应用pCYB1载体表达胰高血糖素

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。

Expression of Recombinant Glucagon Using pCYB1 Vector

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.