金針菇免疫調節蛋白於乳酸菌表現及其免疫調節活性

Abstract

本研究將構築乳酸菌 (LAB) 異蛋白表現系統以獲得 fve 重組蛋白表現菌株，並探討 LAB 表現 fve 重組蛋白之免疫調節活性。 本研究首先構築 LAB 表現異蛋白之載體系統，以穿梭載體 pLP3537 作為骨架，再以 Lactobacillus casei ATCC 393 之 plac 啟動子作為啟動表現元件，搭配大腸桿菌商業載體 pQE30 之 lamda 終止子，建構表現載體 pLP-LT1；分別將 fve 蛋白以及 hisfve 蛋白之表現基因與 pLP-LT1 黏合，獲得表現載體 pLP-LFT1 及 pLP-LHFT1。成功獲得 LAB 重組菌株後，以乳糖培養基進行菌株培養及蛋白表現之誘導。由 SDS-PAGE 電泳結果顯示乳酸菌生產之重組 fve 及 hisfve 表現量極低，但 LAB/hisfve 總蛋白經由 FPLC 系統純化濃縮後，可獲得分子量約 18 kDa 之產物，並以 Anti-His tag 抗體可辨認到此蛋白。 以 BALB/c 小鼠進行免疫活性分析，非特異性免疫反應方面，結果顯示餵食 LAB、LAB/hisfve 及 LAB/fve 對小鼠腹腔巨噬細胞分泌 TNF-alpha 及 NO 以及刺激脾細胞增生及分泌 IFN-gamma 之活化均不顯著 (P＞0.05)。在 OVA 特異性免疫反應方面，LAB、LAB/hisfve 及 LAB/fve 均可顯著提升小鼠脾細胞 OVA 特異性之細胞增生以及 OVA 特異性 IFN-gamma 產量，以及血清中分泌 OVA 特異性IgG (P＜0.05)。然而 LAB/hisfve 及 LAB/fve 組與LAB組之間並無差異 (P＞0.05) 。 本研究成功構築乳酸菌表現載體 pLP-LT1，獲得可表現 fve 蛋白之重組乳酸菌株，結果顯示此表現平台具有實際應用於食品工業之潛力。

The objectives of this study were to construct a heteroprotein expression system of lactic acid bacteria (LAB), to express an immunomodulatory protein from Flammulina velutipes (fve) in recombinant LAB strain, and to study the immunoregulatory activity of fve-expressing LAB strains in vivo. In this study, the LAB expression vector pLP-LT1 was constructed based on shuttle vector pLP3537 as a backbone with a plac promoter from Lactobacillus casei ATCC 393 and a lamda terminator from E.coli vector pQE30. The genes encoding hisfve and fve were cloned to generate the expression vectors pLP-LHFT1 and pLP-LFT1, respectively. After transformation, these two recombinant LAB stains were established and their expression of proteins was produced under lactose- induction. SDS-PAGE analysis showed that the expression levels of hisfve and fve proteins by LAB were too low for detection, although hisfve protein could be blotted and recognized by His-tag antibody after FPLC purification. Different LAB samples (LAB, LAB/hisfve, and LAB/fve) were orally fed (5x109 cells/day) to BALB/c mice for 30 days and their immunomodulatory activities were further evaluated. Results indicated that oral administration of LAB, LAB/fve or LAB/hisfve showed neither significant stimulation on TNF-alpha and NO production by mouse peritoneal macrophage (P＞0.05) nor significant stimulation on cell proliferation and IFN-gamma secretion by spleen cells (P＞0.05). Furthermore, oral administrations of LAB, LAB/fve or LAB/hisfve were effective in increasing OVA-specific cell proliferation and OVA-specific IFN-alpha secretion by spleen cells. The OVA-specific IgG production in mouse sera was also enhanced significantly (P＜0.05). Unfortunately, the low fve-expressing capability of recombinant LAB could lead to insignificant immune activation between LAB, LAB/hisfve and LAB/fve treatments in vivo. Taking together, a LAB shuttle/expression vector pLP-LT1 was constructed and fve-expressing LAB strains were established in this study, which showed potentials for further utilization in food applications.