Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/25470
標題: 人類麩胱甘肽轉移酶基因-Mu、Pi和Theta之選殖及表現
Cloning and Expression of Human Glutathione S-Transferase Genes Mu, Pi and Theta
作者: 林明怡
Lin, Ming-Yi
關鍵字: Human Glutathione S-Transferase
人類麩胱甘肽
Gnen Cloning
Gene Expression
轉移酶
基因選殖
基因表現
出版社: 畜產學系
摘要: 試驗一、人類麩胱甘肽轉移酶-Mu基因之選殖及表現 一、 摘要 麩胱甘肽轉移酶-Mu (glutathione S-transferase Mu, GSTM1),屬於第二期代謝解毒酶,高度表現於人類肝臟和肺臟組織中,其主要催化還原態麩胱甘肽與環氧化合物結合,以達到保護個體對抗細胞毒素和致癌物質之傷害。本試驗之目的乃是選殖人類麩胱甘肽轉移酶-Mu基因,並於哺乳動物細胞、大腸桿菌及酵母菌中表現,期望能生產此蛋白質。首先,設計hGSTM1-Fns與hGSTM1-Rns兩個引子,並應用此引子進行聚合酶鏈鎖反應自人類肝臟cDNA基因庫中增殖出長度為665 bp之片段,之後利用雙去氧核苷酸DNA定序法分析其核苷酸序列。再將此基因轉接入載體內,構築成pCMVhGSTM1ns重組質體,以電孔方式(electroporation) 之方式轉形入中國倉鼠卵巢細胞中表現。另一方面,利用基因重組技術,構築成pEThGSTM1ns及pPICZBhGSTM1ns重組質體,分別於大腸桿菌及酵母菌表現。結果顯示,在哺乳動物細胞表現方面,以RT-PCR分析轉形株CHO M1之mRNA後,可增殖出正確長度之片段(665 bp);以SDS-PAGE和西方吸漬等方法皆偵測到hGSTM1蛋白質表現(31.7 kDa);與受質1-chloro-2,4- dinitrobenzene測定比活性,發現轉形株之比活性為3.72±0.04 units/ mg protein對照,均顯著高於對照組2.64±0.04 units/mg protein (p<0.05)。大腸桿菌表現方面,以SDS-PAGE和西方吸漬等方法亦可偵測到hGSTM1蛋白質表現(49 kDa);與受質1-chloro- 2,4 -dinitrobenzene測定比活性,發現轉形株pEThGSTM1ns之比活性在尚未純化前僅為46.19 units/mg protein,但經GSH-agarose純化後可將比活性提高為376.80 units/mg protein;產量方面以100 ml培養液培養大腸桿菌共可產生20.20 mg的總蛋白質,但經過純化後共可回收1.17 mg具活性的hGSTM1蛋白質,其回收產量為47.25%。酵母菌表現方面,以PCR及RT-PCR皆可增殖出正確之片段;以SDS-PAGE和西方吸漬等方法亦可偵測到hGSTM1蛋白質表現;與受質1-chloro -2,4-dinitrobenzene測定比活性,發現轉形株GS115-pPICZB- hGSTM1ns之比活性在尚未純化前僅為8.17 units/mg protein,但經GSH-agarose純化後可將比活性提高為1134.26 units/mg protein;產量方面,打破20 ml經甲醇誘導2天之重組酵母菌液共可產生12.66 mg的總蛋白質,但經過純化後共可回收0.09 mg具活性的hGSTM1蛋白 質,其回收產量為98.69%。 試驗二、人類麩胱甘肽轉移酶-Pi基因之選殖及表現 摘要 麩胱甘肽轉移酶-Pi (glutathione S-transferase Pi, GSTP1),屬於第二期代謝解毒酶,高度表現於人類呼吸器官和胃腸道中,其主要功能為催化麩胱甘肽與各種親電性物質結合,以達到保護細胞免受細胞毒素和致癌物質造成之傷害。本試驗之目的乃是選殖人類麩胱甘肽轉移酶-Pi基因,並於哺乳動物細胞、大腸桿菌中表現,期望能生產此蛋白質。首先,設計hGSTP1-Fns與hGSTP1-Rns兩個引子,並應用此引子進行聚合酶鏈鎖反應自人類胎盤cDNA基因庫中增殖出長度為641 bp之片段,之後利用雙去氧核苷酸DNA定序法分析其核苷酸序列。再將此基因轉接入載體內,構築成pCMVhGSTP1ns重組質體,以電孔方式 (electroporation) 之方式轉形入中國倉鼠卵巢細胞內表現。另一方面,利用基因重組技術,構築成pEThGSTP1ns及pPICZBhGSTP1ns重組質體,分別於大腸桿菌及酵母菌表現。結果顯示,在哺乳動物細胞表現方面,以RT-PCR分析轉形株CHO P1之mRNA後,可增殖出正確長度之片段 (641 bp);以SDS-PAGE和西方吸漬等方法皆偵測到hGSTP1蛋白質表現 (28 kDa);與受質1-chloro-2,4-dinitrobenzene測定比活性,發現轉形株之比活性為3.42 ±0.17units/mg protein對照,均顯著高於對照組2.64±0.04 units/mg protein (p<0.05)。大腸桿菌表現方面,以SDS-PAGE和西方吸漬等方法亦可偵測到hGSTP1蛋白質表現 (44 kDa);與受質1-chloro- 2,4-dinitrobenzene測定比活性,發現轉形株pEThGSTP1ns之比活性在尚未純化前僅為4.30 units/mg protein,但經GSH-agarose純化後可將比活性提高為128.71 units/mg protein;產量方面以100 ml培養液培養大腸桿菌共可產生19.65 mg的總蛋白質,但經過純化後共可回收 0.45 mg具活性的hGSTP1蛋白質,其回收產量為68.55%。並已構築完成於酵母菌表現之重組質體pPICZBhGSTP1ns。 試驗三、人類麩胱甘肽轉移酶-Theta基因之選殖及表現 摘要 麩胱甘肽轉移酶-Theta (glutathione S-transferase Theta, GSTT1),屬於第二期代謝解毒酶,高度表現於人類肝臟組織中,其主要催化還原性麩胱甘肽與環氧化合物結合,以達到保護個體對抗細胞毒素和致癌物質之傷害。目前,大約有20%的人類缺乏此一基因或是酵素失活,因此會提高罹患癌症之風險。本實驗之目的乃是選殖人類麩胱甘肽轉移酶-Theta基因,並於哺乳動物細胞和大腸桿菌中表現。首先,設計hGSTT1-Fns、hGSTT1-Fns (1)和hGSTT1-Rns等引子,並應用此引子進行聚合酶鏈鎖反應自人類肝臟cDNA基因庫中增殖出長度為720 和737 bp之片段,之後利用雙去氧核苷酸DNA定序法分析其核苷酸序列。