PEP-1-MIT-hSOD2 K29R突变体的筛

发布时间：2018-04-23 19:06

本文选题：PEP-1-MIT-hSOD2 + K29R　；参考：《暨南大学》2017年硕士论文

【摘要】：目的:将蛋白转导域(PTD)PEP-1和线粒体定位的人锰超氧化物歧化酶突变体(hSOD2K29R)进行融合表达,并研究线粒体靶向抗氧化蛋白PEP-1-MIT-hSOD2 K29R突变体对MPP~+损伤SH-SY5Y细胞的帕金森病(PD)细胞模型的保护作用。方法:利用生物软件SYBYL,ClustalX或者在线预测平台Swiss-model,HOT-MUSIC等分析预测出18个活力或者稳定性增大的hSOD2突变体,利用野生型pET-15b-hSOD2质粒为模板,通过PCR构建18个含定点突变的融合表达载体,并分别转入表达宿主,进行百草枯(PQ)抗性筛选,筛选出活力增大的突变体,电感耦合等离子体质谱法(ICPMS)检测Mn~(2+)结合能力,SOD活力试剂盒检测在不同浓度变性剂SDS以及不同温度,不同pH条件下处理1h后的活力变化。并构建pET-15b-PEP-1-MIT-hSOD2突变体融合表达载体。使用E.coliBL21作为宿主进行蛋白表达,使用Ni-NTA纯化蛋白。MTT【3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐】法检测融合蛋白对1000μMMPP~+诱导的SH-SY5Y细胞PD模型的保护作用和融合蛋白对SH-SY5Y细胞的毒性作用。免疫荧光法验证融合蛋白PEP-1-MIT-hSOD2突变体在SH-SY5Y细胞中线粒体定位作用。2'7'二氯荧光素二醋酸盐(DCF-DA)探针检测融合蛋白对PD细胞模型ROS的影响。Real-timePCR检测融合蛋白PEP-1-MIT-hSOD2突变体对PD细胞模型的炎症因子和凋亡因子影响。结果:(1)成功构建了18个pET-15b-hSOD2的突变体表达载体,并利用比较表达不同hSOD2突变体的E.coli的百草枯抗性筛选出活力最可能增强的hSOD2突变体K29R。(2)三维建模发现,hSOD2K29R活性中心Mn~(2+)到与其结合氨基酸H74的N1原子键长变短,由2.05?变到2.01?。ICPMS检测出Mn~(2+)结合量为0.966±0.025mol/molprotein,与野生型相比增加1.05倍。hSOD2K29R在高温、不同pH或不同浓度SDS处理1h后,活力比野生型蛋白大。(3)构建了pET-15b-PEP-1-MIT-hSOD2 K29R融合表达载体,获得分子量大小为29.8kDa的蛋白,产量为每升LB培养基可表达59mg蛋白,纯度在90%以上。(4)MTT检测发现PEP-1-MIT-hSOD2 K29R融合蛋白可以使MPP~+诱导的SH-SY5Y细胞的存活率由53.4%增加到81.6%,且无明显的细胞毒性。(5)PEP-1-MIT-hSOD2 K29R融合蛋白可以穿过SH-SY5Y细胞的细胞膜并进入线粒体,并降低MPP~+诱导产生的ROS。1μMPEP-1-MIT-hSOD2 K29R蛋白可以使MPP~+引起的SH-SY5Y细胞的炎症因子TNF-α,IL-6和凋亡因子Bax的表达下调,使抑制凋亡因子Bcl-2的表达上调,调高Bcl-2和Bax的比例。结论:成功筛选出活力增大的hSOD2突变体hSOD2 K29R。设计并表达出PEP-1-MIT-hSOD2 K29R融合蛋白,它可以较好的清除MPP~+诱导产生的ROS,并且可以穿过细胞膜并定位线粒体定位功能,可以下调PD细胞模型炎症因子TNF-α和IL-6的表达,调高PD细胞模型Bcl-2和Bax的比例,PEP-1-MIT-hSOD2 K29R融合蛋白有望开发成为抗PD的新药。
[Abstract]:Objective: to fuse the protein transduction domain (PTD) PEP-1 and mitochondrial mutants of human manganese superoxide dismutase (hSOD2K29R), and to study the protective effect of mitochondrial targeting antioxidant protein PEP-1-MIT-hSOD2 K29R mutant on the Parkinson's disease (PD) cell model of MPP~+ damaged SH-SY5Y cells. Method: using the biological software SYBYL, Clus. TalX or online prediction platform Swiss-model, HOT-MUSIC and other analyses predicted 18 active or stable hSOD2 mutants. Using the wild type pET-15b-hSOD2 plasmid as the template, 18 fusion expression vectors containing fixed point mutation were constructed by PCR and transferred into the expression host respectively. The resistance screening of paraquat (PQ) was screened and the vitality increased. The mutant, inductively coupled plasma mass spectrometry (ICPMS) was used to detect Mn~ (2+) binding ability. SOD activity kit was used to detect the activity changes of 1h after different concentration of denaturant SDS and different temperatures and pH conditions. The expression vector of pET-15b-PEP-1-MIT-hSOD2 mutant was constructed. E.coliBL21 was used