Showing 24 results for Cloning
Maliheh Esmaeilzadeh Khorasani, Mojtaba Saadati, Khosro Agaei Pour,
Volume 1, Issue 1 (12-2010)
Abstract
Abstract
Introduction: Measles virus (MV) belongs to the morbilivirus genus of paramyxoviridae family, and has single stranded, negative polarity, non segmented RNA genome.
Method: In this research, the total RNA was extracted of measles virus (AIK-C) vaccine strain. The extracted RNA was immediately used in reverse transcription reaction to generate cDNA. The 1st strand cDNA was used to amplify the F gene by specific primers in a reaction PCR. The PCR product with the expected size of 1662 bp was cloned into expression plasmids pET-22b(+) and pET-28a(+).
The recombinant plasmids were transformed into competent E.coli DH5α cells and clonies were screened with direct PCR. The recombinant plasmids were extracted by Alkaline lysis and were compared with non- recombinant plasmids in molecular weight.
Results: Recombinant plasmids were digested with Nde I and Hind III restriction enzymes. The DNA band with an approximate size of 1662 bp was detected on 1.5% agarose gel. The recombinant plasmid pET-28a(+) was sequenced, comparison of this sequence with the coding sequence F protein of measles virus (AIK-C) in Genbank (AF266286) was revealed high degree of homology and showed that F gene is highly conserved.
Conclusion: It was showed that F gene is highly conserved. Thus F gene is important for studing in order to produce recombinant vaccine.
Volume 2, Issue 3 (7-2016)
Abstract
Background: Leptospirosis has been recognized as an important reemerging infectious disease caused by pathogenic Leptospira spp. A major challenge of this disease is the application of a basic research to improve diagnostic method. Outer membrane proteins of Leptospira are potential candidates that could be useful in diagnosis. Among them the lipL41 is an immunogenic protein which is present only in pathogenic serovars. In order to evaluate genetic conservation of the lipL41 gene, we cloned and sequenced this gene from Leptospira interrogans serovar Canicola.
Materials and Methods: Following the DNA extraction from the serovar, the lipL41 gene was amplified and cloned into pTZ57R/T vector and transformed into the competent E. coli (Top10). Recombinant clones were confirmed by colony PCR and DNA sequencing. The related sequences were then analyzed and compared with the sequences in the Genbank database.
Results: PCR amplification of the lipL41 gene resulted in a 1065 bp PCR product. The PCR based on the lipL41 gene detected all the pathogenic reference serovars of the tested Leptospira spp. It was revealed that in Iran the homology of the lipL41 gene between vaccinal and clinical serovars of Canicola was 100%. It also showed >95.9% homology with other pathogenic serovars in Genbank database, which indicates genetic conservation of this gene.
Conclusion: Because of the conservation of lipL41 gene among different strains of Leptospira and its exclusive presence in leptospira, it was revealed that the cloned gene could be further used as a good candidate for developing diagnostic methods such as ELISA and as positive control in diagnostic PCR.
Davoud Farajzadeh, , ,
Volume 6, Issue 1 (10-2015)
Abstract
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.
Volume 7, Issue 4 (11-2021)
Abstract
Backgrounds: Streptomyces is an aerobic filamentous Gram-positive bacterium frequently found in various environments worldwide. Cellulases are a group of glycosyl hydrolase enzymes that are frequently produced by bacteria. Thus, the aim of this study was to detect cellulase-encoding gene (celA) in soil-living Streptomyces strains and evaluate its cloning in Escherichia coli Origami strain.
Materials & Methods: Soil samples were collected from a depth of 5-10 cm in Tehran, Iran. After identification of Streptomyces isolates by morphological and biochemical tests, genomic DNA was extracted. Polymerase chain reaction (PCR) test was employed to identify Streptomyces strains harboring the cellulase gene. The celA gene was positively transmitted to the host bacterium E. coli via a vector and cloned through the TA technique. Real-time PCR was used to measure the overexpression of this gene. ClustalX and Mega5 software were used to draw the phylogenetic tree.
