大鼠DJ-1的表达纯化和复性及抗氧化应激研究

发布时间：2018-01-11 15:38

本文关键词：大鼠DJ-1的表达纯化和复性及抗氧化应激研究　出处：《天津医科大学》2009年硕士论文　论文类型：学位论文

【摘要】：DJ-1是一个高度保守的基因,含有189个氨基酸,以二聚体形式行使功能。不但在体内各种组织中广泛表达,还普遍存在于细胞内的胞核、胞质、线粒体基质和内膜间隙以及哺乳动物细胞外间隙中；因此DJ-1是一种多功能蛋白,有极为重要的研究价值。DJ-1基因突变可导致常染色体隐性早发性帕金森氏病(PD)；DJ-1还是一个依赖Ras的癌基因,可能和几种肿瘤致病机理有关。DJ-1在培养细胞和动物模型中有抗氧化应激作用,通过对抗氧化应激在多种神经病理性损害中有重要保护作用。另外,DJ-1还具有分子伴侣功能,并且与啮齿类动物的精子成熟、受精,转录调节以及细胞凋亡相关。实验目的：第一部分：大鼠DJ-1在E.coli M15[pREP4]中的融合表达和表达条件的优化；表达的融合蛋白的纯化、复性并鉴定其免疫原活性。第二部分：通过大鼠His融合DJ-1及DJ-1活性多肽片段DJ-1IAAICA(102-107)和CAGPTA(106～111)对氧化损伤的保护性作用,探索DJ-1的Cys106残基是否是DJ-1清除活性氧(ROS)的功能位点。实验方法：第一部分：将表达载体pQE-30Xa/DJ-1转化入感受态细胞E.coli M15[pREP4],进行表达条件优化,然后进行融合表达。目的蛋白含有6×His纯化标签,可以经金属鳌合亲和层析吸附于Ni-IDA树脂层析柱,梯度增加冲洗液中咪唑浓度以去除非特异吸附的杂蛋白后,将目的蛋白洗脱,超滤离心法浓缩、复性。经SDS-PAGE、Western blotting鉴定其免疫原活性。第二部分：在稳定的抗氧化应激实验系统中检测钙调神经磷酸酶(CaN)和乳酸脱氢酶(LDH)活性。在20μmol/L过氧化氢作用下His融合DJ-1低浓度和高浓度下测定CaN活性；在1.5μmol/L过氧化氢作用下DJ-1活性多肽片段IAAICA (102～107)和CAGPTA (106～111)下测定LDH活性。实验结果：第一部分：重组pQE-30Xa/DJ-1质粒转化入感受态E.coli M15[pREP4],在37℃C,IPTG浓度为lmmol/L诱导表达6h,产物融合蛋白6xHis-DJ-1表达量最高,目的蛋白可占菌体总蛋白的57.1%。SDS-PAGE、Western blotting证实蛋白质产物确实为融合DJ-1,且融合蛋白具有免疫原活性。第二部分：低浓度和高浓度His融合DJ-1均可以对抗过氧化氢诱导的氧化应激,恢复CaN活性；DJ-1活性多肽片段IAAICA(102-107)和CAGPTA(106-111)均可以部分对抗过氧化氢诱导的氧化应激,提高LDH活性。结论：重组pQE-30Xa/DJ-1质粒转化入感受态E.coli M15[pREP4],在37℃, IPTG浓度为1mmol/L诱导表达6小时产物融合蛋白6xHis-DJ-1表达量最高。Western Blotting鉴定融合表达产物DJ-1具有免疫原活性。融合蛋白的获得为进一步研究其蛋白质功能奠定了基础,并且为以后基因工程生产有生物活性的DJ-1奠定了基础。 DJ-1本身在体外可以对抗过氧化氢诱导的氧化应激；DJ-1多肽片段IAAICA(102-107)和CAGPTA(106-111)均是对抗氧化应激的功能片段,Cys106残基是对抗氧化应激的功能位点之一。DJ-1完整蛋白可能是通过自身C106残基氧化来对抗氧化应激。DJ-1活性多肽IAAICA (102-107)可以完全对抗过氧化氢,为今后对抗过氧化氢的活性多肽发展及应用提供了有力依据。
[Abstract]:DJ-1 is a highly conserved gene, containing 189 amino acids, with two dimer form function. Not only is widely expressed in various tissues in the body, but also exists in the cell nucleus, cytoplasm, mitochondrial matrix and intimal clearance and mammalian extracellular space; therefore DJ-1 is a multifunctional protein that is extremely important research value of.DJ-1 gene mutation can cause autosomal recessive early-onset Parkinson's disease (PD); DJ-1 is a Ras dependent gene, and several tumor may about the pathogenic mechanism of.DJ-1 oxidative stress in cultured cells and in animal models by resisting oxidative stress has an important role in the protection of a variety of neuropathic damage. In addition, DJ-1 also has the function of molecular chaperone, and rodent animal sperm maturation, fertilization, transcriptional regulation and cell apoptosis. Objective experiment:
Part one: the fusion expression and expression condition optimization of rat DJ-1 in E.coli M15[pREP4], purification and renaturation of expressed fusion protein, and identify its immunogenicity.
The second part: through the protective effect of rat His fusion DJ-1 and DJ-1 active peptide fragment DJ-1IAAICA (102-107) and CAGPTA (106~111) on oxidative damage, we explored whether Cys106 residues of DJ-1 are the functional sites of DJ-1 scavenging reactive oxygen species (ROS).
