KLONING GEN ENVELOPE VIRUS DENGUE TIPE 3 ISOLAT BANDUNG DAN KONSTRUKSI PLASMID REKOMBINAN PPICZ?-A-DENV3-E

• Main Author: Rinaldi, Kardian
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/51972
• Institution: Institut Teknologi Bandung

Currently, it is estimated that 390,000,000 cases of dengue virus infection occur every year worldwide. From January to September 2020, there were 84,734 cases of dengue hemorrhagic fever (DHF) occurring throughout Indonesia. Indonesia is one of the endemic areas for dengue virus infection, so an effort is needed to control dengue virus infection in Indonesia. One of the efforts that can be done is through the development of antibody-based rapid detection kit for dengue infection. The initial stages of developing the kit included the cloning, sub- cloning, and bioinformatics analysis of the dengue virus envelope gene by utilizing local virus sequences. The envelope protein plays a role in the infection mechanism and the formation of neutralizing antibodies so that it can be used as an antigen in antibody-based dengue infection detection methods. Dengue virus has four serotypes, namely DENV-1, DENV-2, DENV-3, and DENV-4, of which DENV-3 is the most common serotype found in Bandung. The objectives of this study were to obtain envelope gene clones of dengue virus type 3 isolated in Bandung, to obtain construction of pPICZ?-A-DENV3-E expression vectors, and to analyze the envelope genes bioinformatics. To obtain the DENV-3 envelope gene, a specific primer was designed for the DENV-3 envelope gene and amplification of the gene was carried out. The purified amplicons were then ligated into the pGEM-T Easy cloning vector and used in the transformation of E. coli TOP10 F 'as the host cell for the cloning process. Based on the verification of recombinant plasmids using the colony PCR method, restriction analysis, and DNA sequencing, the DENV-3 envelope gene was successfully obtained with a size of 1173 bp. Phylogenetic analysis of the DENV-3 envelope gene showed that the analyzed DENV-3 belongs to the genotype V. DENV-3 strain has a high similarity with DENV-3 virus isolated from the United States, the Philippines, and China. The results of the amino acid composition analysis showed that the protein translated from the E DENV-3 gene isolated in Bandung consisted of 152 aliphatic nonpolar amino acid residues, 25 nonpolar aromatic amino acid residues, 115 uncharged polar amino acid residues, 54 positively charged amino acid residues , and 45 negatively charged amino acid residues. The results of the secondary structure prediction analysis showed that the dominant conformation of the protein translated from the E DENV-3 gene isolated in Bandung were turn / coil and sheet conformations. The DENV-3 envelope gene that has been restricted from the pGEM-T Easy vector was ligated to the pPICZ?-A expression vector to obtain the recombinant plasmid pPICZ?-A-DENV3-E. Recombinant plasmids were confirmed using colony PCR method, restriction analysis, and DNA sequencing. The recombinant plasmid pPICZ?-A-DENV3-E can then be used for the expression of protein E in Pichia pastoris.