【作者】 岳礼溪；

【导师】 池振明；

【作者基本信息】 中国海洋大学 ， 微生物学， 2009， 博士

【摘要】 以分泌型表达载体pINA1317为基础,分别借助解脂耶罗威亚酵母菌(Yarrowia lipolytica)细胞壁蛋白YlCwp1 C端的131个氨基酸和C端的110个氨基酸,构建了Y. lipolytica表面展示质粒,我们将其分别命名为pINA1317-ylcwp131和pINA1317-ylcwp110。利用新构建的表面展示质粒成功展示了报告蛋白——增强型绿色荧光蛋白(EGFP),展示EGFP的细胞占到观察的细胞总数的100%。用本研究构建的质粒展示外源蛋白不需特殊物质诱导,宿主菌进入生长的稳定期后开始表达。利用pINA1317-ylcwp110分别成功展示了来源于真菌奥默柯达酵母菌(Kodamaea ohmeri)BG3的植酸酶和来自细菌哈维氏弧菌(Vibrio harveyi)SF-1的溶血素蛋白,得到了具有相应活性的菌株,免疫荧光实验结果证明目的蛋白已经展示在Y. lipolytica表面。为了比较表面展示植酸酶与游离植酸酶的异同,利用pINA1317表达载体将K. ohmeri BG3的植酸酶在Y. lipolytica中进行了表达,得到了分泌游离植酸酶的菌株,并利用Ni2+亲和层析对重组酶进行了纯化。SDS-PAGE及Western blotting鉴定结果表明重组酶的分子量约为65.1 kDa;小于天然酶的分子量(98.2 kDa)而大于在大肠杆菌中表达的重组酶分子量(51.0 kDa)。重组酶基本保持了天然酶的性质。表面展示的植酸酶在60.0℃,pH 5.0时酶活是114.7 mU/mg菌体干重。表面展示的植酸酶与天然酶和Y. lipolytica中表达的游离酶相比,最适作用温度由65.0℃降低至60.0℃。与天然植酸酶相比,表面展示酶对温度更为敏感,但与Y. lipolytica中表达的游离酶相比,温度稳定性提高。表面展示植酸酶与另外两者相比,pH稳定范围缩小,在pH3.0～8.0能够保持稳定;最适作用pH范围拓宽,在pH4.0～6.0之间酶活力较高且酶活力相差不大。金属离子对表面展示植酸酶的激活或抑制作用与天然酶及游离重组酶一致,但是作用效果减弱。毒性实验结果表明,表面展示有哈维氏弧菌溶血素蛋白的活的Y. lipolytica对大西洋牙鲆幼鱼是安全的。将该菌株作为活疫苗以5.0×108 cells/ml的浓度免疫大西洋牙鲆幼鱼,每尾0.2ml,并于初次免疫后第7 d和第21 d分别加强免疫;最后一次免疫后第7 d取血,间接ELISA检测到了大西洋牙鲆血清中溶血素特异抗体的产生。由于间接ELISA检测的需要,本研究采用Ni2+亲和层析纯化了在大肠杆菌中表达的重组V. harveyi溶血素,纯化的溶血素具有溶血活性,分子量大小约为45.0kDa;HiTrap rProteinA Sepharose亲和层析纯化了大西洋牙鲆免疫球蛋白IgM,SDS-PAGE结果表明纯化的IgM重链和轻链的大小分别是74.5 kDa和26.3 kDa,这是关于大西洋牙鲆免疫球蛋白IgM的首次报道。利用本研究纯化的IgM制备了小鼠抗大西洋牙鲆IgM多克隆抗体,免疫斑点法测定抗体效价达到1:3200以上。如果进一步改造菌株,增加目的蛋白质在Y. lipolytica表面的表达量,同时减少酵母菌表面其他蛋白质的量,那么该表面展示质粒可以应用于固定化酶、生物转化、生物修复、活疫苗和超高通量筛选等方面。更多还原

