, was obtained.

This simulat can be used as a positive control for the detection of the Burkholderia agent and can further enhance the development of new diagnostic kits in the field.

Inhibitor of apoptosis (IAP) are a family of proteins that block cell death through caspase activity. Survivin is smallest IAPs family member that overexpresses in different cancer types but not in normal tissue except embryonic tissue. Survivin may be used as a new marker to stratify cancer patients for more optimal treatment modalities. The aim of the current study was to investigate survivin DNA cloning into pET-28a and its expression in E.coli.The sequence of survivin gene was amplified by PCR using specific primers and pcDNA-survivin temple. PCR product and pET-28a plasmid were digested by HindIII/NheI restriction enzymes and survivin was ligated into the digested vector. Then, the ligation product was transformed into the E.coli DH5a competent cells and screened by antibiotic selection marker (kanamycin). Positive colonies were selected by colony PCR and screened by double digestion of isolated plasmid. One positive colony was sequenced and confirmed. The recombinant plasmid was transformed into the expression strain of E.coli (BL21) by chemical method. The expression of survivin was induced in the different conditions and expression level investigated by SDS-PAGE.The size of PCR product in agarose gel showed the correctness of amplification. The digested pET-28a plasmid also indicated the correctness of enzymatic reaction. The sequence of the cloned fragment revealed a 100% similarity to the human survivin. In expressing, adding IPTG increased the expression of survivin protein in all conditions, especially 37 ᵒC from 2 h after induction. At all conditions, most of survivin accumulated in the bacteria as inclusion body.

Hsp70 family members are central components of the cellular network of molecular chaperones and folding catalysts. The gene encoding a protein related to Hsp70 or DnaK in the domain bacteria is called dnaK. DnaK proteins are involved in de novo protein folding, formation, and disassembly of protein complexes and degradation of misfolded proteins. The gene dnaJ which codes for Hsp40 in bacteria, modulate the activities of DnaK by acting as co-chaperone. In the present study, we cloned and expressed DnaK from Bacillus halodurans Guj1 were identified. The dnaK gene of B. halodurans was successfully expressed in E. coli BL21 (DE3) using pET-28a+ expression system. The open reading frame of the cloned gene contained 1839bp and encoded 612 amino acid residues. Calculated molecular weight and pI of the protein were 66.18kDa and 4.55 respectively. The deduced amino acid sequence of B. halodurans Guj1 showed about 60% identity with the E. coli counterpart. The 3D structure of dnaK from B. halodurans was constructed using the crystal structure of human HSP70 chaperone BiP as the template, which showed an identity of 88.8% together. Partially purified recombinant DnaK by heat treatment showed a band at approximately 70kDa on SDS-PAGE. Our findings showed that the recombinant DnaK improved the refolding efficiency of the carbonic anhydrase by 27% after 60min at 54°C. According to the results obtained, DnaK from B. halodurans can potentially be used for improving the functional properties of enzymes and proteins in various applications.

Calcitonin is a small peptide hormone that is produced by parafollicular thyroid cells in human and regulates the metabolism of calcium and phosphorus. It is therapeutically used in treatment of calcium-related disorders and osteoporosis. Recombinant calcitonin production encounters with several difficulties due to instability and low molecular weight, and also needs further treatment in prokaryotic systems. Microalgae have recently garnered high attention for their potential in expression of recombinant proteins. The aim of present study was to assess the ability of Chlamydomonas Reinhardtii to express recombinant human calcitonin. The optimized calcitonin coding sequence and carbonic anhydrase secretory signal was cloned in Pchlamy _3 and Pchlamy_4 vectors. The recombinant plasmids were transformed to wild type and also a cell wall deficient strain of Chlamydomonas Reinhardtii by electroporation. Transformed strains were screened by colony PCR method and selected strains were cultivated to produce recombinant calcitonin. Culture media have been collected after cells growth and assayed by ELISA method. Pchlamy_3 vector could not express the target sequence as desired and all the recombinant strains were resulted from Pchlamy_4 vector. The wild type strain also did not show desired yield and only cell wall deficient strain was successfully transformed. The yield of recombinant calcitonin produced by positive strain was about 1 pg/ml. The results of this study show that the used strategy for secretory production of recombinant calcitonin was successful and it could be used in further studies.

Identifying the structure and function of alpha-Synuclein protein can lead to the development of appropriate treatments for Parkinson disease. The aim of the current study was to investigate DNA cloning and the expression of alpha-Synuclein protein in E. coli. In this experimental study, the sequence of encoding alpha-Synuclein in pRK172 recombinant plasmid was amplified by Polymerase Chain Reaction (PCR), using best primers. The synthesized DNA was, then, digested by restriction enzymes and cloned into pET28a and recombinant plasmid was transferred into the expression strain of E. coli (BL21) by Calcium Chloride method. The expression of alpha-Synuclein gene was induced by Isopropyl-Beta-D-Thiogalactoside (IPTG) and the expression of alpha-Synuclein was investigated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) method. Sequencing was done, using the ClustalW algorithm by the BioEdit 5.0.9 program. In products of DNA enzymatic digestive reactions and pET28a plasmid with restriction enzymes, the size of the fragments indicated the correctness of the enzymatic reactions. The synthesized DNA and pET28a plasmid were 407 and 5369 nucleotides, respectively. The translation of the sequence of the cloned fragment revealed a 100% similarity to the human alpha-Synuclein protein. In expressing the recombinant protein in comparison with negative control samples, adding IPTG increased the expression of alpha-Synuclein protein in all samples, especially 2 hours after induction. Most of alpha-Synuclein expressed from the pET28a-alpha-Synuclein plasmid accumulated in the bacteria as incorporated objects. The alpha-Synuclein protein is cloned into the pET28a plasmid and formation of the objects incorporated by alpha-Synuclein is confirmed by the expression of the pET28a-alpha-Synuclein system and paves the way for producing this protein in high scale.

