摘要
以乳酸球菌表現有生物活性的異質蛋白可以降低異質蛋白遭受細菌的蛋白酵素破壞的機會而得到活性較高的蛋白，將異質蛋白與乳酸菌的分泌性蛋白融合表現後，可以直接在培養基或是環境中回收蛋白質產物或是進行分析蛋白活性的實驗。Staphylococcal nuclease (Nuc)是一種可以被乳酸菌表現分泌出胞外的小蛋白質，可以當作乳酸球菌融合表現異質蛋白的reporter。位於革蘭氏陽性菌載體pNuc10上的Nuc蛋白N-端插接了一段九個胺基酸的人工合成胜□序列LEISSTCDA後大幅度強了其分泌效率，若將異質蛋白質融合在Nuc蛋白N-端與LEISSTCDA之間，則可以得到提高異質蛋白產量的效果。

實驗中欲表現的異質蛋白為腸病毒71型的VP1部份外殼蛋白，將其加上GST蛋白作為攜帶分子一起接到Nuc蛋白N-端與LEISSTCDA之間，製成VP1-GST-NucT 融合蛋白，期望藉由乳酸菌進入動物腸道中分泌表現出來以引發黏膜表面的免疫反應，達到作為活菌疫苗的效果。由於缺乏無質體的乳酸菌株，在選殖的時候我們先將pBluescript與pNuc10銜接造成一穿梭載體，利用大腸桿菌的轉殖系統來建構能表現腸病毒VP1蛋白的新載體，待得到可轉殖的乳酸菌時再以電擊法將新載體傳送至乳酸菌宿主中進行表現。

英文摘要
The development of protein secretion systems suitable for use in innocuous lactic acid bacteria could enable these microorganisms to be used for the production by secretion of a number of heterologous proteins. Provided that suitable methods for protein recovery are available, higher initial levels of purity can be obtained when a product is secreted. Staphylococcal nuclease (Nuc protein) is a small, stable, biochemically well-characterized enzyme secreted by numerous gram-positive bacteria.Plasmid pNuc10 encoding LEISSTCDA-NucT is a gram-positive bacteria-cloning vector. The secretion efficiency of proteins fused to the Nuc signal peptide can be improved by the presence of the LEISSTCDA propeptide. In this study, the VP1 peptide of enterovirus 71 was fused with glutathione S-transferase as a heterologous protein. Before introducing of VP1-GST gene into pNuc10, we construct a shuttle vector pBN by fusing vector pBluescript with pNuc10, thus cloning of VP1-GST gene into the site between LEISSTCDA-NucT could be established in E.coli. The VP1-GST expression plasmid is then transferred to the Lactococcus lactis expression strain by electroporation. We hope that the L. lactis transformed with this vector can be used as a life vaccine system.

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