The first part: the expression vector pQE-30Xa/DJ-1 was transformed into competent cells E.coli M15[pREP4] expression, condition optimization, and then fusion expression. Target protein containing 6 x His purification tags by metal chelate adsorption on Ni-IDA resin column affinity chromatography, gradient increase at the imidazole concentration to remove the fluid nonspecific adsorption of proteins after the elution, centrifugal ultrafiltration concentrate, renaturation. By SDS-PAGE, Western blotting to identify its immunogenicity.
The second part: detection of calcineurin in oxidative stress in experimental system stability (CaN) and lactate dehydrogenase (LDH) activity. Under the determination of activity of CaN His fusion DJ-1 low and high concentration of 20 mol/L under the action of hydrogen peroxide; 1.5 mol/L hydrogen peroxide under the action of DJ-1 active polypeptide fragment IAAICA (102 ~ 107) and CAGPTA (106 ~ 111) determination of LDH activity. The experimental results:
The first part: the recombinant pQE-30Xa/DJ-1 plasmid was transformed into competent E.coli M15[pREP4] at 37 DEG C, IPTG concentration of lmmol/L induced expression of 6h fusion protein product, the expression of 6xHis-DJ-1 was highest, the target protein can be accounted for the total bacterial protein 57.1%.SDS-PAGE, Western blotting confirmed that the protein product was indeed fusion DJ-1, and the fusion protein has immunogenicity.
The second part: the oxidative stress of low concentration and high concentration of His fusion DJ-1 can against hydrogen peroxide induced recovery, CaN activity; DJ-1 activity polypeptide fragment IAAICA (102-107) and CAGPTA (106-111) can fight oxidative stress induced by hydrogen peroxide, increase the activity of LDH. Conclusion:
The recombinant pQE-30Xa/DJ-1 plasmid was transformed into competent E.coli M15[pREP4] at 37 DEG C, the concentration of IPTG was induced by 1mmol/L to express fusion protein product of 6 hours the expression of 6xHis-DJ-1 was highest in.Western Blotting identification fusion protein DJ-1 has immunogenicity. The fused protein lays a foundation for further research on the function of protein, and for gene engineering production laid the foundation for the biological activity of DJ-1.
DJ-1 itself can be against hydrogen peroxide induced oxidative stress in vitro; peptide fragments of DJ-1 IAAICA (102-107) and CAGPTA (106-111) is the functional fragment against oxidative stress, Cys106 residue is.DJ-1 complete protein functional sites against oxidative stress may be one of the self oxidation of C106 residues against oxidative stress.DJ-1 peptide IAAICA (102-107) can be completely against hydrogen peroxide, provides a strong basis for the future development and application of bioactive peptides against hydrogen peroxide.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R3416

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