Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52015
標題: 利用大腸桿菌及乳酸鏈球菌表現及純化重組人類第一型三葉因子
Expression and purification of recombinant human trefoil factor 1 by Escherichia coli and Lactococcus lactis
作者: Lu, Meng-Ting
呂孟婷
關鍵字: gastrointestinal healing;腸胃道修復;trefoil factor 1;Escherichia coli BL21(DE3);Lactococcus lactis NZ9000;Caco-2 cell;第一型三葉因子;大腸桿菌;乳酸鏈球菌;人類結腸癌表皮細胞株
出版社: 食品暨應用生物科技學系所
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Cellular localization, binding sites, and pharmacologic effects of TFF3 in experimental colitis in m
摘要: 
Digestive system damages are common problems in people who lived in a stressful life. Mucosal defense in gastrointestinal tract includes local gastric mucosal defense mechanisms and neurohormonal regulation. The surface epithelial cells secrete mucus, bicarbonate and generate prostaglandins, heat shock proteins, trefoil factor peptides (TFFs), and cathelicidins to defense or regulate the mucosal damages. The TFFs mediate mucosal repair by stimulating cell migration, inhibiting apoptosis and inflammation, and promote the barrier function of mucus.
In this study, attempts of expressing recombinant human trefoil factor 1 (TFF1) by well known Escherichia coli and GRAS Lactococcus lactis were proceeded. In the first part of this study, a novel recombinant human TFF1 gene was designed according to the preferred codons of L. lactis. Then the recombinant human TFF1-expressing plasmids were constructed. The recombinant human TFF1-expressed plasmids for L. lactis including constitutive pNZDSASm-sacBATFF1, and nisin-inducible pNZNS-sacBATFF1 and pNZNUB-TFF1. Isopropyl β-D-1-thiogalactopyranoside (IPTG)-indicible pET-sacBATFF1 and pET-TFF1 are the recombinant human TFF1-expressed plasmids of E. coli. Owing to the unstable expression of L. lactis system, the optimized recombinant human TFF1 was purified from fermented E. coli BL21(DE3) transformants and the purified recombinant human TFF1 was identified by native-PAGE, Western blot, and peptide fingerprinting. The bioactivity of recombinant human TFF1 was analyzed by wound healing assay of C2BBe1 cell (clone of Caco-2 cell). Results showed that the recombinant human TFF1 exhibited reclosing up wound of C2BBe1 cell monolayer and could be improved is increased by adding few FBS. The recombinant human TFF1, treated with modified gastrointestinal pH, pH 2.4 and pH 7.0 buffer showed that the recombinant human TFF1 is more active after pH 2.4 treatment. Results suggest that the acidic environment in stomach might be benefitial to active conformation of recombinant human TFF1.

現代人生活壓力大,消化系統不適已經成為普遍的問題之一,導致腸胃發炎、潰瘍、甚至演變為腫瘤。腸胃道黏膜防禦包含局部的黏膜防禦機制與系統性神經調控,而消化道表皮細胞會分泌黏液、HCO3-並生產前列腺素、熱休克蛋白、三葉因子與一些抗菌胜肽修護受損的表皮組織。其中三葉因子可以藉由刺激細胞移行、抑制細胞自殺與發炎,以維持黏膜屏障功能。
本研究嘗試以熟知的大腸桿菌及食品級乳酸鏈球菌表現重組人類第一型三葉因子,首先設計並合成最佳化密碼子之新穎性基因,分別構築重組人類第一型三葉因子之表現質體。乳酸鏈球菌表現重組人類第一型三葉因子之質體包含持續型之pNZDSASm-sacBATFF1,以及以乳酸鏈球菌素誘導之pNZNS-sacBATFF1與pNZNUB-TFF1質體;大腸桿菌使用之表現質體則是以異丙基-β-D-硫代半乳糖苷誘導之pET-sacBATFF1與pET-TFF1質體。由於乳酸鏈球菌之系統表現再現性不高,選用大腸桿菌發酵誘導表現、取菌體之可溶部份純化重組人類第一型三葉因子,可得到0.719 mg/L rTFF1及1.766 mg/L rSTFF1之產量,並經由原態蛋白質電泳、西方墨點及蛋白質身份鑑定確認蛋白質具有單體及雙體結構。最後利用細胞傷口癒合試驗觀察重組人類第一型三葉因子之生物活性,結果顯示重組人類第一型三葉因子具有對於人類結腸癌表皮細胞傷口癒合之效果,且可藉由添加低量胎牛血清提高細胞傷口癒合速度。進一步以pH 2.4與pH 7.0模擬腸胃道之pH環境測試重組人類第一型三葉因子之pH穩定性,由結果可知儲存於pH 2.4之重組人類第一型三葉因子的細胞傷口癒合活性明顯較佳(P < 0.05),推測胃中酸性環境可能有利於重組人類第一型三葉因子之構形及活性。
URI: http://hdl.handle.net/11455/52015
其他識別: U0005-2607201114415200
Appears in Collections:食品暨應用生物科技學系

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