哈维氏弧菌GYC1108-1胞外蛋白酶的制备及免疫原性研究

Preparation and immunogenicity of extracellular Vibrio harveyi GYC1108-1 proteinase of

中文关键词: 哈维氏弧菌  胞外蛋白酶  佐剂  间接ELISA  免疫印迹

英文关键词: Vibrio harveyi  ECPase  Adjuvant  Indirect  ELISA  Western blot

中文摘要:

      外蛋白酶（ECPase）是哈维氏弧菌GYC1108-1胞外产物的主要致病因子，经鉴定其为半胱氨酸蛋白酶，分子量约55 kD，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为由本实验室构建的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，Sephadex G-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3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。3次免疫后制备小鼠血清，间接ELISA测定抗体效价，除空白对照外，其他8组小鼠的免疫血清效价达1∶1.6×104～1∶2.56×105，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55 kD处有明显的反应条带

英文摘要:

      Vibrio harveyi is a kind of important pathogenic bacteria for seawater animals and can bring about severe loss to their culture production. 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. Earlier research in our laboratory demonstrated that the extracellular protease（ECPase）was the major virulent factor of GYC1108-1 and was identified as cysteine protease with a molecular mass of 55 kDa as estimated by SDS-PAGE．The protease can decompose the casein of degrease milk and was named as 1108 ECPase．The method of solid LB culture medium containing 1% skim milk was established to detect relative activities of 1108 ECPase and YZ ECPase (YZ is a recombinant bacteria that can express the target ECPase). The optimum cultivation time of GYC1108 1 and YZ excreting ECPase was 36 h and the optimal concentration of ammonium sulfate for ECPase purification was 70% for the above mentioned method．In this study, 1108 ECPase and YZ ECPase were purified by 70% ammonium sulfate and Sephadex G-25 gel filtration for antigens standby. Three different substances A, B and C were chosen to be adjuvants. A was a common adjuvant which was a mixture of white oil and Tween 80; B was propolis adjuvant and C was Freund's adjuvant. The purified 1108-ECPase and YZ-ECPase antigens were mixed with A, B and C before being applied separately to immunize 3 ICR mouse. The adjuvant free and blank treatments were designed as control. There were nine treatments in the immunization test. The immune sera of the mouse were collected after three immunizations. Except the blank control, multi clone antibodies showed high ELISA titers in the sera of eight treatments, which were between 1∶1.6×104～1∶2.56×105. The titers of immune sera in different treatments were ranked as C>A>B>adjuvant free, and it is suggested that the adjuvants chosen followes this effectiveness sequence. The result of Western blot revealed one clear band in ECPase, which had a molecular mass of 55 kDa as tested by mouse anti serum. These results indicated that the prepared 1108-ECPase and YZ-ECPase had good immunogenicity, and this work has provided some basic knowledge for further study on genetic engineering vaccines.