as the host for protein expression. The Ni-NTA purified protein.MTT [3- (4,5- two methyl thiazole -2) -2,5- two phenyl tetrazolium bromide] was used to detect the protective effect of fusion protein on the PD model of SH-SY5Y cells induced by 1000 mu MMPP~+ and the toxic effect of the fusion protein on SH-SY5Y cells. The effect of.2'7'two chloro fluorescein two acetate (DCF-DA) probe on the effect of fusion protein on the PD cell model ROS. The effect of.Real-timePCR detection of the fusion protein PEP-1-MIT-hSOD2 mutant on the inflammatory factors and apoptosis factors of the PD cell model. Results: (1) the mutant expression vector of 18 pET-15b-hSOD2 was successfully constructed, and the utilization ratio of the mutant protein was successfully constructed. The most likely enhanced hSOD2 mutant K29R. (2) three-dimensional modeling was found to be more likely to be enhanced by the E.coli paraquat resistance of different hSOD2 mutants. It was found that the N1 atomic bond length of the hSOD2K29R active center Mn~ (2+) to its binding amino acid H74 was shorter, from 2.05 to 2.01?.ICPMS. The Mn~ (2+) binding amount was 0.966 +. Compared with the increase of 1.05 times.HSOD2K29R at high temperature, different pH or different concentrations of SDS treated 1H, the activity was larger than that of the wild type protein. (3) the pET-15b-PEP-1-MIT-hSOD2 K29R fusion expression vector was constructed and the protein of the molecular weight of 29.8kDa was obtained. The yield of the protein was expressed as 59mg protein per liter LB culture base, and the purity was above 90%. (4) MTT detection PEP-1-MI was found. T-hSOD2 K29R fusion protein can increase the survival rate of SH-SY5Y cells induced by MPP~+ from 53.4% to 81.6%, without obvious cytotoxicity. (5) PEP-1-MIT-hSOD2 K29R fusion protein can pass through the cell membrane of SH-SY5Y cells and enter mitochondria, and the ROS.1 micron MPEP-1-MIT-hSOD2 K29R protein induced by MPP~+ can cause MPP~+ to cause MPP~+. The expression of TNF- alpha, IL-6 and apoptotic factor Bax was down regulated in the SH-SY5Y cells. The expression of the inhibitor of the apoptotic factor Bcl-2 was up-regulated and the ratio of Bcl-2 and Bax increased. Conclusion: the hSOD2 K29R. of the hSOD2 mutant was successfully screened and expressed the PEP-1-MIT-hSOD2 K29R fusion protein. ROS, which can pass through the cell membrane and locate the mitochondrial localization function, can downregulate the expression of the inflammatory factors TNF- A and IL-6 in the PD cell model, and increase the proportion of Bcl-2 and Bax in the PD cell model, and the PEP-1-MIT-hSOD2 K29R fusion protein is expected to be a new drug for anti PD.

【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915;R96

【相似文献】