建構乳酸菌載體及探討基因轉移的方法

Construct lactic acid bacterial vectors and study methods for gene transfer

劉慧屏(Hui Ping Liu)

指導教授 ： 林志侯

國立清華大學/生命科學院/分子醫學研究所/碩士(2008年)

https://doi.org/10.6843/NTHU.2008.00272

摘要

乳酸菌為一種益生菌，在自然界中廣泛地存在。本實驗利用廣域宿主複製源 pAMβ1 和 pUC origin 建構一個可在乳酸菌及大腸桿菌內複製的穿梭載體，並選用兩無帶質體的乳酸菌希望能做為此穿梭載體的宿主，測試結果可在乳酸球菌(Lactococcus lactis MG1363)複製。此外，亦利用此載體探討不同基因轉移方法如 electroporation、conjugation 和 gene gun 對乳酸菌的應用性。結果發現對 MG1363而言，使用 electroporation 或 conjugation 是可以成功的，但在基因槍法上可能受限於粒子大小而不適合太小的菌種。而在所選的乳酸桿菌(Lactobacillus rhamnosus TCELL-1)利用各種方法都沒有很好的結果，此種桿菌難以轉殖成功的原因不明仍有待後續之研究。

關鍵字

乳酸菌 ； 電穿孔 ； 接合 ； 基因槍 ； 穿梭載體

並列摘要

Lactic acid bacterial is a kind of probiotic bacteria and wide exists in nature. In this research, we constructed a vector, pAC, which can exist between Lactic acid bacterial and E.coli. The pAC vector was constructed by using the pAMβ1 replicon and the pUC origin. In my result, pAC can exist in Lactococcus lactis MG1363 but not in Lactobacillus rhamnosus TCELL-1. In addition, we also use the pAC vector to study methods for gene transfer such as electroporation、conjugation and gene gun on the application of lactic acid bacteria. The result shows that electroporation and conjugation methods were successive in MG1363 but not in TCELL-1. And with gene gun method, it may be restricted by the Au or W particle size and not suitable for the species. The reason that TCELL-1 is difficult to be transformed is not known. It will require further study.

並列關鍵字

Lactobacillus rhamnosus TCELL-1 ； electroporation ； conjugation ； gene gun ； shuttle vector

參考文獻

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