利用乳酸鏈球菌表現純化重組人類第一型三葉因子及人類介白素-10

Abstract

現代人因為飲食習慣不正常以及生活壓力大，常導致腸胃發炎潰爛以致最後形成腫瘤，因此腸胃道不適儼然然成為現代人的文明病。而人類的腸胃道在修復其上皮組織的受損時，常會分泌HCO3-、熱休克蛋白、三葉因子及抗菌胜肽，其中三葉因子 TFF (trefoil factor)可藉由刺激細胞的移動、抑制細胞自殺和發炎，修復腸胃道上皮組織。文明病除了腸胃道問題外又以過敏性疾病為大宗，人體受到細菌或外來物質入侵時，免疫系統將釋放組織胺，並且產生發炎發炎反應，常見的紅腫熱痛便是局部病徵。而急性發炎是免疫系統對於病原入侵策動猛烈攻擊所引發的症狀，不但對病原有殺傷力，對一般正常細胞具有破壞性。介白素-10 (interleukin 10, IL-10)可調節慢性、急性發炎反應如抑制細胞激素IL-1、IL-2和腫瘤壞死因子TFN的產生因此分類為抗發炎性細胞激素。 本論文以 GRAS 級的乳酸鏈球菌表現重組人類第一型三葉因子，首先調整本實驗室先前構築的 MNICE 系統(pNZNS-SacBATFF1)之 nisin 誘導濃度，使MNICE系統能穩定的表現重組人類第一型三葉因子，接下來構築 MPHI 酸誘導系統(pNZAUS-SacBATFF1)，確認其表現具再現性後，兩系統以饋料的發酵方式(Fed batch)在小量發酵槽中嘗試提升其蛋白表現量， MNICE系統可得到418.2 μg/L，而 MPHI 可得到441.7μg/L之重組人類第一型三葉因子，並經由雙體測定、原態蛋白質電泳及蛋白質分子量鑑定，確認蛋白質具有單體、雙體和多聚體。最後利用細胞傷口癒合試驗，測定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，能提高人類胃癌細胞(AGS cell)之細胞癒合效率。為確保重組人類第一型三葉因子也能在胃酸環境中具有活性，進一步以pH 2.4模擬胃酸環境測定其蛋白穩定性，其結果存於 ”pH 2.4” 環境下的重組人類第一型三葉因子，具有較高的細胞癒合活性(P<0.05)，推測在酸性的環境中重組人類第一型三葉因子因構形的改變，使其對細胞的癒合活性提高。 在 IL-10的部分也是以乳酸鏈球菌為宿主，首先設計並合成乳酸鏈球菌最適化之IL-10密碼子，以此構築持續形的 IL-10 表現質體(pNZDSASm-SacBATFF1)。由於在乳酸鏈球菌表現時，上清液並未偵測到 IL-10的表現，因此取乳酸鏈球菌之破菌後菌體可溶部分，以西方墨點法確認後，在目標分子量有明顯的條帶，為確認是否為IL-10，本實驗以市售之 IL-10 antibody作為西方墨點的第一級抗體，結果在目標分子量具有明顯條帶，並與先前之西方墨點結果相似，因此本實驗構築之表現系統，可在胞內表現表現IL-10。 本實驗所構築之MpHI系統，經饋料發酵方式可得到高純度的重組人類第一型三葉因子，且具有生物活性，未來可經工業化生產大量運用於生醫保健市場。

Gastrointestinal dameges are caused by irregular eating habits and life stress, finally result in tumor ulceration. Therefore gastrointestinal diseases have become modern civilized illness. Human gastrointestinal tract epithelial tissue repair their damages,by secreting HCO3-, heat shock proteins, trefoil factor, and antimicrobial peptides. Among these repairing factors trefoil factor can stimulate cell migration, by inhibit cell suicide and inflammation repair gastrointestinal epithelium. Apart from gastrointestinal dameges allergic diseases is also a civilized illness. When the body was invaded by bacteria or foreign objects, the immune system release histamine and cause inflammation. The acute inflammation of the immune system for pathogen invasion caused symptoms, not only destruct pathogens but also make normal cells destructive. Interleukin -10 (interleukin 10, IL-10) can adjust chronic, acute inflammation inhibiting cytokines such as IL-1, IL-2, and tumor necrosis factor production TFN, and interleukin -10 is classified as anti-inflammatory cytokines. In this study, the recombinant human trefoil factor 1(TFF1) were expressed by GRAS grade Lactococcus lactis. In the first part of this study, the induction nisin concentration of MNICE system (pNZNS-SacBATFF1) was re-established, therefore accomplish more stable rTFF1 extracellular expression. Another acid-inducible system(MPHI)(pNZAUS-SacBATFF1)was constructed, and confirmed the expression. The two systems fermented by using fed batch in a 5L fermenter .MNICE obtain 418.2 μg / L and MPHI available 441.7μg / L of recombinant human trefoil factor 1. The purified recombinant human trefoil factor 1 from two systems were identified by homodimer determination, native –PAGE, MALDI-TOF MS. The bioactivity of recombinant human trefoil factor 1 from two systems were analyzed by wound healing assay of AGS cells. Results showed that the recombinant human TFF1 from two systems exhibited wound healing capacities of AGS cell. The recombinant human TFF1, treated with pH 2.4 buffer which showed better active wound healing capacities. This results suggest that the acidic environment in stomach might be benefitial to active conformation of recombinant human TFF1. In another part of this study a novel recombinant human IL-10 gene was designed according to the preferred codons of L. lactis. Then the recombinant human IL-10-expressing plasmids was constructed(pNZDSASm-sacBAIL-10). The rIL-10 was not secreted extracellular but was detected as pre-IL-10 by western blot analysis. In conclusion the MpHI system fermented by using fed batch can obtain high purity and biological activity trefoil factor I. In the near future can be applied to healthcare industry.