【作者】 陈师勇；

【导师】 张培军；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2003， 博士

【摘要】 鳗弧菌（Vibrio anguillarum）是引起水产动物特别是鱼类流行性疾病的一种重要病原。本实验室从山东荣城寻山渔场濒临死亡的患病牙鲆中分离得到一株鳗弧菌致病株M3。该菌的致病性极强（LD50为5.144×103 CFU/ per fish），感染牙鲆时引起出血症和溃烂症。为进一步防治这种严重致病菌，对该菌的主要致病因子进行了研究。毒性实验结果表明脂多糖（Lipopolisacchride，LPS）致病性较弱，而胞外产物（Extracellular product，ECP）有较强毒力，可导致牙鲆死亡，其LD50为3.2μg蛋白/g体重。研究发现M3-ECP具有明胶蛋白酶、淀粉酶、卵磷脂酶和酪蛋白酶活性，可溶解牙鲆红细胞。进一步经超滤、Sephadex G100凝胶层析、DEAE-cellulose离子交换层析和HPLC凝胶色谱层析纯化，从ECP中分离得到一个纯化的毒性蛋白，其对金鱼的半数致死量（LD50）为1.2μg蛋白/g体重。该蛋白可降解azocasein，是一个蛋白酶；其分子量为36.5kD，等电点为5.1；与底物azocasein作用的最适pH为8.0，最适温度为55℃，85℃时作用30min时酶被灭活。该酶可被EDTA、EGTA及1,10-菲咯啉抑制，表明其为金属蛋白酶。底物降解实验发现该酶可以对azocasein、geltin、transferrin、BSA、elastin、fibrinogen、collagen和牙鲆鱼IgM进行降解。将纯化的金属蛋白酶序列测定后，得到N-端的15个氨基酸序列：AGATGTGPGGAGLTG。网上比对发现与鳗弧菌(P43147)金属蛋白酶前体的15个氨基酸系列一致。设计了一对引物，以鳗弧菌M3的基因组DNA为模板进行PCR扩增，PCR产物进行序列测定后得到一个1990bp的序列，含有一个1833bp的阅读框，可编码611氨基酸。所得核酸序列与报道的鳗弧菌金属蛋白酶的相似性为99%，具有保守的Zn2+结合位点，表明该核酸序列编码Zn+结合金属蛋白酶。PCR扩增得到金属蛋白酶基因同源片段后，亚克隆至pNQ705自杀质粒中，通过接合供体菌SY17-1与野生型鳗弧菌M3接合后导入重组质粒，经过同源重组使金属蛋白酶基因插入失活。PCR检测和测序结果表明已成功地构建了鳗弧菌金属蛋白酶基因突变株。该突变株的血清凝集反应与野生型菌株M3相同，但生长速率明显较M3要慢，ECP电泳图谱与M3比有较大区别，ECP中蛋白酶的比活大大下降，突变株与M3相比，不能在血平板上产生明显的溶血现象。为了大量获取菌体和胞外蛋白制备单克隆抗体，研究了M3 菌株的生长条<WP=4>件及其对胞外产物及蛋白酶活力的影响。采用响应面分析方法设计实验，用SAS统计软件分析所获得的数据，得到NaCl浓度、pH值和温度对菌体生长及蛋白酶产量影响的回归模型。在2216E培养基的基础上，添加不同氮源、碳源物质以及不同蛋白胨浓度进行生长研究，结果表明加入脑心浸液与加蛋白胨相比，ECP中蛋白酶活力略低；加入胰大豆蛋白胨，能促进菌体生长及ECP蛋白分泌，但蛋白酶活力未见提高。NH4Cl与酪蛋白水解物可抑制蛋白酶的产生。在培养基中加入牙鲆肌肉匀浆，对菌体、ECP蛋白产量和蛋白酶产生有不同程度的促进作用。培养的菌体量与ECP蛋白含量，在培养基中蛋白胨浓度达4％时达最高值，蛋白酶分泌量则在蛋白胨浓度为2％时已稳定。培养基中添加1％的葡萄糖、蔗糖、甘油，均能显著地提高菌体及ECP蛋白产量，却抑制了蛋白酶的产生。通过采用灭活的鳗弧菌完整菌体免疫Balb/c小鼠，制备了鳗弧菌单克隆抗体。结果获得6株能稳定分泌抗体并与鳗弧菌有较强免疫反应的单抗细胞株。其中，单抗C1C5与其它非鳗弧菌菌株均没有免疫交叉反应。蛋白酶K消化实验表明单抗C1C5、C6C3和C6C32所识别的抗原包含有蛋白质部分。高碘酸氧化实验显示，除了C1C5外的其它单抗都是糖基化的。根据可加性实验判断，C6C3和C6C32应识别相似或相同的抗原决定基，并与单抗C1C5识别的抗原决定基比是在不同位置上。鳗弧菌M3的纯化金属蛋白酶，经浓缩后作为抗原免疫Balb/c小鼠。经细胞融合及筛选，得到三株稳定分泌抗金属蛋白酶的杂交瘤细胞株，分别命名为P2B1、P4A3和P4B1。亚型鉴定结果表明，三株细胞株产生的单抗均为IgG3型。蛋白质印迹(Western Blot)分析显示，所得到的单抗只与分子量为36,500的金属蛋白酶反应，具有较高特异性。三株单抗均能有效抑制蛋白酶活性。更多还原

