摘要
根据文献报道垂体腺苷酸环化酶激活肽 (pituitaryadenylatecyclaseactivatingpolypeptide,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BL2 1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 .
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.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2001年第2期192-197,共6页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金! (30 0 0 0 0 48)
国家重点基础研究发展规划项目! (973项目 ) (19990 5 40 0 5 )资助&&
