فهرست مطالب

Biotechnology - Volume:16 Issue:4, 2018
  • Volume:16 Issue:4, 2018
  • تاریخ انتشار: 1397/09/17
  • تعداد عناوین: 9
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  • Dianoush Falahatgar , Safar Farajnia *, Nosratollah Zarghami, Asghar Tanomand, Shiva Ahdi Khosroshahi, Bahman Akbari, Hadi Farajnia Pages 241-247
    Background
    Epidermal growth factor receptor (EGFR) plays an important role in the progression and tumorigenesis of the various cancers. In this regards, anti-EGFR antibodies are valuable approved therapeutics for the EGFR over-expressing cancers. However, the occurrence of mutations in the EGFR and/or KRAS genes; a common phenomenon which is seen in many cancers, lead to the resistance to the EGFR-directed antibodies. EGFR based immunotoxins are capable of overcoming this limitation by directing the toxin moieties to the cancer cells resulting in cell death.
    Objectives
    In the present study, a novel immunotoxin consisting of the truncated Pseudomonas exotoxin A (PE-40) and anti-EGFR huscFv was developed and evaluated for the induction of cell death in EGFR positive A431tumoral cells.
    Materials and Methods
    PE-40 fragment of the exotoxin A was amplified by using PCR and ligated to pET22b-huscFv. The reaction was confirmed by PCR and restriction digestion. The immunotoxin was expressed in E. coli BL21 (plysS) and then was purified by Ni-NTA affinity column. Subsequently, the toxicity of the purified immunotoxin was evaluated on EGFR over-expressing epidermoid carcinoma of skin, A431 cell line.
    Results
    PCR and restriction digestion experiments have verified the integrity of the immunotoxin construct. Purification by affinity column resulted in a highly purified recombinant immunotoxin. MTT assay revealed the growth inhibitory effect of the huscFv-PE40 immunotoxin on EGFR-over-expressing A431 cells with an IC50 value of 250 ng.mL-1.
    Conclusion
    In conclusion, the results indicated that the immunotoxin developed in this study has a high toxicity on the EGFR-over-expressing tumor cells and could be considered as a promising candidate for the treatment of the EGFR positive cancers.
    Keywords: Cancer target therapy, EGFR, HuscFv, Immunotoxin, Pseudomonas exotoxin A
  • Mohammad Javad Motamedi, Shahla Shahsavandi, Jafar Amani, Rouhollah Kazemi, Somayeh Takrim, Mahyat Jafari, Ali Hataf Salmanian * Pages 248-257
    Background
    Newcastle disease virus (NDV) is a dangerous viral disease, infecting a broad range of birds, and has a fatal effect on the poultry industries. The attachment and consequently fusion of the virus to the host cell membrane is directed by the two superficial glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion (F) which is considered as the important targets for the poultry immune response.
    Objectives
    The principal goal of this investigation was to realize the potential efficacy of the E. coli expression system for the production of the multi-epitopic HN, and F proteins with respect to the ability for the stimulation of the immune system and production of the cross-reactive antibodies in mice.
    Materials and Methods
    The recombinant HN and F (rHN, rF) have accumulated almost 40% of the total bacterial proteins. The presence of rHN and rF proteins recognized by the Western blotting with specific anti-HN, anti-F, anti-Newcastle B1, and anti-poly 6x His-tag antibodies. Furthermore, both rHN and rF have shown the specific reactivity against the Newcastle B1 antiserum as a standard strain.
    Results
    The ELISA analysis showed that the higher dilutions of the antibody against Newcastle B1 could react with the as least quantity as 100 ng of the purified rHN, and rF. Cross-reactivity analysis of the sera from the mice immunized with Newcastle B1 in two time points indicated that the raise of anti-Newcastle B1, anti-HN and anti-F antibodies peaked at 28 days post immunization (dpi). Moreover, temporal variation in IgG titration between both time points was significant at 5% probability level.
    Conclusion
    The results provided valuable information about the cross-reactivity patterns and biological activity of the multi-epitopic proteins compared to the NDV standard strain which was determined by the Western blotting and ELISA.
    Keywords: Affinity purification, Fusion, Hemagglutinin-neuraminidase, Immunization, Newcastle disease virus
  • Nahid Najafikhah, Sameereh Hashemi, Najafabadi *, Saleh Zahediasl, Mohammad Mahdi Nabavimanesh, Khadijeh Farrokhfall Pages 258-263
    Background
    Pancreatic islet transplantation is one of the most promising strategies for treating patients with type I diabetes mellitus.
