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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 purified recombinant human heparanase (HPA). Methods: The heparanase fragment containing no signal peptide was amplified by using the full-length heparanase plasmid pcDNA3.1-HPA. The fragment was inserted into pET-28a (+) vector and transformed into E.coli BL21 , DE3), the inducing conditions were optimized in terms of the density of initial induced bacteria, the temperature of induction, the induction time and the concentration of inducer, and the soluble expression of recombinant human heparanase was achieved. The HisTrapTMcrude affinity chromatography was used to purify the target protein. Results: The purified HPA protein was obtained. Western blotting confirmed that the expressed product was recognized by heparanase and His-tag antibody, which proved that the expressed product had the immunological properties of heparanase. CONCLUSION: The expression of purified human HPA protein provides an experimental material for the preparation and characterization of specific monoclonal antibodies.