胶质细胞源性神经营养因子在链霉菌的分泌表达-东南大学学报医学版

胶质细胞源性神经营养因子在链霉菌的分泌表达

单位：东南大学基础医学院,生物化学教研室,江苏,南京,210009

分类号：Q786

出版年·卷·期（页码）：2002·21·第四期（280-283）

摘要：

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pIJ702连接并转化S.lividans TK24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15000的特异性表达带,与GDNF成熟肽的分子质量吻合.结论:GDNF在MelC1信号肽指导下可以在链霉菌分泌表达.

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.

参考文献：

[1] Lin L-F H, DOHERTY D H, LILE J D. GDNF:a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. 1993. doi:10.1126/science.8493557
[2] Hudson J, GRANHOLM A C, GERHARDT G A. Glial cell line-derived neurotrophic factor augments midbrain dopaminergic circuits in vivo. 1995. doi:10.1016/0361-9230(94)00224-O
[3] CHOI-LUNDBERG D L, LIN Q, CHANG Y N. Dopaminergic neurons protected from degeneration by GDNF gene therapy. 1997. doi:10.1126/science.275.5301.838
[4] Yan Q, MATHESON C, LOPEZ O T. In vivo neurotrophic effects of GDNF on neonatal and adult facial motor neurons. 1995. doi:10.1038/373341a0
[5] Gilbert M, MOROSOLI R, SHARECK F. Production and secretion of proteins by Streptomyces. 1995(1). doi:10.3109/07388559509150530
[6] HOOPWOOD D A, BIBB M J, CHATER K F. Genetic Manipulation of Streptomyces:a laboratory manual, 1985
[7] Nicolas P, KLUEPFEL D, SHARECK F. Increased xylanase yield in Streptomyces lividans:dependence on number of ribosome-binding sites. 1996(6). doi:10.1038/nbt0696-756
[8] Chang S Y, CHANG S. Secretion of heterologous proteins in Streptomyces lividans, 1988

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