فهرست مطالب

Biotechnology - Volume:14 Issue:4, 2017
  • Volume:14 Issue:4, 2017
  • تاریخ انتشار: 1395/10/20
  • تعداد عناوین: 9
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  • Kamran Mousavi Hosseini *, Mehran Ghasemzadeh Pages 213-220
    Context: The major motivation for the preparation of the plasma derived biological medicine was the treatment of casualties from the Second World War. Due to the high expenses for preparation of plasma derived products, achievement of self-sufficiency in human plasma biotechnological industry is an important goal for developing countries.
    Evidence Acquisition: The complexity of the blood plasma was first revealed by the Nobel Prize laureate, Arne Tiselius and Theodor Svedberg, which resulted in the identification of thousands of plasma proteins. Among all these proteins, four of which are commercially important for production due to significant need of patients. These four products are: albumin, IgG, factor VIII, and Factor IX.
    The starting material for the production of biological drugs from plasma is natural which is different from synthetic starting material. So, the quality of plasma as starting material plays an important role in the quality of final product.
    Introducing new techniques for preparation of the biological drugs from human plasma has resulted in the improvements in purity of products, higher safety, and yield noticeably. Still, the backbone of the modern plasma fractionation technique is mainly based on cold ethanol fractionation of the human plasma that is almost the same as fractionation of crude oil, breaking it down into its components.
    The demand for IgG for treating immune deficiencies and coagulation factor VIII for hemophilia A determines how to design the plasma fractionation industry in terms of capacity.
    Nowadays, cold ethanol fractionation has followed by chromatographic methods, since they offer higher purity. In this review, we describe different methods of plasma fractionation such as cold ethanol fractionation, gel filtration, fractionation by salt, and fractionation by polyethylene glycol. There is no doubt that the four main products of human plasma are albumin, IgG, coagulation factor VIII, and IX, which their methods of separation from human plasma have been explained in this paper.
    Conclusions
    It can be concluded that plasma fractionation with ethanol at low temperature for the preparation of the main human plasma biological components including albumin, IgG, coagulation factors VIII, and IX is still the most widely used method at an industrial scale. Nowadays, this method is being used in combination with different chromatographic techniques in order to achieve a higher quality and the yield.
    Keywords: Human plasma, Proteins, IgG, Coagulation factors, Fractionation
  • Nihan, Ouml, Zengin, AyŞ, E. Elmaci* Pages 221-229
    Background
    There is growing interest in the natural and constructed wetlands for wastewater treatment. While nutrient removal in wetlands has been extensively investigated, information regarding the degradation of the pharmaceuticals and personal care products (PPCPs) has only recently been emerging. PPCPs are widely distributed in urban wastewaters and can be removed to some extent by the constructed wetlands. The medium-term (3-5 years) behavior of these systems regarding PPCP removal is still unknown.
    Objectives
    The efficiency of a Leca-based laboratory-scale constructed wetland planted with Phragmites australis (Cav.) Trin. Ex. Steudel in treating an aqueous solution of the pharmaceuticals, namely, carbamazepine, ibuprofen, and sulfadiazine, was to investigate.
    Materials And Methods
    The two pilot-scale constructed wetlands (CW) were operated in parallel; one as an experimental unit (a planted reactor with P. australis) and the other as a control (an unplanted reactor with Leca). Pretreatment and analyses of the carbamazepine, ibuprofen, sulfadiazine, and tissue samples (Leca, P. australis body and P.australis leaf) were conducted using HPLC.
    Results
    The carbamazepine, ibuprofen, and sulfadiazine removal efficiencies for the planted and unplanted reactors were 89.23% and 95.94%, 89.50% and 94.73%, and 67.20% and 93.68%, respectively. The Leca bed permitted an efficient removal. Leca has a high sorption capacity for these pharmaceuticals, with removal efficiencies of 93.68-95.94% in the unplanted reactors.
    Conclusions
    Sorption processes might be of a major importance in achieving efficient treatment of wastewater, particularly in the removal of organic material that are resistant to biodegradation, in which case the materials composing the support matrix may play an important role. The results obtained in the present study indicate that a constructed wetland with Leca as a substrate and planted with P. australis is effective in the treatment of wastewater contaminated with carbamazepine, ibuprofen, and sulfadiazine.
