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標題: Optimization of the expression and purification of a functional recombinant Ganoderma lucidum immunomodulatory protein Ling Zhi-8 in Bacillus subtilis and Lactococcus lactis
作者: 葉鈞凱
Yeh, Chun-Kai
關鍵字: 真菌免疫調節蛋白質;fungal immunomodulatory protein;重組靈芝免疫調節蛋白質;分泌表現;枯草桿菌;乳酸鏈球菌;細胞激素;TH1亞型;recombinant LZ-8;secretory production;Bacillus subtilis;Lactococcus lactis;cytokine;TH1 subsets
出版社: 食品暨應用生物科技學系
Ling Zhi-8 (LZ-8), a fungal immunomodulatory protein (FIP), was isolated from the mycelial extracts of Ganoderma lucidum. The protein consists of 110 amino acid and has a molecular mass of 12,420 Da. LZ-8 is considered as a potential immunomodulating agent and has a potential of applications in medicine and health foods. LZ-8 can be purified from Ganoderma lucidum. Genetic engineering is also an attractive approach for high level production of recombinant LZ-8 (rLZ-8).
Bacillus subtilis and Lactococcus lactis, two species of GRAS (generally recognized as safe) bacteria, are widely used in fermentation industry. In addition, they are attractive hosts for production of heterologous proteins. In previous, our laboratory had established rLZ-8 expression systems for B. subtilis WB800 and B. licheniformis. In this study, we aimed to develop a new expression system for the high level expression of rLZ-8 in L. lactis NZ9000. We also investigated the influence of different media on the expression of rLZ-8 in B. subtilis WB800 and L. lactis NZ9000.
The results showed that two expression vectors pNZSLZ and pFNSLZ used for secretory production of rLZ-8 in L. lactis NZ9000 were successfully constructed. The plasmid pFNSLZ containing a nisin resistance gene nsr as selection marker is a food grade expression vector. L. lactis transformants harboring pNZSLZ and pFNSLZ could increase the secretory expression of rLZ-8. The cell densities of L. lactis NZ9000 and B. subtilis WB800 were enhanced by changing the media composition. The enhanced bacterium density increased the production of rLZ-8 in L. lactis NZ9000 and B. subtilis WB800. In this study, we established a rapid purification procedure for efficient purification of rLZ-8 from culture supernatant of B. subtilis WB800 and L. lactis NZ9000. The purified rLZ-8 exists in the form of homodimer and has the ability to stimulate peripheral blood monocytes and RAW 264.7 macrophages to secrete IL-2, IFN-γ, IL-12, IL-4 and TNF-α. The purified rLZ-8 has a potential to become health-promoting food ingredients with anti-allergic function.

靈芝免疫調節蛋白質Ling Zhi-8 (LZ-8)是由靈芝 (Ganoderma lucidum) 中分離出之真菌免疫調節蛋白質,其含有110個胺基酸,分子量為12,420 Da。研究指出LZ-8為一具有發展潛力之免疫調節劑。若要將其實際運用於醫藥或保健食品中,則首要之任務為取得大量之蛋白質。目前LZ-8之取得多自靈芝菌絲體中分離純化,而利用遺傳工程之方式大量表現生產亦是一可行之策略。本論文乃使用基因重組之方法,大量表現生產重組LZ-8。
枯草桿菌 (Bacillus subtilis)及乳酸鏈球菌 (Lactococcus lactis) 均屬於GRAS (generally recognized as safe) 級之菌種,常被運用於發酵工業。此外在遺傳工程之研究範疇中,亦常被使用作為宿主細胞以表現異源蛋白質。本實驗室先前已成功建立枯草桿菌及地衣芽孢桿菌 (Bacillus licheniformis) 分泌表現重組靈芝免疫調節蛋白質 (recombinant LZ-8; rLZ-8) 之系統。本論文則利用乳酸鏈球菌作為宿主,以建立rLZ-8之分泌表現系統及食品級表現系統,並探討不同培養基對重組蛋白質產量之影響。
論文中已建構出乳酸菌分泌表現質體pNZSLZ及食品級分泌表現質體pFNSLZ。食品級質體中乃利用nisin抗性基因nsr取代抗藥性基因。將這兩個質體轉形入L. lactis後,可觀察到轉形株具有分泌表現rLZ-8之能力,顯示本論文已成功建立乳酸菌分泌表現rLZ-8之系統。進一步探討不同培養基對枯草桿菌及乳酸鏈球菌分泌表現rLZ-8之影響,結果顯示medium X及2GM17B可提升枯草桿菌及乳酸鏈球菌分泌表現rLZ-8之產量。利用本實驗建立之純化流程可自枯草桿菌及乳酸鏈球菌之上清液中純化得到rLZ-8。純化之rLZ-8均具有雙體結構,且於體外活性分析中證實不同微生物生產之rLZ-8皆具有刺激人類週邊血液淋巴細胞分泌IL-2、IFN-γ、IL-12及IL-4等細胞激素與活化小鼠巨噬細胞株RAW264.7之能力,而rLZ-8於適當濃度下能促使體外TH細胞表現傾向TH1亞型之免疫平衡。故以枯草桿菌及乳酸鏈球菌所生產之rLZ-8皆具有開發成為抗過敏健康食品之潛力。
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