再將此基因轉接入載體內,構築成pCMVhGSTT1ns和pCMVhGSTT1ns (1)重組質體,以電孔方式(electroporation) 之方式轉形入中國倉鼠卵巢細胞中表現。另一方面,利用基因重組技術,構築成pEThGSTT1ns及pPICZBhGSTT1ns分別於大腸桿菌和酵母菌中表現。結果顯示,在哺乳動物細胞表現方面,以RT-PCR分析轉形株之mRNA後,可增殖出正確長度之片段 (720 bp);以SDS-PAGE和西方吸漬等方法亦可偵測到hGSTT1蛋白質表現 (32 kDa);與受質1-chloro-2,4-dinitrobenzene測定比活性,發現轉形株之比活性為3.66 ±0.20 units/mg protein對照,均顯著高於對照組2.64±0.04 units/mg protein (p<0.05)。大腸桿菌表現方面,以SDS-PAGE和西方吸漬等方法亦可偵測到hGSTT1蛋白質表現 (50 kDa);與受質1-chloro-2,4- dinitrobenzene測定比活性,發現轉形株pEThGSTT1ns之比活性在尚未純化前僅為 52.26 units/mg protein,但經GSH-agarose純化後可將比活性提高為423.2 units/mg protein;產量方面以100 ml培養液培養大腸桿菌共可產生17.5 mg的總蛋白質,但經過純化後共可回收0.85 mg具活性的hGSTT1蛋白質,其回收產量為39.33 %。並已構築完成於酵母菌表現之重組質體pPICZBhGSTT1ns。
一、 Cloning and Expression of Human Glutathione S-Transferase Gene Mu Abstract Glutathione S-transferase Mu (GSTM1) belong to phase II metabolic detoxify enzymes that is present at a high level in human hepatic tissue. It play an important role in catalyze the conjugation of reduced glutathione with epoxides and may thus protect individuals against cytotoxic or carcinogens. The purpose of the studies were to clone the human GSTM1 gene and express it in mammalian cells, E. coli and P. pastori. Two primers, hGSTM1-Fns and hGSTM1-Rns were designed for the human GSTM1 gene cloning. Cloning was carried out by PCR from human liver cDNA library. A 665 bp fragment of hGSTM1 cDNA was isolated and constructed into vectors to generate pCMVhGSTM1ns recombinant plasmid. The recombinant plasmid had been transfected into Chinese hamster ovary (CHO) cells for gene expression. Furthermore, pEThGSTM1ns and pPICZBhGSTM1ns recombinant plasmids were generated by gene recombinant technique and transfered into E. coli and P. pastori for gene espression. The hGSTM1 mRNA was detected in CHO cell transfected with pCMVhGSTM1ns by RT-PCR, a 665 bp fragment could be observed in agarose gel. A band about 31.7 kDa was represented on the gel detected by SDS-PAGE electrophoresis and western-blotting;GST enzyme specific activity was measured by 1-chloro-2,4-dinitrobenzene analysis, the results showed that the treatment group (3.72±0.04 units/mg protein) was significantly higher than the control (2.64±0.04 units/mg protein). The pEThGSTM1ns was introduced into E. coli and induced by 0.1mM IPTG, the cell lysate was collected for the detected of GST gene expression, a band approximate 49 kDa could be obtained on the gel analyzed by SDS-PAGE electrophoresis and western-blotting;GST enzyme specific activity was also dectermined by 1-chloro- 2,4- dinitrobenzene analysis, the specific activity of GST in the cell lysate unpurified was 46.19 units/mg protein; When the cell lysate was purified by GSH-agarose, the specific activity of GST was increased to 376.80 units/mg protein. The production of total protein about 20.20 mg, after the GSH-agarose purification about 1.17mg, the recovery rate