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研究生:楊琇雅
研究生(外文):Shiu-Ya Yang
論文名稱:人類超氧歧化酶、麩胱甘肽過氧化酶及過氧化氫酶基因在大腸桿菌、CHO細胞及雞生殖道細胞中之表現
論文名稱(外文):Expression of the Human Superoxide Dismutase, Glutathione Peroxidase and Catalase Genes in Escherichia coli, CHO Cells and Chicken Oviduct Cells
指導教授:黃木秋黃木秋引用關係
指導教授(外文):Mu-Chiou Huang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:畜產學系
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:139
中文關鍵詞:人類超氧歧化酶人類麩胱甘肽過氧化酶人類過氧化氫酶雞生殖道細胞
外文關鍵詞:Human superoxide dismutaseHuman glutathione peroxidaseHuman catalaseChicken oviduct cells
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本試驗之目的是選殖出人類銅/鋅超氧歧化酶(hSOD1)、細胞質麩胱甘肽過氧化酶(hGPX1UTR)及過氧化氫酶(hCAT)cDNA序列,並加以構築後轉移入大腸桿菌中,測定其基因之表現。在hSOD1基因部分,根據已發表之hSOD1 cDNA 序列設計出hSOD1-Fns1 primer:5’-TTATGGCGACGAAGGCCGTGTGC-3’及hSOD1-Rns primer:5’-TTGGGCGATCCCAATTACACCAC-3’引子,以人類腎臟cDNA基因庫之cDNA作為模板,增殖出長度464 bp 之片段,將此PCR增殖出之cDNA片段接入載體pcDNA3.1/V5/His-TOPO內,構築出重組質體pCMVhSOD1ns-464。再以限制酶BamHI及XhoI進行切割,將hSOD1ns片段(536 bp)回收後接入載體pET-32a (+)中,構築出重組質體pEThSOD1ns-464。之後將pEThSOD1ns-464重組質體轉移入大腸桿菌(BL21)內,以測定hSOD1之表現。結果顯示,將重組質體pEThSOD1ns-464轉移入大腸桿菌後,以RT-PCR分析,證實有hSOD1 mRNA之表現。在蛋白質表現方面,經SDS-PAGE及西方吸漬分析,發現將pEThSOD1ns-464轉移入大腸桿菌後,有hSOD1蛋白質之表現。活性分析方面,大腸桿菌轉形株pEThSOD1ns-464經誘導後所表現hSOD1之比活性為1850 U/mg顯著高於未誘導組931 U/mg(p<0.05)。在hGPX1UTR基因部分,根據已發表之hGPX1UTR cDNA 序列設計出hGPX1UTR-Fns1 primer:5’-CTGGCTTCTTGGACAATTGCGCC-3’及hGPX1UTR-Rns primer:5’-AGTTCTGCTGACACCCGGCAC-3’引子,依上述方式增殖出長度845 bp之片段,將此PCR增殖出之cDNA片段接入載體pcDNA3.1/V5/His-TOPO內,構築出重組質體pCMVhGPX1UTR-845。再以限制酶BamHI及XhoI進行切割,將hGPX1UTR-845片段(918 bp)回收後接入載體pET-32a (+)中,構築出重組質體pEThGPX1UTR-845。之後將pEThGPX1UTR-845重組質體轉移入大腸桿菌內,以測定hGPX1基因之表現。結果顯示,將重組質體pEThGPX1UTR-845轉移入大腸桿菌後,以RT-PCR分析,證實有hGPX1UTR mRNA之表現。在蛋白質表現方面,經SDS-PAGE及西方吸漬分析,發現將pEThGPX1UTR-845轉移入大腸桿菌後,有hGPX1蛋白質之表現。在hCAT基因部分,根據已發表之hCAT cDNA 序列設計出hCAT-Fns1:5’-GTGTTCTGCACAGCAAACCGCACGC-3’及hCAT-Rns: 5’-CAGATTTGCCTTCTCCCTTGCCGC-3’引子,依上述方式增殖出長度1607 bp 之片段,將此PCR增殖出之cDNA片段接入載體pCRⅡ-TOPO內,構築出重組質體pCRⅡhCATns-1607。再以限制酶SacI及XhoI進行切割,將hCATns-1607片段(1794 bp)回收後接入載體pET-32a (+)中,構築出重組質體pEThCATns-1607。之後將pEThCATns-1607重組質體轉移入大腸桿菌內,以測定hCAT基因之表現。結果顯示,將重組質體pEThCATns-1607轉移入大腸桿菌後,以RT-PCR分析,證實有hCAT mRNA之表現。在蛋白質表現方面,經SDS-PAGE及西方吸漬分析,發現將pEThCATns-1607轉移入大腸桿菌後,有hCAT蛋白質之表現。
試驗二之目的為將雞卵白蛋白基因啟動子構築於pCMVhSOD1ns、pCMVhGPX1UTR及pCMVhCATns中取代CMV啟動子,形成重組載體pOvahSOD1ns、pOvahGPX1UTR及pOvahCATns,待之後轉形於CHO細胞及來航雞之生殖道細胞中以令SOD1、GPX1及CAT基因表現之。以限制酶HindⅢ及BamHI切割pcOvalacz,回收Ova promoter片段並接入經限制酶HindⅢ及BglⅡ切割pCMVhSOD1ns、pCMVhGPX1UTR及pCMVhCATns後回收之片段中,構築出重組質體pOvahSOD1ns、pOvahGPX1UTR及pOvahCATns,將其轉形入CHO細胞及雞生殖道細胞中以測定其表現。結果顯示,將重組質體pOvahSOD1ns、pOvahGPX1UTR及pOvahCATns轉移入CHO細胞後,以RT-PCR分析,證實有SOD1、GPX1及CAT mRNA之表現。在生殖道細胞表現方面,於膨大部細胞之pOvahSOD1ns及pOvahGPX1UTR轉形株分別可增殖出511 bp及846 bp之正確片段,而未轉形株則無;而於峽部、殼腺及腎臟細胞之pOvahSOD1ns及pOvahGPX1UTR轉形株及未轉形株則均分別有增殖出511 bp及846 bp之片段,表示所得之片段應含有雞隻內源性之SOD1及GPX1。但轉形株在峽部及殼腺細胞之mRNA的濃度較未轉形株高,因此可推測此雞卵白蛋白啟動子可令hSOD1及GPX1於雞生殖道中表現。

