融合蛋白CTP-SOD在毕赤酵母中的表达、纯化及抗氧化能力

发布时间：2018-01-08 18:29

本文关键词：融合蛋白CTP-SOD在毕赤酵母中的表达、纯化及抗氧化能力　出处：《西南大学》2010年硕士论文　论文类型：学位论文

【摘要】： 超氧化物歧化酶(Superoxide dismutase SOD)是一种在生物界广泛存在的抗氧化酶,在抗衰老、抗肿瘤、抗免疫疾病和电磁辐射上都起着重要的作用。SOD主要在细胞质中发挥作用,但是由于SOD是生物大分子,没有特异性的受体和通道,不容易跨过细胞膜进入细胞质,大大降低了生物利用度。细胞质转导肽(Cytoplasmic transduction peptide CTP);是在转导肽TAT基础上人工设计的一种新型转导肽,能够携带外源大分子穿过细胞膜,定位于细胞质。本实验利用含有细胞质转导肽基因序列的SOD特异引物,以人总RNA反转录产物为模板,扩增出了CTP和铜锌超氧化物歧化酶(Cu／Zn SOD)的融合基因。将其克隆到毕赤酵母分泌表达载体pPIC9K中,成功地构建重组表达载体CTP-SOD／pPIC9K。通过电激法将重组载体转化巴斯德毕赤酵母GS115,经酵母菌落PCR和SDS-PAGE筛选得到可表达重组蛋白的阳性重组子。大量诱导融合蛋白CTP-SOD表达,收集发酵上清经盐析、透析后,采用镍亲和层析介质纯化重组蛋白,蛋白纯度达到90%以上,比活为676.5U／mg。经Western-Blotting分析,进一步验证了重组蛋白的正确表达。为了分析CTP-SOD的生物功能,我们利用邻苯三酚(pyrogallol)诱导HeLa氧化损伤,研究了重组蛋白对氧化胁迫下HeLa细胞的保护作用。60μmol／L的邻苯三酚处理细胞48h后只有42.2%细胞存活下来；而CTP-SOD预处理HeLa细胞,明显提高了HeLa细胞在氧化胁迫下的存活率,随着CTP-SOD剂量的从1μmol／L增加到4μmol／L,HeLa细胞的存活率比对照组上升了10%到30%。CTP-SOD预处理HeLa细胞,明显提高了HeLa细胞的抗氧化能力,和对照组野生型SOD相比具有显著性差异。结果表明,CTP-SOD融合蛋白比野生型SOD更容易进入细胞,具有更高效的抗氧化功能。
[Abstract]:Superoxide dismutase (Superoxide dismutase SOD) is a kind of widely existed in biological antioxidant enzymes, anti-aging, anti-tumor, anti immune diseases and radiation plays an important role of.SOD play an important role in the cytosol, but because SOD is the biological macromolecules, no specific receptors and channels that is not easy to cross the cell membrane into the cytoplasm, greatly reduces the bioavailability.
Cytoplasmic transduction peptide (Cytoplasmic transduction; peptide CTP) is a new type of transduction peptide transduction peptide in artificial design based on TAT, capable of carrying exogenous macromolecules across the cell membrane, in the cytoplasm of cells. This experiment using the cytoplasmic transduction peptide gene sequence containing SOD specific primers, with total RNA reverse transcription products as template. Amplified CTP and copper zinc superoxide dismutase (Cu / Zn SOD). The fusion gene was cloned into Pichia pastoris expression vector pPIC9K, successfully constructed the recombinant expression vector CTP-SOD / pPIC9K. by electroporation to Pasteur recombinant plasmid was transformed to Pichia pastoris GS115, PCR and SDS-PAGE by yeast colony expression of recombinant protein screened positive recombinant fusion protein. A large number of induced CTP-SOD expression, collecting supernatant by salting out, dialysis, the recombinant protein was purified by nickel affinity chromatography, the purity of protein It was more than 90%, and the specific activity was 676.5U / mg. by Western-Blotting analysis, which further verified the correct expression of the recombinant protein.
In order to study the biological function of CTP-SOD, we use the adjacent benzene three phenol (pyrogallol) induced oxidative damage to HeLa, the research of recombinant protein on oxidative stress of HeLa cells under the protective effect of.60 on mol / L of the adjacent benzene three phenol treated cells 48h after only 42.2% cells to survive; and the pretreatment of CTP-SOD HeLa cells significantly increased HeLa cells under oxidative stress in the survival rate, with the dose of CTP-SOD from 1 mol / L increased to 4 mol / L, the survival rate of HeLa cells than the control group rose 10% to 30%.CTP-SOD pretreated HeLa cells significantly increased the antioxidant capacity of HeLa cells, compared with wild-type controls SOD with a significant difference. The results showed that CTP-SOD fusion protein than the wild type SOD are more likely to enter the cell, with a more efficient antioxidant function.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

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