重组人α降钙素基因相关肽的克隆与表达(英文)  

Cloning and Expression of Recombinant Human α Calcitonin Gene-related Peptide

作　　者：赵丹丹[1] 于晓光[1] 班莺[1] 徐华锋[1] 林平[1] 邹海峰[1] 刘宇[1] 

机构地区：[1]哈尔滨医科大学生物化学与分子生物学教研室,中国黑龙江哈尔滨150086

出　　处：《生命科学研究》2004年第1期22-27,共6页Life Science Research

基　　金：黑龙江省自然科学基金项目(D01-55);哈尔滨市学科后备带头人基金项目(0171218001)~~

摘　　要：通过PCR方法从人类基因组中扩增出编码hαCGRP的片段,将该片段定向克隆到pET-32a(+)载体上,构建pET-hαCGRP重组质粒。转化E.coli JM109菌株,利用酶切和测序方法筛选后,经IPTC诱导再转化E.coli BL21trxB(DE3)pLYsS菌株。SDS-PAGE和Western Blot分析表达产物,最后通过扩张蟾蜍肠系膜微循环血管实验来检测重组hαCGRP扩张血管的活性。结果表明,重组质粒含hαCGRP成熟肽编码基因序列(111bp),符合预想结果;表达出21 kD的融合蛋白,且主要以可溶性形式存在,其粗提物具有扩张血管能力。重组hαCGRP的成功表达为下一步纯化及研制基因工程药物奠定了重要基础。The coding sequence of hαCGRP was amplified by PCR from genomic DNA of human peripheral blood and cloned into pET-32a ( + ) vector to construct recombinant pET-haCGRP. First pET-hαCGRP was transformed to E. coll JM109, screened by restriction enzyme digestion and sequencing, then to E. coli BL21trxB(DE3)pLysS, induced by IPTG. The expression products were analyzed by SDS-PAGE and Western Blot, finally the dilation action was tested by the trial to expand toad s mesentery microcirculation. The results showed that the cloned sequence of the pET-hαCGRP was the same as the coding sequence of mature peptide of hαCGRP (111 bp) as desired. The expression protein was 21 kD fusion proteins as soluble ones, which had enormous action to dilate blood vessels. The successful expression of recombinant haCGRP established an important base for purification of fusion protein and investigating its drug of genetic engineering.

关 键 词：PCR 人类基因组 hαCGRP 融合蛋白 克隆 表达 基因工程 

分 类 号：Q78[生物学—分子生物学]