Biomacromolecules of thermophilic organisms are often thermostable. Apart from high temperature they are also known to withstand denaturants of extremely acidic and alkaline conditions. Thermostable enzymes have capable potentioal in the food and paper industries, detergents, drugs, toxic wastes removal and oil drilling. The enzymes can be produced from the thermophiles through either optimized fermentation of the microorganisms or cloning of fast-growing mesophiles by recombinant DNA technology. Metalloproteases are metal ion dependent protease. Metalloproteases that used in this study was a zinc-dependent endopeptidase. Metalloprotease gene was cloned from Anoxybacillus flavithermus. For the amplification of the gene, PCR was performed. A pair of primers designed with a cleavage site for enzymes XhoI and NheI and vector pET21a() clone was transformed into BL21 host. The results of the cloned gene sequence analysis showed similarity to the protease gene different variants of the species Anoxybacillus flavithermus.

Plant growth promoting bacteria produce ACC deaminase (EC4.1.99.4) which regulates the biosynthesis of ethylene through cleavage of ACC (immediate precursor of Ethylene) into-ketobutyarate and ammonia. Therefore, it has an important role in plant growth promotion via lowering indigenous ethylene levels especially when the plants are exposed to an environmental stress. Therefore, this study aimed to investigate the cloning, expression, purification and determination of biochemical properties of ACC deaminase from Pseudomonas fluorescense. In this regard, the ACC deaminase encoding gene of Pseudomonas fluoresense FY32 was isolated and cloned in pET28 a (+) and the resultant pET28/acdS construct then was transformed into E. coli BL21(DE3). The expressed enzyme was purified by metal-affinity chromatography on Ni(2+)-TED-Sepharose column and then the optimum conditions and biochemical properties of the purified enzyme was examined. This enzyme showed the highest activity at 28 °C, pH 7 in the presence of 30 mM MgSO4. Also, the significant reduction of ACC deaminase activity was observed in 160 ppm of NaCl. The Km and Vmax of enzyme were calculated to be 9.66 mM and 0.11 nM mg-1 h-1, respectively (determined by the concentration of the produced α-ketobutyrate), which were relatively higher than those previously reported.

The thermostable and alkaliphile xylanases are beneficial industrial enzymes in food industry (bakery and alcohol production or fermentation industries) pulp-kraft, and pharmaceutic industry. Xylanases hydrolyse xylan (the hemicellulose of plant cell wall) and produced by different kinds of microorganisms like bacteria, fungi and some algae. In this study, xylanase gene from thermoalkaline Anoxybacillus flavithermus was isolated from hot spring of Meshkinshahr (Iran) and was cloned in E. coli (BL21). At first, total DNA was extracted from bacteria. PCR was then accomplished and xylanase gene was isolated and amplified. Amplification of xylenase coding gene was confirmed by electrophoresis. Cloning was performed by digestion of xylanase gene and pET21a by restriction enzymes (NheI and XhoI), following by ligation and transformation to E. coli (BL21). Finally, the results were confirmed by colony-PCR and sequencing of selected colonies. PCR and sequencing results, demonstrated a high similarity between xylanases of Anoxybacillus species with xylanase of this study.

Aim and Cancer/testis (CT) antigens are a category of tumor antigens that are typically expressed only in the human germ line, and in several types of tumor. MAGEA4 is one member cancer testis antigen family that has relation with other tumors. Since the biological function of MAGEA4 is unclear, the aim of the present study was amplification and cloning of the gene in the expression vector to produce recombinant protein that expresses MAGEA4. Using PCR specific primers including restriction site, MAGEA4 was amplified. The purified PCR products were ligated between the BamH1 and Xho1 sites of pTZ57/R cloning vector and transformed into Escherichia coli Top10 strain and screened by IPTG and X-Gal. The correct orientation of MAGEA4 fragment was identified by restriction enzyme analysis and sequencing of constructed plasmid. Then sub cloning was carried out within pRUF expression vector with the same restriction site. The final confirmation was performed using colony PCR, double digesting and sequence analysis. Therefore the MAGEA4 gene was cloned into the restriction site of pRUF. This study is an important step for producing recombinant proteins and is used to find the function of this gene for therapeutic targets.

