【摘要】 目的:利用杆状病毒表达系统在昆虫细胞(Sf9细胞)中表达人纤维介素蛋白2(hfgl2)凝血酶原酶并检测其蛋白活性。方法:首先将PCR扩增的目的基因hfgl2和pENTR/D-TOPO载体连接,再将pENTR/D-TOPO-hfgl2质粒与BaculoDirectTM线性DNA进行体外基因重组,采用脂质体转染法将重组DNA转染到生长良好的Sf9细胞中进行病毒包装,经过对重组杆状病毒三轮扩增后,用RT-PCR和Western blotting从基因和蛋白水平检测hfgl2表达情况,一期凝固试验检测其蛋白活性。结果:重组杆状病毒感染后,Sf9细胞停止生长,体积变大,细胞开始悬浮,破裂,呈现颗粒样外观。RT-PCR扩增和Western blotting从基因和蛋白水平证实Sf9细胞能够表达hfgl2,蛋白分子量约为63kDa。同时,在细胞上清液中检测到了可溶性hfgl2的表达,蛋白分子量约为50kDa。一期凝固试验证明表达的跨膜型hfgl2具有凝血活性。结论:hfgl2在昆虫杆状病毒表达系统中成功表达并具有生物学活性,为进一步研究hfgl2跨膜型以及可溶性蛋白功能提供了工具,亦可应用于开发重型肝炎蛋白芯片或ELISA诊断试剂盒。更多还原

【Abstract】 Objective:To induce the expression of hfgl2 protein using baculovirus expression system in Sf9 cell.Methods:To express hfgl2 in insect cells,the full length hfgl2 gene was inserted into pENTR/D-TOPO plasmid as interest gene,then through homologous recombination of the plasmid with BaculoDirect~TM linear DNA,the recombinant baculovirus DNA containing the interest gene fragment was obtained.Sf9 cells were transfected with baculovirus DNA using Cellfectin for 4 days.After 3 rounds amplification of recombinant baculovirus,the expression of hfgl2 in Sf9 cell was detected and identified by the RT-PCR and Western blotting.Results:The recombinant hfgl2 protein was stably expressed in Sf9 cell and detected by the Distinct morphological changes of Sf9 cell,RT-PCR and Western blotting.The protein product was determined at 63kDa with capability to induce one-stage clotting assays.Conclusion:The result indicates that hfgl2 gene fragment could be expressed successfully using baculovirus expression system,providing the basic material for its functional study.Also the research took prophase exploration for developing apropos diagnostic reagents using hfgl2 as their antigens.更多还原