[Optimization of refolding of human vascular endothelial growth factor-A121]

Background: vascular endothelial growth factor [VEGF] plays an important role in development of new blood vessel and angiogenesis. Human VEGF121is smallest member of VEGF family. Production of active and correct form of VEGF is the m6st challenging issue

Methods: here we described a method for optimization of refolding of VEGF121 which was expressed in bacterial host. Enzyme linked immunosorbent assay and proliferation assay of human endothelial cells was performed to monitor refolding and functional assay ofVEGFlz1

Results: using described method VEGF was in correct fold and detected by antibody in ELISA. Furthermore, VEGF stimulated proliferation of human endothelial cells in dose-dependent manner

Conclusion: refolded VEGF has potential for stem cell differentiation

An overview on application of phage display technique in immunological studies

Phage display is very strong technique in drug discovery and development. Phage display has many applications in improving the immunological studies. Development of monoclonal antibody, peptides, peptidomimetics and epitope mapping are main application of phage display. Selection of monoclonal antibody or peptides that are displayed on the surface of the phages can be occurred through biopanning process. In biopanning process phage library is incubated with antigen and particular phages can be identified and isolated. Increasing the stringency in the biopanning rounds can be help to select phages with high affinity and specificity. Here, we describe an overview of phage display application with focusing on monoclonal antibody production and epitope mapping.

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

Objective To express human vascular endothelial growth factor121 (VEGF121) in insect cells. Methods A 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. Results Our 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. Conclusions Results from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes.

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

OBJECTIVE@#To express human vascular endothelial growth factor121 (VEGF121) in insect cells.@*METHODS@#A 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.@*RESULTS@#Our 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.@*CONCLUSIONS@#Results from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes.

model to study the phenotypic changes of insect cell transfection by copepod super green fluorescent protein [cop-GFP] in baculovirus expression system

Background: Baculovirus expression system is one of the most attractive and powerful eukaryotic expression systems for the production of recombinant proteins. The presence of a biomarker is required to monitor transfection efficiency or protein expression levels in insect cells

Methods: The aim of this study was to construct a baculovirus expression vector encoding a copepod super green fluorescent protein [copGFP]. In this light, the resultant vector was constructed and used for transfection of Spodoptera frugiperda cells

Results: Expression of the copGFP protein in insect cells was confirmed by fluorescent microscopy and Western-blot analysis

Conclusion: The application of copGFP control bacmid can be considered as an appropriate control for insect cell transfection

Functional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen

Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length transmembrane protein CD20. In this study, we expressed the extra membrane loop of hCD20 [exCD20] consisting of a non-glycosylated 47-amino acids region. The exCD20 coding sequence was amplified by PCR and cloned in pET32a[+] expression vector. The desired protein was expressed in fusion with thioredoxin and 6 His tag in E. coll Origami strain. ELISA and Western-blotting data were performed to indicate the functionality of this protein. We have obtained the exCD20 recombinant protein which can be detected in ELISA and Western-blot experiments. This recombinant fusion protein was soluble and stable without aggregation and misfolding problems. Conclusion: The recombinant extra membrane loop of human CD20 protein in fusion with thioredoxin [exCD20] can be used in function assays and some applications such as ELISA, immuneblotting, affinity purification, immunization, screening, and development of anti-CD20 antibodies

[Production of camel polyclonal antibody against vascular endothelial growth factor receptor-2 and performance evaluation in ELISA test]

In molecular approach, serum of camel contains a unique type of antibodies devoid of light chains since the light chain is missing, the heavy-chain antibodies should bind their antigen by one single domain, the variable domain of the heavy immunoglobulin chain. Vascular endothelial growth factor receptor-2 [VEGFR-2] is one of the important proteins in angiogenesis which over-expressed in many types of tumors. Two male dromedaries [Camelus dromedarius] were immunized against VEGFR-2. ELISA was used to evaluate the immunization process. Camel immune sera were fractionated with protein A and G affinity chromatography. Heavy chain antibodies tested for its specific reactivity in cell-based ELISA in recognition of VEGFR-2 on the cell surface of three cell lines; 293/KDR, HUVECs and A431. In ELISA test, the camel sera in 1/12800 dilution had the acceptable results. Furthermore, cell-based ELISA demonstrated that the polyclonal heavy chain antibodies bind to VEGFR-2 in 293/KDR and HUVECs cell lines. Single chain polyclonal antibodies against VEGFR-2 can be used for detection of soluble form of this protein by ELISA, and also flowcytometry, western blotting and immunohistochemistry

Detection of rifampin resistant tuberculosis meningitis by polmerase chain reaction linked single strand conformation polymorphism analysis of cerebrospinal fluid

To rapidly detect rifampin resistance in Mycobacterium tuberculosis isolates causing meningitis in northeast Iran. This study presents the results of a polymerase chain reaction-single strand conformation polymorphism [PCR-SSCP] analysis for the evaluation of rifampin resistance directly from the CSF of 47 patients strongly suspicious to have tuberculosis meningitis in Emam Reza University Hospital, Mashhad, Iran over 3 years [2002 to 2005]. Each CSF sample underwent microscopic examination, culture and DNA amplification by 2 PCR protocols and subsequent detection of mutations by SSCP analysis. Among these patients, no mutations were revealed in the rpoB segment by SSCP. The SSCP analyses of these samples shows complete susceptibility to rifampin. The use of this method can radically reduce the time needed to provide clinicians with data useful in aiding the selection of appropriate drugs