人肠激酶轻链基因的克隆及其在大肠杆菌中的表达  

Cloning and Fusion Expression of Human Enterokinase Light Chain Gene in Escherichia coli

作　　者：杜广营[1] 

机构地区：[1]烟台大学药学院,山东烟台264005

出　　处：《烟台大学学报（自然科学与工程版）》2008年第1期40-45,共6页Journal of Yantai University(Natural Science and Engineering Edition)

摘　　要：研究目的:克隆表达人肠激酶轻链编码基因,以期应用于融合蛋白的切割与纯化.方法:从人的全基因组中扩增出编码人肠激酶轻链的5个外显子基因片段,经过酶切、连接后得到正确编码的人肠激酶轻链完整基因序列;随后,将目的基因片段插入原核表达载体pBV220中,构建成融合型表达载体pBV-EKL,转化大肠杆菌BL21(DE3),调节温度至42℃诱导重组蛋白表达;通过镍亲和层析纯化得到重组蛋白.结果:所表达的重组蛋白经SDS-PAGE分析,相对表观分子质量约为31 kDa,表达量占菌体总可溶蛋白量的46%;通过镍亲和层析纯化得到的重组蛋白经复性后显示出具有催化切割活性.结论:成功构建了能大量表达重组人肠激酶轻链的菌株,为进一步进行重组人肠激酶活性的研究及其在生产和医学方面的应用研究奠定了基础.Objective ： To clone and express the gene of human the cleavage and purification of fusion proteins. Methods： Fiv obtained by PCR from human genome are ligated into a whole enterokinase light chain which would be used in e exons of human enterokinase light chain gene fragment. Then the fragment is inserted into the pBV220 expression vector and sequenced. The recombinant plasmid pBV220-EKL is transformed into E. coli B1221 （DE3） and induced at 42℃. A pure fusion protein is obtained by nickel chelating chromatography using Ni-NTA agarose. Results： Compared with the sequence deposited in GenBank, the cloned gene sequence is correct. SDS-PAGE analysis indicates that target product is about 31 kDa which occupies 46% of the total soluble proteins. The crude kinase demonstrates cleavage activity. Conclusion： The constructed E. coli can express the recombined human enterokinase light chain successfully, which lays a foundation for human enterokinase activity research and farther application of expression products.

关 键 词：人肠激酶轻链 PBV220 基因工程 原核表达 

分 类 号：Q78[生物学—分子生物学]