Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/25465
標題: 雞溶菌、卵白蛋白基因啟動子與鈣結合蛋白cDNA之選殖與表現
Cloning and Expression of Chicken LyzPro, OvaPro Promoters and Calbindin cDNA Gene
作者: 李志明
Lee, Jhy-Ming
關鍵字: lysozyme
溶菌
ovalbumin
calbindin
卵白蛋白
鈣結合蛋白
出版社: 畜產學系
摘要: 試驗一 雞溶菌啟動子和OPN之選殖、融合基因之構築與表現 摘要 本試驗主要分為雞溶菌基因啟動子、及osteopontin cDNA之選殖與融合基因之構築,並探討雞溶菌基因啟動子和osteopontin在細胞中表現。 在雞溶菌基因啟動子部分,根據發表於NCBI之來航雞溶菌基因啟動子序列設計兩條引子。再用此引子以雞肝臟基因組DNA為模板進行聚合連鎖反應,增殖出2175 bp之來航雞溶菌基因啟動子片段。將啟動子與報導基因lacZ構築在載體pBlue-TOPO以形成重組質體pcLyzlacZ,並進行序列分析比對後發現,有10個核酸不同,但不影響基因啟動功能。再將上述質體轉移至雞殼腺、峽部及膨大部上皮細胞內測定lacZ之表現。以X-gal染色發現殼腺、峽部和膨大部上皮細胞lacZ均有表現,顯示溶菌基因啟動子具有啟動lacZ基因表現之功能。 以來航雞溶菌基因啟動子和osteopontin構築出融合基因方面;將此基因轉移入CHO 細胞株內測定其mRNA之表現。以RT-PCR分析轉形株CHO 細胞之mRNA後,可增殖出正確長度之片段(1112 bp);以SDS-PAGE和西方吸漬等方法皆偵測得66 kDa OPN蛋白質表現。 在溶菌cDNA部分,根據發表於NCBI之來航雞溶菌cDNA序列設計兩條引子。再用此引子對來航雞生殖道膨大部、峽部及子宮總RNA進行RT-PCR有增殖出547 bp。因此可證實生殖道膨大部、峽部及子宮各部位中有來航雞溶菌表現之情形。 在雞溶菌5’端調節序列方面,根據發表於NCBI之來航雞溶菌5’端調節序列設計兩條引子。再用此引子對雞肝臟基因組DNA進行聚合連鎖反應,增殖出2081 bp之片段。將5’端調節序列與報導基因lacZ構築在質體以形成重組質體pcLyzEnlacZ,並進行序列分析比對後發現,有19個核酸不同,但可供日後接入啟動子之前面,測試是否能增加啟動子之功能性。 試驗二 雞卵白蛋白基因啟動子選殖、融合基因之構築與表現 摘要 本試驗之目的是選殖出雞卵白蛋白基因啟動子構築出cOvalacZ融合基因後轉移至雞生殖道膨大部細胞中,測定融合基因之表現。 在雞卵白蛋白基因啟動子方面,根據發表於NCBI之雞卵白蛋白基因組DNA序列設計兩條cOvaPro-F3 ( 5′-TTAAGTCCTCAGACTTGGCAAGGAG-3′ ) 和cOvaPro-R3 ( 5′-GTCTAGAGCAAACAGCAGAACAGTG-3′ )引子。再用此引子以雞肝臟基因組DNA為模板進行聚合 連鎖反應,增殖出2768 bp之雞卵白蛋白基因啟動子片段。將啟動子與報導基因lacZ構築在質體以形成pcOvalacZ。進行序列分比對後發現有61個核苷酸不同,但此種差異並未影響至類固醇結合位置。因此將上述重組質體轉移入來航雞膨大部、峽部及殼腺細胞內測定其lacZ之表現。結果顯示此DNA可在膨大部、殼腺及峽部細胞中產生啟動子導引之lacZ基因表現。顯示此一啟動子在膨大部、殼腺和峽部細胞具有導引其後連結基因表現之功能。 試驗三 雞鈣結合蛋白cDNA選殖、融合基因之構築與表現 摘要 本試驗之目的是選殖出雞鈣結合蛋白cDNA構築出融合基因後轉移至CHO細胞中,測定融合基因之表現。 因此本試驗是根據NCBI來航雞之雞鈣結合蛋白mRNA序列為模板設計引子。以cCalbin-F2 ( 5′-AACATGACGGCGGAGACGCACCTG-3′)和cCalbin-R7 ( 5′-ATTTTCCTCAGCACAGAGAATGAGA-3′)作為引子,以雞十二指腸上皮細胞之total RNA作為模板來進行反轉錄聚合連鎖反應( reverse transcription polymerase chain reaction; RT-PCR ),結果增殖出788 bp的片段。RT-PCR所增殖的雞鈣結合蛋白黏接在表現載體pcDNA3.1/V5/His-TOPO ( Invitrogen )中形成pCMVcCalbinns,經序列分後證明與已發表之序列一致。以RT-PCR證實pCMVcCalbinns在CHO細胞株中有雞鈣結合蛋白cDNA轉錄之情形。經西方吸漬分析亦可證實在細胞中可產生雞鈣結合蛋白質表現。
一、Cloning and Expression of Promoter Sequence of Chicken Lysozyme Gene Abstract The objective of study was to clone and construction fusion gene of the gene promoter sequence of chicken lysozyme and cDNA of chicken osteopontin. The fusion gene of clyzlacZ was constructed and introduced into magnum, isthmus and eggshell cells of chicken oviduct. The fusion gene of cLyzcOPN was constructed and introduced into CHO cells for the detection expression. First, two primers cLyzPro-F5 (5′-AAAAGCCAACCCTGACAGA CATCCC-3′) and cLyzPro-R3 (5′-GCACCTGCCTCTTCTTTTAACTTC CTCCAC-3′) were designed according to genomic DNA sequence of lysozyme gene in chicken published on NCBI GeneBack. The genomic DNA collected from chicken liver was used as template for the promoter sequence cloning. A fragment ( 2175 bp ) containing the promoter sequence of chicken lysozyme gene was cloned and construction with lacZ gene in pBlue-TOPO vector to generate a recombinant plasmid pcLyzlacZ. The cloned sequence was aligned with the published sequence, the result showed that there were ten nucleotides in difference it does not influence function. The recombinant plasmid was introduced into eggshell gland, isthmus and magnum cells of chicken oviduct. The results showed that the gene product of b-galactosidase could be detected in the culture cells, it meant that the cloned of lysozyme promoter sequence could initiate the expression of lacZ gene. Second, the construction fusion gene of lysozyme promoter and osteopontin introduced into CHO cell and detected mRNA and protein expression. The results showed that the OPN mRNA was detected in CHO cell transfected with pCMVcOPN by RT-PCR, a 1112 bp fragment could be observed in agarose. A band about 66 kDa was represented on the gel detected