فصلنامه تازه های بیوتکنولوژی سلولی مولکولی
پیاپی 17 (زمستان 1393)

Aims and Nowadays، monoclonal antibodies (mAbs) became powerful therapeutic and diagnostic tools. Due to showing minimum immunogenic reaction and their properties، human mAbs are important. The purpose of this study was to construct an immune antibody library from a vaccinated donor against tetanus toxin. A whole blood sample was taken from the donor who was vaccinated against tetanus toxoid. PBMC were isolated by using ficol. After RNA extraction، all variation of VH and VL regions by RT-PCR reactions were amplified and linked as a ScFv antibody. The amplicons were inserted in T-vector and transformed to E. coli DH5α strain، followed by an ELISA test. Plasmids were also extracted and sequenced. cDNA quality was confirmed by using HPRT primers. To confirm PCR، insertion and transformation، gel electrophoresis and restriction enzymes digestion were applied. Positive clones were selected based on growth on LB agar which is the blue/white selection method. After plasmid extraction and DNA sequencing، the sequences were aligned using igBLAST at NCBI. The result was shown to have admissible similarity among antibody gene library nucleotide sequences and the antibody genes were deposited in this database. ELISA confirmed this data too. In this study، the immune human antibody library was constructed and confirmed using DNA sequencing and sequences alignment in NCBI database. ELISA test confirms antibody specifically. The next step is to screen the library to find an antibody specifically for tetanus toxin.

Aim and Soil salinity is one of the major abiotic stresses reducing yield crop. SOS (Salt overly sensitive) system in Arabidopsis thaliana act as cellular signaling system in salinity tolerance pathways. SOS1 has an important role in salinity tolerance. In this study, SOS1 gene sequence identified in various plants has been recorded in the databases such as Tair, EBI, NCBI, plant care and EXPASY. We use an in silico analysis in order to determine phylogenetic study and the homology of 13 plant species by bioedit, UPGMA and MEGA5 software. The cluster analysis showed that sos1 protein sequences of these plants classified in three groups. The results of the evolutionary matrix showed that the most distance was between Aegilops tauschii and Arabidobsis thaliana (0.542) and the least distance was between Triticum aestivum and Aegilops tauschii (0.004). Promoter Sequence analysis of SOS1 gene showed that, the ABRE and CAAT boxes were found in all areas of plant promoter. Also, aligning protein sequences showed complete similarity in 15 Amino acids. Due to the presence of ABRE box activated in response to Abscisic acid and CAAT box and increases gene expression in all areas of plant promoter, suggests the potentiality of plants to cope with abiotic stresses. Complete similarity in 15 Amino acids shows conserved region during the evolution process and can have an important structural role in the protein's function and nucleotide sequences of those suggested to design primers.

Aim and Apricot is one of the economically important genera of woody plants in Iran and most of these important varieties grow in Azarbaijan region, because of its Mediterranean ecological factors. Apricot has a large stone seed. Seeds are naturally planted in early autumn and after a prolonged exposure to cold temperatures (winter), seeds germinate in the end of third month of spring or in the beginning of the summer taking sometimes 18 months. Germination occurs more rapidly after cold treatment (vernalization), thus it cannot germinate without prior cold exposure In. this study, an artificial way has been suggested; this method significantly decreases duration of germination. Furthermore, it increases percentage of germination, because seeds in the garden maybe moulded by microorganisms of soil. In this study, 10 seeds of each type of apricot (control) were first planted in the garden and then 10 seeds of each type of apricot were transferred on whattman paper inside each petri dish. All petri dishes were incubated to germinate. The condition of germinator was: 16h day light and 8h dark period with 20˚C and 16˚C respectively. The humidity condition inside germinator was 65%. And the percentage and the rate of germination was measured up to 12 days. The findings showed that the percentage of germination was increased significantly in seeds when compared with control. Also, hundred percent of germination was obtained in D. However, artificial frost affects germination percentage. Therefore, the AVG used germination rate calculation is suggested and Artificial cold period is advised to be done in germination in order to increase percentage and velocity of stone seed germination. It is suggested to use artificial germination for better germination.

