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研究生:李志明
研究生(外文):Jhy-Ming Lee
論文名稱: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
論文名稱(外文):Cloning and Expression of Chicken LyzPro, OvaPro Promoters and Calbindin cDNA Gene
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:2003
畢業學年度:91
語文別:中文
論文頁數:141
中文關鍵詞:溶菌卵白蛋白鈣結合蛋白
外文關鍵詞:lysozymeovalbumincalbindin
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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.
目 錄
文獻檢討 1
一、家禽生殖道構造 1
(一)卵巢 1
(二)漏斗部( infundibulum ) 1
(三)膨大部 ( magnum ) 2
(四)峽部( isthmus ) 2
(五)殼腺( shell gland ) 2
(六)陰道( vagina ) 3
二、蛋殼膜及蛋殼構造 3
(一)蛋殼膜構造 3
(二)蛋殼構造 3
三、蛋殼形成相關基因 6
(一)溶菌 6
(二)卵白蛋白 6
(三)卵白蛋白起動子調節基因結構 9
(四)負的調節要素 11
(五)Osteopontin 11
(六)維生素D受體 13
(七)副甲狀激素相關蛋白 14
(八)鈣結合蛋白 15
試驗一
雞溶菌啟動子與OPN之選殖、融合基因之構築與表現
摘要 18
前言 20
材料與方法 21
一、雞溶菌啟動子與溶菌及OPN cDNA之選殖 21
(一)引子之設計 21
(二)PCR增殖 21
二、雞基因啟動子與cDNA之構築 22
(一)DNA之架接與檢測 22
(二)DNA片段之回收 30
(三)大腸桿菌之轉形作用─氯化鈣(CaCl2)轉形法 31
(四) Cracking method偵測大腸桿菌中重組質體DNA 31
(五)質體DNA之抽取 32
(六)限制切割 33
(七)構築載體之分析 33
三、萃取噬菌體DNA 34
四、測定增殖後之基因庫力價 35
五、初級培養細胞及Chinese hamster ovary(CHO)細胞株培養、轉形及篩選 35
(一)培養條件 35
(二)生殖道中初級培養細胞萃取 36
(三)細胞繼代與冷凍保存 36
(四) 細胞轉形 37
六、雞溶菌啟動子在生殖道細胞中表現之分析 38
(一)細胞固定 38
(二)細胞染色 38
(三)以倒立顥微鏡觀察細胞表現情形(TK/lacZ基因有表現細胞呈深藍色) 38
(四)X-gal染色溶液之配製 38
七、雞osteopontin基因在CHO細胞株中表現之分析 39
(一)RNA分析 39
(二)蛋白質分析 41
八、輸卵管上皮細胞總RNA之萃取 43
(一)總RNA之萃取 43
(二)、生殖道上皮細胞溶菌cDNA之增殖 44
結果與討論 45
一、檢測生殖道各部位溶菌表現之情形 45
二、雞溶菌基因啟動子DNA之選殖 45
三、雞溶菌表現載體之構築 48
(一)載體pcLyzlacZ之構築 48
(二)載體pcLyzcOPN之構築 48
四、雞溶菌於生殖道之初代培養細胞中表現 48
五、雞溶菌增強子基因組DNA之選殖 57
(一)載體pcLyzEnlacZ之構築 57
結論 70
試驗二
卵白蛋白基因啟動子選殖、構築與表現 71
摘要 72
前言 73
材料與方法 74
一、雞卵白蛋白基因啟動子之選殖 74
(一)引子之設計 74
(二)PCR增殖 74
二、雞卵白蛋白基因之構築 76
(一)DNA架接與檢測 76
(二) DNA片段之回收 77
(三)大腸桿菌之轉形作用─氯化鈣(CaCl2)轉形法 78
(四) Cracking method偵測大腸桿菌中重組質體DNA 78
(五)質體DNA之抽取 79
(六)限制切割 80