    Keywords: Carbamazepine, Constructed Wetland, Ibuprofen Leca, Phragmites australis, Sulfadiazine
  • Masoud Reza Seyfi Abad Shapori *, Pezhman Mahmoodi, Dariush Gharibi, Masoud Ghorbanpoor, Sara Yaghoubi, Elham Rezaei, Mohammad Rashno, Neda Mehravar Pages 230-235
    Background
    Staphylococcal protein A (SPA) is a cell wall component of Staphylococcus aureus that binds to different IgG subclasses of human and several animal species. This bacterial protein can be used as an antibody detector in various immunological assays or as an isolation reagent for the purification of antibody molecules via immuno-chromatography procedures.
    Objectives
    Molecular cloning and expression of SPA followed by the purification and conjugation of the recombinant protein to peroxidase enzyme.
    Material and
    Methods
    Encoding DNA fragment of SPA was amplified and inserted into a prokaryotic plasmid vector for the expression of recombinant SPA fused to a maltose binding protein (MBP). The recombinant protein was purified using amylose resin column chromatography and conjugated to horseradish peroxidase (HRP) enzyme. Finally, the reactivity of the recombinant SPA was examined against human IgG molecules in ELISA.
    Results
    The results indicated that the recombinant peroxidase-conjugated SPA has a good recognition capacity for human IgG molecules and it was able to produce significant OD values after reacting with human IgG molecules at a concentration up to 0.06 mg.well-1.
    Conclusions
    This recombinant protein can be very useful in all research laboratories and may decrease some of the expenses, e.g. those for preparing conjugated anti-antibodies.
    Keywords: Horseradish peroxidase, Staphylococcal protein A, SPA, Staphylococcus aureus
  • Yongmin Yan, Wenqian Jiang, Jingwen Liu, Hui Qian *, Yan Xu Pages 236-242
    Background
    Phosphodiesterase 3A (PDE3A) and phosphodiesterase 3B (PDE3B) play a critical role in the regulation of intracellular level of adenosine 3´,5´-cyclic monophosphate (cyclic AMP, cAMP) and guanosine 3´,5´-cyclic monophosphate (cyclic GMP, cGMP). Subsequently PDE3 inhibitors have shown to relax vascular and inhibit platelet aggregation in cardiovascular disease.
    Objectives
    In this study, our aim was to establish a method of expression for the recombinant human PDE3A and PDE3B proteins in insect cells using baculovirus expression system in order to investigate the activity of the expressed PDE3A and PDE3B proteins.
    Materials And Methods
    The full length human PDE3A and PDE3B cDNA were cloned into recombinant baculovirus and transfected into the SF9 insect cells. Recombinant proteins were collected at 48 h, 60 h, 72 h, and 84 h post transfection. Transfection of recombinant baculovirus was verified by the morphological changes of the SF9 cells. Expression of human PDE3A and PDE3B was detected by using RT-PCR and western blot, respectively. The 125I RIA method was used to determine the level of adenosine 3´,5´-cyclic monophosphate cAMP and cGMP, correspondingly. The activity of the expressed PDE3A and PDE3B proteins were investigated by cAMP and cGMP dsgradation with or without addition of milrinone, a potent and selective PDE inhibitor.
    Results
    Recombinant human PDE3A and PDE3B proteins were stably expressed in SF9 cells and could be detected by distinct morphological changes in the SF9 cells, RT-PCR, and western blot at 48 h post-transfection. The molecular weights of the recombinant PDE3A and PDE3B molecular weights proteins were about 120 KDa and 135 KDa, respectively. Results of 125I RIA assay showed that the levels of cAMP and cGMP were significantly decreased after incubation with the expressed PDE3A and PDE3B proteins. Furthermore, degradation of cAMP and cGMP through the activity of PDE3A and PDE3B was suppressed following to the addition of milrinone.
    Conclusions
    Recombinant human PDE3A and PDE3B could be expressed in SF9 cells using baculovirus expression system, and thus provides the basic material for studying human PDE3A and PDE3B activity.