    Objective
    We aimed to assess the immunoisolation properties of the multilayer encapsulated islets using alginate-chitosan-PEG for immunoprotection and insulin secretion from the encapsulated islets induced under different glucose concentrations in vitro.
    Materials and Methods
    In this study, the islets were isolated from Wistar rats. The biological function (insulin secretion) of the immunoisolated islets following to PEGylation and encapsulation in the alginate-chitosan-PEG, separately, in addition to their immuno-protection in a co-culturing with the lymphocytes isolated from the male C57BL/6 mice were investigated, respectively.
    Results
    Alginate-chitosan-PEG decreased IL-2 secretion from the lymphocytes co-cultured with islets. Also, insulin secretion from the encapsulated and PEGylated groups was stimulated by glucose (i.e., 5.6 and 16.7 mM of glucose, respectively); showed insulin secretion similar to the naked islets, without coating, after 30 and 60 min of incubation.
    Conclusion
    In conclusion, encapsulation and PEGylation have no negative effect on the insulin secretion and glucose sensitivity of the islets for all of the groups. Also, encapsulation decreased IL-2 secretion from the lymphocytes.
    Keywords: Alginate, Type I diabetes mellitusAlginate, Chitosan, Type I diabetes mellitus, Islets of Langerhans, Insulin
  • Hossein Javid *, Suthep Wiyakrutta Pages 264-272
    Background
    D-Phenylglycine aminotransferase (D-PhgAT) is highly beneficial in pharmaceutical biotechnology. Like many other enzymes, D-PhgAT suffers from low stability under harsh processing conditions, poor solubility of substrate, products and occasional microbial contamination. Incorporation of miscible organic solvents into the enzyme’s reaction is considered as a solution for these problems; however, native D-PhgAT is not significantly stable in such solvents.
    Objective
    Halophiles are known to survive and withstand unsavory habitats owing to their proteome bios. In the current study, with an eye on further industrial applications, we examined the performance and thermostability of four halophilic peptides fused D-PhgAT variants in reaction mixtures of various proportions of different miscible organic solvents and various temperatures as well as desiccation.
    Materials and Methods
    Plasmid constructs from the previous study (Two alpha helixes and loops between them from Halobacterium salinarum ferredoxin enzyme fused at N-terminus domain of D-PhgAT) expressed in Escherichia coli and then D-PhgAT purified. Purified proteins were subjected to various proportions of miscible organic solvents, different temperatures, and desiccation and then performance and thermostability monitored.
    Results
    Study confirmed increased C50 of all halophilic fused D-PhgAT variants, where the highest C50 observed for ALAL-D-PhgAT (30.20±2.84 %V/V). Additionally, all halophilic fused variants showed higher thermostability than the wild-type D-PhgAT in the presence of different fractions of acetone, N,N-Dimethylformamide and isopropanol in aqueous binary media, while zero activity observed at the presence of methanol.
    Conclusion
    Our results suggest that applying this new technique could be invaluable for making enzymes durable in discordant industrial conditions.
    Keywords: D-Phenylglycine aminotransferase, StabilityD-Phenylglycine aminotransferase, Halophilic peptide fusion, Miscible organic solvents, Stability
  • Aditi Kundu, Bijan Majumdar * Pages 273-278
    Background
    The extracellular xylanase secreted by microorganisms is a hydrolytic enzyme, which arbitrarily cleaves the β-1, 4 backbone of the polysaccharide xylan; an enzyme used in the food processing, bio-pulping and bio-bleaching. The commercial production of the xylanase is limited because of a higher cost involvement, which can be overcome by the cost-effective production of the xylanase through immobilization of the microbial cell by the non-toxic substances.
    Objectives
    In this work, the optimization of the extra-cellular cellulase free xylanase production by the immobilized cell of the Bacillus pumilus IMAU80221 strain using Ca-alginate beads along with standardization of the various parameters for a higher xylanase production were studied.
    Materials and Methods
    Following to sterilization, the Na-alginate solution was mixed with the bacterial suspension of the Bacillus pumilus IMAU80221 and was added drop by drop into the 1 M calcium chloride solution for 1 h for obtaining a uniform sized polymeric bead of the Ca-alginate. For xylanase production, the Ca-alginate beads were then transferred into 100 mL Erlenmeyer flasks with 20 mL of the culture medium containing (w/v) 0.02% NaCl, 0.02% MgSO4, 0.04% (KH4)2PO4, 0.1% peptone, and 0.5% xylan and incubated at 34 °C in an incubator shaker (150 rpm) for 24 h. The resultant supernatant (crude enzyme) was used for enzyme assay.
