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研究生:郭俊宏
研究生(外文):CHUN-HUNG,KUO
論文名稱:重組血紅素 β(67Trp→Phe)- α1M/synoα(29Leu→Phe)蛋白之建構及表現
論文名稱(外文):Construction and expression of recombinant hemoglobin β(67Trp→Phe)-α-1-microglobulin/synoα(29Leu→Phe)
指導教授:黃光策
指導教授(外文):KUANG-TSE,HUANG
口試委員:侯劭毅張岳隆
口試委員(外文):SHAO-YI,HOUYUEH-LONG,CHANG
口試日期:2020-07-29
學位類別:碩士
校院名稱:國立中正大學
系所名稱:化學工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:67
中文關鍵詞:血紅素一氧化氮苯丙胺酸
外文關鍵詞:HemoglobinNOPhenylalanine
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雖然對 B 型肝炎和愛滋病毒的血液檢查已成為血庫的例行檢查。初
期感染的人們具有空窗期無法由陽性檢測出病毒,因此仍然有風險存在。
另外,全血只能在 1〜6°C 下保存 1 個月。因此,除了傳統的輸血方式外,
還必須開發新的血液替代品以應對血液供應不足和因輸血可能引起的病
毒感染。現今,基於血紅蛋白(Hb)的血液替代品的發展已經解決了 Hb
四聚體解離為二聚體的問題,然而,在缺乏 2,3-DPG 調節的情況下,它
們無法釋放適量的氧氣。此外,Hb 與一氧化氮(NO)之間的高反應速
率會導致 metHb 的形成以及血管收縮和血壓的升高。為了降低 Hb-NO 氧
化速率,我們在 α 鏈(29Leu→Phe)和 β 鏈(67Trp→Phe)中進行了點
突變。為了清除超氧化物並將 metHb 還原為 oxyHb,將 α-1-微球蛋白(α1M)
基因與 Hbβ 融合,於是建構了 pETite-Hbβ(67Trp→Phe)- α1M/synoα(29Leu
→Phe)質體。

Although the blood tests for hepatitis B or HIV have become a routine in
blood banks. A window period that people in early infection couldn’t test
positive remains as a health threat. In addition, whole blood can only be
stored for 1 month at 1 ~ 6 ° C. Therefore, in addition to traditional blood
transfusions, new blood substitutes must also be developed to cope with
insufficient blood supply and possible virus infection due to transfusion. The
developments of hemoglobin (Hb)-based blood substitutes today have
resolved the problem of the disassociation of Hb tetramers into dimers,
However, in the absence of 2,3-DPG regulation, they cannot release an
appropriate amount of oxygen. Also, the high reaction rate between Hb and
nitric oxide (NO) leads to the formation of metHb and the increase of
vasoconstriction and blood pressure. In order to reduce the Hb-NO rate, we
performed point mutations in the α chain (29Leu → Phe) and β chain (67Trp
→ Phe). To scavenge superoxide and reduce metHb to oxyHb, the
alpha-1-microglobulin (α1M) gene was fused to Hbβ. In this study, we have
constructed pETite-Hbβ (67Trp→Phe)- α1M/synoα (29Leu→Phe) plasmid
for expressing Hbs.

目錄
摘要....................................................................................................................I
目錄.................................................................................................................IV
圖目錄........................................................................................................... VII
表目錄.............................................................................................................IX
英文縮寫全名對照表...................................................................................... X
壹、緒論........................................................................................................... 1
一、前言................................................................................................... 1
二、文獻回顧........................................................................................... 3
1.Hb 的結構與功能.......................................................................... 3
2.以血紅素溶液進行輸血之副作用................................................ 5
3.化學修飾的血紅素........................................................................ 6
4.基因重組的血紅素........................................................................ 9
5.A1M 合成及蛋白質的結構與功能............................................ 13
三、研究動機......................................................................................... 15
四、實驗設計......................................................................................... 15
貳、實驗儀器、器材與藥品......................................................................... 18
一、實驗儀器......................................................................................... 18
二、實驗器材......................................................................................... 20
三、實驗藥品......................................................................................... 21
參、實驗藥品配製與步驟............................................................................. 28
一、抗生素配製..................................................................................... 28
Kanamycin stock solution(100 mg/mL) ......................................... 28
二、培養液配製..................................................................................... 28
Luria-Bertani broth (LB) medium................................................... 28
LB-kanamycin (100μg/mL) agar plates.......................................... 28
SOC medium................................................................................... 29
V
SOC-kanamycin (50 μg/mL) agar plate.......................................... 30
三、菌種的培養與保存......................................................................... 30
DH5α 之培養.................................................................................. 30
SHuffle T7 E. coli 之培養 .............................................................. 30
菌種保存......................................................................................... 31
重組血紅素的質體純化................................................................. 31
四、聚合酶連鎖反應(polymerase chain reaction;PCR) .................... 32
Primer 的稀釋................................................................................. 32
pETite-β67W-A1M 載體的複製.................................................... 32
synoα 片段的複製.......................................................................... 32
五、質體和 PCR 產物限制酶處理(Restriction enzyme digestion) ..... 34
裁切酵素反應................................................................................. 34
PCR 溶液與 DpnI 酵素反應.......................................................... 34
Exonclease III 接合反應................................................................. 35
六、DNA 電泳 ....................................................................................... 36
0.5X TAE buffer 的配製................................................................. 36
配製瓊脂膠片................................................................................. 36
瓊脂膠純化..................................................................................... 36
七、大腸桿菌轉型................................................................................. 37
DH5α E. coli 轉型 .......................................................................... 37
SHuffle T7 Express E. coli 轉型..................................................... 37
八、蛋白質表達與純化......................................................................... 38
誘導藥品配置................................................................................. 38
SHuffle T7 Express E. coli 小量誘導............................................. 38
高溫破菌......................................................................................... 39
SDS 藥品配置 ................................................................................ 40
SDS-PAGE 蛋白質電泳鑄造及操作............................................. 40
SDS-PAGE 染色及脫色................................................................. 41
肆、實驗結果與討論..................................................................................... 43
一、pETite-Hb(β67W-A1M- synoα-α29F)質體建構............................ 43
1.pETite-Hb(β67W-A1M)載體的複製 .......................................... 43
2.synoα(α29F)片段的複製............................................................. 46
3.β-A1M 載體與 α 片段的接合..................................................... 49
二、pETite-Hbβ(67Trp→Phe)-A1M- synoα-α29F 質體建構............... 50
三、pETite-Hb(β67W-A1M- synoα-α29F)不同溫度下小量誘導之蛋白
質表現..................................................................................................... 53
VI
四、pETite-Hbβ(67Trp→Phe)-A1M- synoα-α29F 不同溫度下小量誘
導之蛋白質表現..................................................................................... 56
五、以 PMSF 溶液對 pETite-Hbβ(67Trp→Phe)-A1M- synoα -α29F 的
保護作用................................................................................................. 58
六、pETite-Hbβ(67Trp→Phe)-A1M- synoα-α29F 定序結果............... 59
伍、結論及未來展望..................................................................................... 61
參考文獻......................................................................................................... 62
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