CLONING AND EXPRESSION OF HPV16 L1 PROTEIN ISOLATE FROM INDONESIA IN ESCHERICHIA COLI BL21(DE3)

• Main Author: Saraswati, Dewi
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/46990
• Institution: Institut Teknologi Bandung

Cervical cancer is a cancer with a high prevalence rate in Indonesia caused by Human papillomavirus (HPV) infection. Type 16 HPV is one of the most dangerous types of HPV and causes 87% of cervical cancer events. One of the most effective prevention to reduce HPV infection is prophylactic vaccine. The prophylactic vaccine currently being developed is composed of L1 proteins that can self-assemble into virus-like particles (VLPs). To date, Indonesia has not yet succeed to produce its own vaccine that can prevent HPV infection, especially HPV strains that circulate in Indonesia. So we need a strategy to produce prophylactic vaccines with relatively lower production costs by producing L1 protein in Escherichia coli BL21 (DE3). Aim of this study was to clone and express L1 protein from HPV type 16 on Escherichia coli BL21 (DE3). The method used was gene 1 of HPV type 16 inserted into the pET-32b (+) vector and expressed on E. coli BL21 (DE3) with the addition of an inducer in the form of isopropyl-?-D-1-thiogalactopyranoside (IPTG) with a concentration of 0, 5 mM and incubated in a shaking incubator at 20?C for 12 hours. To find the optimal conditions of Trx-L1 fusion protein expression, an optimization of the concentration of IPTG with concentration of 0.01, 0.05, 0.1, and 0.5 mM as well as optimization of incubation temperatures of 20?C and 16?C. Recombinant protein expression was analyzed using SDS-PAGE, ImageJ software and western blot. The expressed fusion protein was denatured with 6 M Guanidine hydrochloride (GuHCl) and purified with Ni-NTA agarose. The results of colony PCR, restriction analysis, and sequencing of nucleotide base showed that the l1 gene was successfully constructed in the pET-32b(+) vector. SDS-PAGE and western blot analysis showed that the Trx-L1 fusion protein was successfully expressed with a molecular weight of around 70 kDa but the protein was expressed in the form of an inclusion body. The variation of temperature and concentration of IPTG did not significantly influence the expression of Trx-L1 fusion protein. The Trx-L1 fusion protein was successfully purified as shown by the SDS-PAGE band which was about 70 kDa in size. Based on the results obtained, it can be concluded that the l1 gene has been successfully constructed and expressed in E. coli BL21 (DE3), but the protein was still an inclusion body. Purification of the Trx-L1 fusion protein was successfully conducted showed by a protein band of around 70 kDa.