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研究生:王仕蓉
研究生(外文):Wang, Shih-Rong
論文名稱:豬乳鐵蛋白基因及啟動子之選殖與分析
論文名稱(外文):Cloning and analysis of porcine lactoferrin gene and its promoter
指導教授:林彩雲林彩雲引用關係
指導教授(外文):Jia-Ling Yang
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
畢業學年度:86
語文別:中文
論文頁數:115
中文關鍵詞:乳鐵蛋白基因免疫染色啟動子調控區
外文關鍵詞:Lactoferringeneimmunohistochemicalpromoterregumatory region
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乳鐵蛋白(Lactoferrin)為非血紅素之攜鐵醣蛋白,屬於運鐵蛋白家族(
transferrin fa-mily)之一員,主要存在於乳汁、嗜中性白血球及各種外
泌液中,具有多項重要的生物功能:(1)抗菌及抑菌作用,已有多篇報告
指出,乳鐵蛋白(lactoferrin)能有效抑制大腸桿菌 (Escherichia
coli),鏈球菌 (Streptococus),淋病雙球菌 (Neisseria) 等細菌之生
長; (2)促進白血球之分化生成; (3)可作為發炎現象之指標, 並可和巨
噬細胞等非特異性免疫細胞共同參與免疫反應; (4)在腸胃道中、負責鐵
離子之運送和吸收; (5)可促進淋巴細胞和上皮細胞之增生,並可增進生
殖道與腸道的從小鼠、人乳鐵蛋白之研究可知,在發炎期的嗜中性白血球
及泌乳期的乳腺中含有大量的乳鐵蛋白,腦細胞中則完全沒有此蛋白質;
而在其他體液中則有少量存在,這種表現差異性,使我們對乳鐵蛋白基因
在不同組織中之調控機制,引起了莫大的興趣,對於乳鐵蛋白基因分子層
次之研究,目前以小鼠之資料較完整,乳鐵蛋白在小鼠子宮中的表現是由
雌激素 (estrogen) 所控制,而在乳腺中可能由泌乳激素(prolactin) 所
調節,這些調控機制的探討皆須依賴分子層次,即基因之分析才得以達成
。在基礎研究方面本研究之目的係選殖豬乳鐵蛋白基因並進而瞭解此基因
之調控機制,第一階段先篩選出豬乳鐵蛋白cDNA,我們以設計所得之PCR
片段作為探針篩選豬乳腺基因庫,獲得完整豬乳鐵蛋白cDNA (pLTF)。經
雙向定序, 確定核酸序列全長約2.3 kb,5''端具有ATG轉譯起始點,3''端
具有AATAAA 聚腺核酸化標誌及15個腺核酸。乳鐵蛋白基因之個數經
實驗證實係以低複製數目(一套)存在於豬的基因組內, 此項結果和小鼠
、人之情形相同。另外經由試管內轉錄和轉譯反應 (in vitro
transcription/translation)及免疫沉澱 (immunoprecipitation) 試驗
證實此豬乳鐵蛋白cDNA所產生之~78kD蛋白質產物,其分子量及抗原性和
純化之豬乳鐵蛋白相同。第二階段利用北方印跡(Northern blot
hybridization) 及免疫染色法(immunochemical staining) 檢視豬乳鐵
蛋白基因在豬隻各組織之表現情形。 實驗結果顯示,豬乳鐵蛋白mRNA及
其蛋白質在母豬乳腺及公豬附睪丸皆有高量的表現。豬乳鐵蛋白於泌乳期
之乳腺管腔表皮細胞中表現最強,但在分娩十天後逐漸減弱, 故推測此蛋
白質之表現和乳腺之發育機制有關。另外在腸道細胞中 (十二指腸、空腸
、迴腸) 亦有乳鐵蛋白之表現,其中又以十二指腸表現量居多,顯示此蛋
白質在上消化道可能扮演保護或促進鐵離子運送吸收等功能。而在公豬附
睪丸也發現大量乳鐵蛋白的表現,推第三階段為了探討豬乳鐵蛋白基因之
調控機制,我們以其cDNA 5''端片段為探針篩選出豬乳鐵蛋白基因啟動子
調控區。 核酸序列分析得到約 4 kb 5'' 端上游區, 84 bp 第一轉錄區(
exon 1),2.1 kb 第一內插區(intron 1),及 162 bp第二轉錄區(exon
2)。 又經實驗證實其轉錄起始點是位於ATG轉譯起始點前41個位置之鳥糞
酸 (G)。以電腦軟體分析發現豬乳鐵蛋白基因含有許多常態性和誘發性
之轉錄因子相關區。剪接不同長度區域之核酸片段插入報導基因 (CAT
reporter gene) 上游,藉以測試啟動子功能。 結果顯示在豬的睪丸細胞
株(ST)、腎臟細胞株(PK15)及人的乳腺細第四階段是進一步分析豬乳鐵蛋
白基因之調控區域,藉電泳移動性試驗法 (electrophoretic mo-bility
shift assay) 和足跡試驗法 (DNaseI footprint analysis),探討可能
影響此基因表現之調控因子。 實驗結果顯示與蛋白質SP1, AP2, MGF/
Stat5結合之核酸序列為乳鐵蛋白基因上之重要調控序列, 而MGF/Stat5
則為豬乳鐵蛋白基因於乳腺表現時重要之轉錄因子。
Lactoferrin (LTF) is a member of the transferrin gene family
of non-heme iron binding glycoproteins. This protein notably
found in milk, polymorphonuclear leukocytes and mucosal
secretions. Lactoferrin protein has been reportedto have
multiple functions: (1)LTF has a broad spectrum of antimicrobial
pro-perties, such as Esherichia coli, Streptococcus, Neisseria;
(2)LTF involves indifferentiation process of granulocyte;
(3)LTF modulates nonspecific immune functions and can be used as
an indicator for in Lactoferrin is not expressedin brain but is
constitutively expressed in mature neutrophils. On the
otherhand, lactoferrin is delicately control in the lactation
period of mammary gland. The differential pattern of the
