猫α-synuclein蛋白的表达纯化及生物学特征分析  

Cloning,expression,purification and characterization analysis of cat α-synuclein protein

作　　者：倪斌[1] 张玲[1] 李坚[2] 张金喜[2] 周健[1] 杨可威 张君智 戴一鸣[1] 夏雅琴[1] 鲁燕华[1] 刘俊平[1] 李建峰[1] 

机构地区：[1]杭州师范大学医学院,杭州311121 [2]杭州市萧山区第四人民医院,杭州311121

出　　处：《北京生物医学工程》2015年第5期458-461,508,共5页Beijing Biomedical Engineering

基　　金：浙江省自然科学基金(LQ13C050001);杭州师范大学科研启动基金(PE13002004003);浙江省大学生科技创新活动计划暨新苗人才计划(ZX13005002028);杭州师范大学2013-2014学年学生课外学术科技作品项目(1283XXM165)资助

摘　　要：目的对猫突触核蛋白（α-synuclein）进行克隆、表达及纯化,并探讨其生物信息学特征。方法在Genebank中α-synuclein基因的保守区域内设计引物,从猫脑c DNA文库中PCR扩增得到猫的α-synuclein基因,再将此基因双酶切后克隆到p ET28a原核表达载体中,构建重组质粒,测序正确的重组质粒转化BL21（DE3）大肠杆菌,采用IPTG诱导表达。然后对猫α-synuclein氨基酸的同源性和疏水性进行分析。结果实验成功从猫脑c DNA文库中扩增出α-synuclein基因,基因全长381个碱基,编码126个氨基酸。获得的全长基因成功克隆进入p ET28a,最后转染大肠杆菌BL21（DE3）,获得可溶性表达的α-synuclein蛋白质,蛋白质分子量为13.12k D,与预期分子量一致。生物信息学分析显示猫α-synuclein蛋白与人源及鼠源α-synuclein氨基酸具有很高的同源性,分别为87.35%和83.15%,但是与鼠和人的氨基酸序列比较,猫α-synuclein氨基酸缺失41～54位氨基酸。蛋白质结构预测显示猫α-synuclein具有很好的疏水性,有助于诱导表达时形成可溶性蛋白,这一结果在本研究中得到证实。结论本研究首次克隆了猫α-synuclein基因,并在大肠杆菌中实施了可溶性表达,为后期研究α-synuclein的进化、蛋白晶体结构、生物学功能和帕金森动物模型的构建奠定了一定的基础。Objective To study cloning,expression,purification and characterization analysis of cat α-synuclein protein. Methods According to the conserved regions of α-synuclein gene,primers were designed and cat α-synuclein gene was amplified by PCR from cat brain c DNA. The gene was cloned into p ET28 a and p ET28 a carrying the coding DNA sequence of cat α-synuclein gene was transformed into E. coli BL21（ DE3） to express cat α-synuclein protein by IPTG induction. And the homology and hydrophobicity of cat α-synuclein amino acid were analyzed. Results There was a 381 bp cat α-synuclein gene amplified from the cat brain c DNA and cloned into p ET28 a. The transfected E. coli BL21（ DE3）expressed soluble cat α-synuclein protein. The molecular weight of cat α-synuclein protein was consistent with the expected molecular weight（ 13. 12 k D）. Bioinformaticsanalysis showed that cat,murine and human α-synuclein amino acids had high homology,yet the 41-54 amino acids in cat α-synuclein protein were missing. Protein structure prediction showed that cat α-synuclein had good hydrophobicity,contributing to the formation of soluble protein expression. Conclusions This study successfully cloned cat α-synuclein gene and implemented a soluble expression in E. coli,which provided a basis for the further studies of the evolution of α-synuclein,protein crystal structure,biological function and Parkinson animal models.

关 键 词：Α-突触核蛋白 猫 帕金森病 克隆 原核表达 纯化 

分 类 号：R318[医药卫生—生物医学工程]