【作者】 吴初新；

【导师】 胡成钰；

【作者基本信息】 南昌大学 ， 动物学， 2010， 博士

【摘要】 真核细胞翻译起始因子2α(eIF2α)激酶是在进化中高度保守的丝氨酸／苏氨酸蛋白激酶,存在于哺乳类不同的组织和细胞中,通过调节eIF2α的活性参与机体内多种应激反应。新近报道的鱼类PKZ(包括鲫鱼CaPKZ,斑马鱼DrPKZ,大西洋鲑AsPKZ和稀有鮈鲫GrPKZ)可能是继HRI、PKR、PERK和GCN2等之后的一种新的eIF2α激酶。PKZ结构独特,C端为eIF2α激酶催化区,而在其N端具有2个Z-DNA结合域(Zα)。Z-DNA是不同于B-DNA的左手螺旋DNA,具有特殊的空间构象和重要的生物学功能。为了在体外模拟生理状态下的Z-DNA,我们构建了pMD-18T/d(GC)n(n=6,8,10,13),并利用甲基化抑制实验和抗Z-DNA抗体检测其构象。结果显示,这些插入至质粒的d(GC)n片断能够在负超螺旋维持下形成潜在的Z形构象,且其形成Z-DNA的能力依赖于d(GC)重复序列的数目。表明d(GC)n越长,形成Z-DNA的效率越高。同时,我们构建了pMD-18T/d(TA)n pMD-18T/d(non-GC-repeat)n重组质粒,同样通过抗Z-DNA抗体检测它们形成Z形构象的能力,结果发现它们均不能与抗Z-DNA抗体结合。鲫鱼PKZ(PKR-like)是首次报道的鱼类PKZ。为深入了解鲫鱼PKZ Za的功能,本研究以pET-22b(+)为表达载体,构建了野生型pET-22b(+)/Zα1Zα2和2个替换型pET-22b(+)/Zα1Zα1、pET-22b(+)/Zα2Zα2表达质粒,并运用PCR定点突变方法构建了9个点突变型重组表达质粒(K34A, S35A, N38A, R39A, Y42A, K56A, P57A, P58A和W60A)。这些重组表达载体转化至表达菌BL21(DE3)pLysS并诱导表达,经Ni-NTA亲和层析获得各种纯化的融合多肽。非变性PAGE电泳分析了野生型PZα1Zα2和替换型PZa1Zα1、PZα2Zα2蛋白的二聚化能力。结果显示,纯化的表达多肽PZα1Zα2在体外能够形成二聚体但没有多聚化现象。加入2-ME或SDS后,PZα1Zα2的二聚化则会被抑制,表明二硫键在稳定PZα1Zα2二聚体的过程中起着非常重要的作用。同时发现,pMD18-T/d(GC)6对PZα1Zα2的二聚化没有任何促进作用。与之不同,2个替换型PZα1Zα1、PZp2Zα2蛋白则没有发现的二聚化现象。利用琼脂糖凝胶阻滞实验分别分析了不同的体外表达多肽与pMD-18T/d(GC)n等各种重组质粒的亲和性。结果显示,野生型PZα1Zα2和替换型PZα1zα1对重组质粒pMD18-T/d(GC)n具有很高的亲和性,并且后者的结合能力强于前者。若解除pMD18-T/d(GC)n的负超螺旋,它们间的阻滞效应便会消失,这表明pMD18-T／d(GC)n的Z形构象是为其负超螺旋所维持的。同时,竞争性实验表明PZα1Zα2对于重组质粒pMD18-T/d(GC)n的结合具有特异性。另一方面,替换型PZα2Zα2以及9个点突变型蛋白却丧失了对pMD18-T/d(GC)n亲和能力。与此同时,通过琼脂糖凝胶阻滞实验还检测了PZα1Zα2与重组质粒pMD-18T/d(TA)n. pMD-18T/d(non-GC-repeat)n的亲和性。结果表明,PZα1Zα2与pMD-18T/d(non-GC-repeat)n、pMD-18T/d(TA)n的结合都很弱,尤以pMD-18T/d(TA)n表现为最弱。另外,我们还克隆了草鱼CiPKZ全长cDNA序列及其启动子序列。序列分析表明,草鱼CiPKZ基因序列与鲫鱼CaPKZ的具有极高的同源性。RT-PCR分析结果显示,草鱼CiPKZ具有广谱的组织表达特性,Poly I:C诱导后其表达均会上调。这些结果表明,鱼类PKZ能够为病毒所诱导、并可能参与宿主细胞的免疫应答过程。更多还原

