Abstract
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 (Escherichiacoli)，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 vitrotranscription/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 blothybridization) 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(exon2)。 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 (CATreporter 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-bilityshift 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 familyof non-heme iron binding glycoproteins. This protein notablyfound in milk, polymorphonuclear leukocytes and mucosalsecretions. Lactoferrin protein has been reportedto havemultiple functions: (1)LTF has a broad spectrum of antimicrobialpro-perties, such as Esherichia coli, Streptococcus, Neisseria;(2)LTF involves indifferentiation process of granulocyte;(3)LTF modulates nonspecific immune functions and can be used asan indicator for in Lactoferrin is not expressedin brain but isconstitutively expressed in mature neutrophils. On theotherhand, lactoferrin is delicately control in the lactationperiod of mammary gland. The differential pattern of theexpression of LTF in various tissues aroused the interest ofstudying the regulatory mechanism of LTF expression. Expressionof LTF is regulated by estrogen and prolactin in mouse uterusand mammary gland, respectively. However, the differentialmechanism of regulationof LTF expression. First, we isolated afull length of porcine LTF cDNA clonefrom a sow's mammary glandcDNA library using a DNA fragment of pLTF obtained frompolymerase chain reaction as probe. This porcine LTF was ~2.3kilobase pairs. The porcine LTF gene carries an ATG translationstart codon at 5'-re-gion. Polyadenylation signal (AATAAA) andpolyadenylation tract (A15) at 3'-untranslated region. PorcineLTF was found to be a low-copy (one copy) gene in the haploidgenome as in mice and human. Porcine LTF cDNA, wh UsingNorthernblotting hybridization and immunohistochemical staining,we examined LTF expre-ssion in various organs of pigs. Highamounts of porcine LTF mRNA were detectedin the secretingmammary gland and epididymis. This distribution isconsistentwith that of porcine LTF examined byimmunohistochemistry. In female pigs,porcine LTF mRNAconcentration increased remarkably in the ductal cells of thelactating mammary gland then significantly decreased at day 10after parturi-tion. This pattern imp To elucidate thecontrolling mechanisms of porcine lac-toferrin gene (pLTF), wecloned the 5'-flanking region of the pLTF from a por-cine livergenomic library by using its 5'- end of cDNA clone. The partialse-quence of pLTF gene clone comprises 4 kb of 5' flankingregion, 84 bp of exon 1, 2.1 kb of intron 1, and 162 bp of exon2. The transcription start site was at residue G (-41) from thetranslation start ATG site as assayed by primer ex-tension. ThepLTF promoter possesses several putative cis-acting Detailedan-alysis of this proximal region by electrophoretic mobilityshift assay (EMSA) and DNase I footprint analysis indicated thatthe ubiquitous factors SP1, AP2 and mammary gland factor (MGF/Stat5) might play major roles in regulating the transcription ofpLTF gene.