Expression of the human superoxide dismutase, glutathione peroxidase and catalase genes in Escherichia coli, CHO cells and chicken oviduct cells
Hsiu-Ya Yang
Abstract
The purposes of the studies are is to clone the human SOD1, GPX1 and CAT genes. Expression of these genes in E. coli, CHO cells and chicken oviduct cells were carried out.
A 464 bp of fragment of hSOD1 gene was amplified from the template of human kidney cDNA by PCR using hSOD1-Fns1 and -Rns primers. The fragment was then constructed into the vector pcDNA3.1/V5/His-TOPO to generate the recombinant plasmid pCMVhSOD1ns-464. The 464 bp fragment in pCMVhSOD1ns-464 was isolated and inserted into the BamHI and XhoI sites of pET-32(+) plasmid to generate the pEThSOD1ns-464. The pEThSOD1ns-464 recombinant plasmid was transformed into E. coli BL21 for gene expression. The result of RT-PCR analysis revealed that the mRNA of human SOD1 could be detected in the E. coli carrying pEThSOD1ns-464. The hSOD1 protein were found in E. coli by SDS-PAGE and Western blot analysis. The specific activity of hSOD1 expressed in the induced E. coli carrying pEThSOD1ns-464 was 1857 U/mg.
Second, a 845 bp of fragment of hGPX1 gene was amplified from the template of human kidney cDNA by PCR using hGPX1UTR-Fns1 and -Rns primers. The fragment was then constructed into the vector pcDNA3.1/V5/His-TOPO to generate the recombinant plasmid pCMVhGPX1UTR-845. The 845 bp fragment in pCMVhGPX1UTR-845 was isolated and inserted into the BamHI and XhoI sites of pET-32(+) plasmid to generate the pEThGPX1UTR-845. The pEThGPX1UTR-845 recombinant plasmid was transformed into E. coli BL21 for gene expression. RT-PCR analysis revealed that the mRNA of human hGPX1 could be detected in the E. coli carrying pEThGPX1UTR-845. The hGPX1 protein were found in E. coli by SDS-PAGE and Western blot analysis.
Finally, a 1607 bp of fragment of hCAT gene was amplified from the template of human kidney cDNA by PCR using hCAT-Fns1 and -Rns primers. The fragment was then constructed into the vector pCRII-TOPO to generate the recombinant plasmid pCRIIhCATns-1607. The 1607 bp fragment in pCRIIhCATns-1607 was isolated and inserted into the SacI and XhoI sites of pET-32(+) plasmid to generate the pEThCATns-1607. The pEThCATns-1607 recombinant plasmid was transformed into E. coli BL21 for gene expression. RT-PCR analysis revealed that the mRNA of human hCAT could be detected in the E. coli carrying pEThCATns-1607. The hCAT protein were found in E. coli by SDS-PAGE and Western blot analysis.
