การสกัดและการศึกษาคุณสมบัติของ recombinant dengue envelope protein ที่ผลิตได้จาก Baculovirus Expression Insect Cell Systems

Purification and Characterization of Recombinant Dengue Envelope Protein from Baculovirus Expression Insect Cell Systems

Name: สิริรัตน์ อนันตวรสกุล

Organization : มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

keyword: Dengue virus

; Dengue hemorrhagic fever

; Baculovirus

; Insect cell

; Recombinan

Abstract: Envelope protein is a structural protein of Dengue virus, a major cause of pediatric morbidity and mortality dengue hemorrhagic fever (DHF). This antigenic protein contains epitopes that induced neutralizing, hemagglutinin antibodies and elicited protective immune response in dengue virus patient. This study focused on purification and characterization of recombinant envelope protein produced from Baculovirus Expression Insect Cell System that is aimed to be used as protein antigen for diagnosis of dengue infection.In the Baculovirus Expression Insect Cell System, insect cells were used as host for the production of recombinant envelope protein when being infected by genetic engineered recombinant envelope baculovirus that dengue envelope gene was inserted into baculovirus genome. The recombinant envelope protein was detected in infected cells by Western blot analysis using monoclonal antibody (3H5) specific to dengue envelope protein. The recombinant envelope protein at molecular mass of approximately 57.8 kDa was identified. When compared its molecular weight with recombinant envelope protein produced by E. coli (55.3 kDa), it was suggested that recombinant envelope protein from insect cells might be a glycosylated protein.Purifications of recombinant envelope protein were performed using cation exchange chromatography and metal ion affinity chromatography (IMAC). Recombinant envelope protein bound to cation exchanger under native condition and could be eluted by elute buffer contained 0.25 M NaCl. Recombinant envelope protein was found to bind to Co2+ immobilized resin (IMAC) under denaturing condition and could be eluted by elute buffer contained 0.2 M imidazole. These results suggested that recombinant envelope protein could therefore be partially purified by both methods. The recombinant envelope protein is a glycoprotein as confirmed by N-glycosidase F treatment. The stability of recombinant envelope protein kept at -20?C (in infected Sf-9 cells) for 0-6 months was examined. After 2 months, the stability of recombinant envelope protein declined, to the level of 71% within 6 months. The interactions between recombinant envelope protein and monoclonal antibodies, specific to various epitopes of dengue envelope protein were studied. The recombinant envelope protein from insect cell was shown to be a better antigen than the recombinant envelope protein from bacteria. In addition, the recombinant envelope protein from insect cell was shown to be specifically reacted with pooled convalescent sera but not with pooled normal sera as demonstrated by dot blot analysis. These results indicated the possibility of using the recombinant envelope protein for the development of a new dengue virus diagnostic kit in the future.

มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

Address: กรุงเทพมหานคร (Bangkok)

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

Name: ดร. กนกวรรณ พุ่มพุทรา

Role: อาจารย์ที่ปรึกษา

Created: 2545

Issued: 2005-09-20

Modified: 2006-05-10

วิทยานิพนธ์/Thesis

ISBN: 974963606X

CallNumber: BIT410

tha

DegreeName: วิทยาศาสตรมหาบัณฑิต

Level: ปริญญาโท

Descipline: เทคโนโลยีชีวภาพ

Grantor: มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

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