Recombinant expression and purification of functional vascular endothelial growth factor-121 in the baculovirus expression system

AbstractObjectiveTo express human vascular endothelial growth factor121 (VEGF121) in insect cells.MethodsA gene construct containing VEGF was cloned in the pFastBac-HTA vector, followed by transformation in DH10BAC. The recombinant bacmid was then extracted, and transfected into Sf9 insect cells. The transfected cells were harvested, and then VEGF expression was confirmed by western blotting using specific antibodies. The tube formation assay was used for functional assessment of VEGF.ResultsOur results showed that VEGF could be successfully expressed in the baculovirus system. Purified VEGF was able to stimulate in vitro tube formation of human endothelial cells.ConclusionsResults from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes

Transcription of adaptive-immune genes upon challenge with infectious pancreatic necrosis virus (IPNV) in DNA vaccinated rainbow trout

In the present study, rainbow trout weighting 3±0.3 g were vaccinated with an oral DNA vaccine encoding VP2 gene of a prevalent isolate of IPNV in Iranian trout farms encapsulated in sodium alginate microspheres and Chitosan tripolyphosphate (CS/TPP) nanoparticles. The vaccinated fish were then challenged with a virulent isolate of IPNV at 30 days post-vaccination. The transcriptional changes of adaptive- immune genes (IgM and IgT), as well as the  VP4 gene of IPNV, as an indicators of viral replication were studied 45 days post-challenge. Analysis of RT-qPCR data showed lower levels of VP4 gene expression in the oral DNA vaccinated trout after IPNV challenge compared with the control one. Moreover, the constructed DNA vaccine did not enhance the expression of IgM and IgT genes above the levels observed in the carrier control group but it showed a mimic of viral activity and contributes to maintaining them at appreciable levels in vaccinated group

Cell-specific targeting by engineered M13 bacteriophage expressing VEGFR2 nanobody

Objective(s): Filamentous bacteriophage M13 was genetically engineered to specifically target mammalian cells for gene delivery purpose. Materials and Methods: A vascular endothelial growth factor receptor 2 (VEGFR2)-specific nanobody was genetically fused to the capsid gene III of M13 bacteriophage (pHEN4/3VGR19). A mammalian expression construct containing Cop-green fluorescent protein (Cop-GFP), as a reporter gene, was amplified by PCR and then sub-cloned in the pHEN4/3VGR19 phagemid. The resulting construct was transfected into 293KDR cell. The recombinant phage was extracted and confirmed and then transduced into VEGFR2 expressing cell (293KDR). Results: Seventy-two hr after transfection, green fluorescence was detected in 30% of the cells. About 1% of the cells which transduced by recombinant phages were able to express GFP. Conclusion: It is hoped that the results from this study will help to find potential vectors to improve the efficiency of gene delivery. Taken together, we conclude that this newly-introduced vector can be used in cancer researches

In Silico Analysis of Neutralizing Antibody Epitopes on The Hepatitis C Virus Surface Glycoproteins

Objective: Despite of antiviral drugs and successful treatment, an effective vaccine against hepatitis C virus (HCV)infection is still required. Recently, bioinformatic methods same as prediction algorithms, have greatly contributed tothe use of peptides in the design of immunogenic vaccines. Therefore, finding more conserved sites on the surfaceglycoproteins (E1 and E2) of HCV, as major targets to design an effective vaccine against genetically different virusesin each genotype was the goal of the study. Materials and Methods: In this experimental study, 100 entire sequences of E1 and E2 were retrieved from the NCBIwebsite and analyzed in terms of mutations and critical sites by Bioedit 7.7.9, MEGA X software. Furthermore, HCV-1asamples were obtained from some infected people in Iran, and reverse transcriptase-polymerase chain reaction (RTPCR)assay was optimized to amplify their E1 and E2 genes. Moreover, all three-dimensional structures of E1 andE2 downloaded from the PDB database were analyzed by YASARA. In the next step, three interest areas of humoralimmunity in the E2 glycoprotein were evaluated. OSPREY3.0 protein design software was performed to increase theaffinity to neutralizing antibodies in these areas. Results: We found the effective in silico binding affinity of residues in three broadly neutralizing epitopes of E2glycoprotein. First, positions that have substitution capacity were detected in these epitopes. Furthermore, residuesthat have high stability for substitution in these situations were indicated. Then, the mutants with the strongest affinityto neutralize antibodies were predicted. I414M, T416S, I422V, I414M-T416S, and Q412N-I414M-T416S substitutionstheoretically were exhibited as mutants with the best affinity binding. Conclusion: Using an innovative filtration strategy, the residues of E2 epitopes which have the best in silico bindingaffinity to neutralizing antibodies were exhibited and a distinct peptide library platform was designed

Fiber manipulation and post-assembly nanobody conjugation for adenoviral vector retargeting through SpyTag-SpyCatcher protein ligation

