การสกัดและการศึกษาคุณสมบัติ recombinant NS1 protein ของไวรัสไข้เลือดออกจาก Baculovirus Expression Vector System (BEVS)

Purification and Characterization of Recombinant Dengue Viral NS 1 Protein from Baculovirus Expression Vector System (BEVS)

Name: แคทริยา ผิวขาว

Organization : มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

keyword: Dengue virus

; Dengue hemorrhagic fever

; Insect cell

; Recombinant NS 1 protein

Abstract: Recombinant dengue virus (NS 1) protein was produced from Baculovirus ExpressionVector System (BEVS). Insect cells were used as hosts for the production of recombinant NS1protein when infected with recombinant NS1 baculovirus that was engineered by insertion ofNS 1 gene into the baculovirus genome.NS1 protein is a non-structural protein of dengue virus, one of the major cause ofmorbidity and mortality dengue hemorrhagic fever (DHF). The NS1 protein is known tostimulate immune of dengue virus patient. This study focused on purification andcharacterization of the recombinant NS1 protein of dengue virus from the BEVS system.Techniques and knowledge involve in purification and characterization of the recombinant NS 1protein can be used for production of the purified recombinant NS 1 protein for development of anew rapid diagnosis kit for dengue virus detection.Recombinant NS 1 baculovirus was produced by transfection of genetic engineeredbacmid which contained NS1 gene into insect cells (Sf-9). Recombinant NS1 baculovirus wasthen used to infect into insect cells to produce recombinant NS1 protein. The recombinant NSIprotein from infected cells was detected by Western blot analysis using monoclonal antibody(1B2) specific to dengue NS1 protein and pooled convalescent sera that obtained from patientsinfected with dengue virus. Both antibodies identified a recombinant NS1 protein at molecularweight approximately 48 kDa. Optimization of recombinant NS1 protein production especiallyoptimal amounts of recombinant NS1 baculovirus to infect into insect cells and harvest timewere performed. Sf-9 gmwn in early log phase (1x10' cells/ml) were infected with variousamounts of recombinant NSl baculovirus at MOI 0.1, 1, 5 and 10. Maximum recombinant NS 1protein production was obtained when using MOI 1 and optimal time to harvest recombinantNS 1 protein was at 6 days post-infection.Purification of recombinant NS1 protein was performed using a metal affinitycrkrdmatography. The recombinant NSI protein specifically bound to co2+ immobilized resinunder denaturating condition. The purified and renatured recombinant NS1 obtained from a100 ml insect cell culture was 0.36 mg. Recombinant NS1 purified protein is a glycoprotein asconfirmed by ConA-Affinoblotting and N-glycosidase F treatment. In addition, the purifiedrecombinant NS1 protein has been shown to be specifically react with pooled convalescent serabut not with pooled normal sera as demonstrated by Dot blot analysis. These results indicate theprobability of using the recombinant NSl protein for the development of a new dengue virusdiagnostic kit in the future.

มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

Address: กรุงเทพมหานคร (Bangkok)

Email: info.lib@mail.kmutt.ac.th

Name: ดร.กนกวรรณ พุ่มพุทรา

Role: อาจารย์ที่ปรึกษา

Created: 2543

Issued: 2005-10-28

Modified: 2006-05-30

วิทยานิพนธ์/Thesis

ISBN: 9746249118

CallNumber: BIT314

tha

DegreeName: วิทยาศาสตรมหาบัณฑิต

Level: ปริญญาโท

Descipline: เทคโนโลยีชีวภาพ

Grantor: มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

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