Aim and Fusarium solani, may occur as a mixed infection with HSV and due to similarities in their clinical features, sometimes it leads to misdiagnosis. The aim of this research was the designation and optimization of PCR technique for the diagnosis of F. solani as the main etiological agent or as a mixed infection in corneal samples suspected of having HSV 100 samples of DNA from clinical cases (Include 65 negative and 35 positive samples for HSV), suspected to herpetic keratitis were collected. The PCR test for detecting F.solani in these samples, with specific primers ffuso1 and rfuso2 and mt cytb as the target gene with 330 bp product was optimized. The specificity and sensitivity of the test were then evaluated. Among 65 negative samples for HSV, two samples were positive for Fusarium solani and the PCR 330 bp product were amplified in these two cases. The sensitivity of the test was one copy of Fusarium solani genome and specificity was 100%. The results of PCR test show that, the cases suspected to herpetic keratitis are caused by Fusarium solani., Accurate diagnosis of ethological agent in the cases of clinical features similarity or mix infection, is possible by molecular diagnosis.

Aim and The most destructive types of corrosion are biocorrosion which about 20-40 percent of corrosion recompense are related to it. Natural phenazines in secondary metabolites of bacteria have been receiving increasing attention in recent years due to their potential usage as antibiotics. Steel corrosive bacteria isolated from marine source as the main cause of sea structure corrosion was studied to assay anticorrosion antibacterial material effects on the activity of this microorganism. In vitro antibacterial production of phenazine 1-carboxylic acid (PCA) was evaluated for seven days in batch fermentation. The experiments showed maximum mass production is related to sixth day with 0.313 g/l products, but the antibacterial activity was tested and the inhibition zone on the fourth day was bigger than the other days and analysis of high pressure liquid Chromatography (HPLC) indicates more purity of PCA on this day. According to the result, future increase in PCA production was achieved after four days fermentation. Therefore, in future prospects, we will try to formulate these compounds as anti-corrosive paints for industrial use.

Aim and Tissue engineering is a science using advanced techniques to repair damaged tissue and cause their recovery and reconstruction. The success of this process requires the presence of optimal and effective factors. Among the important factors in this success is to use appropriate scaffolds with wonderful mechanical and biological properties to provide an optimal growth environment and appropriate condition for the differentiation of stem cells. In this research, the evaluation of PRP and PLGA scaffolds efficiency in producing suitable environment for growth of mesenchymal stem cells was carried out. In this study, after preparing PRP and PLGA scaffolds and isolating mesenchymal stem cells from adipose tissue, the cells were seeded separately into two scaffolds and after 48 hours of cell seeding, the cells viability was evaluated by MTT assay. Our findings showed that both PRP and PLGA scaffolds were used as an ideal environment for the culture and proliferation of mesenchymal stem cells. The obtained results of this study using MTT assay indicated that when the active PRP are used with mesenchymal stem cells, its efficiency was higher than PLGA. It seems that using PRP-derived scaffolds as a protective scaffold is better to be considered for mesenchymal stem cells growth to provide effective strategies for the development of tissue engineering and regenerative medicine.

Aim and Genus Linaria Mill. Belonging to the Plantaginaceae family is categorized in Lamiales order. The habitat of this genus is the northern hemisphere and in some parts of Iran. The purpose of this study was to investigate the phylogeny and taxonomy relationships of the mentioned genus in Iran. Molecular studies were performed in accordance with the following procedure: 1-Total DNA extracted from leaves, 2-Amplification and sequencing of the genome of the chloroplast rpl32-trnL fragment area 3-Statistical analysis of evolutionary models to obtain and review the phylogeny of the studied groups,4- Interpretation of cladograms. The data matrix consisted of 71 taxa and 702 characters in Parsimony analysis. The 485 characters are constant, 109 variable characters are parsimony-uninformative, The remaining 108 are potentially parsimony informative. Four major clades including A, B, C and D were detected in Phylogenetic tree analysis. Monophyly of Linaria species was supported in this study. A basal divergence between species with winged and wingless seeds was clearly unsupported, implying the homoplasy of this trait. the results showed that Linaria species constitute a Monophylitic group. According to a set of morphological traits including entire, sessile, pinnately veined leaves; terminal, bracteate, racemose inflorescences and spurred flowers monophyly of Linaria confirmed.