【Abstract】 Eukaryotic initiation factor 2a (eIF2a) kinases, a member of the highly conserved Ser/Thr protein kinases family, are distributed in different mammalian tissues and cells and mainly participated in a variety of emergency responses by regulating the activity of eIF2a. The eIF2a kinases mostly contain HRI, PKR, PERK and GCN2. PKZ, including CaPKZ, DrPKZ, AsPKZ, and GrPKZ, is the most recently discovered member of eIF2a kinase family in fish. PKZ has a special structure, which possessed a conserved eIF2a kinase catalytic domain in C-terminal and two Z-DNA binding domains (Za) in N-terminal. Therefore, PKZ belongs to the Za protein family together with ADAR1, DLM-1 and E3L.Z-DNA, different from B-DNA, adopted unique conformation and to be thougth to have many important biological functions. In order to mimic the Z-DNA under physiological conditions in vitro, we constructed the recombinant plasmids of pMD-18T/d(GC)n (n=6,8,10,13) that were detected by using inhibition of methylation experiments and anti-Z-DNA antibody. The results showed that most of the plasmids containing d(GC)n inserts were maintained in the Z-conformation. Moreover, the ability of the recombinant plasmid to form Z-DNA depended on the number of d(GC) repeats. It suggested that the longer the sequence of d(GC)n the more efficient it can form the Z-conformation. Meanwhile, we have also constructed the recombinant plasmids of MD-18T/d(TA)n and pMD-18T/d(non-GC-repeat)n that were detected by using anti-Z-DNA antibody. The results showed that both of them couldn’t bind to the anti-Z-DNA antibody.CaPKZ is the first identified and reported PKZ in fish. To further investigate the function of CaPKZ Zα, we constructed three recombinant expression vectors of pET-22b(+)/Zα1Zα2, pET-22b(+)/Zα1Zα1 and pET-22b(+)/Zα2Zα2 by using pET-22b(+). Also, we constructed nine mutation vectors of pET-22b(+)/Zα(K34A, S35A, N38A, R39A,Y42A, K56A, P57A, P58A and W60A) by PCR site-directed mutagenesis method. These recombinant expression vectors were transformed into E.coli BL21 (DE3) plysS and then induced with IPTG. All kinds of purified peptides were obtained by using Ni-NTA His-Bind Resin affinity chromatography.The dimerization of PZα1Zα2, Pzα1Zα1 and PZα2Zα2 were analyzed by a 12%native polyacrylamide gel. The results showed that PZα1Zα2 could form dimer other than polymers in vitro. When incubated with 2-ME or SDS, the dimerization of PZα1Zα2 was inhibited. It suggested that disulfide bond could play a major role in stabilization of the dimer of PZα1Zα2.whereas pMD18-T/d(GC)6 had little effect on it. In contrast, no dimerization of PZα1Zα1 and PZα2Zα2 were detected.The recombinant plasmids of pMD18-T/d(GC)n, pMD-18T/d(TA)n and pMD-18T/d(non-GC-repeat)n binding activity of all kinds of fusion peptides were examined by using agarose gel mobility shift assay. At first, we found that both PZα1Zα2 and PZα1Zα1 had high affinity binding to the recombinant plasmids, and that of PZα1Zα1 could be stronger than of PZα1Zα2.If the negative supercoils of pMD18-T/d(GC)n were removed, the band shifts could disappeared. It suggested that the Z-conformation of pMD18-T/d(GC)n might be stabilized by negative supercoiling. At the same time, binding specificity of PZα1Zα2 to the plasmids of pMD18-T/d(GC)n was also examined by a competitive binding assay using the anti-Z-DNA antibody. On the other hand, PZα2Zα2 and nine site-directed mutation PZαcould not bind to pMD18-T/d(GC)n that lost its band shifting activity.In addition, we examined the binding activity of PZα1Zα2 to the recombinant plasmids of pMD-18T/d(TA)n and pMD-18T/d(non-GC-repeat)n by using agarose gel mobility shift assay. The results indicated that PZα1Zα2 has lower affinity binding to them, especially weakest to pMD-18T/d(TA)n.Additionally, the full length cDNA of grass carp(Ctenopharyngodon idellus) CiPKZ and its promoter sequence were cloned. Sequence analysis showed that grass carp CiPKZ shared the highest homology with crucian carp CaPKZ. The RT-PCR analysis showed that CiPKZ had a low level of constitutive expression in spleen, kidney and liver, and it was significantly up-regulated after challenged by Poly I:C. These results showed that the fish PKZ could be induced by virus, and and involved in host defense and immune response.更多还原