Expression and purification of recombinant human trefoil factor 1 and human interleukin-10 by Lactoccus lactis

• Main Authors: Wei-Lun Liu, 劉衛綸
• Other Authors: 葉娟美
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/gg8b48

碩士 === 國立中興大學 === 食品暨應用生物科技學系所 === 101 === 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.