ผลของ Serial Passages ต่อการผลิตแบคคิวโลไวรัส (HaNPV) ในเซลล์แมลง

Name: กุลธิดา พลทวี

Organization : มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

keyword: Baculovirus

; Serial Passages

; Passage Effect

; Two-dimensional Polyacrylamide Gel Electrophoresis

Abstract: Serial passaging of baculoviruses in cell culture leads to rapidly accumulating mutant, also knownas “the passage effect,” which result in changing of virus efficacy. The effect of viral passagingvaries between different types of baculovirus. This study aimed to investigate the effect of serialpassages on baculovirus (Th-HaNPV) productivity in insect cell culture (Hz cell line) and todetermine the overall protein expression (proteome) changes induced by serial passage ofbaculoviruses.The viral efficacy on infection in cell culture and production of extracellular virus (ECV) andocclusion body (polyhedra) were determined throughout ten serial passages. The results showed thatthe percentage of infected cells producing polyhedra decreased to 23% at passage 10, compared tothat of passage 3. The ECV titers increased gradually from passage 2 to 5. The highest ECV titerswas obtained at passage 5 (1.6x1?????9 pfu/ml). From passage 6 onwards, there was a gradual reductionin ECV production. The number of polyhedra produced per cell decreased from 51 polyhedra percell at passage 3 to 5 polyhedra per cell at passage 10, representing a 90% reduction in yield. Theseresults indicated that the viral productivity decreased rapidly during serial passages of Th-HaNPV incell culture and this was similar to HaSNPV and LdMNPV. The information obtained from thisstudy can be applied for baculovirus (Th-HaNPV) production such as serial passage of HaNPVshould not be performed more than 6 passages since very low viral productivity will be obtained.Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to determine the changeof overall proteins expression of extracellular virus (ECV). ECV was obtained from virus stock atpassage 2 (reference passage) passage 5 (high ECV titers) and passage 10 (low ECV titers). Theresults showed that the serial passages resulted in protein profiles changes which could be detectedby 2D-PAGE. Some proteins which have been previous reported to be involved in transcriptionallytransactivating viral gene and augmenting viral DNA replication such as PE38 and 25K FP protein,which was essential for polyhedron formation and viral occlusion, were found to be changedaccording to their efficacy in this study. Moreover, other proteins of baculovirus (HaNPV) which,has not been previously reported, were detected by this technique and may be used as marker protein to predict the viral efficacy.

มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

Address: กรุงเทพมหานคร (Bangkok)

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

Name: ดร. กนกวรรณ พุ่มพุทรา

Role: อาจารย์ที่ปรึกษา

Created: 2546

Issued: 2005-11-08

Modified: 2006-05-09

วิทยานิพนธ์/Thesis

ISBN: 9744563427

CallNumber: BIT460

tha

DegreeName: วิทยาศาสตรมหาบัณฑิต

Level: ปริญญาโท

Descipline: เทคโนโลยีชีวภาพ

Grantor: มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

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