表達外源性吸附蛋白以提高乳酸菌對腸道細胞之吸附性

Increasing the adhesion ability of Lactobacillus to intestinal epithelial cells by heterologously expression of adhesion proteins

薛湘雲(Hsiang-Yun Hsueh)

指導教授 ： 劉嚞睿

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.01388

摘要

益生菌對腸道上皮細胞的吸附能力為菌種篩選要件之一，當其具有高吸附性則更能定殖於腸道中，使腸道上皮形成穩定之正常菌叢，且對於宿主健康有較佳影響。¬而吸附的第一步驟可藉由菌體產生的吸附蛋白與細胞表面接受器進行結合，故吸附蛋白為使菌體具有吸附性的關鍵。
本研究目的即利用基因工程技術，將革蘭氏陽性菌（Gram-positive bacteria）吸附蛋白基因殖入低吸附性的乳酸菌中，讓吸附蛋白表現於乳酸菌菌體表面，增加其吸附能力。首先利用聚合酶鏈鎖反應（polymerase chain reaction, PCR）選殖出Lactobacillus reuteri Pg4膠原吸附蛋白（collagen-binding protein, Cnb）基因與Listeria monocytogenes黏液吸附蛋白（mucus binding protein, Mub）基因，將cnb與mub基因構築於乳酸菌－大腸桿菌穿梭載體pNZ3004後，以電穿孔（electroporation）方式將此重組質體轉殖入Lb. casei ATCC 393中，利用抗生素篩選菌落進行PCR試驗，證實乳酸菌轉殖株Lb. casei pNZ-cnb與Lb. casei pNZ-mub分別帶有外源性吸附蛋白基因cnb與mub。抽取乳酸菌轉形株之RNA並以反轉錄聚酶合鏈鎖反應（reverse transcription-PCR）分析，結果顯示，在乳酸菌轉形株中可觀察到cnb與mub之mRNA表現。西方轉漬分析方法則證實Lb. casei ATCC 393轉形株能表現外源性的29 kDa膠原吸附蛋白或48 kDa黏液吸附蛋白。使用流式細胞儀技術觀察，轉形株菌體表面確實具有膠原吸附蛋白或黏液吸附蛋白存在。進行吸附實驗發現，具有外源性吸附蛋白之乳酸菌轉形株對於腸黏膜Caco-2細胞具有較佳的吸附能力。進一步將乳酸菌與病原菌進行共同競爭（competition）、排斥效應（exclusion）與取代效應（displacement）試驗。結果顯示，Lb. casei pNZ-cnb可藉競爭排斥抑制大腸桿菌O157:H7與Ls. monocytogenes BCRC 15338吸附於Caco-2細胞的能力，Lb. casei pNZ-mub相較於Lb. casei ATCC 393而言，對於Ls. monocytogenes BCRC 15338具有較高的排斥效應以抑制其吸附。

關鍵字

乳酸菌 ； 益生菌 ； 吸附蛋白 ； 競爭排斥

並列摘要

The adhesion ability of bacteria to intestinal epithelial cells has been considered as one of the crucial selection criteria for probiotic strains. The superior adhesion is a prerequisite for colonization of bacteria, therefore, it is an importance effect for the establishment of a stable normal flora in the mammalian intestine and host beneficial healthiness. Initial adhesion is followed by firm attachment where adhesins on the bacterial cell surface recognize receptors on the epithelial cell surface.
As the binding proteins play a key role, this study was aimed to clone the Gram-positive bacterial adhesion protein genes and transformed these genes into a poor-adhesive Lactobacillus strain to increase its adhesion ability to intestinal epithelial cells. The DNA sequences encoding collagen-binding protein (Cnb) of Lactobacillus reuteri Pg4 and the mucus-binding protein (Mub) of Listeria monocytogenes were amplified by polymerase chain reaction (PCR). The PCR fragments encoding Cnb and Mub were subcloned into the E. coli－Lactobacillus shuttle vector pNZ3004 to generate pNZ-cnb and pNZ-mub respectively, and then were used to electroporate into Lb. casei ATCC 393.
The presence of the cnb and mub gene in the Lb. casei transformants (designated Lb. casei pNZ-cnb and Lb. casei pNZ-mub) was demonstrated by direct colony PCR. The total RNA of the Lb. casei ATCC 393 transformants was extracted for estimation of the transcript levels of cnb and mub by reverse transcription-PCR. The results showed that cnb and mub were successfully expressed by Lb. casei ATCC 393 transformants. The heterologous expression of 29 kDa Cnb and 48 kDa Mub were further confirmed by Western blot analysis. Flow cytometric analysis of the Lb. casei ATCC 393 transformants indicated that Cnb and Mub were display on their cell surface. In addition, the intensity of the fluorescent signals measured on Lb. casei ATCC 393 transformants was higher than that on Lb. casei ATCC 393. In the cell adhesion test, the adhesion ability of the Lb. casei ATCC 393 transformants to Caco-2 cells was increased as compared with Lb. casei ATCC 393. Competition, exclusion and displacement of Listeria monocytogenes BCRC 15338 and Escherichia coli O157:H7 by Lb. casei ATCC 393 transformants from adhesion on Caco-2 cell surfaces were further studied. The results indicated that Lb. casei pNZ-cnb could inhibit the adhesion of Ls. monocytogenes BCRC 15338 and E. coli O157:H7 to Caco-2 cells, while Lb. casei pNZ-mub could only inhibit the adhesive ability of Ls. monocytogenes BCRC 15338.
In conclusion, this study demonstrated that Cnb and Mub could be heterologously expressed by Lb. casei ATCC393. The transformed strains Lb. casei pNZ-cnb and Lb. casei pNZ-mub could constitutively express Cnb and Mub on their cell surface, and thus showed a better adhesion ability to Caco-2 cells than Lb. casei ATCC 393 did. Furthermore, Lb. casei pNZ-cnb and Lb. casei pNZ-mub could inhibit the adhesion of pathogens to Caco-2 cells.

並列關鍵字

Lactobacillus ； probiotic ； adhesion protein ； competition exclusion

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