Bacterial lipases are members of α/β hydrolase family that hydrolyzed triacylglycerol at the water- lipid interface. Bacillus thermocatenulatus lipase 2 (BTL2) is a thermoalkalophilic lipase that shows optimal activity at 60–75 oC and pH 8–10. BTL2 is an important research target because of its potential industrial applications. At the present study chimeric Bacillus thermocatenulatus lipase contain the consensus sequence of Candida rugosa lipase (207Gly-Glu-Ser-Ala-Gly211) at the nucleophilic elbow region was cloned and expressed in E. coli as secretion protein. Finally, Catalytic activity of chimeric lipases was evaluated at presence of various triglycerides as substrates and the effects of different parameters such as temperature, pH, detergents, organic solvents and metal ions were evaluated on chimeric enzyme activity using a pH-stat assay. The results showed that the chimeric enzyme is most active to C4 substrate, (pH 9.0) and 60 oC. As well as enzyme activity has increased in the presence of organic solvents, N-Hexane, N-heptane, methanol, chloroform and detergents such as Triton X -100, Tween 20, Tween 40. Also, metal ions, respectively decreased general effect on the enzyme activity in chimeric lipase.

Laccases have many biotechnological applications due to their oxidation ability towards a wide range of phenolic compounds. In this study، the strain QZA2 isolated from Iran soils was used. This strain showed high similarity to Bacillus anthracis strain ATCC 14578 according to 16S rRNA gene sequence. Laccase gene was amplified by cloning primers. then PCR product cloned in the expression vector (pET21a) and transferred to BL21 (DE3) strain of E. coli under the control phage T7 and sequence analysis carried out. The gene of the QZA2 has an open reading frame composed of 1656 bases، which encodes 551 amino acid residues and contain four histidine rich copper-binding domains. The laccase gene from QZA2 showed 16% similarity with CotA from B. subtilis and 57% similarity with multicopper oxidase B. halodurans. The expression was performed under microaerobic condition in order to obtain high amounts of soluble protein. Biochemical properties were investigated using common laccase substrates ABTS.

L-asparaginaseΙΙ (E.C. 3.5.1.1) has effective usage for treatment of acute lymphoblastic leukemia. This enzyme is isolated from bacterial sources and it is commercially available as an anti-cancer drug. This enzyme catalyzes the hydrolysis of L-asparagine to ammonium and aspartate. Unlike Normal cells, the cancer cells strongly require L-Asparagine leading to destruction of these cells. The propose of this project is cloning of L-asparaginaseΙΙ gene from E. coli in B. subtilis to produce the mass of this enzyme.

L-asparaginaseΙΙ gene is isolated from E. coli by PCR approach. The amplified fragment and expression shuttle vector pMR12 are digested by HindΙΙΙ and BamHΙ enzymes. The ligation between DNA fragment and the cloning shuttle vector is done by standard method. In the next step recombinant vector is transferred to E. coli JM101 by cold calcium chloride treatment and finally introduced into B. subtilis by a chemical method.

in this experiment, ansB gene is isolated from E. coli by PCR and cloned into the expression shuttle vector pMR12. Existence of gene with 1047 bp lenght is confirmed by enzymatic analysis and PCR reaction. Then recombinant vector cloned in E. coli at first and then in B. subtilis. Finally the plasmid extracted and compared with the band of expersion shuttle vector containing ansB gene to confirm.

In this study, we tried to clone the ansB gene in B. subtilis using expression shuttle vector pMR12. This is the first report of ansB gene cloning in B. subtilis.

Aim and Influenza A is a major cause of mortality in a year. There has been an efficient vaccine available against it since 1960. Antigenic variation is a preventive agent for yearly vaccine production. Now researchers focus on the constant antigens of viruses. M2 protein makes an ionic channel in the coat of influenza, a virus that is effective on infection by virus. This protein is conserved and it is considered as a proper candidate vaccine for influenza infection. Due to limitations for influenza virus culture at the lab, the sequence of influenza virus H1N1 strain of Iran was synthesized. It was sub-cloned into pET22b vector recombinant protein and expressed in BL21 E.coli and confirmed with specific antibody by SDS-PAGE and western blot techniques. The concentration of recombinant M2 protein was 300μg/ml confirmed by specific antibody. Influenza M2 protein could be expressed in BL21 E.coli host.

To increasing the efficiency of production in livestock industry could use recombinant DNA technology. Recently progress in gene transfer system، genetic modifying of ruminal bacteria with ability of hydrolytic enzyme production has been possible. Resulting this technology reduce of disadvantages of enzyme supplements as well as increase of nutrient flow in rumen. Since transformation in rumen bacteria has low efficiency therefore conjugation could be effective for moving genetic elements. In more previous studies E. coli as a facultative anaerobe bacteria has used because could grow in rumen. The present study، an amylase gene previously clone in pET28a and amplified by polymerase chain reaction then subcloned in shuttle vector pGFK114. 1 following transformed in E. coli DH5α. Conjugation was performed between two E. coli DH5α and TG1 donors containing pGFK114. 1 and pRK231 helper plasmid respectively and one recipient E. coliBL21 DE3 using pattern of digestive system. We have successfully transformed cloned amylase gene to recipient bacteria such rumen environment as reported before.