DEVELOPMENT OF AN ANTI-DIMERIZATION SUBSTANCE SELECTION SYSTEM OF THE HUMAN IMMUNODEFICIENCY VIRUS (HIV) PROTEASE FROM AN INDONESIAN ISOLATE

• Main Author: Nurfajri R, Rifki
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/48425
• Institution: Institut Teknologi Bandung

Human Immunodeficiency Virus (HIV) is a virus that affects the human immune system. It has been known that the virus can cause a complication in the immune system which is known as Acquired Immune Deficiency Syndrome (AIDS). There are two dominant types of HIV-1 which are circulating in Indonesia, HIV-1 subtype B (HIV-1B) and circulating recombinant form (CRF) 01_AE. In the previous studies, a system has been developed to select a drug candidate. The system is called the dimer based screening system (DBSS). The system utilizes the anti-dimerization activity against HIV protease by a drug candidate. In said system, a plasmid with a fused gene of DNA binding domain of AraC (DBD AraC), protease HIV and a reporter gene, green fluorescent protein (GFP). This method, along with said plasmid, has been successfully applied for the HIV strain, HIV-1 B. However, even until now, said system has not been applied for the HIV strain, HIV CRF01_AE (HIV ID). Thus, the aim of this research is to optimize the DBSS method for HIV CRF01_AE. The plasmid of the desired gene is confirmed by the polymerase chain reaction (PCR) method and DNA sequencing. Protein expression of the cultivated cells is analyzed through the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Escherichia coli BL21 (DE3) with only DBD Ara C in its plasmid is used as the positive control while the culture with the desired plasmid without the drug candidate molecule is used as the negative control in the DBSS system. The drug which is tested in the method is Darunavir. The concentrations of the drug which are tested are 1, 5, and 10 ppm. Fluorescence level test is done after 16 hr of cultivation with the temperature of 37ºC. In- silico analysis using stretcher local alignment technique shows that nucleotide and amino acids sequences between HIV ID and HIV-1 B proteases show high (>80%) similarity. Analysis of a possible three-dimension structure of HIV ID protease shows that there is a high probability that the three-dimension structure of HIV ID protease resembles HIV-1 B protease. This indicates that there is also a high probability that the interactions between darunavir and HIV CRF01_AE will resemble darunavir’s interactions with HIV-1B. The result of electrophoresis of the PCR result shows a band with the weight ~1075bp. That band is confirmed to be comprised of the desired gene since the result of the alignment between the sequencing result and the referenced DNA to be 98% similar. The SDS-PAGE method shows a band with the weight ~24,2 kDa. This band is hypothesized to be comprised of the fused protein. The DBSS method shows that the most effective concentration to stop dimerization of protease HIV ID is 1 ppm. This is shown by the results of the normalized fluorescence and ratio of normalized fluorescence scores shown by the culture which is given 1 ppm Darunavir to be higher than any other concentration in the treated group. Thus, it can be concluded that the DBSS system can be applied to select a drug candidate for an HIV of an Indonesian isolate.