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研究生:羅鴻基
論文名稱:TAT-HA-RhoA融合突變蛋白的表現、純化、及其在血管平滑肌細胞之胞骨骼重組的角色
論文名稱(外文):The expression and purification of TAT-HA-RhoA mutant fusion proteins and^^preliminary characterization of their roles in cytoskeletal reorganization of vascular smooth muscle cells
指導教授:江美治江美治引用關係
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
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
中文關鍵詞:TAT-HA-RhoA 融合突變蛋白
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細胞的多種功能,包括形狀的維持、移動力,收縮、胞噬及胞飲作用、及細胞質分裂等,均是經由肌動蛋白纖維的動態重組來調節。小分子GTP結合蛋白Rho家族成員已被證實為肌動蛋白纖維動態重組的主要調節因子。在Swiss 3T3纖維母細胞及氣管平滑肌細胞中, RhoA的活化能導致 stress fiber形成。本研究的目標有二:一為表現並純化出能自由通透細胞膜,以處理大量細胞的兩種TAT-HA-RhoA突變蛋白的融合蛋白:持續活化態的TAT-HA-RhoA V14與不活化的TAT-HA-RhoA N19;其次為觀察TAT-HA-RhoA V14/N19 在收縮態及合成態的大白鼠主動脈平滑肌細胞中對肌動蛋白細胞骨架造成的影響。
為表現TAT-HA-RhoA融合蛋白,我們首先利用聚合鏈反應取得rhoA V14/N19 cDNA,並與pTAT-HA質體鑲嵌而構築pTAT-HA-rhoA V14/N19的混合質體(hybrid plasmid)。將混合質體送入表現蛋白的大腸桿菌株BL-21後,以isopropyl-β-thiogalactoside(IPTG)誘導蛋白表現並先後利用親合性、離子交換、及膠體過濾三種層析法純化TAT-HA-RhoA V14/N19兩種融合蛋白。純化所得的融合蛋白以SDS-polyacrylamide電泳分析的分子量約為35~40 kDa。將不同濃度的融合蛋白(100-800 nM)與平滑肌細胞作用1或2小時後,以西方點墨法分析,發現兩種融合蛋白均能進入細胞內,且其量與融合蛋白的處理濃度及時間呈正相關。我們利用Swiss 3T3細胞來鑑定純化出的融合蛋白在細胞內是否有功能:以TAT-HA-RhoA V14(25-100 nM)處理Swiss 3T3細胞1小時可誘導stress fiber的形成; 然而TAT-HA-RhoA N19(100-400 nM)的前處理並無法抑制血清(10 %)對Swiss 3T3細胞stress fiber的誘導作用,顯示V14融合蛋白有活性而N19融合蛋白則無作用。
為了探討融合蛋白對合成態及收縮態平滑肌細胞肌動蛋白細胞骨架的作用,我們將平滑肌細胞培養在無血清培養液中6或72小時,以分別代表合成態及收縮態的細胞。以100及200 nM RhoA V14融合蛋白處理血清剝奪6小時的大白鼠主動脈平滑肌細胞,對肌動蛋白纖維並無明顯的作用;但400及800 nM則能增加stress fiber的密度。在血清剝奪72小時的平滑肌細胞,上述四種濃度的RhoA V14融合蛋白均能增加stress fiber的密度。類似的現象在10 % FBS/DMEM 及2U/ml thrombin處理組亦可偵側到。Rho kinase的抑制者,Y27632(10μM),的處理則造成肌動蛋白纖維局部或完全消失,並導至細胞變形及去黏著的現象。
總結上述,我們表現並純化出兩種RhoA突變蛋白的融合蛋白:TAT-HA-RhoA V14與TAT-HA-RhoA N19。在Swiss 3T3纖維母細胞的功能測試顯示RhoA V14融合蛋白有活性而RhoA N19則無。無論在合成態及收縮態的平滑肌細胞,活化的RhoA均能增加stress fiber的密度。

摘 要…………………………………………………………………………3
Abstract………………………………………………………………………..5
第一章 緒 論…..………………………………………………………7
第二章 材 料…..……………………………………………………..22
I. hybrid plasmid (pTAT-HA-rhoA V14 /N19) cloning相關材料……….23
II. 融合蛋白表現(fusion protein expression)的相關材料………………...24
III. 融合蛋白在Swiss 3T3及RASMCs處理的相關材料………………..27
第三章 方 法…..……………………………………………………..29
壹、含有RhoA V14與RhoA N19基因的載體建構…………………………..31
( 一 ) 蛋白質表現載體(protein expression vector)的製備………………..31
( 二 ) insert cDNA(rhoA V14 及 rhoA N19)的製備……………………..33
( 三 ) insert cDNA 與pTAT-HA的接合……………………………………36
( 四 ) TOP-10 E.coli 的轉殖(transformation)…………………………………..37
( 五 ) 篩選出具有hybrid plasmid 的 TOP-10 菌株……………………...39
( 六 ) hybrid plasmid 的分離、純化、及定序………………………………42
( 七 ) hybrid plasmid 轉殖至蛋白質表現系統……………………………..43
貳、融合蛋白的表現、純化、與功能的探討………………………………..45
( 八 ) 融合蛋白的表現與純化……………………………………………..45
( 九 ) 融合蛋白進入細胞的條件測試:檢測所需濃度和時間……………50
( 十 ) TAT-融合蛋白的功能測試…………………………………………...53
第四章 結 果…..……………………………………………………..55
蛋白質表現載體(pTAT-HA)與insert cDNA (rhoA V14 /N19)的鑑定………56
蛋白質表現載體與insert cDNA的接合(ligation)……………………………56
TOP-10菌株的轉形(transformation)及篩選………………………………….58
Hybrid plasmid的純化及定序………………………………………………...59
Hybrid plasmid轉殖(transform)至蛋白質表現系統………………………….59
融合蛋白的表現及純化……………………………………………………….60
融合蛋白進入細胞的條件測試:檢測所需濃度和時間……………………...62
融合蛋白的功能測試………………………………………………………….63
TAT-HA-RhoA V14/N19對RASMCs actin filament的影響…………………64
第五章 討 論…..……………………………………………………..66
使用in-frame TAT-融合蛋白的優點………………………………………….67
TAT-融合蛋白的表現…………………………………………………………70
TAT-融合蛋白的純化…………………………………………………………71
TAT-融合蛋白能穿透細胞並顯示功能………………………………………73
第六章 參考資料…..…………………………………………………91
第七章 附 錄……………………………………………………… 100

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