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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 clone, express and purify α-synuclein and explore the bioinformatics characteristics. Methods Primers were designed in the conserved region of α-synuclein gene in Genebank. The α-synuclein gene of cat was amplified by PCR from cat brain c DNA library. The gene was double digested and cloned into pET28a prokaryotic expression vector. Recombinant plasmids were constructed and transformed into E. coli BL21 (DE3) with the correct recombinant plasmid and induced by IPTG. Then, the homology and hydrophobicity of α-synuclein amino acids were analyzed. Results The α-synuclein gene was successfully amplified from the brain c DNA library. The gene was 381 bp in length and encoded 126 amino acids. The full-length gene was successfully cloned into pET28a and finally transfected into E. coli BL21 (DE3) to obtain the soluble α-synuclein protein with the molecular weight of 13.12kD, which is consistent with the expected molecular weight. Bioinformatics analysis showed that the α-synuclein protein was highly homologous to the α-synuclein amino acids of human and murine origin, accounting for 87.35% and 83.15%, respectively. However, compared with the amino acid sequences of both human and mouse, α-synuclein Amino acids 41 to 54 amino acids are missing. Protein structure prediction shows that the α-synuclein in cats has good hydrophobicity and helps to induce the formation of soluble proteins when expressed. This result was confirmed in this study. Conclusions The α-synuclein gene was cloned for the first time and expressed in Escherichia coli, which provided a basis for further study on the evolution of α-synuclein, the crystal structure of the protein, the biological function and the construction of Parkinson’s animal model .