人扭转蛋白A的基因克隆表达及蛋白质纯化
发布时间：2018-09-18 08:59
【摘要】：原发性扭转痉挛是一组由于躯干、肢体、颈部或颜面肌肉协调功能失调而出现各种姿势的异常或肢体的扭转，已有14种类型的扭转痉挛被定位。常染色体显性早发性扭转痉挛定位于9q34，是一种最常见、最为严重的扭转痉挛，1989年Ozelius等将该基因命名为DYT1，1997年又克隆出该基因，它编码332个氨基酸组成的DYT1扭转蛋白A(Torsin A)，该蛋白是腺苷三磷酸酶AAA家族的成员，一种ATP结合蛋白，它与许多细胞活动有关。DYT1基因突变可引起9q连锁的扭转痉挛。 在实验中，我们从人扭转蛋白A的编码序列中设计引物，以人肝cDNA文库作模板，成功地扩增到编码DYT1蛋白的基因片段，该片段全长939bp。将所得片段与pMD18-T载体连接，转化到JM109大肠杆菌中，从转化平板上挑出菌落，用碱裂法提取质粒，，通过PCR和酶切分析，成功地筛选到阳性克隆，测序结果与文献报道结果一致。 提取质粒，用BamH I和Xho I酶切，回收目的片段，分别克隆到原核表达载体pET28a(+)和pGEX-6P-1中，转化JM109受体菌，从JM109受体菌中提出质粒，再转化到BL21(DE3)菌中，筛选出阳性克隆，成功地构建了人扭转蛋白A原核表达载体。将该工程菌接种于含有适当抗生素的LB培养基中，37℃振荡培养过夜，次日将此菌液按1：100的比例接种于250mL LB液体培养基中，37℃振荡培养，当细菌长至适当的密度时，加入IPTG(终浓度为1mM)诱导，继续培养约5h，离心收集菌体，进行SDS-PAGE，结果在两种载体中均得到了高效表达。 在pET28a(+)中表达的DYTI蛋白为包含体(IBS)，经过IBS制备、蛋 白质复性、金属鳌合层析纯化、superdex75凝胶过滤纯化，得到了纯的DYTI 蛋白;在pGEX一6P一1中表达的DYTI蛋白与GST的融合蛋白也为IBS，但此融 合蛋白复性不好，只得到极少量的融合蛋白。 关键词:人;OYTI基因;扭转蛋白A;cDNA;克隆;原核表达;蛋白质纯化
[Abstract]:Primary torsion spasm is a group of abnormal posture or limb torsion due to the malcoordination of torso, limb, neck or facial muscles. Fourteen types of torsion spasm have been located. Autosomal dominant early torsion spasm, located at 9q34, is one of the most common and severe torsion spasms. In 1989, Ozelius et al named the gene DYT1, 1997 and cloned the gene. It encodes a 332-amino acid DYT1 torsion protein A (Torsin A), a member of the AAA family of adenosine triphosphatase, a ATP binding protein, which is associated with many cellular activities and causes 9q-linked torsion spasm by mutations in the .DYT1 gene. In the experiment, we designed primers from the coding sequence of human torsion protein A and used human liver cDNA library as template to amplify the gene fragment encoding DYT1 protein successfully. The fragment was ligated with pMD18-T vector and transformed into JM109 Escherichia coli. The colony was isolated from the transformed plate and the plasmid was extracted by alkali cleavage method. The positive clones were successfully screened by PCR and enzyme digestion. The results of sequencing were consistent with those reported in the literature. The plasmids were extracted and digested with BamH I and Xho I, and the target fragments were cloned into prokaryotic expression vectors pET28a () and pGEX-6P-1, respectively. The plasmids were transformed into JM109 receptor bacteria, then transformed into BL21 (DE3) bacteria, and the positive clones were screened out. The prokaryotic expression vector of human torsion protein A was successfully constructed. The engineered bacteria were inoculated in LB medium containing appropriate antibiotics for the night at 37 鈩
本文编号：2247415
[Abstract]:Primary torsion spasm is a group of abnormal posture or limb torsion due to the malcoordination of torso, limb, neck or facial muscles. Fourteen types of torsion spasm have been located. Autosomal dominant early torsion spasm, located at 9q34, is one of the most common and severe torsion spasms. In 1989, Ozelius et al named the gene DYT1, 1997 and cloned the gene. It encodes a 332-amino acid DYT1 torsion protein A (Torsin A), a member of the AAA family of adenosine triphosphatase, a ATP binding protein, which is associated with many cellular activities and causes 9q-linked torsion spasm by mutations in the .DYT1 gene. In the experiment, we designed primers from the coding sequence of human torsion protein A and used human liver cDNA library as template to amplify the gene fragment encoding DYT1 protein successfully. The fragment was ligated with pMD18-T vector and transformed into JM109 Escherichia coli. The colony was isolated from the transformed plate and the plasmid was extracted by alkali cleavage method. The positive clones were successfully screened by PCR and enzyme digestion. The results of sequencing were consistent with those reported in the literature. The plasmids were extracted and digested with BamH I and Xho I, and the target fragments were cloned into prokaryotic expression vectors pET28a () and pGEX-6P-1, respectively. The plasmids were transformed into JM109 receptor bacteria, then transformed into BL21 (DE3) bacteria, and the positive clones were screened out. The prokaryotic expression vector of human torsion protein A was successfully constructed. The engineered bacteria were inoculated in LB medium containing appropriate antibiotics for the night at 37 鈩
本文编号：2247415
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