by SDS-PAGE electrophoresis and western-blotting. Three, two primers cLyz-F1 ( 5′- GTGTGTACGACACTGGCAAC ATGAG -3′ ) and cLyz-R1 ( 5′- GATGCGTTTAAAACTGCCAAGCGG G -3′) were designed according to the cDNA sequence of lysozyme gene in chicken published on NCBI GeneBack. The cDNA collected from chicken oviduct was used as template for the cDNA sequence cloning. A fragment ( 547 bp ) containing tne cDNA sequence of chicken lysozyme gene was cloned. The results showed that the mRNA product of lysozyme could be detected in magnum, isthmus, and uterus of the chiken oviduct. Finally, the primers cLyzPro-F2 ( 5′-CGGCTTCCTATGCGTGCTC AGAAAAC-3′ ) and cLyzPro-R1 ( 5′-AGCGCTGGTAATCTTCATAAA AATG-3′ ) were designed according to the genomic DNA sequence of lysozyme 5’ region of regulation sequence in chicken published on NCBI GeneBack. The genomic DNA collected from chicken liver was used as template for the 5’ region of regulation sequence cloning. A fragment ( 2081 bp ) containing the 5’ region of regulation sequence of chicken lysozyme gene was cloned and construction with lacZ gene in pBlue-TOPO vector to generate a recombinant plasmid pcLyzEnlacZ. The cloned sequence was aligned with the published sequence, the result showed that there were nineteen nucleotides in difference. But it can be ligated to the promoter front and tested the promoter function. 二、Cloning and Expression of Promoter Sequence of Chicken Ovalbumin Gene Abstract The objective of this study was to clone the promoter sequence of chicken ovalbumin gene. A fusion gene of cOvalacZ was construction and introduced into the magnum, isthmus and eggshell gland cells of chicken oviduct for the detection of gene expression. Two primers cOvaPro-F3 ( 5′-TTAAGTCCTCAGACTTGGCAAG GAG-3′ ) and cOvaPro-R3 ( 5′-GTCTAGAGCAAACAGCAGAACAGT G-3′ ) were designed according to the genomic DNA sequence of ovalbumin gene in chicken published on NCBI GeneBack. The genomic DNA collected from chicken liver was used as template for the promoter sequence cloning. A fragment ( 2768 bp ) containing the promoter sequence of chicken ovalbumin gene was cloned and construction with lacZ gene in pBlue-TOPO vector to generate a recombinant plasmid pcOvalacZ. The cloned sequence was aligned with the published sequence, the result showen that there are sixty-one nucleotides in difference it does not influence the steroid hormone binding sits. The recombinant plasmid was introduced into magnum, isthmus and eggshell gland cells of chicken oviduct . The results showed that the gene product of b-galactosidase could be detected in the cultural cells, it meant that the cloned ovalbumin promoter sequence could initiate the expression of lacZ gene. 三、Cloning, Construction and Expression of Chicken Calbindin Gene Abstract The objective of study was to clone cDNA sequence of chicken calbindin. After constructing, the fusion gene were introduced into the CHO cells and then we may assay for the expression of fusion gene expression. The primers of cCalbin-F2 ( 5′-AACATGACG GCGGAGACGCAC CTG -3′) and -R7 ( 5′-ATTTTCCTCAGCACAGAGAATGAGA -3′) had been designed according to the laying chicken calbindin mRNA sequence reported by NCBI for cDNA cloning. The total RNA collected from epithelial cells in chicken duodenum was used as a template for cDNA cloning by reverse transcription polymerase chain reaction ( RT-PCR ). A fragment of RT-PCR product in the length of 788 bp was cloned and inserted into the vector of pcDNA3.1/V5/His-TOPO to generate the recombinant plasmids pCMVcCalbinns. The mRNA product could be detected in CHO cells by RT-PCR. The protein of calbindin was found in CHO cells by Western analysis.
URI: http://hdl.handle.net/11455/25465
Appears in Collections:動物科學系

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