人N端脂多糖结合蛋白基因在sf21昆虫细胞中的表达

Expression of human N-lipopolysaccharide binding protein gene in sf21 insect cells

目的表达重组人 N端脂多糖结合蛋白 (truncated lipopolysaccharide binding protein,t L BP)。方法通过病毒鉴定、SDS- PAGE、western blot、毛细管电泳、体外结合实验、细胞活性实验对重组病毒及重组蛋白 t L BP的分子量、纯度和生物学活性进行分析。结果重组病毒空斑分析确定 MOI(multiplicity of infection)为 8～ 10 ,SDS- PAGE显示感染时间 6 5～ 72 h为最佳表达条件 ;凝胶扫描显示特异表达蛋白占总产物的 9.8% ,纯化蛋白的分子量约 2 70 0 0 u,纯度达 95 %以上 ;毛细管电泳显示表达产物呈单一峰型 ;L owry法测定约 1L 上清液可获 9m g纯化蛋白 ;Western blot间接显示表达产物反应带与预期值相符 ;酶联体外结合实验证实该蛋白可特异结合 L PS。应用 U937细胞 ,经 L PS(1ng/ m L)刺激与 L PS(1ng/ m L)刺激加纯化蛋白 (1μg/ m L)对比观察发现 ,加入纯化蛋白后不同时相点 TNF- α含量均有所下降。结论人 N端脂多糖结合蛋白在 sf2 1昆虫细胞中获得高效表达 。

Objective To express the recombinant human NH terminal lipopolysaccharide binding protein MethodsThe recombinant baculovirus were identified and purified by plaque assay, SDS PAGE, western blot, capillary electrophoresis, conjugation in vitro , and cell activity assay respectively ResultsThe results showed that MOI of recombinant virus was 8-10 by plaque assay It was found that the best expression time was 65-72 hours and the expressed protein was a 27 000 u glycoprotein by SDS PAGE and western blot, its purity was more than 95% Gel scan and capillary electrophoresis indicated that the quantity of specific protein was 9 8% and expression products displayed a single peak It was determined that 1L expression supernatant could produce 9 mg purified protein The bioactivity assay showed that the product could bind LPS in vitro and products of TNF α dropped down after U937 cells were stimulated by LPS (1 ng/mL) or LPS (1 ng/mL) +tLBP (1 μg/mL) respectively ConclusionThe human NH terminal lipopolysaccharide protein was effectively expressed in sf21 insect cells and it was observed that the product could bind or neutralize LPS in vitro