鸭疫里默氏菌dnaK基因功能初析

发布时间：2019-05-08 23:25

【摘要】：鸭疫里默氏菌(RA)可感染鸭、鹅、火鸡等多种禽类,引起一种急性败血性或慢性传染病,常给养鸭业带来巨大的经济损失。为了研究RA的DnaK是否具有分子伴侣的基本特征,以及在热胁迫等应激条件下是否发挥作用,本文进行了一系列的研究,获得如下结果。1、将本实验室分离保存的RA野毒株RA-CH-1株dnaK、dnaJ和grpE全基因序列分别克隆到表达载体pET32a(+)载体中,构建原核表达质粒pET32a (+)dnaK、 pET32a(+)dnaJ和pET32a (+)grpE并将其分别转入大肠杆菌BL21中,成功表达出DnaK、 DnaJ和GrpE重组蛋白,用亲和层析的方法获得纯度较高的重组蛋白。2、用超微量ATP测试盒分别测定纯化的重组蛋白DnaK、DnaJ和GrpE在K+/Na+或Mg2+/Ca2+环境中水解ATP的活性。结果显示,重组蛋白DnaJ和GrpE不具有ATPase活性；而重组蛋白DnaK具有微弱的ATPase活性且DnaJ和GrpE可增强DnaK的ATPase活性,反应温度50℃时DnaK的ATPase活性最高。3、用纯化的DnaK蛋白免疫小鼠制备鼠抗DnaK多克隆抗体,间接ELISA检测血清效价达到1：102400。Western blot检测鼠抗DnaK与RA-CH-1全菌蛋白及纯化的DnaK蛋白的反应性,结果显示该抗体能特异性地识别RA的DnaK蛋白。4、以不同应激条件处理RA-CH-1,提取RNA，，荧光定量PCR检测其dnaK的mRNA水平,结果表明,当RA受到热、H202、氨基糖苷类抗生素胁迫及渗透压的作用时,其dnaK的转录水平显著升高；受到酸碱胁迫时,dnaK的mRNA水平无明显变化。5、根据RA的DnaK与大肠杆菌的DnaK氨基酸序列一致性达60%以上,利用λred重组的方法获得缺失了dnaK的大肠杆菌XL1-Blue △dnaK株,同时构建的RA DnaK原核表达载体pBAD24dnaK转化XL1-Blue AdnaK获得缺失回补株,在不同温度的应激条件下测定亲本株E. coliXL1-Blue.缺失株E. coli XL1-Blue △dnaK和缺失回补株E.coliXLl-Blue AdnaK pBAD24dnaK的生长曲线,结果亲本株E. coliXLl-Blue生长良好,缺失株E.coli XL1-Blue AdnaK在42℃时不生长,而回补了RA DnaK的E. coliXL1-Blue AdnaK pBAD24dnaK生长速率得到一定的恢复。表明RADnaK不仅能在一定程度上增强大肠杆菌XL1-Blue△dnaK对温度的耐受,在RA受到各种理化因子的刺激时,可能也扮演非常重要的角色。综上,RA的DnaK蛋白具有ATPase活性,DnaJ和GrpE能有效提高其ATPase的活性,且DnaK在RA抗高温、抗氧化、抗渗透压和抗氨基糖苷类抗生素等应激时具有重要的作用,但在抗酸碱协迫时并不发挥功能；RA DnaK在一定程度上能替代大肠杆菌的DnaK在热休克等应激反应中增强其耐受性。
[Abstract]:Riemerella (RA) can infect ducks, geese, turkeys and other birds, causing an acute septicemia or chronic infectious disease, which often brings great economic losses to the duck industry. In order to study whether the DnaK of RA has the basic characteristics of molecular chaperone and whether it functions under stress conditions such as heat stress, a series of studies have been carried out in this paper, and the following results have been obtained. The dnaK,dnaJ and grpE gene sequences of RA field virus strain RA-CH-1 isolated in our laboratory were cloned into the expression vector pET32a () respectively, and the prokaryotic expression plasmid pET32a () dnaK, was constructed. PET32a () dnaJ and pET32a () grpE were transformed into E. coli BL21, respectively. The recombinant proteins of DnaK, DnaJ and GrpE were successfully expressed, and the recombinant proteins with higher purity were obtained by affinity chromatography. The activity of purified recombinant proteins DnaK,DnaJ and GrpE to hydrolyze ATP in K / Na or Mg2 / Ca2 environment was determined by ultra-micro ATP test box. The results showed that the recombinant proteins DnaJ and GrpE had no ATPase activity. The recombinant protein DnaK had weak ATPase activity and DnaJ and GrpE could enhance the ATPase activity of DnaK. The ATPase activity of DnaK was the highest at reaction temperature of 50 鈩

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