广东省教育厅211工程专项基金资助项目(4209601).
This work was supported by a grant from The Special Funds for 211 Project of Guangdong Department of Education (4209601).
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-T simple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,TAT PTD-Ngb融合蛋白可以转运入皮质神经元内,在48 h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TAT PTD-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了一个新的治疗策略.
The fused gene (TAT PTD-Ngb) which included nine amine 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.
周国钰,周盛年,楼之茵,朱灿胜,胡学强.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[J].生物化学与生物物理进展,2007,34(6):634-641
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