F磁珠对血液样本中EB病毒的富集方法

An enrichment method of EBV in blood samples by F beads

目的设计表达一种全新的重组蛋白(重组Ficolin-2蛋白,F蛋白)并包被磁珠,实现多种抗凝血中EB病毒的富集效果。方法通过体外基因合成重组F蛋白基因片段,该片段包括人IL-2信号肽、his标签、人IgG1 Fc、人Ficolin-2颈区及纤维蛋白原样区域(fibrinogen-like domain,FGD);并整合进表达质粒pCDNA^(TM)3.1(+)载体中,通过转染HEK293F细胞,实现上清中的分泌表达;通过镍柱纯化蛋白后包被到蛋白A磁珠上构建F磁珠;最后,收集三种不同抗凝剂(分别为ETDA、肝素、柠檬酸钠)抗凝的临床EB病毒血样本,并且比较了F磁珠和课题组前期开发的重组甘露聚糖结合凝集素磁珠(M磁珠)对三种抗凝剂抗凝血液中EB病毒富集的效果。结果M磁珠对肝素和柠檬酸钠抗凝血液中EB病毒富集效果均较好(P<0.01),但是对EDTA抗凝血EB病毒没有富集作用;而F磁珠对三种抗凝血中EB病毒的富集效果均较好(P<0.01)。结论F磁珠可以实现多种抗凝血中EB病毒的富集作用。

Objective To achieve the effect of EBV enrichment from different anticoagulants,a novel recombinant protein F was designed and produced.Methods A novel recombinant protein F was designed,which contained human IL-2 signal peptide,his,human IgG1 Fc,human Ficolin-2 neck region and Fibrinogen-like domain(FGD).The synthetic gene segment was cloned into pCDNA^(TM)3.1(+)vector,which was then transfected into HEK293F cells.The recombinant protein(F protein)was then produced in supernatant.F beads was constructed by coating F protein onto protein A beads.At last,EBV positive whole blood samples anticoagulated by three kinds of anticoagulants were collected.F beads and M beads(previously studied)were compared to be applied to enrich EBV from these blood samples.Results M beads were effective to enrich EBV from heparin or sodium citrate anticoagulated blood(P<0.01),not for EDTA anticoagulated blood.While,F beads were effective for all three anticoagulated blood(P<0.01).Conclusions F beads could enrich EBV from different anticoagulated blood.