Makara Seri Sains (Nov 2011)
CLONING OF DENGUE VIRUS TYPE 3 (INDONESIAN STRAIN D3-1703) NON STRUCTURAL-1 GENE INTO pYES2/CT VECTOR
Abstract
Dengue is an infectious disease caused by dengue virus. Dengue endemic region includes America, Western Pacific,Africa, East Mediterranian, and South East Asia including Indonesia. An early diagnostic system specific for Indonesiais needed to control dengue in Indonesia. In this research, cloning of Non Structural 1 (NS1) gene from dengue virustype 3 (Indonesian strain D3-1703) into pYES2/CT vector was performed. In the long run, NS1 recombinant proteinwill be expressed in Saccharomyces cerevisiae for diagnostic materials. Polymerase Chain Reaction (PCR)amplification of NS1 gene fragments were done with optimal annealing temperature at 55 ºC. NS1 gene fragment andpYES2/CT were cut by BamH I and Not I enzymes. The digested pYES2/CT was dephosphosrylated using CalfIntestine Alkaline Phospatase enzyme. Ligation with the vector:insert ratio of 1:12 and 1:20 resulted in 6 and 5recombinant colony candidates respectively. Restriction enzyme and PCR verifications showed that 5 recombinantplasmids contained NS1 gene. Sequencing of the first 600 bp of one recombinant plasmid was performed. The blastnanalysis showed that it had a 99% identity with dengue virus type 3 strain FW06. Finally, it was shown that NS1 clonewithin pYES2/CT was in the correct Open Reading Frame and ready to be expressed in S. cerevisiae
