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研究生:林雪芳
研究生(外文):Hsueh-Fang Lin
論文名稱:利用核醣體去氧核糖核酸之內轉錄區間鑑定昆蟲細胞株和細胞株的交互污染
論文名稱(外文):Identification of insect cell lines and cell line cross-contamination (CLCC) by internal transcribed spacer region of ribosomal DNA
指導教授:王重雄
指導教授(外文):Chung-Hsiung Wang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:昆蟲學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:64
中文關鍵詞:昆蟲細胞株內轉錄區間核醣體去氧核糖核酸細胞株交互污染懸浮培養
外文關鍵詞:insect cell linesinternal transcribed spacer (ITS)ribosomal DNA (rDNA)cell line cross-contamination (CLCC)suspension culture
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昆蟲細胞株 (insect cell lines) 為研究工業化之體外 (in vitro) 增殖病毒或生產重組蛋白的有效工具,特別適用於昆蟲桿狀病毒表現載體 (baculovirus expression vector system, BEVS)。就多數的細胞培養研究室而言,昆蟲細胞被經常性的使用、操作和維持時,需要定期的偵測和鑑定,以避免發生細胞株交互污染 (cell line cross-contamination, CLCC) 的事件。因此我們利用分子標示法,針對核醣體去氧核糖核酸 (ribosomal DNA, rDNA; or nuclear rDNA) 之內轉錄區間 (internal transcribed spacer, ITS),進行昆蟲細胞株 (NTU-LY, NTU-PN, IPLB-LD-652Y, and Sf9 cell lines) 之種源鑑定和細胞株交互污染 (Cell line cross-contamination, CLCC) 的檢測。係以增幅的 ITS 加上 5.8S 區間,若蟲體與其同源細胞株的序列相似度 (identity) 高達 95% 至 99% 者,如此這些被增幅的複製子序列 (amplicon’s sequences),可用於證實該細胞株的物種來源。根據這些增幅的複製子序列,配合兩種限制酶,PstI 和 HindII 的篩選,利用聚合酶連鎖反應—限制酶片段長度多態型 (PCR-restriction fragment length polymorphism, PCR-RFLP) 的技術,建立一套快速鑑定昆蟲細胞株的方法。同時,也透過DNA 逢機增幅多態型聚合酶連鎖反應 (random amplified polymorphic DNA-PCR, RAPD-PCR) 的分析,利用隨機的核苷酸引子—OPU-10,進行本研究之細胞株的篩選和分析。另外,設計了各別的專一性引子對:Ly-ITS1/Ly-ITS2、ITS1-1/Ld-ITS1 及 Sf9-F2/ITS4,分別針對 LY、LD 及 Sf9 等三細胞株,進行種特異性聚合酶連鎖反應 (species-specific polymerase chain reaction, SS-PCR) 的鑑定。上述的三個分子標示法:PCR-RFLP、RAPD-PCR 和 SS-PCR的技術輔助確認,可鑑別細胞種源和避免發生細胞株交互污染。除此之外,在確認 NTU-LY 細胞株的種源無誤,並無發生細胞株交互污染後,則利用細胞株進行大量生產的工作。之後透過昆蟲細胞的懸浮培養,進行昆蟲病毒的產量工作,最適條件分別為:細胞密度為 1 x 106 cell/ml,培養總體積為 200 ml,轉速為 65 rpm,培養於 28℃。透過懸浮培養量病毒的平均每顆細胞的黑角舞蛾核多角體包體產量 (LyxyNPV OBs),高達 70.5 ~ 97 OBs/cell,遠高於單層培養的 27.7 OBs/cell。
Insect cell lines are useful tools in the study of virus basic biology and production of recombinant proteins, especially by baculovirus expression vector system (BEVS). In most cell culture laboratories, several different cell lines are routinely used, maintained, or handled, these cell lines should be regularly monitored to avoid cell line cross-contamination (CLCC). We developed a technique for identification of species