(七)構築載體之分析 80
三、萃取噬菌體DNA 81
四、測定增殖後之基因庫力價 82
五、初級培養細胞培養、轉形及篩選 82
(一)培養條件 82
(二)生殖道中初級培養細胞萃取 82
(三)細胞繼代 83
(四)細胞轉形 84
六、雞卵白蛋白啟動子在生殖道細胞中表現之分析 85
(一)細胞固定 85
(二)細胞染色 85
(三)以倒立顥微鏡觀察細胞表現情形(TK/lacZ基因有表現細胞呈深藍色) 85
(四)X-gal染色溶液之配製 85
結果與討論 87
一、雞卵白蛋白基因啟動子DNA之選殖 87
二、雞卵白蛋白表現載體之構築 87
(一)、載體pcOvalacZ之構築 87
(二)卵白蛋白在生殖道膨大部、峽部及殼腺細胞表現之情形 87
結論 98
試驗三
雞鈣結合蛋白cDNA選殖、構築與表現 99
摘要 100
前言 101
材料與方法 102
一、雞鈣結合蛋白cDNA基因之選殖 102
(一)引子之設計 102
二、十二指腸組織上皮細胞總RNA之萃取 102
(一) 總RNA之萃取 102
(二)、十二指腸中鈣結合蛋白cDNA之增殖 103
三、雞鈣結合蛋白基因之構築 104
(一)DNA架接與檢測 104
(二) DNA片段之回收 106
(三)大腸桿菌之轉形作用─氯化鈣(CaCl2)轉形法 107
(四) Cracking method偵測大腸桿菌中重組質體DNA 107
(五)質體DNA之抽取 108
(六)限制切割 109
(七)構築載體之分析 109
四、Chinese hamster ovary(CHO)細胞株培養、轉形及篩選 110
(一)培養條件 110
(二)細胞繼代與冷凍保存 110
(三) 細胞轉形 112
五、雞鈣結合蛋白基因在CHO細胞株中表現之分析 113
(一)RNA分析 113
(二蛋白質分析 116
結果與討論 118
一、雞鈣合蛋白cDNA之選殖 118
二、雞鈣結合蛋白表現載體之構築 118
(一)載體pCMVcCalbinns之構築 118
三、雞鈣結合蛋白cDNA在細胞中之表現 122
結論 128
圖 次
圖1. 家禽生殖道圖式概要 4
圖2. 完整或喪失溶菌起動子其活化位置之模式 8
圖3. 雞卵蛋白基因5’側區之圖譜 10
圖4. 在雞肝臟和生殖道中不同長度之卵白蛋白基因起動子其後接CAT
之活性 12
圖5. 雞溶菌基因啟動子與osteopontin cDNA之選殖、構築與表現測
定 試驗流程 23
圖6. 雞溶菌5’端調節序列之選殖與構築 24
圖7. 雞溶菌基因組DNA序列及引子設計 25
圖8. 雞溶菌cDNA序列及引子設計 26
圖9 雞溶菌5’端調節序列及引子設計 27
圖10. 雞OPN cDNA序列及引子設計 28
圖11. 利用RT-PCR以引子cLyz-F1及-R1 偵測生殖道中溶菌基因之
mRNA轉錄表現。以cLyz-F1和-R1可增殖出549 bp之cLyz cDNA
片段 46
圖12.以來航雞基因組基因庫DNA為模板,利用cLyzPro-F5及-R3引子以
PCR增殖出之cLyzPro (2.1 kb)產物之電泳圖譜 47
圖13.構築於pBluTOPO載體上之雞溶菌基因起動子基因序列與Rapp
(2001)發表之序列比對。”-”代表與前人發表之序列相同 50
圖14.質體pcLyzLacZ 經限制切割後之電泳圖譜(A)。膠片中之DNA轉移
至尼龍膜後,與DIG - cLyz探針雜交之顯影圖(B) 51
圖15.重組質體pcLyzlacZ之構築 52
圖16.應用PCR以cLyzPro-F5、cLyzPro-R3、T7及LacZ引子偵測pcLyzLacZ
之cLyzPro基因組DNA 53
圖17.pcLyzlacZ重組質體於雞生殖道殼腺細胞中經類固醇內泌素誘導3天
後之表現。 為表現lacZ基因產物之細胞 54
圖18.pcLyzlacZ重組質體於雞生殖道峽部細胞中經類固醇內泌素誘導3天
後之表現。 為表現lacZ基因產物之細胞 55
圖19. pcLyzlacZ重組質體於雞生殖道膨大部細胞中經類固醇內泌素誘導
3天後之表現。 為表現lacZ基因產物之細胞 56
圖20.構築於pcDNA3.1載體上之蛋雞OPN-2基因序列與Castagnola et al.
