Document Type : Original Article
Pasteur Institute, Tehran, Iran
Introduction: Human T cell Lymphotropic l.irus type I is responsible for Adult T cell Leukemia/Lymphoma and HTLV-I associated myelopathy. The aim of this study was constructing two reporter plasmids to enable us to evaluate the effects of HTLV-I Tax protein upon intra cellular signalling pathways which recruit CREB and NFkB proteins.
Material and Methods: A complete coding region of bacterial betagalactosidase gene was subcloned into pUC18 , followed by inserting a poly adenylation signal downstream to it. Promoter regions of HTLV-I long terminal repeat and Interlukin 2 receptor alpha (which were stimulated by CREB and NFkB respectively) were amplified by PCR and separately inserted upstream to betagalactosidase gene, leading to construction of two reporter plasmids. The effect of cotransfection of a Tax expressing plasmid with each of these plasmids was evaluated by X-gal staining, beta galactosidase ELlSA or beta galactosidase activity assay with CPRG substrate.
Results: Results clearly showed that both reporter plasmids responded well to stimulation of their promoters by Tax and the produced beta galactosidase could successfully be detected by all three methods. Results of ELlSA and assay tests for betagalactosidase showed a high correlation (r=0.949).
Conclusion: Both reporter plasmids constructed in this study are able to produce considerably more betagalactosidase after stimulation by HTLV-I Tax. Advantages and disadvantages of three evaluating method for detection of betagalactosidase are studied and discussed.