Introduction and Human cytomegalovirus infection has high incidence worldwide. This virus is an important mortality factor in some blood donors. The aim of this investigation was study of seroprevalence of anti-cytomegalovirus IgG and IgM in voluntary blood donors of Ardebil province. In this cross-sectional study after blood samples collection and serum separation, the specific anti-cytomegalovirus IgG and IgM antibodies were evaluated on sera using ELISA method and the relation of the obtained results with different parameters including gender, age, blood group and Rh factor were analyzed with appropriate statistical method using SPSS 17 version software. From 182 collected sera, 180 samples were positive for anti-cytomegalovirus IgG antibody (98.9%) whereas only 10 samples were positive for IgM antibody (5.5%) and 1 sample was suspicious. There was not statistical significance between blood donor’s sexuality and IgG class anti-cytomegalovirus prevalence, meanwhile, concerning IgM class anti-cytomegalovirus prevalence, it showed statistical significance (P<0.05) as the prevalence of anti-cytomegalovirus IgM antibody in females was higher than males. In addition, among age and prevalence of anti-cytomegalovirus IgM and IgG antibodies, statistical difference was not significance. It has not been shown any statistical significance between blood group, Rh factor and mentioned antibodies (P>0.05). In recent study, the high seroprevalence of anti-cytomegalovirus IgG and IgM antibodies was found in voluntary blood donors of Ardebil province resemble of other parts of country.

new nano-carrier platforms based on natural biological building blocks offer great promises in revolutionizing medicine. The usage of specific protein cage structures such as virus-like particles (VLPs) for drug packaging and targeted delivery has attracted much attention. versatile chemical and genetic modifications on the outer surfaces and inner cavities of VLPs facilitate the preparation of new materials requirements for drug delivery. A full evaluation on the toxicity, biodistribution and immunology of these materials are envisaged to boost their application potentials. VLPs can easily offer the requirements that are needed for a drug delivery, such as biocompatibility, solubility in water, and high uptake efficiency.

Cisplatin (Cispt) is an anti-cancer drug with a low level of solubility. One of Cispt`s solvents is dimethyl sulfoxide (DMSO) which can be substituted with chlorine of drug as Cispt`s solvent. Using this solvent is impossible in biological studies due to intense reduction in activity. On the other hand, it is specified that Cispt`s stability is increased in aqueous media by increasing sodium chloride (NaCl) concentration up to 0.9%. Consequently, we intended to study the effect of DMSO on cytotoxicity of Cispt in the presence of sodium. G-292 cells and MTT assay was used to study cytotoxicity of different compounds in culture media. FTIR was employed to investigate chemical structure of Cispt and Cispt dissolved in DMSO. cytotoxicity in dilutions of 300 and 9 (p<0.01) of Cispt in Cispt+NaCl+DMSO formulation was equal to 78 and 7%. These numbers were estimated 79 and 18% for Cispt+ DMSO formulation. Studying chemical structure of Cispt and Cispt dissolved in DMSO showed that sodium chloride cannot inhibit inactivating effect of DMSO on Cispt and effect of this solvent on Cispt is independent from presence of sodium chloride. This study showed that effect of solvent on Cispt is independent from presence of sodium chloride. Our findings suggest more studies to use this solvent for Cispt.

Aim and Vulvovaginal candidiasis is one of the most common female genital tract infections that is caused by overgrowth Candida species, especially Candida albicans and sometimes becomes chronic and recurrent form. Candida glabrata is the second or third leading cause of candidiasis after C. albicans. Following the widespread and increased use of immunosuppressive therapy along with broad-spectrum antimycotic therapy, the frequency of mucosal and systemic infections caused by C. glabrata has increased significantly. Fluconazole is one of the most common drugs used for treatment of this type of candidiasis but resistant is also seen. The first step in infection is adherence to epithelial cells. This study was designed to evaluate the adherence of fluconasole sensitive or resistant Candida albicans and Candida glabrata to Vagina and Intestine cell lines. In this study, 160 vaginal swabs were collected from women with vaginal candidiasis. Then samples were cultured on Sabaouraud Dextros Agar and Chromagar for morphologic analysis. For final identification of Candida species the PCR-RFLP was done and C. albicans and C. glabrata were selected for our study. Hella and HT29 cell line cultured in DMEM medium supplemented with FBS. The yeast cells were adjusted to 1×106 cell and then cultured with definite number of each cell line separately in 96- well micro plate and incubated in 37°C for 24 hours. After this period, the number of adherent and non-adherent cells was counted by culture on Sabaouraud Dextros Agar and colony count assay. According to our result, the clinical isolates are resistant to fluconazole and C. glabrata isolates had more ability to adhere to vaginal epithelial cells compared with C. albicans; but, in contrast, C. albicans had more ability to adhere to intestinal epithelial cells rather than C. glabrata. This research showed that C. glabrata had more ability to adhere to vaginal cells. As regards, there is little information about the reaction between C. glabrata and innate immune system, Therefore, recognition of the reactions and factors that mediate adherence to host epithelial surfaces has a particular importance for finding new aspects in the pathogenesis and a more suitable treatment for the disease.

