检索规则说明:AND代表“并且”;OR代表“或者”;NOT代表“不包含”;(注意必须大写,运算符两边需空一格)
检 索 范 例 :范例一: (K=图书馆学 OR K=情报学) AND A=范并思 范例二:J=计算机应用与软件 AND (U=C++ OR U=Basic) NOT M=Visual
机构地区:[1]清华大学生物科学与技术系,北京100084 [2]清华大学教育部蛋白质科学实验室,北京100084
出 处:《生物化学与生物物理进展》2009年第12期1569-1577,共9页Progress In Biochemistry and Biophysics
基 金:supported by a grant from National Hi-Tech Research and Development Program of China (2003AA624150)~~
摘 要:高保真PCR克隆出编码大鼠海马组织N型钙通道α1B亚基ⅢP区的DNA序列,分别与表达载体pET28b和pGEX-4T-1连接,转化入能补充稀有密码子tRNAs的大肠杆菌Rosetta菌株中,通过优化表达条件,实现了高效诱导表达.再通过低温诱导,将包涵体用尿素溶解再稀释后透析法复性、酶切、亲和柱层析等方法得到较纯的可溶性目的蛋白HIS-Cav22P、GST-Cav22P和Cav22P,紫外吸收光谱检测实验证明了重组蛋白HIS-Cav22P能和钙离子可逆地结合.最后通过GST沉降实验证明了芋螺毒素SO3与Cav22P存在体外相互结合作用.上述结果为揭示芋螺毒素SO3特异性阻断大鼠海马组织N型钙流的分子机制提供了直接的依据,也为建立筛选新的非吗啡型天然镇痛药物的技术平台提供了基础.The DNA fragment coding the third P region of α 1B subunit of N-type voltage-gated calcium channel ofRattus norvegicus (Cav22P for short) was amplified by high fidelity PCR, inserted into vectors pET28b and pGEX-4T-1 respectively, and expressed in Escherichia coli Rosetta. The expressed product HIS-Cav22P mostly deposited in an inclusive body. The inclusive body of HIS-Cav22P dissolved in urea buffer. After dilution and dialysis, the refolded protein HIS-Cav22P was finally purified via Histrap chelating HP column. Ultraviolet spectroscopy results demonstrated that HIS-Cav22P protein can bind to calcium ions reversely. The expressed product GST-Cav22P was purified from the supernatant of cell lysate using Glutathione Sepharose 4B column. However, GST-Cav22P degraded severely, which caused it difficult to purify the protein Cav22P and the GST pull-down assay. All the results suggest that the active recombinant proteins of Cav22P may act as a molecular target for high through screen of non-narcotic analgesic drugs.
正在载入数据...
正在载入数据...
正在载入数据...
正在载入数据...
正在载入数据...
正在载入数据...
正在载入数据...
正在链接到云南高校图书馆文献保障联盟下载...
云南高校图书馆联盟文献共享服务平台 版权所有©
您的IP:216.73.216.249