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垂体腺苷酸环化酶激活肽基因的合成表达与活性研究
引用本文:陈哲宇,柴延丰,何成,路长林,吴祥甫.垂体腺苷酸环化酶激活肽基因的合成表达与活性研究[J].生物化学与生物物理进展,2001,28(2):192-197.
作者姓名:陈哲宇  柴延丰  何成  路长林  吴祥甫
作者单位:陈哲宇(第二军医大学神经生物学教研室,上海 200433)       陈哲宇(中国科学院上海生物化学与细胞生物学研究所,上海 200031)       柴延丰(第二军医大学神经生物学教研室,上海 200433)       何成(第二军医大学神经生物学教研室,上海 200433)       路长林(第二军医大学神经生物学教研室,上海 200433)       吴祥甫(中国科学院上海生物化学与细胞生物学研究所,上海 200031)
基金项目:国家自然科学基金(30000048)和国家重点基础研究发展规划项目(973项目)(1999054005)资助.
摘    要:根据文献报道垂体腺苷酸环化酶激活肽(pituitary adenylate cyclase activating polypeptide, PACAP)的氨基酸序列,推导出其核苷酸序列并设计成部分互补的6条寡核苷酸片段,利用DNA合成仪人工合成、纯化这6条寡核苷酸片段,通过片段退火、连接、克隆及测序鉴定获得了PACAP基因.将PACAP基因克隆至pGEX-4T-3质粒中转化BL21进行表达分析,融合蛋白约占细胞总蛋白的30%,其中部分为可溶性,部分以包涵体形式存在.通过亲和层析纯化GST-PACAP融合蛋白,该蛋白质能促进PC12细胞轴突生长及脊髓神经元存活.

关 键 词:垂体腺苷酸环化酶激活肽,基因克隆,基因表达,PC12细胞,脊  髓神经元
收稿时间:3/2/2000 12:00:00 AM
修稿时间:2000年3月2日

Gene Cloning and Expression of PACAP and Study of Its Biological Activity
CHEN Zhe-Yu,CHAI Yan-Feng,HE Cheng,LU Chang-Lin and WU Xiang-Fu.Gene Cloning and Expression of PACAP and Study of Its Biological Activity[J].Progress In Biochemistry and Biophysics,2001,28(2):192-197.
Authors:CHEN Zhe-Yu  CHAI Yan-Feng  HE Cheng  LU Chang-Lin and WU Xiang-Fu
Abstract:In order to study the expression and the feasibility of scaled production of neuropeptide in the routine expression system such as E.coli with the pituitary adenylate cyclase activating polypeptide(PACAP) as an example, the following experiments were carried out. First, on the basis of the reported amino acid sequence of PACAP, DNA sequence of PACAP was deduced and six partially complementary oligonucleotide fragments were designed. The coding region of PACAP was obtained by renaturing the DNA fragments and ligation and identified by DNA sequencing. The coding region of PACAP was cloned into plasmid pGEX-4T-3 and transformed into E.coli BL21(DE3). An expression strain BLPACAP was selected. SDS-PAGE analysis revealed that the GST-PACAP fusion protein was highly expressed and accumulated to about 30% of the total bacterial proteins. By affinity chromatography, up to 90% GST-PACAP was purified by one step from bacterial lysate. The purified protein could promote neurite outgrowth of PC12 cells and the survival of spinal cord neurons.
Keywords:pituitary adenylate cyclase activating polypeptide  gene cloning  gene expression  PC12 cells  spinal cord neurons
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