Backgrounds and This research is to develop new systems for drug release systems based on sol - gel systems. The objective of this study is to develop controlled release formulation of water insoluble Olanzapine (OZ), using Glycerol monooleate (GMO) and Polyethylene glycol (PEG 300). A Box- Behnken response surface methodology was applied to design gel system with 3 factors with an initial drug containing, weight ratio of GMO/water (w/w) and weight ratio of PEG 300/GMO (w/w). Therefore, in this study Design-Expert® software with Box- Behnken response surface methodology has been used specifically to determine the relationships between variables and responses. According to the results of experiments, a quadratic model as an appropriate equation has been selected to fit the percentage of loading. Percentage of release at 12th and 168th hr a cubic model is selected. Kinetic release profile of OZ was investigated with several models, from which Higuchi model showed the best fitting and highest correlation. Optimization of gel system was carried out based on statistical concept of experimental design. Validation test was carried out under optimum conditions of the parameters predicted by the polynomial model.

Aim and Granulomatous skin infections probably originated from an environment and through skin wounds, scratching, trauma, surgery into the inoculated skin, and abscess pus appears and sometimes is in form of Ulcerativa. Infection is limited to the skin and in case of immunosuppression would transmit and spread in form of infection. PCR is a rapid sensitive, specific and accurate method for diagnosis of bacterial infections and pathogens in the shortest time. In this study atypical Mycobacterium granulomatous skin infections diagnosed by PCR, using specific primers design for Mycobacterium genes (16srRNA) and confirmation by sequencing methods (sequencing of nucleotides of) atypical mycobacterium and comparing with genome sequencing of 16srRAN genes atypical mycobacterium in Gene Bank and application of this technique in the production of atypical mycobacterium skin infections diagnostic kits. Among 58 studied paraffin blocks samples of skin tissue, 18 samples were in the range of positive control and established bands showing positive PCR results. Out of 18 positive samples of Mycobacterium granulomatous skin lesions, 83% were men and 17% women. In 13 cases (72%) there were occupational causes and in (28%) lack of occupational causes. 66% of mycobacterium granulomatous skin infections (NTM) were in adults over age 40 years. PCR is a rapid, specific, sensitive and accurate method for the diagnosis of bacterial infections and pathogens in the shortest time. This method can recognize a few atypical Mycobacterium genomes in samples, which is the superior advantage of this method, compared to conventional pathological methods such as culture and differential biochemical experiments.

Aim and Nocardia spp are Gram-positive, partially acid-fast causing various infections. Due to the complex structure of the cell wall of the microorganism that is similar to mycobacteria, DNA extraction from this bacterium is different from other bacteria. The boiling method with STET buffer solution was used for DNA extraction of Nocardia. Nocardia colonies were suspended in 200 µl STET buffer and was boiled for 30 minutes. Suspension was centrifuged and transferred to another micro tube and Ethanol %95 was added and saved at -20 °C for 30 minutes. After this stage, sample was centrifuged and the supernatant was discarded. In the first stage, distilled water was added and stored at -20 °C for molecular works. To confirm the presence and quality of the DNA extract, the electrophoresis on a %1 agarose gel was carried out and PCR reaction 16S rRNA gene was used. DNA was extracted on agarose gel and the purity and quality was appropriate. 16S rRNA gene PCR was then performed and observed to be 1500 bp.