origin and CLCC detection for insect cell lines (NTU-LY, NTU-PN, IPLB-LD652Y, and Sf9 cell lines) based on the sequence of internal transcribed spacer (ITS) of ribosomal DNA (rDNA). The identity of the amplicon’s sequences between insect and its homologous cell line was up to 95 %~99 %, thus these amplicon’s sequences could be used to confirm the species origin of a cell line. Two endonucleases, PstI and HindII based on these sequences of amplicons, were selected for rapid identification of insect cell lines by PCR-RFLP (PCR-restriction fragment length polymorphism). The homologous cell lines could be examined by RAPD-PCR (random amplified polymorphic DNA – PCR) method with a selected oligonucleotide primer, OPU-10. Three species-specific primer sets, Ly-ITS1/Ly-ITS2, ITS1-1/Ld-ITS1, and Sf9-F2/ITS4, were then designated for identification of LY, LD, and Sf9 cells, respectively, by SS-PCR (species-specific PCR). Therefore three methods, RFLP-PCR, RAPD and SS-PCR, can be used to define cell origin and avoid CLCC. Otherwise, the mass production of NTU-LY cells was also undertaken by suspension culture after confirmations of cell origin and free from CLCC. The optimal condition of LY cell suspension culture was 1 x 106 cells/ml, in 200 ml culture volume and 65 RPM at 28℃. The LyxyNPV OBs (occlusion bodies) production reached to 70.5 ~ 97 OBs/cell that was much higher than monolayer infection (27.7 OBs/cell).
目 錄
中文摘要 …………………………………………………………………i
英文摘要 ………………………………………………………………iii
目錄………………………………………………………………………v
表次……………………………………………………………………viii
圖次………………………………………………………………………ix
壹、緒言…………………………………………………………………1
貳、往昔研究 ……………………………………………………………5
參、材料與方法…………………………………………………………16
一、供試之幼蟲蟲體和細胞株…………………………………………16
二、基因組 DNAs 萃取與純化…………………………………………16
三、DNA 片段增幅………………………………………………………17
四、DNA 定序……………………………………………………………17
五、序列分析……………………………………………………………18
六、聚合酶連鎖反應-限制酶片段長度多態型………………………18
七、去氧核糖核酸逢機增幅多態型聚合酶連鎖反應…………………18
八、同功異構酶…………………………………………………………19
九、聚合酶連鎖反應序列特異性引子…………………………………19
十、細胞次選殖…………………………………………………………20
(一) 細胞型態…………………………………………………………20
(二) 細胞倍增時間……………………………………………………21
(三) 病毒性細胞病變…………………………………………………21
(四) 細胞對病毒感受性………………………………………………22
十一、細胞株交互污染對細胞之病毒接受力的影響…………………23
十二、懸浮培養…………………………………………………………24
十三、病毒量產…………………………………………………………24
肆、結果…………………………………………………………………26
一、聚合酶連鎖反應.…………………………………………………26
二、聚合酶連鎖反應-限制酶片段長度多態型………………………26
