计算机辅助设计提高人NMDA受体主亚基M3-M4环在E.coli中的表达  

Computor-aided design to raise the expression level of the M3-M4 loop in the essential subunit of human NMDA receptor in E.coli

作　　者：张玉梅[1] 孙长凯[1,2,3] 范明[4] 李伍举[4] 赵杰[1] 韩大跃[5] 王吉庆[5] 吴加金[4] 

机构地区：[1]大连医科大学脑疾病研究所 [2]军事医学科学院基础医学研究所,北京100850 [3]解放军大连210医院,大连116011 [4]军事医学科学院基础医学研究所 [5]解放军大连210医院

出　　处：《基础医学与临床》2003年第5期499-502,共4页Basic and Clinical Medicine

基　　金：国家自然科学基金 (30 0 70 2 6 7);中国博士后科学基金 (2 0 0 1);辽宁省重点科技攻关 (2 0 0 12 2 6 0 0 5 )

摘　　要：基于对螺旋区随机堆积的RNA二级结构的预测和密码子偏性计算 ,建立了pBV2 2 0载体中外源基因高效表达的判别模型。该模型认为 ,外源基因的表达水平与其 5′端 30～ 39区域 ,以及 3′端 30～ - 39区域的RNA二级结构自由能具有显著的统计学意义 ;其次与 3′端 9bp的局部密码子偏性相关。据此模型 ,使用RNAfold和Goldkey软件优化改构设计了用于克隆表达人NMDA受体主亚基M3 M4环的引物。将PCR扩增的基因克隆入pBV2 2 0载体中 ,在大肠杆菌DH5α宿主中获得了高效表达 (表达水平在 2 0 %以上 )。To improve the expression of foreign gene in pBV220 vector, a model was established by predicting RNA secondary structure based on helical regions random stacking and calculating the codon adaptation. The model showed a statistically significant linkage between high(low) expression and the free energy of secondary structure in the regions 30～39 of 5′end and 30～-39 of 3′end, while the local codon adaptation of 9 bp in the 3′ end was also related to expression level. Guided by this model , a pair of PCR primers was designed with RNAfold and Goldkey software. The primers were used for expressing the M3 M4 loop gene in the essential subunit of human NMDA receptor which was amplified and ligated into pBV220 vector. By transforming DH5α with this recombinant plasmid , M3 M4 loop was highly expressed in DH5α transformant with expression level over 20%.

关 键 词：计算机辅助设计 人NMDA受体 E-coli 基因克隆 主亚基 M3-M4环 N-甲基-D-门冬氨酸受体 

分 类 号：R346[医药卫生—基础医学]