哈维氏弧菌GYC1108-1胞外蛋白酶的制备及免疫原性研究.pdfVIP

哈维氏弧菌GYC1108-1 胞外蛋白酶的制备及免疫原性研究 郝贵杰，沈锦玉，潘晓艺，尹文林，曹 铮 (浙江省淡水水产研究所，浙江湖州 313001) 摘要：胞外蛋白酶（ECPase ）是哈维氏弧菌GYC1108-1 胞外产物的主要致病因子，经鉴定其为半胱胺酸蛋白酶， 分子量约 55KD，能够分解脱脂奶中的酪蛋白，命名为 1108-ECPase。本实验建立了胞外蛋白酶活性平板法检测 1108-ECPase 和YZ-ECPase 的相对酶活（YZ 为能够分泌目的蛋白酶的重组菌），并利用这个方法确定了细菌分泌 ECPase 达最高峰的培养时间为36h，粗提ECPase 最适的饱和硫酸铵浓度为70% 。 GYC1108-1 和YZ 培养上清经 硫酸铵盐析， SephadexG-25 凝胶过柱后，得到较为纯化的 1108-ECPase 和YZ-ECPase 。选择白油、蜂胶和弗氏 三种不同佐剂与适量抗原混匀，分别免疫3 只ICR 小鼠，并设不加佐剂免疫组及空白组作为对照。三次免疫后制 4 5 备小鼠血清，间接ELISA 测定抗体效价，除空白对照外，其它8 组小鼠的免疫血清效价达1:1.6×10 ～1:2.56×10 ， 免疫组的效价从高到低依次为弗氏＞白油＞蜂胶＞不加佐剂，不同佐剂的免疫效果依次为：弗氏＞白油＞蜂胶。 免疫印迹实验结果表明：小鼠血清在55KD 处有明显的反应条带。以上结果表明，ECP 具有很好的免疫原性，这 为下一步研究基因工程疫苗奠定了基础。 关键词：哈维氏弧菌；胞外蛋白酶；佐剂；间接ELISA ；免疫印迹 Preparation and Immunogenicity of Extracellular Proteinase from Vibrio harveyi GYC1108-1 HAO Guijie, SHEN Jinyu, PAN Xiaoyi, YIN WenLin, CAO Zheng (Zhejiang Institute of Freshwater Fisheries, Huzhou 313001, China) Abstract: Vibrio harveyi is a kind of important pathogenic bacteria for seawater animals and brings severe loss to their culture. V .harveyi strain, GYC1108-1, which was isolated from diseased Pseudosciaena crocea in Zhejiang province in 2003, was identified after morphological observation, biochemical characteristic analysis, 16sRNA and HSP60 gene sequence detection.The prophase research in our lab demonstrated that the extracellular proteases （ECPase ）was the major virulent factor of GYC1108-1 and was detemined to be cysteine protease of having a molecular mass of 55 kDa as estimated by SDS．The protease can decompose the casein of degrease milk and was named with 1108-ECPase．The method of solid LB culture medium containing 1% degrease milk was established to