Iranian Journal of Biotechnology
Volume:13 Issue: 2, Spring 2015

Tumor associated antigens can be viably used to enhance host immune response. The immunomodulatory effect of biogenic selenium nanoparticles (SeNPs) was compared between treated and untreated mice with crude antigens of 4T1 cells. Female inbred BALB/c mice (60) were injected by cancinogenic 4T1 cells causing breast cancer. After 10 days, all tumor bearing mice were divided into 4 groups. Group 1 was daily provided oral PBS and injected by the same buffer after tumor induction and was considered as control. Group 2 received only 100 mg/day SeNPs as an oral supplement for 30 days. Group 3 was only injected with 4T1 cells crude antigens with nil supplementation of SeNPs. Group 4 animals were supplemented 100 mg/day SeNPs for 30 days and simultaneously injected with crude antigens of 4T1 cells. All antigens or PBS injections were introduced at 7, 14 and 28 days following tumor induction. Oral PBS and SeNPs supplementation initiated from the first day of tumor induction and continued up to 30 days. During tumor growth, animal weights and survival rates were monitored and at the end of the study the concentrations of different cytokines and DTH responses were measured. Data clearly showed that the levels of cellular immunomodulatory components (granzyme B, IL-12, IFN-g, and IL-2) significantly increased (P < 0.05) in mice treated with both SeNPs and crude antigens of 4T1 cells in comparison to the other groups. In contrast, the levels of TGF-b in these mice decreased. Although SeNPs showed a noticeable boosting effect for the immune response in mice bearing tumor exposed to crude antigens of 4T1 cells, further complementary studies seem to be inevitable.

Follicle stimulating hormone (FSH) plays an essential role in reproductive physiology and follicular development. A new variant of the equine fsh (efsh) gene was cloned, sequenced, and expressed in Pichia pastoris (P. pastoris) GS115 yeast expression system. The full-length cDNAs of the efshα and efshβ chains were amplified by reverse transcription polymerase chain reaction (RT-PCR) using the total RNA isolated from an Iranian Turkmen-thoroughbred horse's anterior pituitary gland. The amplified efsh chains were cloned into the pPIC9 vector and transferred into P. pastoris. The secretion of recombined eFSH using P. pastoris expression system was confirmed by Western blotting and immunoprecipitation (IP) methods. The DNA sequence of the efshβ chain accession number JX861871, predicted two putative differential nucleotide arrays, both of which are located in the 3′ UTR. Western blotting showed a molecular mass of 13 and 18 kDa for eFSHα and eFSHβ subunits, respectively. The expression of desired protein was confirmed by protein G immunoprecipitation kit. eFSH successfully expressed in P. pastoris. These findings lay a foundation to improve ovulation and embryo recovery rates as well as the efficiency of total embryo-transfer process in mares.

Rabies virus (RABV) is a deadly neurotropic virus that causes the disease of rabies in humans and animals. L protein is one of the large structural protein of rabies virus, which displays multiple enzymatic activities, and is required for viral transcription and replication. A truncated L protein of Rabies virus is being cloned, expressed and purified to produce relevant polyclonal antibody. The gene fragment of L protein of RABV was subcloned into prokaryotic expression vector p Rabies virus (RABV) is a deadly neurotropic virus that causes the disease of rabies in humans and animals. L protein is one of the large structural protein of rabies virus, which displays multiple enzymatic activities, and is required for viral transcription and replication. The results of PCR and sequencing confirmed that the fragment of L gene of RABV was successfully cloned into the expression vector. The expression of recombinant L protein fragment induced by IPTG was confirmed by the band of 43 kDa in SDS-PAGE and western blot. The antiserum of purified L protein immunized mice was reacted with RABV infected N2a cells and suckling mouse brain tissue lysates. Our data showed that the recombinant L protein produced by pET-28a vector was very successful, and the purified L protein could efficiently induce the antibody response in mice. The antiserum could recognize the virus in RABV infected cells and tissue very well.ET-28a and transformed into E.coli Rosetta DE3 host strain. The recombinant L protein of RABV was expressed and characterized by SDS-PAGE and western blot analysis using anti-his tag antibody. Mice were immunized with the purified recombinant L protein, the reaction of the anti-serum was checked by immunofluorescence and dot-blot, respectively. The results of PCR and sequencing confirmed that the fragment of L gene of RABV was successfully cloned into the expression vector. The expression of recombinant L protein fragment induced by IPTG was confirmed by the band of 43 kDa in SDS-PAGE and western blot. The antiserum of purified L protein immunized mice was reacted with RABV infected N2a cells and suckling mouse brain tissue lysates. Our data showed that the recombinant L protein produced by pET-28a vector was very successful, and the purified L protein could efficiently induce the antibody response in mice. The antiserum could recognize the virus in RABV infected cells and tissue very well.

