TAT-BDNF载体构建及融合蛋白表达

Construction of Vector pTAT-BDNF and Expression of Fusion Protein

目的构建重组表达载体TAT-BDNF,在E.coli BL21中高效表达并纯化融合蛋白,为研究TAT携带BDNF穿透血脑屏障治疗中枢神经系统疾病奠定了基础。方法经RT-PCR获得编码人BDNF的全基因序列,连接到原核表达载体pTAT上,得到重组表达载体pTAT-BDNF,转化大肠杆菌,IPTG诱导TAT-BDNF融合蛋白的表达。表达产物用SDS-PAGE鉴定,组氨酸亲和层析柱纯化融合蛋白。结果成功构建了TAT-BDNF融合蛋白的原核表达载体,在诱导下获得了高效表达并纯化了融合蛋白。结论为进一步研究TAT蛋白转导作用奠定了基础。

Objective To construct recombinant expression vector pTAT - BDNF and ex- press the Fusion Protein for study if the TAT - BDNF can penetrate the blood - brain barrier and treat disease of CNS. Methods BDNFcDNA fragment were amplified by RT - PCR. The product was diges- ted with restriction enzymes then was inserted into pTAT which contain protein transduct domain of TAT. The recombinant vector was transformed into E. coli BL and induced with IPTG. The expression fusion protein was analyzed by using SDS - PAGE method. The highly expressed product was purified by affinity chromatographyo Results The recombinant expression vector pTAT - BDNF was correctly constructed and fusion Proteion TAT - BDNF was successfully expressed in procaryotic cells. Conclu- sion This has laid the foundation for studying the protein transduction of TAT.