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

Construction of Pichia pastoris Expression Vector of Codon Optimized αB-crystallin Gene and Expression Optimization

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。

αB-crystallin is a member of the small heat shock protein family, which has the molecular chaperones activity. The expression of αB-crystallin increases obviously in astrocytes in the brains of Alzheimer＇ s disease （AD） patients, and colocalizes with β-amyloid （Aβ） protein deposition densely. In order to study the mechanism of extracellular αB-crystallin in Alzheimer＇ s disease at molecular level, need to obtain a large amount of active αB-crystallin. According to the Pichia pastoris codon preference, the codons of αB-crystallin gene and added six histidine residues at the 3＇ terminal of the gene . Then the optimized gene is claned into pPIC9K and transferred the vector into Pichia pastoris GS115 is optimized through the electroporation method. The best expression conditions are as follows： the transferring proportion of BMGY and BMMY is 2：1 ; the expression time is 72h and adding the methanol to the final concentration to 0.5%. There are three protein fragments by Western blotting, and the molecular weights of them are： 20.0kDa, 18.6kDa and 13.1 kDa. The recombination protein was partially degraded and obtained the purified 20kDa fragment, which has molecular chaperones activity using insulin reduction method. The result may make a base line for further studing protection mechanism of exogenous αB-crystallin to astrocytes in Alzheimer＇ s disease.