本文關(guān)鍵詞：大鼠DJ-1的表達純化和復性及抗氧化應(yīng)激研究　出處：《天津醫(yī)科大學》2009年碩士論文　論文類型：學位論文

  更多相關(guān)文章： DJ-1 融合表達 生物活性 活性多肽片段 氧化應(yīng)激

【摘要】：DJ-1是一個高度保守的基因,含有189個氨基酸,以二聚體形式行使功能。不但在體內(nèi)各種組織中廣泛表達,還普遍存在于細胞內(nèi)的胞核、胞質(zhì)、線粒體基質(zhì)和內(nèi)膜間隙以及哺乳動物細胞外間隙中；因此DJ-1是一種多功能蛋白,有極為重要的研究價值。DJ-1基因突變可導致常染色體隱性早發(fā)性帕金森氏病(PD)；DJ-1還是一個依賴Ras的癌基因,可能和幾種腫瘤致病機理有關(guān)。DJ-1在培養(yǎng)細胞和動物模型中有抗氧化應(yīng)激作用,通過對抗氧化應(yīng)激在多種神經(jīng)病理性損害中有重要保護作用。另外,DJ-1還具有分子伴侶功能,并且與嚙齒類動物的精子成熟、受精,轉(zhuǎn)錄調(diào)節(jié)以及細胞凋亡相關(guān)。實驗目的： 第一部分：大鼠DJ-1在E.coli M15[pREP4]中的融合表達和表達條件的優(yōu)化；表達的融合蛋白的純化、復性并鑒定其免疫原活性。 第二部分：通過大鼠His融合DJ-1及DJ-1活性多肽片段DJ-1IAAICA(102-107)和CAGPTA(106～111)對氧化損傷的保護性作用,探索DJ-1的Cys106殘基是否是DJ-1清除活性氧(ROS)的功能位點。實驗方法： 第一部分：將表達載體pQE-30Xa/DJ-1轉(zhuǎn)化入感受態(tài)細胞E.coli M15[pREP4],進行表達條件優(yōu)化,然后進行融合表達。目的蛋白含有6×His純化標簽,可以經(jīng)金屬鰲合親和層析吸附于Ni-IDA樹脂層析柱,梯度增加沖洗液中咪唑濃度以去除非特異吸附的雜蛋白后,將目的蛋白洗脫,超濾離心法濃縮、復性。經(jīng)SDS-PAGE、Western blotting鑒定其免疫原活性。 第二部分：在穩(wěn)定的抗氧化應(yīng)激實驗系統(tǒng)中檢測鈣調(diào)神經(jīng)磷酸酶(CaN)和乳酸脫氫酶(LDH)活性。在20μmol/L過氧化氫作用下His融合DJ-1低濃度和高濃度下測定CaN活性；在1.5μmol/L過氧化氫作用下DJ-1活性多肽片段IAAICA (102～107)和CAGPTA (106～111)下測定LDH活性。實驗結(jié)果： 第一部分：重組pQE-30Xa/DJ-1質(zhì)粒轉(zhuǎn)化入感受態(tài)E.coli M15[pREP4],在37℃C,IPTG濃度為lmmol/L誘導表達6h,產(chǎn)物融合蛋白6xHis-DJ-1表達量最高,目的蛋白可占菌體總蛋白的57.1%。SDS-PAGE、Western blotting證實蛋白質(zhì)產(chǎn)物確實為融合DJ-1,且融合蛋白具有免疫原活性。 第二部分：低濃度和高濃度His融合DJ-1均可以對抗過氧化氫誘導的氧化應(yīng)激,恢復CaN活性；DJ-1活性多肽片段IAAICA(102-107)和CAGPTA(106-111)均可以部分對抗過氧化氫誘導的氧化應(yīng)激,提高LDH活性。結(jié)論： 重組pQE-30Xa/DJ-1質(zhì)粒轉(zhuǎn)化入感受態(tài)E.coli M15[pREP4],在37℃, IPTG濃度為1mmol/L誘導表達6小時產(chǎn)物融合蛋白6xHis-DJ-1表達量最高。Western Blotting鑒定融合表達產(chǎn)物DJ-1具有免疫原活性。融合蛋白的獲得為進一步研究其蛋白質(zhì)功能奠定了基礎(chǔ),并且為以后基因工程生產(chǎn)有生物活性的DJ-1奠定了基礎(chǔ)。 DJ-1本身在體外可以對抗過氧化氫誘導的氧化應(yīng)激；DJ-1多肽片段IAAICA(102-107)和CAGPTA(106-111)均是對抗氧化應(yīng)激的功能片段,Cys106殘基是對抗氧化應(yīng)激的功能位點之一。DJ-1完整蛋白可能是通過自身C106殘基氧化來對抗氧化應(yīng)激。DJ-1活性多肽IAAICA (102-107)可以完全對抗過氧化氫,為今后對抗過氧化氫的活性多肽發(fā)展及應(yīng)用提供了有力依據(jù)。
[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.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R3416

【參考文獻】

相關(guān)期刊論文 前3條

1 王健;氧化應(yīng)激與帕金森病研究進展[J];國外醫(yī)學(內(nèi)科學分冊);2001年08期

2 官志忠;齊曉嵐;;氧化應(yīng)激和神經(jīng)遞質(zhì)受體在阿爾茨海默病發(fā)病機制中的作用[J];貴陽醫(yī)學院學報;2006年01期

3 隋廣超，胡美浩;影響大腸桿菌中外源基因表達的因素[J];生物化學與生物物理進展;1994年02期

，

本文編號：1410170