expression of LTF in various tissues aroused the interest of
studying the regulatory mechanism of LTF expression. Expression
of LTF is regulated by estrogen and prolactin in mouse uterus
and mammary gland, respectively. However, the differential
mechanism of regulationof LTF expression. First, we isolated a
full length of porcine LTF cDNA clonefrom a sow''s mammary gland
cDNA library using a DNA fragment of pLTF obtained from
polymerase chain reaction as probe. This porcine LTF was ~2.3
kilobase pairs. The porcine LTF gene carries an ATG translation
start codon at 5''-re-gion. Polyadenylation signal (AATAAA) and
polyadenylation tract (A15) at 3''-untranslated region. Porcine
LTF was found to be a low-copy (one copy) gene in the haploid
genome as in mice and human. Porcine LTF cDNA, wh Using
Northernblotting hybridization and immunohistochemical staining,
we examined LTF expre-ssion in various organs of pigs. High
amounts of porcine LTF mRNA were detectedin the secreting
mammary gland and epididymis. This distribution is
consistentwith that of porcine LTF examined by
immunohistochemistry. In female pigs,porcine LTF mRNA
concentration increased remarkably in the ductal cells of the
lactating mammary gland then significantly decreased at day 10
after parturi-tion. This pattern imp To elucidate the
controlling mechanisms of porcine lac-toferrin gene (pLTF), we
cloned the 5''-flanking region of the pLTF from a por-cine liver
genomic library by using its 5''- end of cDNA clone. The partial
se-quence of pLTF gene clone comprises 4 kb of 5'' flanking
region, 84 bp of exon 1, 2.1 kb of intron 1, and 162 bp of exon
2. The transcription start site was at residue G (-41) from the
translation start ATG site as assayed by primer ex-tension. The
pLTF promoter possesses several putative cis-acting Detailed
an-alysis of this proximal region by electrophoretic mobility
shift assay (EMSA) and DNase I footprint analysis indicated that
the ubiquitous factors SP1, AP2 and mammary gland factor (MGF/
Stat5) might play major roles in regulating the transcription of
pLTF gene.
封面
目錄 (Table of Contents)
Acknowledgements
List of Talbes
List of Figures
Abbreviation
中文摘要
英文摘要
Chapter I. Literature review
1. Iron homeostasis
2. Discovery and properties of lactoferrin
3. Proposed functions of lactoferrin
4. Implication and significance
Chapter II. Characteriaztion of procine lactoferrin cDNA
1. Introduction
2. Materials and methods
3. Results
4. Discussion
Chapter III. Spatial and temproal expression of porcine lactoferrin
1.Introduction
2. Materials and merhods
3. Results
4. Discussion
Chapter IV. Analysis of the promoter activity of procine lactoferrin gene
1. Introduction
2. Materials and methods
3. Results
4. Discussion
Chapter V. Molecular regulation of procine lactoferrin gene
1. Intriduction
2. Materials and methods
3. Results
4. Disscussion
Chapter VI. References
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