|摘要: ||枯草桿菌為一革蘭氏陽性、兼性厭氧之產孢桿狀細菌。在遺傳工程上經常被用來作為表現異源蛋白之宿主，其原因如下：(1) 培養容易，(2) 被認可為 GRAS 級菌種，(3) 具有 DNA 轉型能力，(4) 具備強力之蛋白分泌能力。|
生物活性胜肽指的是來自於動物或植物之蛋白質，經由蛋白酶水解後所釋出之短鍊胜肽，其具有調節人體生理機能之活性。自大豆中分離出來之抗氧化胜肽：Leu-Leu- Pro-His-His (LLPHH)，經研究發現具有抑制脂質過氧化之能力，且其抗氧化活性與胜肽 C 端之 -His-His 有著密切之關聯。Ile-Ile-Ala-Glu-Lys (IIAEK)，名為 Lactostatin，為一自牛乳 β-乳球蛋白中所分離出來之胜肽。此胜肽具有降低膽固醇之活性，其作用機制為活化肝臟細胞中特異性存在之酵素：cholesterol 7α-hydroxylase 基因之表現，該酵素負責膽固醇與膽酸轉換過程之速率決定步驟。
靈芝免疫調節蛋白(Ling-Zhi 8；LZ-8)為一自靈芝(Ganoderma lucidum)菌絲體中分離出來之蛋白。其生理活性包括有免疫調節、抗腫瘤、減緩器官移植之排斥作用等。研究指出其免疫調節活性與其雙體結構有密切之關聯性。本實驗室先前已成功於枯草桿菌表現重組靈芝免疫調節蛋白(recombinant LZ-8；rLZ-8)，並且證實重組靈芝免疫調節蛋白具有刺激多種細胞激素分泌之活性。之後本實驗室進一步嘗試於rLZ-8上融合抗氧化胜肽LLPHH或降膽固醇胜肽IIAEK，以期能獲得多重生理活性之蛋白，而在設計上也於胜肽與rLZ-8間導入蛋白酶切位以使胜肽能透過蛋白酶之水解而自融合蛋白上釋出。先前之設計為融合三重複之LLPHH於 rLZ-8上以及三重複之LLPHH與三重複之IIAEK於 rLZ-8上，並且導入連接子、必需胺基酸及蛋白酶切位胺基酸，但此兩蛋白卻不易在枯草桿菌表現及純化，推論其原因可能為所接上之胺基酸序列影響到rLZ-8 之構型，使融合蛋白不易被表現或被枯草桿菌胞內之蛋白酶降解系統所分解。
本實驗嘗試改進融合蛋白所接上之胺基酸序列。LLPHH及IIAEK均融合一重複或二重複於 rLZ-8上，並導入trypsin或pepsin切位胺基酸，將四種融合蛋白命名為rLZHO、 rLZHO2、rLZHC 及rLZHC2。結果顯示此四個融合蛋白均能於枯草桿菌表現並被純化到，分別為130μg/ml、30μg/ml、270μg/ml、220μg/ml。在經由蛋白酶作用後生物活性胜肽能自融合蛋白上釋出，之後將兩種生物活性胜肽以高效能液相層析分析後發現兩者之身分均與化學合成之標準品相同。抗氧化胜肽之活性測試結果顯示來自融合蛋白 rLZHO 之 LLPHH 可有效抑制亞麻油酸之自氧化反應，且於 10-4 M 下其抑制效果較 BHA 佳。
Bacillus subtilis is a Gram-positive, facultative anaerobic, endospore forming rod-shaped bacterium. It has been widely used in production of recombinant protein due to it’s characteristics such as easy to culture, as a GRAS bacterium, has DNA transforming ability and powerful protein secretion capacity.
Bioactive peptides are designated as peptides released from animal or plant proteins after enzymatic digestion, with regulatory functions in human body. Leu-Leu- Pro-His-His (LLPHH), isolated from soybean protein, is an antioxidative peptide which can inhibit lipid peroxidation. Its activity is highly related to the C-terminal –His-His. Ile-Ile-Ala-Glu-Lys (IIAEK), termed as lactostatin, is a hypocholesterolemic peptide isolated from bovine milk β-lactoglobulin. Its hypocholesterolemic mechanism is to activate the gene expression of a liver specific enzyme, cholesterol 7α-hydroxylase, which is the rate-limiting enzyme in the pathway of cholesterol and bile acid conversion.
Ling-Zhi 8 (LZ-8) is a protein isolated from the mycelium of Ganoderma lucidum. Its biological activities include immunomodulatory responses, antitumor activity, alleviation of transplant rejection. Research showed that the activity is highly related to its dimer form. In our previous study, recombinant LZ-8 was successfully expressed by our Bacillus subtilis system, and the cytokines’ secretion stimulation and immunomodulatory activity were proved. After that we tried to fuse rLZ-8 with LLPHH or IIAEK, and also introduced protease cleavage sites between bioactive peptides and rLZ-8 so that the bioactive peptides can be released from the protein by protease digestion. Previously we fused rLZ-8 with three copies of LLPHH in one designation and three copies of LLPHH together with three copies of IIAEK in another, and also added linker, essential amino acids and protease cleavage sites in the fusion sequence. But the expressions were not so successful by the B. subtilis system. It is possible that the amino acid sequence fused with rLZ-8 may interfere the conformation of rLZ-8, so the fusion proteins were hard to express in B. subtilis or degraded by its intracellular protease system.
In this study, we tried to change the amino acid sequence fused to rLZ-8. We fused one copy or two copies of LLPHH to rLZ-8 with trypsin or pepsin cleavage sites, and these two fusion proteins were designated as rLZHO and rLZHO2. In IIAEK we constructed same copies and designated as rLZHC and rLZHC2. These four fusion proeins were successfully expressed in B. subtilis WB800 and could be purified to obtain 130μg/ml, 30μg/ml, 270μg/ml, 220μg/ml proteins, respectively. After protease treatment the peptides could be released from the fusion proteins. The HPLC analysis proved that the bioactive peptides were the same as standards, and the rLZ-8 was in a dimer form that indicated the bioactivity of rLZ-8. The result of antioxidative activity assay showed that the LLPHH from rLZHO can inhibit the autoxidation of linoleic acid, and the antioxidative activity is higer than BHA in 10-4 M.
Keywords: Bacillus subtilis, Ling-Zhi 8, dimer form, antioxidative peptide, hypocholesterolemic peptide, fusion protein