Findings: Out of 12 Streptomyces isolates, 25% were found to carry the celA gene. After cloning the celA gene, the cloned strains were chosen by colony selection (blue/white). The real-time PCR test showed the expression of the celA gene in the transformed strains. Phylogenetic analysis results using the neighbor-joining assay showed that Streptomyces spp. with 81% bootstrap were located in the same clade, indicating their close relatedness.
Conclusion: Soil is one of the high-potential sources of the production of secondary metabolites, which could be used as a valuable source of various biological products such as cellulase.
Volume 8, Issue 3 (10-2004)
Abstract
Mohmoud Sadeghi
Assistant Professor of Law, Tarbiat Modarres University
After a description of human cloning, and a review of various divine religious authorities point of views (Christian, Jewish and Islamic) and a report of legal attitudes and global policies on human cloning, both reproductive and therapeutic, we shall discuss this matter according to the Islamic, especially Shiat jurisprudence. As a general result of this research, human reproductive cloning, if could not intrinsically be prohibited, it seems unjustifiable because of its various bad consequences, such as disorder of family system, confusion of clones family relations, possibility of body and mental diseases for clones and suffers of egg donors women and surrogate mothers, regarding to a number of Islamic jurisprudence rules, such as rule of self preservation and necessity respect for human beings and the leading rule of “la zarar” (ban of causing harm to oneself and other) and prevention of confusion of social system order, etc. However use of cloned human embryos for research and exploitation of stem cells to produce transplantable tissues and organs, seems, acceptable, regarding to the important promises of these researches for scientific development in the field of biomedicine, with high benefits for the mankind and bearing in mind that reasons in the authentic Islamic texts for respect of human embryo are related to its post implantation stages.
S. Sheykhi , M. Amininasab, B. Saffari, S. Abdi,
Volume 9, Issue 1 (1-2018)
Abstract
Aims: 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.
Materials and Methods: 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.
Findings: 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.
Conclusion: 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.
S. Hemati, F. Dehghan Nayeri,
Volume 9, Issue 2 (9-2018)
Abstract
Aims: Antioxidants in sesame oil, including tocopherols and sesamin have greatly increased the shelf life of it against heat. Following the increase in the expression of the cytochrome P450 enzyme encoder (CYP81Q1), the content of sesame is increased in different stages of development of sesame seeds. The aim of this study was cloning, sequencing, and bioinformatics study of CYP81Q1 gene of Iranian sesame (Seamum indicum L.) cultivar.
Materials and Methods: In the present experimental research, DNA was extracted from leaves and stems of Karaj1 sesame cultivar and the target gene was amplified by PCR. Gene was cloned in binary vector pBI121 and confirmed by 3 methods, including enzymatic digestion, PCR, and sequencing. Then bioinformatics characterization of this gene was studied and the Ramachandran plot was drawn on the three-dimensional structure of the gene.
Findings: Cloning was confirmed. DNA sequencing results confirmed the cloned segment. Molecular weight and predicted isoelectric point of the protein were 57021.3 Dalton and 8.46, respectively. The three-dimensional structure of the protein had a good stroke chain. The sequencing result of this gene showed a difference in the 23 nucleotides of this gene in sesame seeds of Karaj 1 (access number KP771974.1) with a reported sequence in the NCBI gene bank (access number AB194714.1), which resulted in the sequencing of the CYP81Q1 gene in Iranian sesame (Karaj 1) at this database.
Conclusion: Based on nucleotide sequencing, the target gene has 1521 base pairs, and differs from 23 nucleotides with the sample registered at the NCBI World Bank. This gene encodes a protein length of 506 amino acids. This protein is very similar with the registered protein in NCBI.
H. Naghoosi, H. Ofoghi, Z. Amini-Bayat , N. Moazami,
Volume 10, Issue 1 (3-2019)
Abstract
Aims: 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.
Materials & Methods: 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.
Findings: 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.
Conclusion: 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.