of protein was 46.68%. In the yeast trail, the pPICZBhGSTM1ns was introduced into P. pastori and induced by 0.5% methanol, the cell lysate was collected for the detected of GST gene expression, a band approximate 29.2 kDa could be obtained on the gel analyzed by SDS-PAGE electrophoresis and western-blotting;GST enzyme specific activity was also dectermined by 1-chloro-2,4 -dinitrobenzene analysis, the specific activity of GST in the cell lysate unpurified was 8.17 units/mg protein; When the cell lysate was purified by GSH-agarose, the specific activity of GST was increased to 1134.26 units/mg protein. The production of total protein about 12.66 mg, after the GSH-agarose purification about 0.09 mg, the recovery rate of protein was 98.69%. 二、Cloning and Expression of Human Glutathione S-Transferase Pi Gene Abstract Glutathione S-transferase Pi (GSTP1) belong to phase II metabolic detoxify enzymes that is present at a high level in human respiratory organs and gastrointestinal tract. It can catalyze the conjugation of reduced glutathione with electrophilic groups of a wide variety of compound and sever to protect cell from damage caused by cytotoxic and carcinogenic agents. The purpose of the studies were to clone the human GSTP1 gene and express it in mammalian cells and E. coli. Two primers, hGSTP1-Fns and hGSTP1-Rns were designed for the human GSTP1 gene cloning. Cloning was carried out by PCR from human placenta cDNA library. A 641 bp fragment of hGSTP1 cDNA was isolated and constructed into vectors to generate pCMVhGSTP1ns recombinant plasmid. The recombinant plasmid had been transfected into Chinese hamster ovary (CHO) cells for gene expression. Furthermore, pEThGSTP1ns and pPICZBhGSTP1ns recombinant plasmids were generated by gene recombinant technique and transfered into E. coli and P. pastori for gene expression. The hGSTP1 mRNA was detected in CHO cell transfected with pCMVhGSTP1ns by RT-PCR, a 641 bp fragment could be observed in agarose gel. Analysis of cell lysate, a band about 28 kDa was represented on the gel detected by SDS-PAGE electrophoresis and western-blotting;GST enzyme specific activity was measured by 1-chloro-2,4-dinitrobenzene analysis, the results showed that the treatment group (3.42±0.17 units/mg protein) was significantly higher than the control (2.64±0.04 units/mg protein). The pEThGSTP1ns was introduced into E. coli and induced by 0.1mM IPTG, the cell lysate was collected for the detection of GST gene expression. A band approximate 44 kDa could be obtained on the gel analyzed by SDS-PAGE electropgoresis and western-blotting;GST enzyme specific activity measured was also determined by 1-chloro-2,4-dinitrobenzene analysis, the activity of GST in the cell lysate unpurified 4.30 units/mg protein;When the cell lysate was purified by GSH-agarose, the specific activity of GST was increased to 128.71 units/mg protein. The production of total protein about 19.65 mg, after the GSH-agarose purification about 0.45 mg, the recovery rate of protein was 68.55%. In the yeast trail, the cloning and construction of recombinant gene pPICZBhGSTP1ns have been carried out. 