For adenoviruses (Ads) to be optimally effective in cancer theranostics, they need to be retargeted toward target cells and lose their natural tropism. Typically, this is accomplished by either engineering fiber proteins and/or employing bispecific adapters, capable of bonding Ad fibers and tumor antigen receptors. This study aimed to present a simple and versatile method for generating Ad-based bionanoparticles specific to target cells, using the SpyTag-SpyCatcher system. The SpyTag peptide was inserted into the HI loop of fiber-knob protein, which could act as a covalent anchoring site for a targeting moiety fused to a truncated SpyCatcher (SpyCatcherΔ) pair. After confirming the presence and functionality of SpyTag on the Ad type-5 (Ad5) fiber knob, an adapter molecule, comprising of SpyCatcherΔ fused to an anti-vascular endothelial growth factor receptor 2 (VEGFR2) nanobody, was recombinantly expressed in Escherichia coli and purified before conjugation to fiber-modified Ad5 (fmAd5). After evaluating fmAd5 detargeting from its primary coxsackie and adenovirus receptor (CAR), the nanobody-decorated fmAd5 could be efficiently retargeted to VEGFR2-expressing 293/KDR and human umbilical vein endothelial (HUVEC) cell lines. In conclusion, a plug-and-play platform was described in this study for detargeting and retargeting Ad5 through the SpyTag-SpyCatcher system, which could be potentially applied to generate tailored bionanoparticles for a broad range of specific targets; therefore, it can be introduced as a promising approach in cancer nanotheranostics

Response of Spring Safflower Cultivars to Irrigation Intervals in Birjand Condition

Abstract In order to evaluate the effect of irrigation intervals on yield and yield components of three spring safflower cultivars (Carthamus tinctorius L.) in Birjand, an experiment was conducted at research farm of faculty of Agriculture of Birjand University in 2007. Experiment was a split plot on the basis of randomized complete block design with four replications. Three irrigation intervals, including: 7, 14 and 21 days were randomized in main plots and three spring safflower cultivars, including: Kooce, P1 and IL111 were allocated to subplots. The highest yield was obtained from Irrigation with 7 days and cultivars of Kooce with mean seed yield of 2653.2 kg/ha. In this irrigation treatment yield of cultivars P1 with 16% reduction was not different from Kooce, but cultivar IL111 with approximately 30% lower yield showed a significant difference with P1. In all three cultivars the lowest seed yield was obtained from 21-day irrigation interval. Mean seed yield in this treatment was 1069.46 kg/ha and reduced by 53% compared to yield in 7-day irrigation intervals. The influence of irrigation intervals in this situation on cultivar IL111 was considerably more than two other cultivars. The results showed that cultivar Kooce had the best yield with 7-day irrigation intervals in Birjand condition. Keywords: Irrigation interval, Safflower, Yiel

Design of a humanized anti vascular endothelial growth factor nanobody and evaluation of its in vitro function

Objective(s): Nanobodies, the single domain antigen binding fragments of heavy chain-only antibodies occurring naturally in camelid sera, are the smallest intact antigen binding entities. Their minimal size assists in reaching otherwise largely inaccessible regions of antigens. However, their camelid origin raises a possible concern of immunogenicity when used for human therapy. Humanization is a promising approach to overcome the problem.   Materials and Methods: Here, we designed a humanized version of previously developed nanobody (anti vascular endothelial growth factor nanobody), evaluated and compared its predicted 3D structure, affinity and biological activity with its original wild type nanobody. Results: Our in silico results revealed an identical 3D structure of the humanized nanobody as compare to original nanobody. In vitro studies also demonstrated that the humanization had no significant visible effect on the nanobody affinity or on its biological activity.  Conclusion: The humanized nanobody could be developed and proposed as a promising lead to target pathologic angiogenesis

Phylogenetic analysis of metalloprotease from transcriptome of venom gland of Hemiscorpius lepturus

Hemiscorpius lepturusis a dangerous scorpion and referred to health concern issue in Khuzestan, Iran. The venom of H.lepturus is cytotoxic and its effect is similar to spider Loxosceles reclusa. Metalloproteinases are the important class of enzymes in the venom that has hemorrhagic activity. The early finding suggests the existence of metalloproteases in the transcriptome of venom gland of H.lepturus. Phylogenetic analysis was accomplished to reveal the evolutionary relationship of identified metalloproteases. The phylogenetic tree was constructed by Molecular Evolutionary Genetics Analysis software and neighbor-joining method. Results showed among three sequences, two metalloproteinases named HLMP1 and HLMP3 of H.lepturus were most close to spider P. tepidariorum. The third sequence named HLMP2 was different and formed an independent clade in the phylogenetic tree. The results suggest that the sequence of metalloproteases in the venom component of H.lepturus is similar to the spider than the scorpion