選題：αB-晶狀體蛋白基因　視角：巴斯德畢赤酵母　引自：《中國生物工程雜志》2017年07期

【摘要】：αB-晶狀體蛋白屬于小熱休克蛋白家族,具有分子伴侶活性。在阿爾茨海默病大腦中,星形膠質(zhì)細胞表達的αB-晶狀體蛋白明顯上調(diào),并且這些上調(diào)的αB-晶狀體蛋白緊密地分布在β-淀粉樣蛋白沉積周圍。為了在分子水平上研究胞外αB-晶狀體蛋白在阿爾茨海默病中的作用機制,需要獲得大量具有生物學(xué)活性的αB-晶狀體蛋白。根據(jù)畢赤酵母密碼子偏好性對人αB-晶狀體蛋白基因進行優(yōu)化,并在目的基因3'端加上6個組氨酸標(biāo)簽,優(yōu)化后的基因克隆到巴斯德畢赤酵母(Pichia pastoris)表達載體p PIC9K上;電轉(zhuǎn)入畢赤酵母GS115中;優(yōu)化確定最佳表達條件為:BMGY與BMMY的轉(zhuǎn)接比為2∶1,表達時間為72h,甲醇補加終濃度為0.5%;經(jīng)Western blotting鑒定,獲得20kDa的目的蛋白,以及18.6kDa和13.1kDa兩個蛋白質(zhì)片段,重組目的蛋白出現(xiàn)了部分降解,純化獲得20kDa目的蛋白;利用胰島素還原法測定該片段具有分子伴侶活性,為進一步研究外源αB-晶狀體蛋白在阿爾茨海默病中對星形膠質(zhì)細胞的保護機制奠定基礎(chǔ)。
[Abstract]:偽 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 偽 B-crystallin in astrocytes was significantly up-regulated, and these up-regulated 偽 -B-crystallins were closely distributed around the deposition of 尾 -amyloid proteins.In order to study the mechanism of extracellular 偽 B crystallin in Alzheimer's disease at the molecular level, a large number of bioactive 偽 B crystalline proteins need to be obtained.Based on the preference of Pichia pastoris codon, the human 偽 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 偽-B-crystallin on astrocytes in Alzheimer's disease.
【作者單位】： 西南大學(xué)生命科學(xué)學(xué)院;
【分類號】：Q78;R749.16

【相似文獻】

相關(guān)碩士學(xué)位論文 前1條

1 胡繼元;常染色體顯性遺傳性粉塵狀白內(nèi)障一家系的相關(guān)基因突變篩查[D];安徽醫(yī)科大學(xué);2009年

，

本文編號：1730812