[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

Production of recombinant human T lymphotropic virus type 1 tax protein in Rosetta [DE3] bacterial host

HTLV1 is the first detected retrovirus causing disease in human. The physiopathology of HTLV1 related diseases was mainly linked with its Tax protein characteristics. Use of mutant Tax proteins accompanied by immune regulator drugs could help treating HTLV1 associated myelopathy patients as a modulator of potent immune response against this viral protein. Since Tax protein is not commercially available, production of recombinant Tax protein was targeted for this study. Coding sequence of Tax protein [containing R222K mutation] in the pcDNA3.1[+] was digested with BamHI and XhoI restriction enzymes, and then removed and inserted into the expression vector pET32a[+] within the same cutting sites and cloned into E.coli DH5alpha. Recombinant vector was confirmed with enzymatic digestion, colony PCR, and sequencing of cloned gene. E.coli Rosetta [DE3] was transformed with the recombinant plasmid and the expression was induced. The expression of protein was assayed with SDS-PAGE and western blot using monoclonal antibodies against Tax and 5His epitope. Finally, antigenic characteristic of the recombinant protein was evaluated by western blotting against patient sera. Presence of Tax protein band in the SDS-PAGE and western blot was confirmed. Western blotting of the recombinant protein with patient sera showed the band related to Tax protein. The recombinant protein is well produced and could be detected by patients' sera, making it eligible to be used as a recombinant viral antigen for future purposes