H. Mamandi , B. Golestani Eimani , R. Pilehchian Langroudi ,
Volume 10, Issue 1 (3-2019)
Abstract
Clostridium perfrinjens is an anaerobics, Gram-positive, rod-shaped and heat resistant bacterium of genus clostridium. C. perfringens is a spore-forming bacterium and widely occurring pathogen. The organism is grouped into 5 types (A, B, C, D, and E) on the basis of the production of 4 major toxins alpha, beta, epsilon, and iota toxins. Tpel Clostridium perfringens (C. perfringens) toxin have identified with A, B, and C types by cytotoxin activity in recent years. In this study C. perfringens type B had been used. Tpel caused to intestinal disease especially intestinal infections in human and necrotic enteritis in birds. In this study, perfect genomic DNA extracted by phenol-chloroform and Polymerase Chain Reaction (PCR) method used to isolation Tpel gene by a couple exclusive primer of perfect bacterium genomic DNA. PCR product after joining to pTZ57RL/T vector by TA cloning method in E. coli strain TOP10 susceptible became cloned and then colony PCR method used to screening transforming bacterium colonies with recombinant plasmid. Presence of fragment close to 2469bp on 1% agarose gel indicated that Tpel gene in E. coli strain TOP10 have be cloned.
F. Vahdani, H. Ghafoori , S. Sarikhan,
Volume 10, Issue 2 (7-2019)
Abstract
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.
S. Shamriz, H. Ofoghi,
Volume 10, Issue 2 (7-2019)
Abstract
Microalgae are microscopic algae found in a wide range of habitats including freshwater and marine systems. Over the last decades, biotechnological processes based on microalgae have been receiving increasing interest due to their potential to produce large quantities of valuable products used as human food supplements, pharmaceuticals and animal feed. Microalgae have also been proved as an efficient and cost-effective platform for recombinant protein production. Most progress in this field has been achieved using Chlamydomonas reinhardtii, a photosynthetic unicellular alga which has been mostly considered as a model organism in different studies. High growth rate, ease of cultivation, well-established genetics and the ability to perform post-translational modifications are the most beneficial attributes that have made C. reinhardtii an attractive system for the expression of recombinant proteins. In this review, we focus on C. reinhardtii as a novel platform for the development of advanced recombinant products and compare them with other commonly used expression systems. We also present a comprehensive overview of its structure, life cycle, culture systems, and media in detail and then discuss the strategies for engineering its three different genomes to produce recombinant cells. Finally, algal culture collections in the world are introduced.
Volume 10, Issue 2 (4-2021)
Abstract
Piscidin has a wide range in killing microorganisms including bacteria, fungi, viruses, parasites and has strong anti-tumor activity and plays a role in increasing innate immunity and also does not provide resistance against bacteria; Therefore, it is of great importance in aquaculture. In this study, piscidin gene of Sparus aurata in vector pTZ57R / T was cloned. In this research, ligation product was transferred to component cell of E. coli DH5α strain. Plasmid extraction was performed from single colonies observed in ampicillin plate. Confirmation of the accuracy of single colonies grown in this research was performed by direct PCR and sequencing. The amplified cDNA fragment of the gilthead seabream piscidin gene consists of 310 nucleotides and 57 amino acids. The results of this research show that piscidin gene has been successfully cloned in pTZ57R / T vector. Comparison of nucleotide sequence of piscidin gene in this study showed high similarity with piscidin 5 of Morone chrysops. The comparison of the amino acid sequence of signal peptide piscidin is quite similar to Dicentracin-like of that species registered in the genebank, and mature peptide piscidin sequence is similar in only three amino acids to Pleorocidin-like of Poesila farmosa and Dicentracin-like of Sphaeramia orbicularis. This study could be a step towards further studies of piscidin peptide.
Volume 11, Issue 0 (10-2009)
Abstract
Objective: Leishmaniasis is caused by parasitic protozoa of the genus Leishmania which in the infected host is obligate intracellular parasite. LACK gene is conserved among related Leishmania species. LACK is the immuno-dominant antigen of L.major which is considered as the most promising molecule for a recombinant or DNA vaccine against leishmaniasis.
