含蛋白质转导域的Ngb融合蛋白的原核表达、纯化及其生物学活性的初步研究  

Prokaryotic Expression, Purification of Ngb Fusion Protein Containing Protein Transduction Domain and Its Biologic Activity

作　　者：周国钰[1] 周盛年[2] 楼之茵[1] 朱灿胜[1] 胡学强[1] 

机构地区：[1]中山大学附属第三医院神经病学科,广州510630 [2]山东大学齐鲁医院神经内科,济南250012

出　　处：《生物化学与生物物理进展》2007年第6期634-641,共8页Progress In Biochemistry and Biophysics

基　　金：广东省教育厅211工程专项基金资助项目(4209601)~~

摘　　要：以SD大鼠脑组织RNA为模板,利用RT-PCR技术,将HIV-1反式激活蛋白(TAT)中具有蛋白质转导功能的9个氨基酸序列,即蛋白质转导域(PTD)基因与脑红蛋白(Ngb)基因融合,应用T-A克隆技术将融合基因与pMD19-Tsimple载体连接,经测序正确后克隆至表达载体pET28b中,转化感受态E.coli BL21(DE3)plysS,得到的转化子经IPTG诱导后获得可溶性表达,并经蛋白质印迹检测进一步鉴定.表达产物经Ni2+亲和纯化层析、脱盐后在原代培养的大鼠皮质神经元进行生物学活性检测,结果显示,TATPTD-Ngb融合蛋白可以转运入皮质神经元内,在48h内可检测到其存在,并能提高缺氧条件下皮质神经元存活率、减少缺氧诱导的皮质神经元的凋亡.这一结果为进一步研究TATPTD-Ngb的神经保护机制提供了线索,并可能为神经系统疾病尤其是脑血管疾病、神经系统退行性疾病提供一个新的治疗策略.The fused gene （TAT PTD-Ngb） which included nine amino acid transactivator of transcription （TAT） protein transduction domain （RKKRRQRRR） of HIV-1 and neuroglobin gene was amplified by RT-PCR from rat brain RNA and cloned into the expression plasmid pET28b. The recombinant plasmid pET-PN was transformed into the E.coli. BL21 （DE3）plysS, which was induced with IPTG （0.4 mmol/L） to express TAT PTD-Ngb fusion protein. Ni-NTA resin was used to purify the product, which was identified by SDS-PAGE and Western blot subsequently. After being purified and desalted, the biological activity of TAT PTD-Ngb was deteced in primary cultured cortical neurons. The results showed that TAT PTD-Ngb could transduced into cortical neurons, increase cell viability under hypoxia and attenuate apoptosis induced by hypoxia. The present study provides a clue for the research of neuroglobin and seems to provide a protein therapy strategy for CNS diseases,especially in cerebrovascular diseases and neurodegenerative diseases.

关 键 词：蛋白质转导 脑红蛋白 蛋白质治疗 

分 类 号：Q25[生物学—细胞生物学]