人GDNF在甲醇酵母及家蚕幼虫中的表达

将人胶质细胞源性神经营养因子(GDNF)基因克隆入酵母分泌型表达载体pPIC9K中，酶切线性化后电穿孔导入酵母细胞进行整合，经G418筛选得到多拷贝转化子，甲醇诱导表达。将人GDNF基因克隆入昆虫病毒转移载体pBacPAK8中，与线性化Bm-BacPAK6修饰病毒基因组DNA共转染家蚕细胞，经体内重组，筛选到重组病毒。用重组病毒感染家蚕幼虫，5d后收集血淋巴。SDS-PAGE和蛋白质印迹杂交结果证实了酵母培养上清液及家蚕幼虫血淋巴中含有GDNF蛋白。活性研究表明，甲醇酵母及家蚕幼虫表达的GDNF蛋白能促进多巴胺能神经元的存活和突起生长。

Expression of Human GDNF in Methyltrophic Yeast Pichia pastoris and Silkworm Larvae

The cDNA encoding glial cell derived neurotrophic factor (GDNF) was cloned into the Pichia expression vector pPIC9K and then transformed into his4 mutant yeast GS115 by electroporation. Multicopy transformants were screened by various G418 concentrations and induced by methanol. The human GDNF gene was cloned into the baculovirus transfer vector pBacPAK8. The recombinant transfer vector pBacPAK-GDNF was coinfected with linear Bm-BacPAK6 DNA into BmN cells. The recombinant virus was screened and plaque-purified. The silkworm larvae were infected with the recombinant virus and collected 5 days later. SDS-PAGE and Western blot confirmed that GDNF was expressed in Pichia culture medium and silkworm larvae hemulymph. The GDNF protein expressed in Pichia and silkwom larvae could significantly promote the survival and neurite outgrowth of dopaminergic neurons.