(1991)發表之序列比對。” - ”代表與發表之序列相同 59
圖21.質體pcLyzcOPN經限制切割後之電泳圖譜 60
圖22.重組質體pcLyzcOPN之構築 61
圖23.應用RT-PCR以引子cOPN-P1, cOPN-P2 偵測轉形入質體pcLyzcOPN
之CHO細胞mRNA之cOPN cDNA 。cOPN-P1和cOPN-P2可增殖出
1.1 kb片段 62
圖24.轉形入pcLyzcOPN的CHO cell之培養液以不同飽和濃度硫酸銨沈澱出
之蛋白質以SDS-PAGE (A) 與 Western blot (B)分析cOPN基因表現
63
圖25.以來航雞基因組DNA為模板,利用cLyzPro-F2及cLyzPro-R1引子以
PCR增殖出之cLyzProEnhancer (2.0 kb)產物之電泳圖譜 64
圖26.載體pcLyzEnLacZ經限制切割後之電泳圖(A)。膠片中之DNA轉移
至尼龍膜後,與DIG - cLyz探針雜交之顯影圖(B) 65
圖27.構築於pcLyzEnlacZ載體中之雞溶菌基因起動子增強子基因序列
(B)與McReynolds et al. (1978)發表之序列(A)比對。”-”代表
與發表之序列相同 67
圖28.應用PCR以cLyzPro-F2、cLyzPro-R1、T7及lacZ引子偵測
pcLyzEnlacZ之cLyzPro基因增強子序列( 2081 bp ) 68
圖29.重組質體pcLyzEnlacZ之構築 69
圖30.雞卵白蛋白基因組DNA序列及引子 75
圖31.試驗總流程 86
圖32.以來航雞基因組基因庫DNA為模板,利用cOvaPro-F3及cOvaPro-R3引
子以PCR增殖出之cOvaPro DNA( 2.7 kb )片段產物之電泳圖譜88
圖33構築於pcOvalacZ載體中之雞卵白蛋白基因起動子基因序列(cOvaPro)
與McReynolds et al. (1978)發表之序列)比對。”-”代表與發表
之序列相同 90
圖34載體pcOvalacZ經限制切割後之電泳圖譜(A)。膠片中之DNA轉移至
尼龍膜後,與DIG - cOva探針雜交圖(B) 91
圖35.重組質體pcOvalacZ之構築 92
圖36.應用PCR 以OvaPro-F3、cOvaPro-R3、T7及lacZ引子偵測pcOvalacZ
之cOvaPro基因組DNA 93
圖37.pcOvalacZ重組質體於雞生殖道膨大部細胞中經類固醇內泌素誘導3
天後之表現。 為表現lacZ基因產物之細胞 95
圖38.pcOvalacZ重組質體於雞生殖道峽部細胞中經類固醇內泌素誘導3
天後之表現。 為表現lacZ基因產物之細胞。 96
圖39.pcOvalacZ重組質體於雞生殖道峽部細胞中經類固醇內泌素誘導3
天後之表現。 97
圖40.雞鈣結合蛋白cDNA序列及引子 105
圖41.試驗總流程 111
圖42.以來航雞十二指腸上皮細胞之總RNA為模板,利用cCalbin-F2及
cCalbin-R4引子以PCR增殖出之cCalbin cDNA ( 0.78 kb )產物之電
泳圖譜 119
圖43.構築於pCMVcCalbinns載體中之鈣雞鈣結合蛋白基因序列與
Hunziker (1986)發表之序列比對。”-”代表與發表之序列相同
120
圖44.所選殖出之雞鈣結合蛋白胺基酸序列。與Hunziker ( 1986 )發表之
序列比對。”-”代表與發表之序列相同 121
圖45.重組質體pCMVcalbinns之構築 123
圖46.應用PCR以cCalbin-F2、cCalbin-R7、T7及BGH引子偵測
pCMVcCalbinns之cCalbin cDNA序列( 788 bp ) 124
圖47.體pCMVcCalbinns經限制切割後之電泳圖譜(A)。膠片中之DNA
轉移至尼龍膜後,與DIG - cCalbin探針雜交之顯影圖(B)125
圖48.應用RT-PCR以引子cCalbin-F2和cCalbin-R7 偵測轉形入質體
pCMVcalbinns之CHO細胞mRNA之cCalbin cDNA 。cCalbin-F2和
cCalbin-R7可增殖出0.78 kb片段 126
圖49.pCMVcCalbinns轉形入CHO細胞株,培養1-6天,以SDS-PAGE (A)和
Western blot (B)分析cCalbin基因之表現。利用質體上anti-V5抗
體進行西方吸漬,可產生約35.7 kDa之蛋白質 127
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