三、去氧核糖核酸逢機增幅多態型聚合酶連鎖反應…………………27
四、利用同功異構酶測試細胞株交互污染……………………………28
五、聚合酶連鎖反應序列特異性引子…………………………………28
六、細胞株交互污染對細胞之病毒接受力的影響……………………29
七、細胞次選殖…………………………………………………………29
(一) 細胞型態…………………………………………………………29
(二) 細胞倍增時間……………………………………………………30
(三) 細胞對病毒感受性………………………………………………30
八、懸浮培養……………………………………………………………31
九、量產昆蟲病毒………………………………………………………31
伍、討論…………………………………………………………………32
一、核醣體去氧核糖核酸之內轉錄區間………………………………32
二、細胞株交互污染……………………………………………………36
三、病毒感染……………………………………………………………39
四、細胞次選殖…………………………………………………………40
五、懸浮培養……………………………………………………………41
陸、結論…………………………………………………………………44
柒、參考文獻……………………………………………………………45
捌、誌謝…………………………………………………………………66
玖、圖表…………………………………………………………………68
壹拾、附錄………………………………………………………………90

表次
表一、增幅 rDNA 使用之 PCR 引子序列……………………………68
表二、幼蟲蟲體和細胞株之部份 18S rDNA 加 ITS 區段之相似度.69
表三、限制酶HindII 和 PstI酶切部份18S rDNA 加ITS 區段 PCR-RFLP結果…………………………………………………………………70
表四、RAPD-PCR 電泳圖譜,以內差法計算片段大小………………71
表五、供試黑角舞蛾之四個次選殖株,各細胞型態大小尺寸(μm)72
表六、利用黑角舞蛾核多角體病毒 2 號及黑角舞蛾 5 號多角體分離病毒株,分別感染黑角舞蛾1 號細胞,進行單層細胞培養和 200 毫升懸浮培養的病毒感染後,第 14 天之多角體產量的測試…………73

圖次
圖一、rDNA 之結構和增幅 ITS 區段所使用各引子對………………74
圖二、以限制酶 HindII (A) 和 PstI (B) 酶切部份18S rDNA 加ITS 區段之 PCR-RFLP 結果……………………………………………75
圖三、針對黑角舞蛾和四株次選殖的同源細胞株 (NTU-1 to -4) (A, C, D),和榕樹透翅毒蛾的兩株同源細胞株 (NTU-PN-HH, -HF) (B, E, F, G, H),透過 OPU-03 (C, E)、-06 (D)、-09 (F)、-10 (A, B)、-19 (G) 和 OPBD-06 (H) 等之逢機引子進行 RAPD 增幅.76
圖四、利用三種昆蟲細胞株(NTU-LY-1、IPLB-LD-652Y、Sf9),與各別混合其中二種昆蟲細胞株之同功異構酶圖譜………………………78
圖五、利用種特異性引子對,針對不同的細胞株之部份 ITS 區段,進行 PCR 增幅的結果…………………………………………………79
圖六、利用種特異性引子對,針對不同種混合的細胞之部份 ITS 區段,進行 PCR 增幅的結果……………………………………………81
圖七、利用核多角體病毒感染不同細胞株 (NTU-LY-1、IPLB-LD-652Y、Sf9),兩週後含有包含體之感染細胞之比例…………………83
圖八、黑角舞蛾細胞株的四個次選殖株 (NTU-LY-1, -31, -32, -4 cells) 之不同型態佔有細胞的百分比………………………………84
圖九、黑角舞蛾細胞的四個次選殖株 (NTU-LY-1, -31, -32, -4 cells),在20℃(黃色) 和 28℃ (綠色) 培養溫度下之細胞倍增時間………………………………………………………………………85
圖十、黑角舞蛾細胞的四個次選殖株 (NTU-LY-1, -31, -32, -4 cells),培養於恆溫 28℃ 下,以含 4 種不同血清濃度培養液 (0, 4, 8, 16%) 培養之細胞倍增時間……………………………………86
圖十一 黑角舞蛾細胞的四個次選殖株 (NTU-LY-1, -31, -32, -4 cells),培養於不同血清濃度下 (0, 4, 8, 16%) 之多角體產量 (M.O.I.=1)……87
圖十二 黑角舞蛾細胞株 (NTU-LY-1) 之 100 ml懸浮培養,在各種轉速細胞數之變化…………………………………………………………88
圖十三 吉普賽舞蛾細胞株 (IPLB-LD-652Y) 之懸浮培養,在各種不同轉速之細胞數變化……………………………………………………89

附錄
附錄一、供試之幼蟲體和細胞株………………………………………90
附錄二、同功異構酶之電泳與染色方法………………………………91
附錄三、細胞倍增時間…………………………………………………96
附錄四、半數細胞感染量測定法………………………………………97
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