抗微生物胜肽普遍存在於各生物體中，為非專一性先天免疫系統的一環，在近年來因抗生素的濫用，造成許多具抗藥性致病性微生物的出現，而抗微生物胜肽因其獨特的生物活性及不同於傳統抗生素的作用機制，成為科學家開發研究的目標。但從天然物中萃取過程複雜且產量極少，化學合成肽類成本又過高，因此利用基因工程來生產為目前主要發展的方向。本論文利用大腸桿菌原核表現系統將抗微生物胜肽以融合蛋白B2-C-AMP 的方式在大腸桿菌中表現，再利用化學裂解反應取代蛋白酶的分解將融合蛋白中的抗微生物胜肽片段切下。從資料庫中選取三種活性有差異且長度不同之抗微生物胜肽apidaecin Ia、calcitermin 與CAP 7，將其對應核酸序列接在NNVB2基因之3' 端，並在連結處加上化學裂解切位Met(M)、Asp(D)與Asn-Gly(NG)之相對核酸密碼，得到B2-M-Cal、B2-D-Cal、B2-NG-Cal、B2-M-Api、B2-D-Api與B2-M-CAP 共六個重組基因，將其轉殖至E. coli BL21(DE3)中表現得到融合蛋白，所有融合蛋白皆以包涵體的形式表現。融合蛋白表現量為73.2~247.1 mg/L，表現量與抗微生物胜肽本身活性成負相關。在化學裂解反應方面，我們選用常用的三種化學藥劑溴化氰、蟻酸與鹽酸羥胺來做胜肽切割，比較三種化學藥劑裂解的效率，發現溴化氰之裂解效率最佳(達90%以上)。使用高效液相層析儀以C18 逆相管柱純化化學裂解產物可得到抗微生物胜肽，並做抑菌圈等分析確定其具有抗菌活性；從抑菌圈分析發現apidaecin Ia與CAP 7 皆可抑制大腸桿菌生長而形成抑菌圈，而calcitermin 可短期抑制大腸桿菌生長，且其活性與從鼻腔分泌液萃取的calcitermin 相同。本論文成功地利用大腸桿菌表現系統，以NNVB2 融合蛋白方式，生產形成包涵體的B2-抗微生物胜肽融合蛋白，其生產率比其他已發表的生產系統高，再加上使用化學裂解法，可大大降低生產成本，當可進一步發展成為大量生產抗微生物胜肽的方法。

AMPs (antimicrobial peptides) are an important part of innate immunity in all living things. In recent years, there was increase drug resistance of pathogenic microbes to many antibiotics due to antibiotics abuse. Researchers then change their research targets to develop AMPs which have unique bioactivity and different function mechanism compared to antibiotics. Extraction of antimicrobial peptide from organisms is complicated and little amount was obtained. The cost of chemical synthesis of peptides is high. To obtain large amount of AMPs, recombinant expression of AMPs would be the best choice. In this research, we have developed an E. coli fusion expression system to produce AMPs. NNVB2 was used as the fusion partner. The coding sequence of AMPs was cloned into pET29b(+) vector and expression as a B2-C-AMP (C presents a chemical cleavage site) fusion protein in E coli. Three different chemical cleavage reagents, cyanogen bromide, formic acid and hydroxylamine were used instead of protease to cleave off AMPs from fusion proteins, and the use of chemical cleavage reagents was cost less. Three AMPs, apidaecin Ia, calcitermin and CAP 7 (expression as fusion protein B2-M-Cal, B2-D-Cal, B2-NG-Cal, B2-M-Api, B2-D-Api and B2-M-CAP) have been successfully expressed using this expression system and the resulting expression level of fusion proteins reached up to 73.2~ 247.1 mg per liter cell culture. All fusion proteins were expressed as inclusion bodies in E. coli. 90% AMPs can be cleaved off from fusion protein by cyanogen bromide and 46% by formic acid. No cleavage product was found treated with hydroxylamine. The antimicrobial activities of the AMPs, purified by C18 reverae-phase column FPLC, were measured and are identical to that of the native extracted peptides. Recombinant apidaecin Ia and CAP 7 both inhibited the growth of E. coli and clear inhibition zones can be seen in inhibition zone assay. Recombinant calcitermin inhibited the growth of E. coli temporarily similar to native extracted calcitermin. In this research, we have developed successfully a high production system of antimicrobial peptides using NNVB2 as fusion partner. E. coli expression system that expression of antimicrobial peptides by a fusion partner reinforcing formation of inclusion bodies can increase the level of productions.

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