重组蛋白ES-Kringle5的表达、纯化及活性检测  

Expression of Fusion Protein ES-Kringle5 and Its Purification and Biological Analysis

作　　者：陈悦[1] 付中平[1] 李景荣[1] 殷晓进[1] 

机构地区：[1]江苏先声药业有限公司,南京210042

出　　处：《中国生物工程杂志》2014年第5期60-65,共6页China Biotechnology

基　　金：国家"重大新药创制"资助项目(2009ZX09103)

摘　　要：目的:利用基因工程方法原核表达重组融合蛋白ES-Kringle5并进行纯化及活性检测。方法:ES-Kringle5是将内皮抑素N端的前27个氨基酸与Kringle5通过连接肽相连的重组融合蛋白,合成该重组蛋白的基因片段并插入载体pMD18-T中,然后克隆至大肠杆菌表达载体pET25b中并转化E.coli BL21(DE3)。乳糖诱导表达后经Ni-NTA亲和层析纯化后获得目的蛋白。通过抑制HUVEC细胞增殖实验检测其生物学活性。结果:重组质粒构建正确。利用乳糖诱导表达并降低诱导温度能增加目的蛋白的产量及可溶性表达。纯化后的重组蛋白纯度大于95%。生物学活性证明该重组蛋白具有抑制HUVEC的增殖能力。结论:具有生物学活性的重组蛋白ESKringle5可在大肠杆菌中高效表达,为研究其体内药效、药代及安全性评价奠定了基础。Objective： To express recombinant ES-Kringle5 fusion protein in prokaryotic cells, purify and identify the bioactivity of expressed product. Method：The nucleotide sequence encoding the 27-amino-acid peptide corresponding to the NH2-terminal domain of endostatin and Kringle 5 via a peptide linker were synthesized and inserted to pMD18-T, then the sequence cloned into vector pET25b. And this recombinant plasmid was transformed to E. coli BL21 （DE3） for expression by lactose induction. The expression product was purified by Ni-NTA. The abilities of ES-Kringle biological activity assay. Results： The ES-Kringle5 5 to inhibit the proliferation of HUVECs was used for its coding sequence was correctly cloned into pET-25b vector. Use lactose and lower the induction temperature can increase the expression level and soluble protein expression. The recombinant protein reached a purity of more than 95 % after purification. Bioactivity assay result shows that ES-Kringle5 could inhibit the proliferation of HUVECs. Conclusion：The recombinant ES-Kringle5 fusion was successfully expressed in E. coli with high biological activity, which lay the material foundation protein for its pharmacology study in vivo.

关 键 词：原核表达 抗血管生成 抗肿瘤 KRINGLE5 内皮抑素 

分 类 号：Q782[生物学—分子生物学]