【Abstract】 An extracellular protease produced by Vibrio anguillarum strain M3 was purified following the steps of ultrafiltration, gel filtration with Sephadex G100, ion-exchange chromatography with DEAE-cellulose A52, HPLC gel filtration. The purified protease, with an isoelectric point of about 5.1 and molecular weights of about 36.5KDa, was lethal for goldfish at LD50 value of 1.2ug protein/g body weight. It was inhibited by EDTA、EDGA and 1,10-phenanthroline, which proved to be family of metalloproease, and showed maximal activity at pH8.0 and 55℃. It was found that this protease could degrade several protein substrates, including azocasein, geltin, transferrin, BSA, elastin, fibrinogen, collagen and fish IgM. Partial N-terminal amino acid sequence of the protease of Vibrio anguillarum strain M3 was AGATGTGPGGAGLTG.In order to demonstrate the role of metalloprotease in the pathogenesis of Vibrio anguillarum, we designed primers and successfully cloned the metalloprotease gene. According to this gene, two primers were designed and a internal homologous fragment was cloned. The fragment was subcloned into a suicide vector pNQ705, and then was transformed into the conjugation donor strain E.coli S17-1. Insertion mutation in Vibrio anguillarum chromosomal gene are isolated by mating the recombinant into M3 strain. PCR and sequence were used to validate the correct mutation. The mutant was analyzed to keep the original outmembrane antigens with serology test, but was found to grow obviously more slowly than the wild strain. Moreover, the SDS-PAGE pattern showed that extracellular products of mutant were<WP=6>dramatically different from those of M3, and the typical 36kD electrophoresis band disappeared in the pattern. The protease activity was also founded to have a greatly drop. No hemolysis phenomenon was found when the mutant was cultured in blood plate. In conclusion, the construction of metalloprotease gene mutant will surely be beneficial to further study of the pathogenesis of Vibrio anguillarum.The growth, production of extracellular products and protease of Vibrio anguillarum strain M3 cultured on solid or liquid medium were studied. An experiment was designed with Response Surface Methodology to assay the effects of NaCl concentration, pH value and temperature on growth of bacteria and protease production, and two regression models were established with SAS statistical software to evaluate the data. Protease activity was lower on the brain heart infusion medium than on the peptone medium. Compared with the peptone medium, strain growth and ECP production were stimulated on the trypticase soy broth medium, but protease production remained the same level. The presence of NH4Cl or casamino acids significantly decreased protease production. Muscle homogenate of flounder fish was able to stimulate the bacterial growth, ECP and protease production at different levels. The quantity of bacteria and ECP arrived at a maximal value at 4％ peptone in the medium, and protease production was steady at 2% peptone. The bacterial growth and ECP production increased, whereas the protease production decreased at the presence of 1％ glucose, sucrose or glycerin.Monoclonal antibodies (Mabs) against V.anguillarum strain M3 were produced, and their isotype were also characterized. Among them, C1C5 is the only Mab which did not cross-react with other eleven non-V.anguillarum strains. The proteinase K digestion test showed that the epitopes recognized by C1C5, C6C3 and C6C32 Mabs contain a protein in. While, the periodate oxidation test showed that the epitopes recognized by Mabs except C1C5 were glycosylated. In addition, results of additivity test indicated that the<WP=7>epitopes recognized by C6C3 and C6C32 Mabs were similar, which were quite different from that of Mab C1C5.The metalloprotease purified from the extracellular products(ECP) of Vibrio amguillarum M3, was used as antigen to immunize Bab/c mouse. Three monoclonal antibodies(MAbs), P2B1, P4A3 and P4B1, against the purified metalloprotease have been obtaine更多还原