纤维素结合域的酿酒酵母表面展示及其黏附位点初探

Cell Surface Display of Cellulose Binding Domain on Saccharomyces cerevisiae and Analysis of Its Adhesion Sites

利用酿酒酵母表面展示系统表达产琥珀酸丝状杆菌S85纤维素结合域家族2(CBD2),分析其纤维黏附位点。将CBD2基因插入质粒pYD1的AGA2 3′端,构建pMCBD2重组质粒,化学转化pMCBD2至酿酒酵母EBY100中,2%半乳糖诱导CBD2表达于酿酒酵母表面。利用免疫组化技术检测重组酵母细胞在稻草茎细胞壁上的黏附位点。结果表明:利用酿酒酵母表面展示系统可成功表达产琥珀酸丝状杆菌S85的CBD2基因;重组CBD2的pMCBD2-EBY100与稻草茎孵育后,经抗V5异硫氰酸荧光素标记抗体处理可在稻草茎的薄壁、厚壁和维管组织均检测到荧光。产琥珀酸丝状杆菌S85的CBD2黏附稻草多个组织。结果提示:酿酒酵母表面展示技术与免疫组化技术联用研究瘤胃细菌CBD的黏附位点是可行的。

To analyze the adhesion sites of cellulose binding domain（CBD） family 2 of Fibrobacter succinogenes S85 on fiber using a cell surface display system of Saccharomyces cerevisiae strain EBY100,the CBD2 encoding fragment was inserted into the 3′ terminal of AGA2 fragment in pYD1 vector to construct a recombinant plasmid,pMCBD2.Then pMCBD2 was transformed into strain EBY100 chemically.The 2% galactose was used to induce the expression of CBD2 on the cell surface of EBY100.The adhesion sites of the recombinant strain EBY100 to cell wall of rice straw stem were detected using immunohistochemistry.The results showed that CBD2 can potentially be expressed on the surface of strain EBY100.After incubating EBY100-pMCBD2 with rice straw stem slice,fluorescence was detected on the sclerenchyma,parenchyma and vascular tissues of rice straw stem with Anti-V5-FITC.It suggests that CBD2 can potentially adhere to most tissues of rice straw stem.Cell surface display system of S.cerevisiae along with immunohistochemistry can be suitable for the study on the adhesion sites of rumen bacterial CBD.