应用pCYB1载体表达胰高血糖素

Expression of Recombinant Glucagon Using pCYB1 Vector

目的 研制基因工程胰高血糖素。方法 利用pCYB1表达载体,亚克隆胰高血糖素基因后转化于BL21（DE3）菌种,并对阳性克隆的质粒进行DNA测序。检测不同条件下,IPTG诱导胰高血糖素-intein-几丁质结合结构域（CBD）组成的融合蛋白表达量,确定工程菌表达最适条件。利用几丁质结合柱进行亲和层析,在DTT作用下由intein在柱上进行自我切割,纯化胰高血糖素。SDS-PAGE检测融合蛋白表达量,SDS-PAGE在Tris-Tricine缓冲系统中电泳检测纯度,Lowry法检测蛋白质含量,Western blot检测免疫原性,升血糖实验检测活性并进行 N-末端测序。结果DNA测序显示表达产物基因序列正确,纯化胰高血糖素具有免疫原性和生物学活性,N-端15个氨基酸与天然产品相同,电泳呈单一谱带,产率为1.25mg/L。结论 获得分泌胰高血糖素基因工程菌,适于快速大规模生产。

Objective To prepare recombinant Glucagon.Methods Clucagon gene was subcloned to pCYB1 expression vector and transformed to BL21(DE3) cell, then positive clones were sequenced. The expression levels of Glucagon - Intern - Chition Binding Domain fusion proteins under the induction of IPTG were detected for determining the optimal expression conditions . Recombinant Glucagon was purified by affinity chromatography using chitin column and Intein- self- clc;avage buffer.The expressed fusion protein was identified by Western blot,and the N - terminal of it was sequenced.The expression level, purity and content of it were detected by common SDS - PAGE, SDS - PAGE in Tris - Tricine buffer and The Lowry method respectively . Results DNA sequencing showed correct sequence of recombinant plasmid. The expressed product after being purified showed immunogenicity and biological activity of Glucagon. The 15 amino acids at N - terminal of the expressed product were identical to those of natural Glucagon. Conclusion A recombinant strain suitable for the rapid and large - scale production of Glucagon was constructed.