摘要
为增强神经型乙酰胆碱受体(n AChR)蛋白的稳定性和表达量,以满足晶体学研究的要求。根据同源蛋白的氨基酸序列比对结果,设计了胞内5个切除位点,设计合适的引物,利用分子克隆的方法扩增出相应的改造后c DNA序列并构建到BacMam表达载体,最后利用BacMam杆状病毒表达系统构建表达突变型重组杆状病毒,并转染到HEK293F哺乳动物细胞中,诱导表达重组蛋白。采用免疫印迹法小规模表达检测后,选取合适突变型进行表达,并利用MBP亲和层析和分子排阻法进行两步纯化后,通过电子显微镜负染技术检测纯化后的蛋白质量。结果显示,成功克隆并表达了乙酰胆碱神经受体α7的5种不同的胞内区域突变型,且蛋白表达量与野生型相比均有所提高,其中P5突变型表达量最高。经表达纯化后,能够获得毫克级别的乙酰胆碱受体蛋白,并且电子显微镜观察的结果表明蛋白呈现出相对稳定的五聚体形貌,大小尺寸与理论相符。
In order to enhance the stability and expression of protein,was selected neural acetylcholine receptor（ n AChR） to meet requirement of crystallology. Based on amino acid sequence alignments with homologous protein,were designed five suitable intracellular excision sites,primers and expanded mutant c DNA sequence constructind into Bac Mam expression vector. Finally,was transfected the mutant virus into HEK293 f cells and induced to express the mutant protein. By western blotting,was selected the appropriate mutant type and developed into large-scale expression. Then it was purified through MBP affinity column and by molecular exclusion method,using electron microscope to observe the protein status. The results showed that all the mutant types have larger expression quantity compared with the wild type,especially the P5 mutant. Through large scale expression and purity,milliequivalent and high purity protein was gained. The electron microscope showed a steady pentamer flower ring structure,and the size was consistant with that of theory.
出处
《药物生物技术》
CAS
2016年第2期95-98,共4页
Pharmaceutical Biotechnology
基金
美国文安德基金项目
中国科学技术委员会资助项目(No.2013CB910600)
上海科学技术委员会资助项目(No.13ZR1447600
No.13JC1406300)
