弧菌琼胶酶和丝氨酸蛋白酶的筛选、分析以及免疫应用

IOCAS-IR > 海洋环流与波动重点实验室

	弧菌琼胶酶和丝氨酸蛋白酶的筛选、分析以及免疫应用
其他题名	The Application of Agarase and serine protease from Vibrio sp in the Immunoprotect of the Japenese Flounder
	张卫卫
学位类型	博士
	2009-05-21
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	琼胶酶丝氨酸蛋白酶分泌序列牙鲆病原菌
摘要	从山东黄岛海水养殖场分离到一株弧菌V134，从中克隆得到琼胶酶基因agaV，并将其在大肠杆菌中表达，纯化得到重组的琼胶酶AgaV。酶活分析发现该琼胶酶的最适温度在40℃左右，对pH比较敏感，pH 7.0时具有最高的琼胶裂解活性。对AgaV进行了两种应用性探索：（1）利用AgaV从琼脂糖凝胶中回收DNA，回收效率可达90%以上；（2）利用agaV作为报告基因构建了捕获分泌序列的载体pBU，并用其从革兰氏阳性细菌（G+菌）和革兰氏阴性细菌（G-菌）中筛选出了一系列分泌蛋白。将利用pBU从一株哈维氏弧菌T4中筛选出的6个分泌蛋白分别进行基因克隆、蛋白表达纯化和牙鲆免疫实验，发现其中一个蛋白，命名为DegQVh，具有免疫保护效应，其免疫保护率（RPS）可达64%。为了提高DegQVh的免疫保护效应，将AgaV的分泌结构域与DegQVh融合，构成融合抗原AgaV－DegQVh。利用大肠杆菌作为载体菌构建了AgaV－DegQVh融合抗原递呈系统，用其作为疫苗进行免疫，发现其RPS可达到95%。酶活分析表明DegQVh在50℃、pH 8.0时具有最高的活性。突变分析表明83位的组氨酸、113位的天冬氨酸和188位的丝氨酸以及两个PDZ结构域是DegQVh活性所必需的。表达分析发现degQVh表达受温度和细胞浓度调控，并且其上游有一个受E调控的启动子。进一步的分析发现DegQVh能够与大肠杆菌的DegP功能互补。
其他摘要	Vibrio sp V134 was isolated from a fish farm in Huangdao Shandong Province. agaV gene was cloned and expressed in BL21(DE3), the recombinant protein AgaV was purified with nichel-nitrilotriacetic acid agarose under native conditions. The purified protein was analyzed using agarose as substrate to establish the optimum temperature and pH of the agarase, which was around 40℃ and pH 7.0, with a narrow agarolytic range of pH respectively. AgaV was demonstrated in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for selection of genes encoding secretion proteins from both G+ and G- bacteria. Of the six signal sequences selected by pBU from fish pathogen T4, a Vibrio harveyi strain, one characterized to be a serine protease and named degQVh, was tested to be an immunoprotective protein agaist T4. The protease exhibited highly RPS (Relative Percentage Survival) ≈65% aganist the disease caused by T4. To enhance RPS, AgaV-DegQVh fusion protein was expressed in DH5α to form the antigen delivery system, which was proved to increase the RPS about 30%. degQVh was cloned into PET258 and expressed in BL21 (DE3), the recombinant DegQvh was purified and characterized, which showed the highest protelytic activity around 50℃ and pH 8.0. Five mutations concluding three site mutations and two PDZ deletion mutations demonstrated that the protease activity of DegQVh need His（83）, Asp (113) and Ser (188), the three typical catalytic amino acid and both of the PDZ domains. Analysis indicated that the expression of degQVh was regulated by temperature and cell density. The sequence alignment of up stream of degQVh indicated that it contained one E –dependent promoter. Further study indicated that degQVh could complement the function of DegP in E. coli.
页数	122
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/851
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	张卫卫. 弧菌琼胶酶和丝氨酸蛋白酶的筛选、分析以及免疫应用[D]. 海洋研究所. 中国科学院海洋研究所,2009.

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