The purposes of the experiment Ⅱ is construct the Ova promoter into the pCMVhSOD1ns, pCMVhGPX1UTR and pVMVhCATns to generate pOvahSOD1ns, pOvahGPX1UTR and pOvahCATns instead of CMV promotor, and then transfect into the CHO cells and chicken oviduct cells for protein production. RT-PCR analysis revealed that the mRNA of human SOD1, GPX1, CAT can be detected in the CHO cells transfected with pOvahSOD1ns, pOvahGPX1UTR and pOvahCATns. In addition, the mRNA of human SOD1, GPX1, CAT can be detected in the magnum cells transfected with pOvahSOD1ns、pOvahGPX1UTR and pOvahCATns, and the non-transfected group not detected. The mRNA of human SOD1 and GPX1 can be detected in the isthmus cells, gland cells and kidney cells transfected with pOvahSOD1ns and pOvahGPX1UTR or not, so it revealed that we obtained the fragments were endogenous from SOD1 and GPX1 of chicken. But the mRNA concentration of isthmus cells and gland cells of transfected group were higher then non-transfected group. For this reason, we can presume the Ova promoter advance the expression of SOD1 and GPX1 in chicken oviduct.

目錄 頁次
一、氧化自由基.......................................................1 (一)自由基的種類 .......................................1
(二)自由基的產生 1
(三)常見的自由基傷害...................................2
二、細胞內抗氧化酵素.....................................4
三、細胞內抗氧化酵素.....................................5
(一)超氧歧化酶(superoxide dismutase, SOD).............5
(二)麩胱甘肽過氧化酶(glutathione peroxidase, GPX).....8
(三)過氧化氫酶(catalase, CAT).......................11
三、大腸桿菌表現重組蛋白系統............................14
試驗一
摘 要...................................................18
前言....................................................20
材料與方法..............................................21
一、人類SOD1、GPX1UTR及CAT cDNA之選殖...................21
(一)引子設計..........................................21
(二)PCR增殖...........................................29
二、人類SOD1、GPX1UTR及CAT cDNA之構築...................29
(一)pCMVhSOD1ns-464、pCMVhGPX1UTR-845及PCRⅡhCATns- 1607重組質體之構築築.........................29
(二)pEThSOD1ns-464、pEThGPX1UTR-845及pEThCATns-1607
重組質體之構築....................................35
三、pEThSOD1ns-464、pEThGPX1UTR-845及pEThCATns-1607在
大腸桿菌中表現之分析................................36
(一)RNA分析...........................................36
(二)蛋白質分析........................................39
結果與討論..............................................44
一、人類銅/鋅超氧歧化酶之選殖、構築及在大腸桿菌之表現...44
(一)人類銅/鋅超氧歧化酶 cDNA之選殖....................44
(二)人類銅/鋅超氧歧化酶表現載體之構築.................44
(三)人類銅/鋅超氧歧化酶於大腸桿菌中之表現.............45
二、人類細胞質麩胱甘肽過氧化酶之選殖、構築及在大腸桿菌
之表現..............................................56
(一)人類細胞質麩胱甘肽過氧化酶 cDNA之選殖.............56
(二)人類細胞質麩胱甘肽過氧化酶表現載體之構築..........56
(三)人類細胞質麩胱甘肽過氧化酶於大腸桿菌中之表現......57
三、人類過氧化氫酶之選殖、構築及在大腸桿菌之表現........67
(一)人類過氧化氫酶 cDNA之選殖.........................67
(二)人類過氧化氫酶表現載體之構築......................67
(三)人類過氧化氫酶於大腸桿菌中之表現..................68
結論....................................................78
試驗二
摘要....................................................80
前言....................................................81
材料與方法..............................................82
一、pOvahSOD1ns、pOvahGPX1UTR及pOvahCATns之構築.........84
(一)限制酶切割作用....................................84
(二)DNA片段之回收.....................................84
(三)限制酶切割後DNA片段之接合作用.....................84
(四)大腸桿菌之轉形作用................................85
(五)確認構築之重組質體DNA .............................85
二、Chinese hamster ovary(CHO)細胞株及雞生殖道初級細胞
之培養、轉形及篩選..................................85
(一)培養基之配製.................................... .85
(二)雞生殖道中初級細胞萃取............................89
(三)細胞繼代與冷凍保存................................89
(四)細胞轉形..........................................90
三、pOvahSOD1ns、pOvahGPX1UTR及pOvahCATns在Chinese
hamster ovary(CHO)細胞株及雞生殖道初級細胞中
表現之分析..........................................91
(一)RNA分析...........................................91
結果與討論..............................................93
一、pOvahSOD1ns在CHO細胞及雞生殖道細胞之表現............93
二、pOvahGPX1UTR在CHO細胞及雞生殖道細胞之表現..........102
三、pOvahCATns在CHO細胞及雞生殖道細胞之表現............111
結論...................................................116
參考文獻...............................................117
英文摘要...............................................129

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