【Abstract】 In this study, two surface display plasmids (pINA1317-ylcwp131 and pINA1317-ylcwp110) were constructed in Yarrowia lipolytica using C-terminals of YlCwp1 with different lengths from Y. lipolytica based on plasmid pINA1317, a pre-existing auto-cloning system for heterologous protein production in Y. lipolytica. When the gene encoding enhanced green fluorescent protein (EGFP) was cloned into the newly constructed surface display plasmids and expressed in cells of Y. lipolytica, respectively, we found that the target protein was successfully displayed on the yeast cells and 100% of the yeast cells had found anchoring target protein. The target gene cloned on the plasmids can be expressed when the cell growth is at stationary phase without adding any inducer to the medium. To our knowledge, this work constitutes the first report of surface display expression systems in Y. lipolytica.The genes encoding phytase derived from a marine yeast Kodamaea ohmeri BG3 and haemolysin from Vibrio harveyi SF-1 were expressed in the yeast. Localization of the expressed phytase and haemolysin on the cell surface were confirmed by immunofluorescence microscopy. The phytase displayed on the yeast surface exhibited activity toward sodium phytate. The yeast cells displaying haemolysin had haemolytic activity on erythrocytes from Atlantic flounder.The gene encoding mature phytase was also cloned into pINA1317 expression vector and expressed in Y. lipolytica. The recombinant phytase secreted into the medium was purified by Ni2+ affinity chromatography. The purified phytase was analyzed by SDS-PAGE and western blotting. A specific band with molecular mass of approximatly 65.1 kDa was found. The found molecular mass was less than that of native phytase and more than that of recombinant enzyme expression in E. coli BL21 (DE3). The recombinant phytase maintained the general properties of the native enzyme. A maximum activity of 114.7 mU/mg dry cell weight was obtained from yeasts harboring phytase. Optimal pH and temperature of the displayed phytase were 5.0 and 60.0℃, respectively. The phytase displayed on the cell surface was found little more sensitive to temperature as compared with the native enzyme and more stable than recombinant phytase secreted into the medium. Surface displayed phytase was stable at pH3.0～8.0, and exhibited high and similar activity at pH 4.0～6.0. The effect of cations on displayed phytase was similar to that on secreted recombinant phytase expression in Y. lipolytica and native phytase of K. ohmeri BG3.The serum immunoglobulins of Atlantic flounder (Paralichthys dentatus) were purified by means of HiTrap rProtein A Sepharose affinity chromatography. SDS-PAGE analysis of the immunoglobulins showed that the heavy chain (H chain) and the light chain (L chain) of the purified serum IgM had molecular mass of the purified serum IgM were 74.5 kDa and 26.3 kDa, respectively. A mouse polyclonal antibody against Atlantic flounder immunoglobulin was produced.The anti-serum titer evaluated by immunodotting assay was 1:3200.The recombinant haemolysin with 6×His tag expression in E. coli JM109 was purified by Ni2+ affinity chromatography. The purified haemolysin with a molecular mass of about 45.0 kDa according to SDS-PAGE had haemolytic activity on the erythrocytes collected from Atlantic flounder.The yeasts displaying haemolysin were suspended in PBS and cell density of the suspension was adjusted to 5.0×108 cells/ml. 0.2 ml of the yeast cell suspension was administered i.p. to healthy Atlantic flounder. The immunization was carried out three times. After prime, each fish was immunized at both the second and the fourth week. No dead fish was found among all the fish vaccinated with the yeast cells displaying haemolysin, although the yeast cells had high haemolytic activity. This means that the yeast cells displaying haemolysin were safe to the marine animal tested. Indirect ELISA analysis indicated that the Atlantic flounders immunized with the yeast cells having the haemolytic activity had produced specific antibody against haemolysin in the serum.This demonstrates that the yeast cells displaying haemolysin may be used as the live vaccine in marine fish. Because the surface display system has many unique characteristics, increasing the amount of the target proteins and reducing the number of native cell wall proteins, the yeasts displaying target proteins will be useful in different fields such as immobilized biocatalyst, bioconversion, bioremediation, live vaccine development and ultra-high-throughput screening for the identification of novel biocatalysts.更多还原