    Keywords: Baculovirus Expression System, PDE3A, PDE3B, SF9 cells
  • Warawan Eiamphungporn *, Sakda Yainoy, Virapong Prachayasittikul Pages 243-249
    Background
    Human Cu/Zn superoxide dismutase (hSOD1) is an antioxidant enzyme with potential as a therapeutic agent. However, heterologous expression of hSOD1 has remained an issue due to Cu2 insufficiency at protein active site, leading to low solubility and enzymatic activity.
    Objectives
    The effect of co-expressed human copper chaperone (hCCS) to enhance the solubility and enzymatic activity of hSOD1 in E. coli was investigated in the presence and absence of Cu2.
    Materials And Methods
    pETDuet-1-hSOD1 and pETDuet-1-hCCS-hSOD1 were constructed and individually transformed into E. coli strain BL21(DE3). The recombinant hSOD1 was expressed and purified using immobilized metal affinity chromatography. The yield and specific activity of hSOD1 in all conditions were studied.
    Results
    Co-expression with hCCS increased hSOD1 solubility at 37°C, but this effect was not observed at 25°C. Notably, the specific activity of hSOD1 was enhanced by 1.5 fold and greater than 3 fold when co-expressed with hCCS at 25°C with and without Cu2 supplement, respectively. However, the chaperone co-expression did not significantly increase the yield of hSOD1 comparable to the expression of hSOD1 alone.
    Conclusions
    This study is the first report demonstrating a potential use of hCCS for heterologous production of hSOD1 with high enzymatic activity.
    Keywords: Cu, Zn superoxide dismutase, Co-expression, Human copper chaperone
  • Kamal Abdellatif, Ragaa Abdelfattah Hamouda, Mostafa Sayed Mostafa El Ansary Pages 250-259
    Background
    Root-knot nematodes are known to cause significant damage to eggplants. New approaches by green silver nanoparticles (GSN) are used to control plant-parasitic nematode to avoid chemical nematicide hazards.
    Objectives
    Analyses of the incorporation of different concentrations of nanoparticles on two different algae (Ulva lactuca and Turbinaria turbinata) were carried out. Furethermore, the effect of GSN on the eggplant DNA profile was studied using RAPD and EST molecular markers.
    Materials And Methods
    Green Silver Nanoparticles (GSN) have been synthesized and characterized using the algal extract solution prepared from two algal genera. Nematicidal effect of the GSN was evaluated in greenhouse on eggplants (Solanum melongena cv. Login). Genomic DNA was extracted for use in molecular analysis. Both RAPD and EST molecular markers were used to study the GSN effect on eggplant DNA modification.
    Results
    GSN (17 mg.mL-1) obtained from U. lactuca was more effective in reducing second-stage juveniles (J2s) of M. javanica (69.44%) population in soil. All treatments improved eggplants growth parameters. Change in DNA profile using of both RAPD and EST markers was noted.
    Conclusions
    GSN (12.75 mg.100 mL-1) were effective on controlling the root-knot nematode (both T. turbinataand and U. lactuca algae), similar to chemical control in eggplants. GSN did not cause any phototoxicity in eggplants under treatment.
    Keywords: Eggplant, EST, Green Silver Nanoparticles, Molecular markers, Root, Knot Nematode, RAPD
  • Biocontrol Activities of Gamma Induced Mutants of Trichoderma harzianum against some Soilborne Fungal Pathogens and their DNA Fingerprinting
    Sakineh Abbasi, Naser Safaie*, Masoud Shams Bakhsh, Samira Shahbazi Pages 260-269
    Background
    Random induced mutation by gamma radiation is one of the genetic manipulation strategies to improve the antagonistic ability of biocontrol agents.
    Objectives
    This study aimed to induce mutants with more sporulation, colonization rate leading to enhanced antagonistic ability (in vitro assay) comparing to wild type (WT) and the assessment of genetic differences (in situ evaluation) using molecular markers. The superior mutants could be appropriate biocontrol agents against soil borne fungal diseases.
    Materials And Methods
    In this research sampling and isolation of Trichoderma isolates were performed from soils with low incidence of soil borne disease. T. harzianum 65 was selected and irradiation was conducted with gammacell at optimal dose 250 Gray/s. Mutants (115) were obtained from the WT. The antagonistic abilities of twenty-four mutants were evaluated using dual culture and culture filtrate tests.
