嗜水气单胞菌温敏胞外蛋白酶活性片段的表达和对小鼠的免疫试验  

Expression of the Active Fragment of an Extracellular Temperature-labile Protease from Aeromonas hydrophila and Its Immune Effect on Mice

作　　者：任燕[1] 陆承平[1] 姚火春[1] 

机构地区：[1]南京农业大学农业部动物疫病诊断与免疫重点实验室,南京210095

出　　处：《农业生物技术学报》2006年第6期875-878,共4页Journal of Agricultural Biotechnology

基　　金：江苏省攻关(三药)项目(No.BE2004609)资助。

摘　　要：为确定嗜水气单胞菌(Aeromonashydrophila,Ah)J-1株温敏胞外蛋白酶EprJ1的表达产物是否具有酶活性和抗原性及测定对小鼠的相对保护率,根据eprJ1基因ORF阅读框的序列设计1对引物,扩增出不含信号肽序列的EprJ1成熟蛋白结构基因(850bp),经EcoRⅠ/SalⅠ双酶切后连接pET-32a(+),转化大肠杆菌(Escherichiacoli)BL21。在30℃经IPTG诱导后,重组菌冻融液在脱脂奶平板上经28℃和24h孵育检测有透明溶蛋白圈出现,经56℃和30min处理后则无。纯化的重组蛋白免疫小鼠后,用1.0×107cfu的AhJ-1(50LD50)腹腔注射攻击小鼠,重组蛋白对小鼠的相对保护率可达60%。免疫印迹显示,AhJ-1胞外产物的抗血清可以识别该融合蛋白,但AhJ-1的热稳定胞外蛋白酶ECPase54抗血清不能识别该融合蛋白。重组蛋白分子量约53.2kD,有温敏蛋白酶活性和抗原性。To define whether the expressed protease of Aeromonas hydrophila （Ah） J-1 strain has proteolytic activity and antigenicity and determine the relative percentage survival （RPS） for the mice, a pair of primers were designed according to the gene of an extracellular temperature-labile protease （EprJ1） of Ah J- 1 strain. With the specific primer, the 850 bp fragment encoding peptide of the EprJ1 without signal sequence was amplified from the Ah J-1 genome. After digesting the fragment with EcoR Ⅰ ＋Sal Ⅰ, the gene was inserted into pET-32a （＋）. The recombinant pET-eprJ1 vector was transformed into the expressing strain BL21 and induced expression by 1 mmol/L IPTG at 30 ℃. The molecular weight and the antigenicity of recombinant protein were detected by SDS-PAGE and Western blot, respectively. At the same time the proteolytic activity was assayed through skim milk plate and SDS-PAGE gel containing 0.1% casein, respectively. The mice were immunized with the recombinant protein, then injected intrapetitoneally with 1.0 ×1^07cfu/mouse of Ah J- 1 （50LD50）. An expected 53.2 kD protein was expressed. The proteolytic activity of the expressed product could be detected, but it could not be actived when treated at 56 ℃ for 30 rain. The recombinant protein could be detected by Western blot with Ah J-1 ECPs antiserum, but not with Ah J- 1 ECPase54 antiserum. The results revealed that in vitro expression of the fragment of eprJ1 had the critical antigenitic epitopes of Ah J-1 ECPs. The immuned mice were injected intraperitoneally with 1.0 ×10^7cfu/mouse of Ah J-1 （about 50LD50） and the RPS could achieve 60%. The recombinant protein has the temperature-labile protease activity and the high antigenicity.

关 键 词：嗜水气单胞菌 温敏胞外蛋白酶 融合表达 相对保护率 

分 类 号：S188[农业科学—农业基础科学]