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研究生:黃鳴捷
研究生(外文):Ming-Chieh Hung
論文名稱:利用無血清培養液於昆蟲桿狀病毒操作系統中表現分泌性的重組蛋白質
論文名稱(外文):Production of secreted recombinant proteins in serum-free medium by baculovirus expression system
指導教授:蘇建國
指導教授(外文):Jyan-Gwo Su
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:55
中文關鍵詞:桿狀病毒無血清
外文關鍵詞:baculovirusserum-free
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利用無血清培養液於昆蟲桿狀病毒操作系統中表現分泌性的重組蛋白質
中文摘要
桿狀病毒是一種廣泛存在於環境中的病毒,然而其宿主專一性高,只會感染節肢動物,安全性高,所以常用來作為生物防治之生物農藥等應用。另外桿狀病毒有獨特的感染週期,所以使用其末期基因的啟動子來表現重組蛋白質核多角蛋白質及p10蛋白質。另外,由於昆蟲細胞一般皆培養於有胎牛血清的培養液中,成本較高,且血清有可能干擾到重組蛋白質的純化,若要使用市面上所售之無血清培養液則成本更高,所以本實驗使用低濃度或不加胎牛血清的Grace 培養液,來培養昆蟲細胞,以求降低製造重組蛋白質的成本。
本論文中以桿狀病毒操作系統表現分泌性蛋白質Resistin及非分泌性的蛋白質BIG-3,以作為測試的對象。實驗之結果顯示低濃度或不含胎牛血清的Grace 培養液也能表現重組蛋白質Resistin及BIG-3。並且於0﹪、1﹪的低濃度血清中,Resistin約為病毒感染後60小時表現量較多,而BIG-3則是於病毒感染後24~36小時表現量較多,另外為了將來在培養液內能直接得到純化的蛋白質,故使用不同容量的無血清培養液於T-75 flask中表現重組蛋白質,最低能表現蛋白質的容量為2ml.
所以綜合以上的結果可知,利用桿狀病毒操作系統表現重組蛋白質時,可將完整含胎牛血清的Grace 培養液換成低濃度或無血清的Grace 培養液,並且仍然可表現目標蛋白質。

Production of secreted recombinant proteins in serum-free medium by baculovirus expression system
Abstract
Baculoviruses are virtually ubiquitous in the environment. They specifically infect Arthropods and have never been found to cause disease in any organism outside the phylum Arthropods. Therefore, it is safe to use baculovirus as bio-pesticides. In addition, baculovirus has a special life cycle for its infection and it’s late promoters, polyhedrin and p10, are useful for large expression of recombinant proteins.
Insect cell has been cultured in the complete Grace medium containing 10﹪fetal bovine serum (FBS). However, there are two disadvantages: (1) the FBS is expensive and (2) FBS is likely to interfere the purification procedure of recombinant proteins. In order to avoid these disadvantages, we would like to test whether the insect cells still produce recombinant protein largely in the culture using serum-free or low-serum Grace medium, instead of using commercial serum-free medium.
In this dissertation, expressions of resistin and BIG-3 in baculovirus expression system were used as samples for the test of culture conditions. The results indicated that cell cultured in both low-serum and serum-free Grace medium still can produce resistin and BIG-3. The quantity of recombinant resistin and BIG-3 are higher at 60 hours and 24~36 hours, in contrast, after infection in the cells cultured in serum-free Grace medium and medium with 1% serum. In order to obtain the concentrated recombinant secreted protein in the medium, the infected cells were cultured in T-75 flask with different volume of serum-free medium. It showed that the recombinant proteins were still produced; even cells were cultured with 2 ml medium.
According to the experimental results, we can have the conclusion that, in baculovirual expression system, insect cells still can produce recombinant proteins when they were cultured in Grace medium with non-serum or littler serum, instead of with 10% serum.

目錄
中文摘要.………………………………………………………………I
英文摘要.………………………………………………………………III
目錄…………………………………………………………………….V
圖目錄…………………………………………………………………VII
一、緒論………………………………………………………………..1
1.1桿狀病毒的分類與特性……………………………………….1
1.2 桿狀病毒的感染週期………………………………………….2
1.3 桿狀病毒操作系統簡介……………………………………….3
1.4質體的構築………………………………………………………4
1.5實驗目的…………………………………………………………4
二、材料………………………………………………………………….6
2.1 藥品…………………………………………………………….6
三、方法…………………………………………………………………8
3.1 構築含BIG-3的轉移質體…………………………………….8
3.1.1小量抽取質體DNA………………………………..8
3.1.2 限制酵素處理……………………………………..9
3.1.3 電泳並回收DNA片段……………………………9
3.1.4 接合反應………………………………………….10
3.1.5 形質轉移…………………………………………11
3.1.6 DNA定序………………………………………...12
3.2桿狀病毒操作系統……………………………………………12
3.2.1昆蟲細胞(SF-9)培養………………………………12
3.2.2共同轉染昆蟲細胞……………………………….13
3.2.3 噬菌斑測試……………………………………….13
3.2.4製造高濃度的重組病毒液………………………..14
3.2.5病毒濃度測定……………………………………..15
3.2.6以不同濃度的胎牛血清培養細胞並進行蛋白質表現測試…………………………………………...15
3.2.7 蛋白質電泳和西方點墨法……………………….16
3.2.8 以收Time course的方法檢測被重組病毒感染後各時期蛋白質表現情形………………………17
3.2.9 以不同容量的培養液培養細胞並作蛋白質表現及檢測……………………………………………...18
四、結果…………………………………………………………………20
五、討論…………………………………………………………………22
六、參考文獻…………………………………………………………...23
附錄……………………………………………………………………..52
圖目錄
圖一 BIG-3基因片段構築流程………………………………………29
圖二 SF-9細胞,病毒感染前後細胞病變的情形…………………..30
圖三 Plaque assay 選殖重組病毒圖………………………………….31
圖四 plaque assay選殖重組病毒照片………………………………..32
圖五 以SDS-PAGE 檢測在不同濃度血清下BIG-3表現情形…….33
圖六 以SDS-PAGE 檢測在不同濃度血清下Resistin表現情形…..34
圖七 以西方點墨法檢測在不同濃度血清下Resistin表現情形……35
圖八 以無血清培養液及含血清培養液培養感染BIG-3、Resistin及
β-gal病毒之SF-9細胞並檢測蛋白質表現情形……………36
圖九 以Time course的方法,檢測在1﹪血清培養液下BIG-3蛋白質表現情形……………………………………………………….38
圖十 以Time course的方法,檢測在0﹪血清培養液下BIG-3蛋白質表現情形…………………………………………………….40
圖十一 以Time course的方法,檢測在1﹪血清培養液下Resistin蛋白質表現情形……………………………………………….42
圖十二 以西方點墨法檢測在1﹪血清培養液下Resistin蛋白質表現情形…………………………………………………………….44
圖十三 以Time course的方法,檢測在0﹪血清培養液下Resistin蛋白質表現情形………………………………………………46
圖十四 以西方點墨法檢測在0﹪血清培養液下Resistin蛋白質表現情形……………………………………………………………48
圖十五 以SDS-PAGE檢測在不同容量的無血清培養液下Resistin表現情形……………………………………………………….50
圖十六 以西方點墨法檢測在不同容量的無血清培養液下Resistin表現情形……………………………………………………….51

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