    Results
    The maximum xylanase production by the free cell (1.9 U.mL-1.min-1) was recorded at 48 h which was 40.5% lower than the xylanase production by the immobilized cell (2.67 U.mL-1.min-1) at the same time. The beads containing the immobilized cells could be reused up to eight fermentation cycles for xylanase production and retained 83.5% of the productivity at the fourth cycle. The entrapped cells were stable after six months of storage at 4 °C and retained 68% of the xylanase productivity.
    Conclusion
    Cellulase free xylanase production from the immobilized Bacillus pumilus IMAU80221 was optimized. The xylanase production by the immobilized cells of Bacillus pumilus was higher by 40.5 and 132.6 % over the free cells respectively after 48 and 72 h of the incubation.
    Keywords: Bacillus pumilus, Cellulase free xylanase, Calcium alginate, Immobilization
  • Leila Firouzi Dalvand, Farzaneh Hosseini *, Shahram Moradi Dehaghi, Elham Siasi Torbati Pages 279-286
    Background
    Based on the increase in antibiotic-resistant pathogens, it is necessary to have various effective compounds, so as to prevent the proliferation of these pathogens. For this purpose, nano-materials such as bismuth oxide nanoparticles can be used.
    Objectives
    The aim of this study was to produce bismuth oxide nanoparticles by Bacillus licheniformis PTCC1320 and to determine the antimicrobial effects on methicillin-resistant Staphylococcus aureus species compared with some antibiotics.
    Materials and Methods
    In this study, 200 bacterial samples were collected from hospitalized patients with burn infections from the Burn Rescue Hospital in Tehran. Thereafter, 65 strains of methicillin-resistant Staphylococcus aureus were identified by their phenotype and genotype. A total of 92% of strains with the highest resistance to antibiotics were isolated. Bismuth oxide nanoparticles were synthesized by Bacillus licheniformis PTCC1320. FTIR spectroscopy, X-ray diffraction, and scanning electron microscopy (SEM) were used to analyze the extracellularly produced nanoparticles. Finally, the antibacterial properties of nanoparticles produced on the biofilm of some pathogens were examined.
    Results
    In the present study, cube-shaped bismuth oxide nanoparticles were formed in the size range of 29-62 nm. They were found to have antimicrobial activity on 16% of the strains. The FTIR results showed the vibrational frequencies of bismuth oxide at 583, 680, 737, and 1630 nm. The XRD results also confirmed the structure of nanoparticles. Compared with antibiotics such as Ciprofloxacin, bismuth oxide nanoparticles had less affectivity on this resistant hospital pathogen. Increasing the concentration of bismuth oxide nanoparticles, increased its antimicrobial effect and decreased bacterial growth rate.
    Conclusion
    Compared with heavy metals, bismuth nanoparticles have very low toxic effects. Considering this feature, the use of less antibiotics can be achieved with bismuth nanoparticles in the treatment of infections, thereby reducing antibiotic resistance.
    Keywords: Bacillus licheniformis, Methicillin-Resistance Staphylococcus aureus, Nanoparticles
  • Banafsheh Douzandeh, Mobarrez, Mehdi Ansari, Dogaheh, Touba Eslaminejad, Maryam Kazemipour, Mojtaba Shakibaie* Pages 287-293
    Background
    Magnetic nanoparticles (MNPs) loaded by various active compounds can be used for targeted drug delivery.
    Objectives
    In the present study, the Fe3O4 magnetic nanoparticles that contained gentamicin were prepared and their antibacterial activities were studied.
    Materials and Methods
    MNPs containing gentamicin (G@SA-MNPs) were prepared using sodium alginate (SA) as a surface modifier. After and before coating, the prepared MNPs were characterized using transmission electron microscopy (TEM), X-ray diffraction spectroscopy (XRD), Fourier transform infrared spectroscopy (FTIR), and vibrating sample magnetometer (VSM). Finally, the antibacterial effect of the MNPs was investigated by a conventional serial agar dilution method.
    Results
    Particle size distribution analysis showed that the size of MNPs, before and after coating, was in the range of 1-18 nm and 12-40 nm, respectively. The magnetization curve of G@SA-MNPs (with saturation magnetization of 27.9 emu.g-1) confirmed ferromagnetic property. Loading gentamicin on the surface of MNPs was qualitatively verified by FTIR spectrum. Quantitative analysis measurements indicated the gentamicin loading on SA-MNPs as 56.7 ± 5.4%. The measured MICs of G@SA-MNPs for Pseudomonas aeruginosa (PTTC 1574) was 1.28 μg.mL-1. The sub-MIC (0.64 μg.mL-1) concentration of G@SA-MNPs in nutrient broth could successfully inhibit the growth of P. aeruginosa for 14 hours.
