密码子优化后的αB-晶状体蛋白基因毕赤酵母重组质粒的构建及表达的初步研究

发布时间：2018-04-10 10:21

选题：αB-晶状体蛋白基因　视角：巴斯德毕赤酵母　引自：《中国生物工程杂志》2017年07期

【摘要】：αB-晶状体蛋白属于小热休克蛋白家族,具有分子伴侣活性。在阿尔茨海默病大脑中,星形胶质细胞表达的αB-晶状体蛋白明显上调,并且这些上调的αB-晶状体蛋白紧密地分布在β-淀粉样蛋白沉积周围。为了在分子水平上研究胞外αB-晶状体蛋白在阿尔茨海默病中的作用机制,需要获得大量具有生物学活性的αB-晶状体蛋白。根据毕赤酵母密码子偏好性对人αB-晶状体蛋白基因进行优化,并在目的基因3'端加上6个组氨酸标签,优化后的基因克隆到巴斯德毕赤酵母(Pichia pastoris)表达载体p PIC9K上;电转入毕赤酵母GS115中;优化确定最佳表达条件为:BMGY与BMMY的转接比为2∶1,表达时间为72h,甲醇补加终浓度为0.5%;经Western blotting鉴定,获得20kDa的目的蛋白,以及18.6kDa和13.1kDa两个蛋白质片段,重组目的蛋白出现了部分降解,纯化获得20kDa目的蛋白;利用胰岛素还原法测定该片段具有分子伴侣活性,为进一步研究外源αB-晶状体蛋白在阿尔茨海默病中对星形胶质细胞的保护机制奠定基础。
[Abstract]:伪 B-crystallin belongs to the small heat shock protein family and has molecular chaperone activity.In the brain of Alzheimer's disease, the expression of 伪 B-crystallin in astrocytes was significantly up-regulated, and these up-regulated 伪 -B-crystallins were closely distributed around the deposition of 尾 -amyloid proteins.In order to study the mechanism of extracellular 伪 B crystallin in Alzheimer's disease at the molecular level, a large number of bioactive 伪 B crystalline proteins need to be obtained.Based on the preference of Pichia pastoris codon, the human 伪 B-crystallin gene was optimized. The gene was cloned into Pichia pastoris expression vector p PIC9K by adding 6 histidine tags to the 3 'end of the target gene.The optimal expression conditions were as follows: the ratio of Western BMGY to BMMY was 2: 1, the expression time was 72 h, and the final concentration of methanol was 0.5%. The target protein of 20kDa and two protein fragments of 18.6kDa and 13.1kDa were obtained by Western blotting.The recombinant target protein was partially degraded and purified to obtain the 20kDa target protein, and the fragment was determined by insulin reduction method to have molecular chaperone activity.To further study the protective mechanism of exogenous 伪-B-crystallin on astrocytes in Alzheimer's disease.
【作者单位】：西南大学生命科学学院;
【分类号】：Q78;R749.16

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