【作者】 杨慧；

【导师】 陈吉祥；

【作者基本信息】 中国海洋大学 ， 遗传学， 2007， 博士

【摘要】 鳗弧菌(Vibrio anguillarum)能引起世界范围内的淡水、海水鱼类及其它养殖动物发病,其病症为快速的爆发性败血症及组织损伤,病鱼在短期内死亡,常给水产养殖业造成巨大的经济损失。鳗弧菌W-1分离自山东莱州湾海区发病的鲈鱼,已有的研究表明,W-1胞外金属蛋白酶是其重要的毒力因子之一,在细菌致病过程中发挥了重要的作用。编码金属蛋白酶的empA基因已被克隆和测序。本研究将鳗弧菌W-1金属蛋白酶基因empA分别克隆到原核表达载体pBV220、pBAD24和pET24d(+)中,得到的三种重组质粒分别转化大肠杆菌JM109、TOP10和BL21(DE3)。比较empA基因在三种重组菌JM109/pBV220/empA、TOP10/pBAD24/empA和BL21(DE3)/pET24d(+)/empA中的表达情况,最终选择重组菌BL21(DE3)/pET24d(+)/empA来表达、纯化及定点突变研究empA基因。对重组菌培养及诱导条件进行优化后发现,25℃时经1 mM IPTG诱导重组菌体表达的EmpA金属蛋白酶可溶性蛋白表达量最多、包涵体形成最少,并且分泌到培养上清液中的EmpA金属蛋白酶量也最大;Western-blot检测重组表达的EmpA金属蛋白酶具有抗原性。进一步用Ni亲合柱从重组菌菌体及其培养上清液中纯化了EmpA金属蛋白酶,SDS-PAGE电泳测得分子量都为36 kDa,而未经加热处理的蛋白分子量为44.6 kDa;用Sephadex G-200测得蛋白分子量都为44.6 kDa,表明在大肠杆菌中表达的EmpA金属蛋白酶为单体蛋白。纯化的EmpA金属蛋白酶对牙鲆鳃细胞系(FG)有毒性作用,用5μg的蛋白作用于FG,在12 h内即可观察到细胞的死亡;酶对大菱鲆也表现出致死毒性,其症状与感染鳗弧菌的病鱼症状相似,半致死剂量LD50为8.1μg/鱼。利用PCR方法对推测的EmpA金属蛋白酶活性位点氨基酸的碱基序列进行了定点突变,将突变质粒转化到大肠杆菌BL21(DE3)中进行表达,并用Ni亲合柱分别纯化了各突变蛋白。比较EmpA金属蛋白酶及其突变蛋白的蛋白水解活性和细胞毒性发现,突变蛋白都不同程度地丧失了其蛋白水解活性和细胞毒性,表明突变位点的氨基酸对酶活性起着十分重要的作用。将没有蛋白水解活性和细胞毒性的m-EmpA7蛋白的基因序列m-empA7克隆到真核表达载体pEGFP-N1中,得到真核重组表达质粒pEGFP-N1/m-empA7。用磷酸钙共沉淀法将重组质粒转染至CHO和HEK293T细胞中,通过荧光显微镜、RT-PCR检测和Western-blot检测证实目的基因在真核细胞内获得了表达。对牙鲆肌肉注射50μg/尾的重组质粒pEGFP-N1/m-empA7,于接种后不同时间分别取对照组和试验组牙鲆各组织做冷冻切片,在荧光显微镜下观察可见,在试验组牙鲆的注射点周边肌肉、注射点对面体侧肌肉、脾脏、前肾、体肾、后肠、心、鳃和肝脏等部位均可见绿色荧光,对照组组织没有荧光。RT-PCR检测和Western-blot检测的结果也证实目的基因在牙鲆体内获得了表达。为研究真核重组表达质粒pEGFP-N1/m-empA7对牙鲆的免疫效果,分别以5μg/尾、20μg/尾和50μg/尾的不同剂量对牙鲆进行肌肉注射免疫,同时设置注射PBS和空质粒的对照组。ELISA检测结果显示,各免疫组牙鲆体内均产生了抗m-EmpA7抗原的特异性抗体,且抗体水平随接种剂量的增加而升高;而对照组牙鲆血清基本没有抗体凝集发生,在不同时间内抗体水平无明显变化。免疫后第5周,用鳗弧菌W-1对各组进行攻毒实验,各免疫组的免疫保护率(RPS)分别为57.1%,71.4%和85.7%,与对照组相比,免疫组产生了显著的保护作用(p < 0.001)。更多还原