    Conclusions
    Loading gentamicin on the SA-MNPs exhibited reasonable antibacterial effects against P. aeruginosa.
    Keywords: Antibacterial activities, Fe3O4, Gentamicin, Magnetic nanoparticles
  • Surya Sukumaran, Haridas Madhathilkovilakathu * Pages 294-302
    Background
    The ethno-medical significance of Clerodendrum genus raises the interest towards the characterization of its seed lectin by inexpensive and most effective technique.
    Objective
    The focus of this study is the purification, characterization, and evaluation of the antioxidant and antiproliferative potential of a galactose-specific lectin from Clerodendrum infortunatum L. seeds.
    Materials and Methods
    The crude extract, homogenized in 6 volumes of the saline containing 10 mM β-mercaptoethanol was subjected to pigment removal by Toyopeal HW-55 column prior to ammonium sulfate fractionation (40-80 %).The crude protein extract was then loaded to the gel filtration column Sephadex G-200 followed by affinity chromatography using activated galactose coupled Sepharose-4B.
    Results
    The SDS-PAGE analysis showed a single band of about 30 kDa which further determined by MALDI-TOF analysis. The MALDI-TOF spectra revealed that Clerodendrum infortunatum lectin (CIL) is a homo-tetramer of 120 kDa consisting of four identical subunits of 30 kDa. The haemagglutination inhibition assay was done with purified lectin by many sugars, among which N-acetyl-D-galactosmine (NAG), D-galactose and lactose exhibited high inhibition. NAG showed the highest inhibition amongst the tested sugars, having the minimum inhibitory concentration of about 0.97 mM. The lectin exhibited a moderate antioxidant activity with an IC50 value of 6.1 ± 0.1 mg.mL-1 and induced cell death with IC50 of 82.8 μg.mL-1 against human gastric cancer cell line, AGS, indicated the potential of CIL for clinical and therapeutic applications.
    Conclusion
    The present study demonstrated the moderate ability of the CIL to inhibit the growth of human gastric cancer cells, AGS either by causing cytotoxic or anti-proliferative effects. Thus, CIL due to its remarkable properties may be considered as a potential bio-molecule in tumor research and glycobiology.
    Keywords: Anti proliferative, Clerodendrum. infortunatum Lectin, Glycoproteins, Haemagglutination, Lectins
  • Naeimeh Shibaei, Jafar Majidi *, Nemat Sokhandan Bashir, Ali Asghar Karkhaneh, Khadijeh Razavi Pages 303-310
    Background
    Expression of virus coat protein (CP) in Escherichia coli often leads to production of partially folded aggregated proteins which are called inclusion bodies. Grapevine fanleaf virus (GFLV) is one of the most serious and widespread grapevine virus diseases around the world and in Iran.
    Objective
    The main objective of this study was to find a simple and brief method for producing polyclonal antibodies (PAbs) to be used for immunodiagnosis of GFLV. Material and Methods: An antigenic determinant in GFLV CP gene was inserted into pET-28a bacterial expression vector and the construct (pET-28a CP42) was cloned into E. coli strain BL21 (DE3).
    The recombinant coat protein of GFLV (CP42) was expressed and characterized by SDS-PAGE and western blot analysis using commercial anti-GFLV antibody. Expression of the CP was detected in the form of inclusion bodies in insoluble cytoplasmic fraction. Then, the inclusion bodies were isolated from the bacterial cells and injected into rabbits for PAbs production. The reaction of the antiserum was checked by ELISA assay. In order to analyze efficiency of the produced PAbs, first the infected and uninfected grapevine samples were confirmed based on morphological symptoms then the indirect plate- trapped antigen Enzyme-linked Immunosorbent Assay (IPTA-ELISA) was applied using the commercial anti GFLV antibody. In the next ELISA assay, efficiency of the raised polyclonal antibody was compared with commercial one.
    Results
    The expression of recombinant CP42 induced by IPTG was confirmed by the band of 42 kDa in SDS-PAGE and western blot. The antiserum of purified inclusion body immunized rabbit was reacted with CP42 and GFLV infected Grapevine samples. The results revealed an acceptable efficacy for prepared antibodies compared to that of commercial antibody.
    Conclusions
    It was evident that the recombinant coat protein in the form of inclusion bodies can be prepared and used as the antigen for immunizing animals in order to produce PAbs.
    Keywords: Polyclonal antibodies, inclusion body, recombinant coat protein, GFLV, Enzyme-Link Immunosorbent Assay (ELISA)