Materials and Methods: In this study the genomic DNA of an Iranian standard strain of Leishmania major (MRHO/IR/75/ER) was ertracted and the LACK gene was amplilified by PCR. Then the PCR product was cloned into pTZ57R/T cloning vector, The PT-LACK recombinant plasmid was extracted from transformed E.coli bacteria (TG1 strain) and sequenced.
Results: The LACK gene (Accession no LmjF28.2740) of MRHO/IR/75/ER & L. major was amplified using PCR method. LACK gene was cloned into pTZ57R/T coloning vector. Sequence analysis of the cloned LACK gene showed high homology 89% with LmjF28.2740 (LACK gene).
Conclusion: The LACK gene of L.major was cloned in pTZ57R/T vector successfully. Recombinant plasmid was confirmed and could be used for the production of recomiomort antigens is DNA vaccines, for further studies.
Volume 11, Issue 0 (10-2009)
Abstract
Objective: Toxoplasmosis, caused by an intracellular protozoan parasite, and the Toxoplasma gondii, is widespread throughout the world. In recent years, significant progress has been made in the identification of vaccine candidates which could induce a protective response.
GRA7, an excretory 29 kDa Toxoplasma gondii a dense granular antigen released by infected host cells. In tachyzoite-infected cells, p29 accumulates within the parasitophorous vacuole and co-localizes with its delimiting membrane.
Materials and Methods: In the present work, first genomic DNA of Toxoplasma gondii was extracted and used for amplifying of GRA7 gene as a template. Then PCR product was extracted from agarose gel and cloned into TOPO vector. The plasmid containing GRA7 gene was extracted from the transformed bacteria (TOP10 strain) and sequenced.
Results: Sequence analysis of GRA7 gene cloned into TOPO vector showed only one base difference when composed with the gene bank sequence for RH strain was only one base.
Conclusion: The results indicated that this clone is suitable for subcloning in Prokaryotic and Eukaryotic plasmid.
Mahsa Tirmomenin, Farangis Ataei, Saman Hosseinkhani,
Volume 11, Issue 3 (10-2020)
Abstract
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.
Volume 12, Issue 4 (10-2010)
Abstract
Objective: The secreted aspartic proteinases (Sap2) of Candida albicans has prominent role on Candida adherence, invasion, and pathogenicity. The aim of this study was cloning, expression and characterizing of Sap2 enzyme. Also in this study for the first time, the expression system P. pasturis was used for expressing the recombinant protein.
Materials and Methods: C. albicans Sap2 gene was amplified by PCR with sticky ends, EcoR1 and SacII, and it was subcloned into the T/A vector. The sequencing of this gene was done with universal primers and then the Sap2 gene was cloned into pGAPZαA expression vector. The construct was transformed into P. pasturis yeast; the Sap2 gene integration into the yeast genome was accomplished by the homologous recombination. The expressed protein was confirmed by western blotting using monoclonal antibody against Sap2 protein. Finally, the recombinant protein was purified by Ni-NTA chromatography column, and the activity of the enzyme was confirmed.
Results: In this study, we successfully amplified C.albicans Sap2 gene and subsequently integrated into the yeast pichia pasturis genome by homologous recombination. Moreover, we were able to identify a yeast clone secreting the recombinant protein. The optimum over expression of sap2 protein was obtained after 96 h, at 30ْ C.
Conclusion: Expression of Sap2 gene in P. pasturis, in comparison to bacterial expression system, leads to a high-level expression, and also need for post translation modifications, that might be required for the activity of enzyme, is obviated in the yeast system. Based on our results, the purified acid aspartyl proteinase purified from P. pasturis was capable of degrading BSA as a substrate in-vitro. The recombinant Sap2 protein had maximum activity in an acidic pH.
Zahra Aghaei Jeshvaghani, Ramin Hosseini,
Volume 13, Issue 1 (3-2022)
Abstract
Introduction: Proteases are the most important industrial enzymes. Bacillus bacteria are commonly used to produce these enzymes. The aim of this study was cloning, sequencing, expression and bioinformatics study of aprX serine protease gene extracted from Bacillus licheniformis.