【Abstract】 Vibrio anguillarum is the causative agent of vibrosis, one of the major bacterial disease affecting marine and freshwater fishes in the world. The infected fish showed a rapid fulminating hemorrhagic septicemia and wide-spread tissue damage, and died in several days. The distribution of vibrosis is worldwide, which cause great economic loss to the aquaculture industry. Vibrio anguillarum strain W-1 was originally isolated from diseased seabass (Lateolabrax japonicus). Previous studies revealed that the extracellular protease EmpA of Vibrio anguillarum was one its important virulence factors. The gene coding EmpA was cloned and sequenced.In this study, empA was cloned into three prokaryotic expression plasmids pBV220, pBAD24 and pET24d(+) respectively, resulting three recombinant Escherichia coli strains JM109/pBV220/empA、TOP10/pBAD24/empA and BL21(DE3)/pET24d(+)/empA. We finally chose BL21(DE3)/pET24d(+)/empA to express, purify and mutate empA since comparisons of the expression of empA in this three recombinant strains showed that BL21(DE3)/pET24d(+)/empA had the best expression profile. The expression conditions of the recombinant E. coli BL21(DE3)/pET24d(+)/empA were optimized. It was found that the soluble productions of EmpA expressed in recombinant cells and secreted to the medium were both maximal when E. coli BL21(DE3)/pET24d(+)/empA was induced with 1 mM IPTG at 25℃. The expressed reombinant EmpA could react with the anti-EmpA serum in Western-blot. EmpAs expressed in recombinant E. coli and secreted to the medium were purified by metal chelating affinity chromatography. Estimated molecular weights of purified EmpAs were both 36 kDa by SDS-PAGE, but both 44.6 kDa if the samples were not heated in boiling water. And they were both determined to be 44.6 kDa by Sephadex G-200 gel filtration. Thus, EmpA expressed in E. coli was a monomer polypeptide. Purified EmpA had cytotoxicity to flounder gill cell line (FG). The development of CPE-like morphologic damage of the cells was visible by light microscopy within 12 h of incubation of the cells with 5μg of purified EmpA. It could also cause death of turbot when injected intraperitoneally, which showed similar symptoms with diseased fish infected by Vibrio anguillarum. The LD50 of EmpA to turbot was established as 8.1μg protein/g of fish.The nucleotides of the amino acid residues in the active sites of EmpA were mutated by PCR site-directed mutagenesis. All recombinant plasmids with mutated empAs were transformed into E. coli BL21(DE3), and mutated EmpAs were expressed and purified to characterize the effects of alterations of these amino acid residues on enzymatic activity. It was found that all of the single point mutations at the conservative residues reduced the proteolytic activity and cytotoxicity of the enzyme with different levels, indicating that they play an important role in enzymatic activity.To construct DNA vaccine, mutated empA (m-empA7) was subcloned into eukaryotic expression vector pEGFP-N1, resulting the recombinant plasmid pEGFP-N1/m-empA7. It was then transfected into CHO and HEK293T cells and the expression of m-empA7 was detected with fluorescent microscope, RT-PCR and Western-blot, which indicating that m-empA7 was expressed in CHO and HEK293T cells.Flounder were intramuscularly injected with 50μg of the DNA vaccine solution to examine if m-empA7 could be expressed. Fluorescent microscope, RT-PCR and Western-blot detection revealed that m-empA7 was expressed in the muscle, spleen, kidney, hind intestines, heart, gill and liver of flounder. To study the protective efficacy of the recombinant plasmid pEGFP-N1/m-empA7 on flounder, fish were respectively immunized with three doses of DNA vaccine (5μg/fish, 20μg /fish and 50μg /fish). The results of ELISA revealed that the immunized fish harvested much higher special antibody against m-EmpA7 in relation to negative controls of PBS buffer and pEGFP-N1, and the antibody titer increased as the immunized dose increased. Five weeks after vaccination, fish were challenged by intramuscularly injection of Vibrio anguillarum W-1 cell suspension. Mortalities following exposure to the bacteria were much lower in fish vaccinated with pEGFP-N1/m-empA7 compared to those of the control groups injected with PBS buffer and pEGFP-N1 alone. DNA vaccine of three doses protected fish from the bacterium with 57.1%, 71.4% and 85.7% of RPS respectively.更多还原