嗜酸乳杆菌S-层蛋白表面展示系统的构建及酶切位点分析  

The Construction of S-Layer Protein Surface Display System of L.acidophilus and the Analysis on Restricted Enzyme Sites

作　　者：刘琼[1] 王春凤[2] 杨桂连[2] 秦守涛[2] 刘曼[2] 

机构地区：[1]吉林工程技术师范学院食品工程学院,吉林长春130052 [2]吉林农业大学动物科学技术学院,吉林长春130118

出　　处：《安徽农业科学》2011年第30期18421-18423,18434,共4页Journal of Anhui Agricultural Sciences

基　　金：国家"863"计划项目(2007AA10Z322;2006AA10A205);国家自然科学基金项目(30671573;30700602;30870116);吉林省科技发展计划项目(20080104)

摘　　要：[目的]克隆和表达嗜酸乳杆菌S-层蛋白基因,用其构建能承载外源抗原表位的乳酸菌细胞表面展示系统。[方法]以染色体DNA为模板克隆S-层蛋白基因SlpA,通过酶切、连接将其插入大肠杆菌-乳酸菌穿梭表达载体pW425et,构建表面展示系统pW425et-S,并对酶切位点进行分析。[结果]将pW425et-S转化入thyA基因缺陷型E.coli X13感受态细胞,SDS-PAGE、Western-blotting、全细胞ELISA检测表明,在重组菌表面表达出S-层蛋白,构建出表面展示系统,确定BstEⅡ作为融合外源保护性抗原基因的酶切位点。[结论]克隆出嗜酸乳杆菌S-层蛋白基因,成功构建表面展示系统pW425et-S,为开发乳酸菌活载体口服活菌疫苗提供了可行性操作平台。[Objective] The aim was to clone and express S-layer protein gene of L.acidophilus to construct a novel expression system capable of locating antigen molecules on the cell surface of L.actobacillus.[Method] The SlpA gene including N-terminal signal sequence,variable domain and C-terminal domain anchors sequence was amplified via PCR from genome extract of the L.acidophilus.This sequence was digested and ligated into an E.coli-L.actobacillus shuttle secretion vector pW425et,and a cell surface display system called pW425et-S was obtained with a restriction endonuclease recognition site qualifying for integration of exogenous genes.[Result] Recombinant pW425et-S was transformed into competent cell E.coli X13 which thymidylate synthetase（ThyA） was deficiency.SDS-PAGE and Western-blotting analysis were used to detect the expression condition of S-layer protein.S-layer protein of L.acidophilus was expressed by recombinant and this protein can be detected by whole-cell ELISA.[Conclusion] S-layer protein gene of L.acidophilus was cloned and surface display system pW425et-S was constructed and that vector can be used as an applicable operate platform for exploitation of L.actobacillus vivi-vector oral vaccine.

关 键 词：嗜酸乳杆菌 S-层蛋白 表面展示系统 

分 类 号：S188[农业科学—农业基础科学]