乳酸菌為不產生孢子之革蘭氏陽性球菌、球桿菌或桿菌，能利用碳水化合物進行發酵，產生以乳酸為 主之代謝產物。有關乳酸菌之應用十分多元化，例如作為發酵乳製品之菌酛或益生菌。某些乳酸菌如乳酸 鏈球菌 (Lactococcus lactis) 亦被開發成為用於生產重組蛋白質之表現宿主。然乳酸菌基因表現系統之建立仍遭 遇多項瓶頸，例如無適當載體可供使用與無合適之基因轉形方法。本研究之主要目的在於利用胚芽乳酸桿 菌 (Lactobacillus plantarum) ATIT-023無性狀質體pLP-8之複製區域建構一種新型大腸桿菌/乳酸桿菌穿梭載體， 以作為乳酸菌基因工程之操作工具。新型穿梭載體pBRLP-8係結合大腸桿菌載體pBR322與L. plantarum ATIT- 023質體pLP-8之複製區域建構而成。利用電轉形法可將pBRLP-8轉形至L. plantarum ATIT-018與鼠李糖乳酸桿菌 (Lactobacillus rhamnosus GG, LGG) 中。即時定量聚合酶連鎖反應結果顯示，pBRLP-8於L. plantarum ATIT-018與 LGG之複製數分別為37與27。進一步將乳酸鏈球菌之P23表現訊號與增強型綠螢光蛋白質基因嵌入pBRLP-8， 建構表現載體pBRLP-8-P23-GFPT。將pBRLP-8-P23-GFPT轉形至L. plantarum ATIT-018與LGG後發現乳酸桿菌轉 形株可表現增強型綠螢光蛋白質。以上結果說明，利用pBRLP-8所衍生之表現載體搭配L. plantarum ATIT-018或 LGG可建立新型基因表現系統。

Lactic acid bacteria (LAB) are a group of non-spore forming Gram-positive cocci, coccobacilli or rods that have the ability to produce lactic acid as the major metabolic product of carbohydrate fermentation. These bacteria have numerous uses in a wide range of applications, for example, as a starter culture in dairy fermentation or as probiotic for humans and animals. Some LAB such as Lactococcus lactis have also been developed into the expression hosts for production of recombinant proteins. However, construction of gene expression systems for use in LAB still encounters some bottlenecks such as lack of suitable vector and transformation method. The objective of this study was to construct a new Escherichia coli/Lactobacillus shuttle vector based on the replicon of a cryptic plasmid pLP-8 from Lactobacillus plantarum ATIT-023 for use as a tool in genetic engineering. The new shuttle vector pBRLP-8 was constructed with combination of a replicon from pBR322 and a replicon from pLP-8. The pBRLP-8 can be transformed into L. plantarum ATIT-018 and Lactobacillus rhamnosus GG (LGG) by electro-transformation. The relative copy numbers of pBRLP-8 in L. plantarum ATIT-018 and LGG were estimated to be about 37 and 27 by real-time PCR, respectively. Furthermore, the lactococcal P23 expression signal and the gene encoding enhanced green fluorescence protein (GFP+) were inserted in pBRLP-8 to generate pBRLP-8-P23-GFPT. Then, the pBRLP- 8-P23-GFPT was transformed into L. plantarum ATIT-018 and LGG, respectively. The expression of GFP+ in L. plantarum ATIT-018 and LGG transformants were determined. The results showed that L. plantarum ATIT-018 and LGG harboring pBRLP-8-P23-GFPT have the ability to express GFP+. As mentioned above, the combination of pBRLP-8-derived expression vectors and L. plantarum ATIT-018 or LGG can be used to establish new expression systems.