人GDNF-TAT真核表达载体的构建及其在毕赤酵母中的表达

目的：在毕赤酵母中表达带有TAT多肽的人胶质细胞源性神经营养因子（glial cell line-derived neurotro-phic factor,GDNF）,为探讨GDNF-TAT用于帕金森病的治疗奠定基础。方法：用密码子优化法合成人GDNF基因序列连在pPICZαA,用PCR方法分别在上游引入TAT位点与XbaⅠ酶切位点,下游设计扩增pPICZαA上的α-因子序列。然后插入pPICZαA载体中,转化大肠杆菌TOP10,筛选阳性克隆,对其进行PCR和双酶切并测序鉴定,构建GDNF-TAT在毕赤酵母表达质粒pPICZαA-GDNF-TAT。电击转化毕赤酵母GS115,对阳性克隆进行诱导表达后经SDS-PAGE和Western印迹鉴定目的蛋白。结果：双酶切pPICZαA-GDNF-TAT后,琼脂糖电泳可分别见到大小约为3.1kb和432bp片段,表明目的片段已插入载体中,经序列测定其含有完整的GDNF-TAT基因片段,Western印迹结果显示含有pPICZαA-GDNF-TAT的毕赤酵母GS115能分泌表达GDNF-TAT。结论：成功构建了毕赤酵母表达载体pPICZαA-GDNF-TAT,并能在毕赤酵母GS115中分泌表达GDNF-TAT。

Construction and expression of a eukaryotic vector for human GDNF-TAT in Pichia pastoris

Objective： To construct and express a Pichia pastoris expression vector of glial cell line-derived nenrotrophic factor（GDNF）-TAT fusion protein in order to explore the application of GDNF-TAT protein to the treatment of Parkinson＇s disease. Methods：The GDNF gene was synthesized by codon optimization and was ligated to pPICZαA. The GDNF-TAT gene with TAT gene site and Xba I restriction enzyme sites and α-factor of pPICZαA were obtained by PCR and subcloned into the pPICZαA vector. After being identified by restrictive enzymes digestion and sequencing, reconstructed plasmid was transformed into P. pastoris GSll5 by the method of electroporation. Expression of GDNF-TAT protein was assayed in P. pastoris GS115 culture supernatant by SDS and Western blotting. Results ：The target gene GDNF-TAT fragment about 432 bp was integrated into the genome of P. pastoris GSll5. The recombinant GDNF-TAT protein with relative molecular mass of 17×10^3 was secreted into the culture supernatant and was confirmed by Western blotting. Conclusion：P. pastoris expression vector pPICZαA-GDNF-TAT was constructed successfully and recombinant GDNF-TAT protein was expressed in P. pastoris GS115.