抗血管表皮生长因子VEGF纳米抗体在E.coil中的表达和纯化

Poster – [A-10-817-2] Structural studies on interaction of lysozyme with cationic detergents, cetyl- and dodecyl-trimethylammonium bromides Sadegh Farhadian, Behzad Shareghi, Majid Farhadian, Mohammadreza Karimi Shahrekord University, Shahrekord, Iran E-mail addresses: sadeghfarhadian@gmail.com (S. Farhadian), share.beh@sci.sku.ac.ir (B. Shareghi), majidfarhadian@yahoo.com (M. Farhadian), rezakarimi682@yahoo.com (M. Karimi) Lysozyme (1,4-b-N-acetylmurmidase) belongs to the class of enzymes that lyse the cell walls of bacteria as the bond between the C-1 of N-acetylmuramic acid and the C-4 of N-acetylglucosamine of the peptidoglycan is cleaved. Native hen lysozyme comprises 129 amino acids (E.C. 3.2.1.17) which are organized into two structural domains (α-helical domain and β-sheet domain) and stabilized by four disulfide bonds. Interaction between lysozyme and cetyl- and dodecyl-trimethylammonium bromides was investigated at different pHs and temperatures by fluorescence and UV–Vis spectroscopies. The results showed that (1) addition of DTAB and CTAB decreases the melting temperature of lysozyme. (2) CTAB has very stronger effect on decreasing the thermal stability of the enzyme. (3) By increasing the pH, lysozyme melting temperature (Tm) shifts to lower temperatures. Keywords: Lysozyme, Cetyl-trimethylammonium bromides (CTAB), Dodecyl-trimethylammonium bromides (DTAB) doi:10.1016/j.clinbiochem.2011.08.610 E.poster – [A-10-843-1] Cloning of the R1 and R2 subunits of ribonucleotide reductase from Helicobacter pylori Ali Afrasyabi, Reza Rofougaran Institute of Biochemistry and Biophysics, Tehran University, Tehran, Iran E-mail addresses: aliafrasyabi60@yahoo.com (A. Afrasyabi), rofougaran@ibb.ut.ac.ir (R. Rofougaran) Introduction: Ribonucleotide reductase (RNR) is an enzyme that catalyzes the formation of deoxyribonucleotides from ribonucleotides. The RNR protein consists of two non-identical subunits, the R1 and R2 proteins. This enzyme has been targeted for drug design against pathogens. Helicobacter pylori (H. pylori) is the human pathogen and associated with the gastric diseases such as gastric inflammatory and gastric cancer. Method:We aimed to clone the RNR subunits from this bacterium. To achieve this goal, at first we amplified the R1 and R2 genes for 30 cycles after a hot start at 94 °C for 5 min. Each cycle for the R1 gene includes denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 7 min. The same procedure was applied for the R2 gene but annealing temperate was 59 °C instead. In our generated primers for the R1 and R2 genes we designed restriction sites for BamHI and NdeI to facilitated subcloning. The single PCR products of the R1 and R2 genes were digested with BamHI and NdeI and subcloned into pET-3a digested with the same enzymes. Then we transformed the recombinant vector into Escherichia coli XL-1 blue cells. After confirmation by sequencing, the corresponding proteins was purified by affinity chromatography from the E. coli Rossetta cells containing the transformed construct. Hopefully, the characterization and allosteric regulation of the Helicobacter pylori (H. pylori) RNR can be used for rational new drug design against this pathogen. The detailed result is presented. Keywords:Ribonucleotide reductase (RNR), Allosteric regulation, Cloning doi:10.1016/j.clinbiochem.2011.08.611 Poster – [A-10-867-2] Expression and purification of a specific Nanobody against Vascular Endothelial Growth Factor (VEGF) Fatemeh Rahbarizadeha, Bahram Kazemib, Zahra Farajpourc aDept. of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box: 14115-331, Tehran, Iran bDep. Of Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, PO Box: 19395-4719, Tehran, I.R. Iran cDepartment of Pharmaceutical Biotechnology, Faculty of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran E-mail addresses: rahbarif@modares.ac.ir (F. Rahbarizadeh), bahram-14@yahoo.com (B. Kazemi), zfarajpour@hotmail.com (Z. Farajpour) Introduction: The inhibition of angiogenesis by blocking antibodies against Vascular Endothelial Growth Factor (VEGF) and its receptors becomes a potent therapeutic strategy for cancer treatment. Nanobody (Nb), variable domain of Heavy chain antibodies isolated from Camelidae family, is the smallest antibody fragment which has high homology to variable domain of human antibodies. Therefore the aim of this study was to produce and purify a VEGF-specific Nanobody selected from Phagemid library. Methods: We prepared a panel of Nbs against VEGF165 by biopanning of Phagemid library constructed from immunized One- humped camel. Screening of the mixture of antigen-specific clones is performed by phage-ELISA. Expression of Nbs in soluble formwas done utilizing a non-suppressor Escherichia coli strain. To determine the best medium for protein expression, E. coli was grown in LB, 2YT and SB. Optimization of protein expression was performed using different concentrations of IPTG at 22 °C and 37 °C aswell. The level of expression was monitored by ELISA and protein was detected by western immunoblotting. Nb was purified by affinity chromatography. Results: The best medium for protein expressionwas LB. IPTG at the concentration of 1 mM resulted in high level of Nb expression. When culture was induced at 22 °C, the level of proteinwas significantly high. Conclusion: Expression of Nb was optimal when host cells were grown in LB medium at 37 °C and induced with 1 mM IPTG at 22 ° C. 17 kDa protein was successfully purified by affinity chromatography. Keywords: VEGF, Cancer, Expression, Purification doi:10.1016/j.clinbiochem.2011.08.612 Poster – [A-10-883-3] Study of the effect of substitution in the C4 cytosine by Density Functional Theory Mozhgan Mahmoodi Aval, Mahbob Jamali, Jalal Shakhs Emampour, Homa Moghadam Department of Chemistry, Faculty of science, Islamic Azad University, Mashhad Branch, Mashhad, Iran E-mail addresses: mahmoodi.chem@gmail.com (M.M. Aval), mahbobjamali@yahoo.com (M. Jamali), J-Emampour@yahoo.com (J.S. Emampour), homa.moghadam44@gmail.com (H. Moghadam) Cytosine is oneof themajor structural units amongnucleic acidbases in DNA. In this work, we have used Density Functional Theory (DFT) to study the effect of substitution as electron donor and electron acceptor Abstracts S247