The production of waste pollutants has become a major problem for many food and oil industries. However, oil wastes can provide alternative substrates for industry, which could help to solve environmental pollution problems. Furthermore, oil wastes can be used as substrates to produce unsaturated fatty acids, which are important for health. The production of fatty acids in fungi using oil wastes and renewable substrates were investigated. Oil waste sources were obtained from food factories and restaurants (F1, F2, F3, R1, and R2). Cunninghamella echinulata DSM1905 and Rhizopus stolonifer DSM2194 were used to treat the wastes. Changes in lipid and fatty acid contents, biomass, and pH were monitored. C. echinulata produced about 18.4 and 20.1% gamma linolenic acid (GLA) from the R1 and R2 oil wastes, respectively. It also produced 9.3% and 12.4% linolenate from the F2 and F3 wastes. R. stolonifer produced 21% GLA from R1 and 9.3% linolenate from F3. C. echinulata reduced biological oxygen demand (BOD) and chemical oxygen demand (COD) by 67%-74% and 50%-98%, respectively. R. stolonifer reduced BOD by 36%-74% and COD by 10%-78%. This study emphasized the abilities of oleaginous fungi to utilize oil wastes as carbon sources to reduce BOD and COD of the wastes, producing essential fatty acids.

Stresses such as heat shock, starvation, or osmotic is essential to lead isolated microspores towards embryogenesis. Despite the effectiveness of stresses in embryogenesis, they exert adverse effects on metabolism and growth of the regenerated plants. The effects of heat shock and 2,4-D treatment on total protein content of treated microspores, morphological and physiological characteristics of the doubled haploid (DH) plants were assessed. Buds containing mid- to late- uninucleate microspores were used for microspore culture. Microspores were isolated and cultured in NLN-13 medium and incubated at 30ºC for 14 days or treated with 2,4-D (35 mg.L-1) for 30 min to induce embryogenesis. Microspore-derived embryos were transferred onto B5 medium for plantlet regeneration. Ploidy level of the regenerated plantlets was determined using Partec flow cytometry. Spectrophotometric readings were carried out at 490, 663 and 645 nm to determine Chl a-b and carotenoids contents. TRIzol and cetyl-three-ethyl-ammonium bromide (CTAB) were used for protein extraction from microspores and leaves. Length and width of stomata and pollen grains were also photographed using light microscope (Olympus). Applied stressors significantly reduced total protein content of treated microspores however, protein content and concentration of chlorophyll a and b of the DH plants were only increased by heat shock treatment when compared with the donor plant ‘Hyola 420’. In contrast, carotenoids were not affected by applied stressors. Longer and wider stomata were observed by 2,4-D treatment but, the length of pollen grains was significantly decreased following heat shock and 2,4-D treatment. Total protein content of cultured microspores, concentration of chlorophyll a and b, length and width of stomata of microspore-derived doubled haploid plants were significantly affected by the type of inductive stresses. However, carotenoids were more stable and not affected by applied stressors.

The tan spot disease of wheat caused by Pyrenophera tritici-repentis has become a major disease in most wheat growing areas worldwide. Here we used ISSR and RAPD markers to study the genetic diversity of 34 P. tritici-repentis isolates collected from North of Iran. The leaves having the typical symptoms of tan spot disease were collected and after fungal isolation, purification and identification, DNAs were extracted. After PCR amplification using each primer, PCR products were run in agarose gels, and the resulting bands were scored. Cluster analysis was performed using Un-Wighted Nighbor Joining method. A total of 178 reproducible bands were scored. Out of which 115 (65%) were polymorphic corresponding to an average of 8 polymorphic bands per primer. The average PIC values for ISSR and RAPD markers were 0.38 and 0.43, respectively. A high degree of genetic variability among Iranian P. tritici-repentis isolates was identified. Cluster analysis based on un-weighted neighbor-joining method using the combined molecular data revealed five distinct clusters. The results from the cluster analysis indicated that the genetic similarity among the Iranian P. tritici-repentis isolates could be partly explained by geographic origins where the isolates were collected. Genetic variability of P. tritici-repentis along with relatively high level of geographic diversity observed in this study may suggest longer evolutionary period for the isolates from the Middle East, wheat center of origin, as opposed to other places.

