EXPRESSION AND PURIFICATION OF DENV3-NS1 RECOMBINANT PROTEIN OF DENGUE VIRUS IN Pichia pastoris KM71
Dengue fever is an epidemic disease caused by dengue virus in the tropics and subtropics in which Indonesia is a country with the highest dengue fever case in Southeast Asia. There is no medicine and licensed vaccine yet for dengue disease, hence an early diagnose is highly required for early treatm...
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| Format: | Theses |
| Language: | Indonesia |
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| Online Access: | https://digilib.itb.ac.id/gdl/view/38242 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Dengue fever is an epidemic disease caused by dengue virus in the tropics and subtropics in which Indonesia is a country with the highest dengue fever case in Southeast Asia. There is no medicine and licensed vaccine yet for dengue disease, hence an early diagnose is highly required for early treatment of the dengue infected patients. Currently, several commercial diagnostic kits are available, however they are all imported, and unfortunately the kits were developed using foreign dengue virus nucleotide sequences. The objectives of this research were to produce and to purify the DENV-3 NS1 recombinant protein derived from Indonesian dengue virus nucleotide sequence. A recombinant plasmid pPICZ?A- DENV3-NS1 which had a synthetic gene encoding for NS1 protein from dengue virus serotype 3 (DENV-3) was used to transform Pichia pastoris KM71. The DENV3-NS1 gene was inserted between AOX1 promoter and AOX1 transcription terminator. DENV3-NS1 gene together with the whole plasmid was integrated into the P. pastoris chromosome through single cross over homologous recombination in the AOX1 locus. The selection of P. pastoris transformants carrying DENV-3 NS1 was undertaken by colony PCR using a pair of AOX1 primers. In this recombinant plasmid, the DENV-3 NS1 coding sequence was fused with an oligonucleotide for pre-pro-? factor signal peptide to allow the formation of DENV-3 NS1 which can be secreted into the culture medium. The DENV-3 NS1 expression was induced by 3% (v/v) methanol for 72 hours at 30
oC. The presence of DENV-3 NS1 recombinant protein in the culture medium was
analyzed by SDS-PAGE. The DENV-3 NS1 was found as an extracellular protein
with molecular size of ~43 kDa, and interestingly the protein was also detected intracellularly. Furthermore, both the extracellular and intracellular DENV-3
NS1s were purified by Ni-NTA affinity chromatography with protein yield of 0.7 mg/L and 16.1 mg/L, respectively. Since the purified DENV-3 NS1 was found to be able to interact with anti-NS1 monoclonal antibodies in commercial diagnostic kit Dengue ICT strip, hence the resulted DENV-3 NS1 could be used for the development of dengue diagnostic kit.
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