Purification of HIV-1 Reverse Transcriptase and evaluation of anti-HIV-1 RT activity of selected medicinal plants through fluorescence-based assay
Human Immuno Deficiency virus is a lentivirus (a subgroup of Retrovirus family Retroviridae) which attacks the immune system of humans. Due to rapid evolution of HIV owing to error-prone replication, emergence of drug resistant strains is inevitable. As there is no permanent cure for this deadly disease, development of new drugs is necessary. HIV-1 Reverse Transcriptase has long been an obvious anti HIV-1 drug target. Many secondary metabolites and bioactive molecules from plant sources have been evaluated as an antiviral drug. HIV-1 RT is a heterodimer containing a p66 (560 residue) subunit and a p51 (440 residue) subunit. In the present research, we aimed to identify novel compounds from selected plants which can potentially inhibit the activity of HIV-1 RT in vitro. E. coli BL21 strain having plasmid inserted with p66 were cultured and induced by IPTG for overexpression of p66. The cells were harvested and p66-6X His tagged protein was purified by using Immobilized Metal Ion Affinity Chromatography. Then dialysis was carried out and finally SDS PAGE run to confirm the presence of purified protein. We then focused on an in vitro study to evaluate anti HIV-1 RT activity of selected plant extracts by using PICOGREEN dsDNA quantification reagent. In this assay, RT activity in a biological sample generates long dsDNA which are detected by PicoGreen reagent and samples can be read in a Fluorometer for evaluating the activity of RT. To assess the effect of inhibitors (plant derived crude extracts), HIV-1 RT assay was performed in presence of different concentrations of the plant extracts followed by detection of fluorescence intensity. Evaluation of anti HIV-1 RT activity was done through the comparison of fluorescence intensities between the control and test samples.
Keywords: SDS-PAGE, IPTG, Immobilized metal ion affinity chromatography, Picogreen, dsDNA.