Downy mildew caused by Plasmopara halstedii is a devastating disease in sunflower worldwide. Several dominant resistance genes designated as Pl have been identified and linked molecular markers have been demonstrated. However, no information on theresistance genes is available forIranian lines. The presence of three map-based molecular markers previously proved to be linked to different resistance genes were evaluated in sunflower inbred lines. Using PCR-based and CAPS molecular markers, 26 sunflower inbred lines with different responses to P. halstedii race 100 were used to detect the presence of three resistance loci including Pl1, Pl6 and Pl13 within the lines. Molecular marker linked to Pl13 was present in some of the sunflower lines but was not correlated with the phenotypic reaction of the lines to race 100. Despite the use of three markers linked to Pl6, PCR failed to amplify any corresponding product. This data may suggest that none of the genotypes possessed Pl6 locus. Pl1-linked cleaved amplified polymorphic sequences (CAPS) were present in several resistance lines and effectively differentiated susceptible and resistant sunflower lines. Applicability of molecular markers in breeding programs revisited in disease management.

Identification and quantification of mycotoxins produced by Fusarium species are important in controlling fungal diseases. Potential of zearalenone, butenolide and fusarin C production was investigated in five Fusarium graminearum and five F. culmorum isolates at molecular level. Presence of PKS13, FG08079.1 and PKS10 genes, associated with production of zearalenone, butenolide and fusarin C, respectively, were confirmed by PCR. In addition, expression levels of them together with housekeeping gene (b-tubulin) were detected by real time PCR. PKS13 and FG08079.1 transcripts were determined in all isolates, while PKS10 specific primers failed to amplify any product, indicative of no expression. DDCT of PKS13 was ranged between 1.79E-03-3.97E-03 and for FG08079.1 was between 0.25E-03 and 6.02E-03. The highest PKS13 expressions were 3.86E-03 in F. graminearum F9 and 3.97E-03 in F. culmorum F16. Maximum FG08079.1 expressions were calculated as 6.02E-03 and 3.81E-03 in F. graminearum 2F and F. culmorum F2, respectively. We revealed that ten Fusarium isolates produced zearalenone and butenolide under culture conditions. However, fusarin C was not generated by them in these conditions.

Splicing by overlap extension (SOE) PCR is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein’s structure and function. We introduced a nested-SOE-PCR (N –SOE-PCR) in order to increase the specificity and generating mutations in a gene by SOE-PCR. Genomic DNA from Bacillus thermocatenulatus was extracted. Nested PCR was used to amplify B. thermocatenulatus lipase gene variants, namely wild type and mutant, using gene specific and mutagenic specific primers, followed by cloning in a suitable vector. Briefly in N-SOE-PCR method, instead of two pairs of primers, three pairs of primers are used to amplify a mutagenic fragment. Moreover, the first and second PCR products are slightly longer than PCR products in a conventional SOE. PCR products obtained from the first round of PCR are used for the second PCR by applying the nested and mutated primers. Following to the purification of the amplified fragments, they will be subject of the further purification and will be used as template to perform the third round of PCR using gene specific primers. In the end, the products will be cloned into a suitable vector for subsequent application. In comparison to the conventional SOE-PCR, the improved method (i.e. N-SOE-PCR) increases the yield and specificity of the products. In addition, the proposed method shows a large reduction in the non-specific products. By applying two more primers in the conventional SOE, the specificity of the method will be improved. This would be in part due to annealing of the primers further inside the amplicon that increases both the efficiency and a better attachment of the primers. Positioning of the primer far from both ends of an amplicon leads to an enhanced binding as well as increased affinity in the third round of amplification in SOE.