    Results
    The results of in vitro assays revealed that Th15, Th11 and Th1 mutants exhibited stronger growth inhibition (GI) and colonization rate on Macrophomina phaseolina and Rhizoctonia solani AG4 compared to the wild type. Th15 and Th11 mutants exhibited stronger GI and colonization rate on Sclerotinia sclerotiorum in dual culture and culture filtrate tests and Th1 and Th11 mutants exhibited stronger GI on Fusarium grminearum in culture filtrate test.
    The DNA fingerprinting was carried out using RAPD and rep-PCR markers. Two (Th9 and Th17) out of the 24 mutants categorized distantly from the rest based on different polymorphism obtained by molecular markers. However, Th9 was different in GI% from Th17. RAPD analysis separated WT from mutants, Th9 from Th17 and also phenotypically superior mutants from other mutants. Meanwhile, rep-PCR analysis categorized WT isolate and mutants according to their antagonistic properties.
    Conclusions
    The latter marker (rep-PCR) appeared to be reproducible and simple to distinguish mutants from a single isolate of T. harzianum. Mutants (3 isolates) were phenotypically and genotypically distinct from WT. These mutants demonstrated a pronounced biocontrol activities against soilborne fungal phytopathogens.
    Keywords: Enhancement of antagonistic properties, Random Mutagenesis, RAPD, rep-PCR
  • Majid Talebi *, Haleh Salimi, Masoud Bahar, Aghafakhr Mirlohi Pages 270-277
    Background
    Potato has a narrow genetic base which is due to its development, as it takes its genetic root from a few genotypes originated from South America.
    Objectives
    The objective of this study was to assess the genetic relationships among potato (Solanum tuberosum L.) genotypes originated from different geographical regions.
    Materials And Methods
    This study has rendered 25 useful SSRs and EST-SSRs that were located in pre-existing genetic maps, fingerprinted in a collection of the 47 potato genotypes from America, Europe and Iran.
    Results
    The number of alleles per locus ranged from 2 to 9 with an average of 6.22 alleles per locus. UPGMA dendrogram, constructed from microsatellite data based on Jaccard similarity coefficient slightly clustered the American and European potatoes according to their geographical distribution. Iranian genotype, “Istanbuli”, joined to a group with American genotype. The results indicated that American genotypes show the highest expected heterozygosity compared to the European genotype. This result was expected due to the narrow genetic base of European potatoes considering their origin from a limited number of introductions.
    Conclusions
    It could be concluded that SSR is an appropriate marker for evaluating genetic diversity within and among potatoes from different geographical regions.
    Keywords: EST, SSR, Genetic diversity, Microsatellite, Potato
  • Salim Morammazi, Ali Akbar Masoudi *, Rasoul Vaez Torshizi, Abbas Pakdel Pages 278-285
    Background
    Milk proteins genes have been the focus of the researches as the candidate target genes that play a decisive role when animal breeding is desired.
    Objectives
    In the present study, the transcriptional levels of Beta-lactoglobulin (BLG) and Alpha S1 casein (CSN1S1) genes were investigated during prenatal, milking and drying times in mammary glands of the Adani goats which showed high and low breeding values.
    Materials And Methods
    The breeding values of the animals were estimated first by applying multi-trait random regression model. Using the biopsy gun, the mammary gland samples were taken and real-time PCR was applied to search the expression of the genes. Fixed factors of the model were the breeding value groups, sampling times and their interactions.
    Results
    The interactions were significant for both genes. At milking time, the high breeding value group exhibited more transcriptional levels for BLG and less transcriptional levels for CSN1S1 gene compared with the low breeding value group. The expression patterns of these genes were also different between the two breeding value groups. The maximum level of BLG and CSN1S1 transcriptions were found to occur at drying time.
    Conclusions
    A difference in the gene expression was observed between the two groups which indicate the change in the nucleotide sequence for transcription factor binding sites, or miRNA binding sites, otherwise in the coding regions. Therefore, the variations in the coding and promoter regions of this gene should be investigated in the further studies.
    Keywords: Adani goats, Alpha S1 casein, Beta-lactoglobulin, Gene expression, Mammary gland, Milk yield breeding value