三、Cloning and Expression of Human Glutathione S-Transferase Theta Gene Abstract Glutathione S-transferase Theta (GSTT1) belong to phase II metabolic detoxify enzymes that is present at a high level in human hepatic tissue. It play an important role in catalyze the conjugation of reduced glutathione with epoxides and may thus protect individuals against cytotoxic or carcinogens. There were 20% individual absent this gene or lack of enzyme function and maybe increased cancer risk. The purpose of the studies were to clone the human GSTT1 gene and express it in mammalian cells and E. coli. Three primers, hGSTT1-Fns 、hGSTT1-Fns (1) and hGSTT1-Rns were designed for the human GSTT1 gene cloning. Cloning was carried out by PCR from human liver cDNA library. The 720 and 737 bp fragments of hGSTT1 cDNA was isolated and constructed into vectors to generate pCMVhGSTT1ns and pCMVhGSTT1ns (1) recombinant plasmids. The recombinant plasmid had been transfected into Chinese hamster ovary (CHO) cells for gene expression. Furthermore, pEThGSTT1ns and pPICZBhGSTT1ns recombinant plasmids were generated by gene recombinant technique and transferred into E. coli and P.pastori for gene expression . The hGSTT1 mRNA was detected in CHO cell transfected with pCMVhGSTT1ns by RT-PCR, a 720 bp fragment could be observed in agarose gel. Analysis of the cell lysate, a band about 32 kDa was represented on the gel detected by SDS-PAGE electrophoresis and western-blotting;GST enzyme specific activity was measured by 1-chloro-2,4-dinitrobenzene analysis, the results showed that the treatment group (3.66±0.20 units/mg protein) was significantly higher than the control (2.64±0.04 units/mg protein). The hGSTT1ns was introduced into E. coli and induced by 0.1mM IPTG, the cell lysate was collected for the detected of GST gene expression. A band approximate 50 kDa could be obtained on the gel analyzed by SDS-PAGE electrophesis and western-blotting;GST enzyme specific activity was also determined by 1-chloro-2,4-dinitrobenzene analysis, the specific activity of GST in the cell lysate unpurified was 52.26 Unit/mg protein;When the cell lysate was purified by GSH-agarose, the specific activity of GST was increased to 423.20 units/mg protein. The production of total protein about 17.5 mg, after the GSH-agarose purification about 0.85 mg/, the recovery rate of protein was 39.33%. In the yeast trail, the cloning and construction of recombinant gene pPICZBhGSTT1ns have been carried out.
URI: http://hdl.handle.net/11455/25470
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