人与鼠源内皮抑制素不同标签重组蛋白的表达纯化及活性分析  

Expression and Anti-agiogenesis Activity of Human and Mouse Endostatin

作　　者：杨益金 陆俊佳[1] 覃霞[1] 畅荣妮[1] 赖青鸟[1] 华荣[1] 方玲[1] 吴华裕[1] 侯伟[1] 吴飞翔[2] 谢彤[2] 袁志刚[1] 舒伟[1] 

机构地区：[1]广西医科大学细胞生物学与遗传学教研室,广西南宁530021 [2]广西壮族自治区肿瘤医院,广西南宁530021

出　　处：《过程工程学报》2014年第4期649-654,共6页The Chinese Journal of Process Engineering

基　　金：国家自然科学基金资助项目(编号:81260479);广西自然科学青年基金资助项目(编号:2013GXNSFBA019143)

摘　　要：根据文献报道和NCBI数据库确定人源内皮抑制素(HE)和鼠源内皮抑制素(ME)DNA序列,以DNA合成法分别获得长度为554 bp的人源内和长度为565 bp的鼠源内皮抑素基因;分别构建携带谷胱甘肽巯基转移酶(GST)表达标签的原核表达质粒pGEX-4T1-HE和携带硫氧还蛋白A(TrxA)表达标签的原核表达质粒pET-32a-ME,在大肠杆菌BL21(DE3)中诱导表达,获得不同表达标签的HE、ME重组蛋白.最后通过鸡胚绒毛尿囊膜(CAM)试验验证重组蛋白的抗血管生成活性.结果表明,构建了人与鼠源内皮抑制素基因不同标签表达载体,在大肠杆菌中成功诱导表达.获得具有活性的人与鼠源ES重组蛋白.纯化复性后的重组蛋白能明显抑制新生血管的生长.GST和TrxA标签对增进重组ES可溶表达没有显著差异,两组标签的重组蛋白都主要存在于包涵体内.Human endostatin （HE） and mouse endostatin （ME） cDNA sequences were determined based on the reports and NCBI database. Their 554 bp HE and 565bp ME cDNA sequences were obtained through DNA synthesis, respectively. Prokaryotic expression plasmids pGEX-4T1-HE and pET-32a-ME with different protein labels of GST and TrxA were constructed, then expressed through induction in Escherichia coli BL21 （DE3）, HE and ME recombinant proteins were obtained and their anti-angiogenesis activity was analyzed by chick chorioallantoic membrane （CAM） test. The human and mouse endostatin gene expression vectors with different labels were successfully constructed and used in induced expression in Escherichia coli. CAM testing results indicated that the purified refolded recombinant protein had a strong anti-angiogenesis activity. The human and mouse ES recombinant proteins were obtained successfully. TrxA and GST labels showed no difference in the increase of soluble expression of recombinant ES, both of the recombinant protein with different labels mainly existed in inclusion body.

关 键 词：内皮抑素 DNA序列 重组蛋白 纯化 鸡胚绒毛尿囊膜试验 诱导表达 

分 类 号：Q786[生物学—分子生物学]