胶质细胞源性神经营养因子在链霉菌的分泌表达

Expression and secretion of glial cell line-derived neurotrophic factor in Streptomyces lividans

目的 :构建胶质细胞源性神经营养因子 (GDNF)表达质粒 ,实现GDNF在链霉菌的分泌表达。方法 :从人基因组中克隆GDNF成熟肽编码基因 ,与链霉菌酪蛋白酶melC1启动子和信号肽DNA序列融合。melC1信号肽DNA与GDNF融合后无翻译框架的漂移 ,且GDNF的转录受控于melC1启动子。融合基因与链霉菌载体pIJ70 2连接并转化S .lividansTK2 4原生质体。结果 :经抗性筛选及限制性酶切分析获得重组质粒pIJMG。携带pIJMG的链霉菌培养上清存在约 15 0 0 0的特异性表达带 ,与GDNF成熟肽的分子质量吻合。结论

Objective To construct the expression vector harborring human glial cell line-derived neurotrophic factor(GDNF) and test its ability to produce secreted GDNF in Streptomyces.Methods The gene encoding mature peptide of GDNF was cloned from human genomic DNA.GDNF cDNA was fused with the signal squence and upstream regulatory element of melC1,a gene essential for the production of functional tyrosinase in Streptomyces.Transcription of GDNF cDNA was under the control of melC1 promoter and no frameshift occurred in the conjunction of melC1 signal DNA and GDNF coding sequence.The fusion fragment was further transferred into Streptomyces vector pIJ702.Results The recombinant plasmid pIJMG capable of replication in Streptomyces was obtained.Culture supernatant of S.lividans TK24 containing pIJMG produced a unique protein band of 15000,which was in agreement with molecular weight of GDNF mature peptide.Conclusion Streptomyces MelC1 signal peptide is effective in directing the secretion of GDNF in S.lividans.