摘要
目的:表达纯化重组的人乙酰肝素酶(heparanase,HPA)。方法:以表达乙酰肝素酶全长质粒pcDNA3.1-HPA为模板,扩增不包括信号肽的乙酰肝素酶片段,将其插入pET-28a(+)载体,转化到大肠杆菌BL21(plysS,DE3)中,在起始诱导菌密度、诱导温度、诱导时间、诱导剂浓度4个方面对诱导条件进行了优化,实现了重组人乙酰肝素酶的可溶性表达。采用HisTrapTMcrude亲和层析对目的蛋白进行了初步纯化。结果:得到了初步纯化的HPA蛋白,Western印迹证实表达产物可被乙酰肝素酶抗体和His标签抗体识别,证明表达产物具有乙酰肝素酶的免疫学特性。结论:表达纯化的人HPA蛋白为特异性单抗的制备和鉴定提供了实验材料。
Objective：To express,purify and identify the recombinant human heparanase. Methods：The cDNA of truncated human heparanase （without signal peptide） was cloned by PCR using pcDNA3.1-HPA as template and subcloned into pET-28a （ ＋ ） vector. The expression plasmid was transformed into Escherichia coli BI2,1 （plysS, DE3 ） to produce soluble protein. The protein was purified by means of affinity chromatography. Results： The result showed that the expressed protein reacted with anti-His antibody and anti-beparanase antibody respectively. Conclusion：We obtain a soluble expression protein in prokaryotic expression system, which has immunological characteristics of heparanase.
出处
《军事医学科学院院刊》
CSCD
北大核心
2009年第5期434-437,共4页
Bulletin of the Academy of Military Medical Sciences
基金
科技部支撑计划重大项目"常见恶性肿瘤分子标志的临床评价"(2006BA102A14)
关键词
乙酰肝素酶
大肠杆菌
表达优化
蛋白纯化
heparanase
Escherichia coli
expression optimization
protein purification
