在大腸桿菌表現系統表現 Streptococcus zooepidemicus HAS蛋白質

Abstract

透明質酸透明質酸(hyaluronic acid)，又稱玻尿酸或醣醛酸，由葡糖醛酸-N-乙醯氨基葡糖為雙糖單位組成的直鏈高分子量之生物多醣，普遍存在於動物結締組織，常於組織中的細胞外間(extracellular matrix)結合而形成一具有支撐性及保護性的立體結構存在於細胞周圍，為結締組織多醣體的一種。它對於組織結構上整體的保養和細胞間的輸送都具有很重要的功能。目前透明質酸主要商業應用的範圍有眼科手術、骨關節炎、創傷、運動醫學、整容外科、化妝品、藥物遞送、外科手術防黏劑、牙科及腸科。目前主要生產商品用的透明質酸是以動物組織為原料的提取法和微生物發酵法。但這兩種方法基於安全性及經濟成本的理由仍有改善的空間。近年來則有提倡以分子生物技術生產透明質酸的趨勢。在本研究中，我成功的藉由genomic-PCR獲得 Streptococcus zooepidemicus hasA的轉錄區域片段，並選殖於大腸桿菌載體，並在 E. coli中做表現。重組蛋白上並帶有 HA或HIS 的tag，因此可使用Anti-HA-HRP或Anti-HIS-HRP抗體以Western blotting偵測HAS重組蛋白質的表現，我可在大腸桿菌加IPTG誘導四小時的pellet部分，觀察到重組蛋白質的表現。而且可以用nickel column純化帶有HIS-tag之重組蛋白質。

Hyaluronic acid exists in many mammalian connective tissues such as joints, vitreous bodies, umbilical cords, cartilages, skins, and combs of fowls as a constituent; it fulfills important functions such as flexibility and structure maintenance of tissues. Hyaluronic acid is a polymer made of repeating units of glucuronic acid and N-acetyl-glucosamine bound by alternating β-1,3 and β-1,4 bonds. Its molecular weight is in the range of 10 kD~ 1000 kD. It has a wide variety of uses in cosmetics, medicine for wounds, eye drops, and medicines for ophthalmic surgery and arthritis. Currently, the production of HA comes from extraction animal tissue or fermentation of HA-producing pathogens. Both have issues concerning health and safety. Introduction of molecular recombinant DNA technique to the production of HA is a new trend to solve the problems. In this study, I have successfully constructed recombinant plasmid expressing hasA encoding protein of Streptococcus zooepidemicus in E. coli. The recombinant protein has contained in-vivo-expressing tag of either HA (influenzae hemagglutinin) or HA-HIS (influenzae hemagglutinin-polyhistidine). Recombinant proteins of HAS fusing with HA-HIS tag can be detected in the pellet fraction of the E. coli expression system by Western blotting analysis. Meanwhile, Purification using nickel column of the recombinant protein of HAS with HIS tag was also achieved.