益生性乳酸菌之篩選及穿梭載體系統之建構

Screen of Probiotic Lactic Acid Bacteria and Constructing Shuttle Vector of Lactic Acid Bacteria

張效銘(Shiao-Ming Chang)

指導教授 ： 顏聰榮

大同大學/工程學院/生物工程學系所/博士(2008年)

摘要

本研究結果分為益生性乳酸菌之篩選及乳酸菌穿梭載體系統之建構兩個部分。第一部分為篩選具有益生菌特性的乳酸菌，以提供腸道保健及當做第二部分的穿梭載體系統之宿主。第二部分為篩選一內源性的乳酸菌質體及其序列分析。利用乳酸菌質體的質體建構一乳酸菌穿梭載體系統，可穩定地存在第一部分所篩選的益生性乳酸菌中及進行異質蛋白基因之表現。在益生性乳酸菌之篩選上，從發酵蔬菜及動物排泄物的乳酸菌分離菌落中，成功地篩選出21個乳酸菌株具有可以抵抗酸、高膽鹽、抗菌、利用異麥芽寡糖及龍膽寡糖及誘導巨噬細胞RAW 264.7產生細胞激素(IL-12 p40p70及IL-10)等益生菌特性，可當做腸道保健使用的益生菌。菌種經由API 及16S rRNA鑑定為Lactobacillus acidophilus、Lactobacillus casei、Lactobacillus plantarum及Lactobacillus rhamnosus。乳酸菌株E1、E33、E40及E55為不含質體，可當做乳酸菌載體系統之宿主使用。在乳酸菌穿梭載體系統之建構上，成功地從乳酸球菌(Lactococcus lactis subsp. lactis)篩選一個cryptic質體pL2，質體完整序列大小為5,299-bp (GenBank accession no. DQ917780)。質體複製型態鑑定為theta-type且屬於pAMβ1 family。pL2質體含有兩個起始區域(轉移起始區域及複製起始區域)及五個推測的開放可讀框架(ORF 1-5)。ORF1可轉譯成386個胺基酸序列，為pL2質體的起始複製蛋白基因(repB)。利用pL2質體的複製元(包括複製起始區、複製啟動子、複製蛋白及複製起始區域上游之結構區域)與大腸桿菌選殖載體，建構一乳酸菌-大腸桿菌穿梭載體pUL6erm。穿梭載體pUL6erm可以容易地轉形及穩定地存在於大腸桿菌、乳酸桿菌、乳酸球菌及嗜熱性鏈球菌宿主中。利用穿梭載體pUL6erm為基礎，建構的氯誘導型乳酸菌表現載體pUL6erm-gadR-GUS，可以在0.3 M NaCl及50 mM glutamate 誘導下，表現2.37 Unit/mg的β-D-glucuronidase酵素活性。

關鍵字

thet-type複製型態質體 ； pL2質體 ； 益生菌 ； 乳酸菌 ； 穿梭載體系統

並列摘要

The results are divided into two parts with screen of probiotic Lactic acid bacteria and constructing shuttle vector of Lactic acid bacteria. In part I, the probiotic Lactic acid bacteria (LAB) was screened and used for provide intestinal health and host of shuttle vector of LAB. In part II, a cryptic LAB plasmid was isolated and the plasmid sequence was determined. Based on LAB plasmid, a shuttle vector was constructed, it could maintain stably in probiotic LAB and express heterologous protein gene. In screen of probiotic LAB, the twenty-one of LAB strains which could withstand high acid and bile salt conditions were selected. These strains meanwhile could tolerate digestive system, restrain pathogenic bacteria growth, metabolize isomaltooligosaccharides and gentiooligosaccharides, and induce cytokine production (IL-12 p40p70 and IL-10) by RAW 264.7 macrophage cell. All of these strains could be used as a candidate for probiotics. The species were identified as Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus rhamnosus by API and 16S rRNA. Strain E1, E33, E40, and E55 were plasmid free and selected as the host of shuttle vector system of LAB. In constructing shuttle vector of LAB, a cryptic plasmid pL2 was isolated from Lactococcus lactis subsp. lactis and its complete nucleotide sequence was determined (5,299-bp, GenBank accession No. DQ917780). Its replication mode was identified as a theta-type belonging to the pAMβ1 family. Analysis of the nucleotide sequence revealed that pL2 contained a transfer origin, a replication origin, and five putative open reading frames (ORF 1-5). ORF1 (386 amino acids) was homologous to replication protein RepB. The shuttle vector pUL6erm was constructed by using a replicon from pL2, a multiple cloning site, colE1 ori, the origin of Gram-negative bacteria from vector pUC19, and the erythromycin resistance gene from pVA838 as a selective marker. The pUL6erm could be transformed easily and maintained stably in E. coli, Lactobacillus casei, Lactobacillus plantarum, Lactococcus lactis, and Streptococcus thermophilus. The expression plasmid pUL6erm- gadR-GUS was constructed base on pUL6erm and a chloride-inducible gene expression cassette encoding gadR and the Pgad promoter. Growth in the presence of 0.3 M sodium chloride and 50 mM glutmate, the beta-D-glucuronidase was induced and expressed with 2.37 Unit/mg by plasmid pUL6erm-gadR-GUS.

並列關鍵字

theta-type replicating plasmid ； pL2 plasmid ； Probiotics ； Lactic acid bacteria ； shuttle vector system

參考文獻

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