Materials and Methods: In this study, after extraction of bacterial DNA, aprX serine protease genes was isolated from Bacillus licheniformis and cloned into pTG19-T vector and then subcloned in pET28a vector. The molecular structure, its biochemical and phylogenetic properties were investigated and three-dimensional structure of the cloned enzyme was predicted. For the induction of gene expression and protein production of the recombinant serine protease IPTG was used at different concentrations, different temperatures and different time periods. Confirmation of aprX gene expression was performed by SDS-PAGE and dot blot analysis. Then, the activity of recombinant protease enzyme was measured at different temperatures and pHs.
Results: Cloning was confirmed by sequencing. Based on the results of phylogenetic studies, the obtained protein sequence showed a high similarity to the sequences of other Bacillus species. After evaluating the drawn models, it was found that the models provided by RAPTROX and I-TASSER software were desirable models for predicting the three dimentional structure of this protease. The recombinant protein production was successfully induced by IPTG induction in the host containing the plasmid pET28a-aprX. The highest expression values were obtained at 25 ° C for 20 hours with 0/5 mM IPTG. Also, the recombinant protein produced showed the highest activity at 50 ° C and pH 8.
Volume 13, Issue 2 (4-2010)
Abstract
Objective: Helicobacter pylori is a widely distributed Gram negative bacterium that infects the human stomach and duodenum. Some antibiotic regimens are subjected to cure the infection but the cost of drugs, poor patient compliance and emerging of antibiotic-resistant strains are limiting the usefulness of these antibiotic therapies. Therefore, interest in developing a H. pylori vaccine is growing up rapidly. The aim of this study was to construct a recombinant vector containing fusion genes encoding a fragment of B subunit from Helicobacter pylori (H. pylori) urease (UreB332) and Helicobacter pylori adhesion A (HpaA) and expressed it in E. coli BL21, as well as determining its antigenicity as a vaccine candidate of H. pylori.
Materials and Methods: The target genes encoding UreB332 and HpaA amplified from standard H. pylori chromosome by PCR, digested by restricted endonuclease enzyme and inserted into the prokaryotic expression vector pET28a(+) which was digested by corresponding restricted endonuclease enzyme. The target fusion protein was expressed in the BL21 (DE3) E.coli. Furthermore, UreB332-HpaA antigenicity was studied by western blotting after Ni-NTA agarose resin purification.
Results: Enzyme digestion analysis, PCR and sequencing showed that the target genes were inserted correctly into the recombinant vector. The fusion protein UreB332-HpaA was recognized by the rabbit anti H. pylori polyclonal antibody and the human sera infected with H. pylori.
Conclusion: Our results in addition to favorable properties of HpaA and UreB antigens, support the application of rUreB332-HpaA fusion protein, as a good candidate for the development of H. pylori vaccine.
Roghaye Hamidi, Farangis Ataei, Saman Hosseinkhani,
Volume 13, Issue 3 (1-2023)
Abstract
Aims: 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.
Methods: 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.
Finding: 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.
Conclusion: 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.
Volume 14, Issue 2 (7-2010)
Abstract
In human history, if a detection or revolutionary idea has emerged, once cause anger Palladian system of its age. The question of human cloning is analogical with such statues. Perspective of making human by cloning technology has caused widespread solicitude among religious, ethic and law thinkers.
Cloning is the birth of chrysalis homogeneous with original by non- sexual way. The main subject of this paper is the argument of parentage in human cloning. Yet two view points have been proposed about parentage in human cloning by current jurists. The first view point states that there is no parentage in human cloning because of non-normal zygosis. The second view point confirms the existence of parentage in human cloning, because of traditional understanding. They believe that if cell padrone was male, it is the father of the cloned child and if cell padrone was female, it is the mother. Our point in this paper is the firstly there is parentage in human cloning and secondly the father and mother of cell padrone are the father and mother of the cloned child. In other word, cell padrone and child are twins.
