重组人PLCζ蛋白在昆虫细胞/杆状病毒表达系统内的表达、纯化及活性测定

Expression, purification and characterization of recombinant PLCζ protein in baculovirus-insect cell expression system

摘要:

PLCζ是PLC家族的一种新型同工酶，在哺乳动物卵母细胞激活中起着极其重要的作用。近年来，体外大量表达和纯化有活性的PLCζ蛋白用于结构生物学研究一直未能获得成功。本研究首次在杆状病毒表达系统中表达和纯化重组人PLCζ蛋白，首先将人PLCζ基因克隆至pFastBac-HTA质粒构建重组载体，转化DH10Bac发生位点特异性转座，经抗性和蓝白斑筛选，获得重组穿梭质粒Bacmid-PLCζ；在脂质体介导下将穿梭质粒转染Sf9昆虫细胞产生重组病毒，扩增病毒感染Sf9昆虫细胞进行蛋白表达；利用Ni2+亲和柱及分子筛来纯化蛋白，并通过考马斯亮蓝染色、Western blotting及飞行时间质谱对蛋白进行鉴定，并进行酶活性测定。结果显示重组蛋白在Sf9昆虫细胞感染杆状病毒后72 h达到峰值并以分泌形式表达在细胞培养基中，Ni2+亲和柱及分子筛纯化后的重组蛋白经Western blotting及电离飞行时间质谱鉴定为PLCζ蛋白，酶活性可达326.8 U/mL。该实验结果为重组人PLCζ蛋白大规模生产和生物医学应用研究提供了可参考利用的技术。

Abstract:

PLCζ is a new isoenzyme of the PLC family which plays an important role in activating mammalian oocytes. In recent years, large-scale expression and purification of active PLCζ protein in vitro for structural biology research has not been successful. In this study, the recombinant human PLCζ protein was expressed and purified in the baculovirus expression system. First, the full length of human PLCζ gene was cloned into the pFastBac-HTA plasmid to form the recombinant donor plasmid that was further transformed into DH10Bac Escherichia coli cells to construct the recombined bacmid by the site-specific transposition that was screened by resistance and blue-white spots. Then the bacmid was transfected to Sf9 insect cells via cellfectin to package the recombinant baculovirus. After the amplification of the recombinant baculovirous, the recombinant protein was expressed from the cells transduced by the recombinant baculovirus and was purified by Ni-NTA resin. Purified protein was identified by Western blotting and time-of-flight mass spectrometry and the enzyme activity was determined. The results showed that the recombinant PLCζ protein in the Sf9 cells was achieved at 72 hours after baculovirus infection and expressed in secreted form in cell culture medium. The recombinant protein purified by Ni2+ affinity column was identified as PLCζ by Western blotting and ionization time-of-flight mass spectrometry and the enzyme activity was up to 326.8 U/mL. The experimental results provide a reference for the large-scale production and biological application of recombinant human PLCζ protein.