CLONING AND EXPRESSION OF NON - STRUCTURAL 1 (NS1) GENE DENGUE VIRUS SEROTYPE 4 I N ESCHERICHIA COLI

• Main Author: RAMDHAN ANSHARI NIM: 10514047, ZAID
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/31876
• Institution: Institut Teknologi Bandung

iii <br /> <br /> <br /> <br /> Abstra <br /> <br /> <br /> <br /> ct <br /> <br /> <br /> <br /> Indonesia has the second <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> highest epidemic dengue in the <br /> <br /> <br /> <br /> w <br /> <br /> <br /> <br /> orld. Health Ministry of Republic <br /> <br /> <br /> <br /> Indonesia <br /> <br /> <br /> <br /> reported that <br /> <br /> <br /> <br /> there <br /> <br /> <br /> <br /> was <br /> <br /> <br /> <br /> 0,67% <br /> <br /> <br /> <br /> mortality rates <br /> <br /> <br /> <br /> in January <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> May 2017 <br /> <br /> <br /> <br /> . <br /> <br /> <br /> <br /> An <br /> <br /> <br /> <br /> Accurate <br /> <br /> <br /> <br /> and rapid <br /> <br /> <br /> <br /> diagnosctic test <br /> <br /> <br /> <br /> is needed to <br /> <br /> <br /> <br /> allow <br /> <br /> <br /> <br /> the proper handling <br /> <br /> <br /> <br /> of the <br /> <br /> <br /> <br /> dengue virus infect <br /> <br /> <br /> <br /> ed <br /> <br /> <br /> <br /> patients. One of the important component in diagnostic test is NS1 protein. NS1 protein is an <br /> <br /> <br /> <br /> antigen that can interact with IgG and IgM antibody in dengue virus infect <br /> <br /> <br /> <br /> ed <br /> <br /> <br /> <br /> patients. NS1 <br /> <br /> <br /> <br /> protein ha <br /> <br /> <br /> <br /> s <br /> <br /> <br /> <br /> hydrophob <br /> <br /> <br /> <br /> ic <br /> <br /> <br /> <br /> area <br /> <br /> <br /> <br /> about <br /> <br /> <br /> <br /> ~31%, therefore <br /> <br /> <br /> <br /> it has low <br /> <br /> <br /> <br /> solubility <br /> <br /> <br /> <br /> w <br /> <br /> <br /> <br /> hen expressed in <br /> <br /> <br /> <br /> Escherichia coli <br /> <br /> <br /> <br /> . <br /> <br /> <br /> <br /> T <br /> <br /> <br /> <br /> he s <br /> <br /> <br /> <br /> olubility of DENV <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> 4 <br /> <br /> <br /> <br /> NS1 <br /> <br /> <br /> <br /> can be improved by fusion with thioredoxin <br /> <br /> <br /> <br /> protein. <br /> <br /> <br /> <br /> The purposes of this research were to <br /> <br /> <br /> <br /> construct pET32b <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> DENV4 <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> NS1 recombinant <br /> <br /> <br /> <br /> plasmid <br /> <br /> <br /> <br /> and <br /> <br /> <br /> <br /> to <br /> <br /> <br /> <br /> produc <br /> <br /> <br /> <br /> e <br /> <br /> <br /> <br /> DENV <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> 4 <br /> <br /> <br /> <br /> NS1 <br /> <br /> <br /> <br /> recombinant protein <br /> <br /> <br /> <br /> in <br /> <br /> <br /> <br /> Escherichia coli <br /> <br /> <br /> <br /> . A <br /> <br /> <br /> <br /> gene <br /> <br /> <br /> <br /> fragment of <br /> <br /> <br /> <br /> 1,059 base pair (bp) encoding DENV4 <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> NS1 <br /> <br /> <br /> <br /> was amplified by PCR technique <br /> <br /> <br /> <br /> using pET16b <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> DENV4 <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> NS1 as a DNA template. The DNA fragment encoding DENV4 <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> NS1 <br /> <br /> <br /> <br /> was <br /> <br /> <br /> <br /> subcloned <br /> <br /> <br /> <br /> into <br /> <br /> <br /> <br /> pE <br /> <br /> <br /> <br /> T32b <br /> <br /> <br /> <br /> expression vector <br /> <br /> <br /> <br /> between <br /> <br /> <br /> <br /> restriction site <br /> <br /> <br /> <br /> s <br /> <br /> <br /> <br /> of <br /> <br /> <br /> <br /> Eco <br /> <br /> <br /> <br /> RI and <br /> <br /> <br /> <br /> Xho <br /> <br /> <br /> <br /> I to <br /> <br /> <br /> <br /> obtain a 6,931 bp recombinant plasmid pET32b <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> DENV4 <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> NS1. <br /> <br /> <br /> <br /> E. coli <br /> <br /> <br /> <br /> BL21(DE3) was <br /> <br /> <br /> <br /> transformed by pET32b <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> DENV4 <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> NS1 using heat shock method <br /> <br /> <br /> <br /> and was <br /> <br /> <br /> <br /> then <br /> <br /> <br /> <br /> selected on LB <br /> <br /> <br /> <br /> media <br /> <br /> <br /> <br /> containing <br /> <br /> <br /> <br /> ampicil <br /> <br /> <br /> <br /> lin. <br /> <br /> <br /> <br /> E. coli <br /> <br /> <br /> <br /> BL21(DE3)/pET32b <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> DENV4 <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> NS1 transformant was <br /> <br /> <br /> <br /> grown in <br /> <br /> <br /> <br /> 2x YT <br /> <br /> <br /> <br /> media <br /> <br /> <br /> <br /> (tryptone 1.6%, YE 1%, <br /> <br /> <br /> <br /> and NaCl 0.5%) <br /> <br /> <br /> <br /> and <br /> <br /> <br /> <br /> IPTG <br /> <br /> <br /> <br /> 0, <br /> <br /> <br /> <br /> 2 mM <br /> <br /> <br /> <br /> was then <br /> <br /> <br /> <br /> added to induce the expression of <br /> <br /> <br /> <br /> DENV4 <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> NS1 gene <br /> <br /> <br /> <br /> for <br /> <br /> <br /> <br /> 4 hours at 37 <br /> <br /> <br /> <br /> o <br /> <br /> <br /> <br /> C <br /> <br /> <br /> <br /> . SDS <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> PAGE <br /> <br /> <br /> <br /> a <br /> <br /> <br /> <br /> nalysis <br /> <br /> <br /> <br /> showed <br /> <br /> <br /> <br /> that <br /> <br /> <br /> <br /> Trx <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> DENV4 <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> NS1 <br /> <br /> <br /> <br /> fusion <br /> <br /> <br /> <br /> protein is produced with molecular mass of ~ <br /> <br /> <br /> <br /> 55 <br /> <br /> <br /> <br /> kDa. <br /> <br /> <br /> <br /> The DENV <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> 4 NS1 aggregat was unfolded in 8M urea with recovery of about 100% <br /> <br /> <br /> <br /> . <br /> <br /> <br /> <br /> The <br /> <br /> <br /> <br /> unfolded DENV <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> 4 NS1 was <br /> <br /> <br /> <br /> then <br /> <br /> <br /> <br /> refolded in Ni <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> NTA column chromatography by serial <br /> <br /> <br /> <br /> dilution step. <br /> <br /> <br /> <br /> The refolded <br /> <br /> <br /> <br /> Trx <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> DENV <br /> <br /> <br /> <br /> 4 <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> NS1 fusion protein was <br /> <br /> <br /> <br /> digested with <br /> <br /> <br /> <br /> thrombin <br /> <br /> <br /> <br /> to <br /> <br /> <br /> <br /> remove Trx from the fusion protein. <br /> <br /> <br /> <br /> SDS <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> PAGE analysis <br /> <br /> <br /> <br /> s <br /> <br /> <br /> <br /> howed <br /> <br /> <br /> <br /> that <br /> <br /> <br /> <br /> the resulted <br /> <br /> <br /> <br /> DENV <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> 4 <br /> <br /> <br /> <br /> NS1 <br /> <br /> <br /> <br /> has <br /> <br /> <br /> <br /> molecular mass of ~45 kDa. <br /> <br /> <br /> <br /> The recombinant <br /> <br /> <br /> <br /> DENV <br /> <br /> <br /> <br /> - <br /> <br /> <br /> <br /> 4 <br /> <br /> <br /> <br /> NS1 protein <br /> <br /> <br /> <br /> recognize <br /> <br /> <br /> <br /> s <br /> <br /> <br /> <br /> NS1 <br /> <br /> <br /> <br /> monoclonal antibody <br /> <br /> <br /> <br /> on <br /> <br /> <br /> <br /> the <br /> <br /> <br /> <br /> commerc <br /> <br /> <br /> <br /> ial <br /> <br /> <br /> <br /> NS1 diagnostic kit test