登革2型病毒结合血管内皮细胞分子机制的初步研究

The Study of Molecular Mechanism of DEN2 Binding to Endothelial Cells

以ECV304细胞为对象分析登革病毒感染血管内皮细胞的机制。2型登革病毒(DEN2)吸附后微量蚀斑法测定ECV304细胞上清释放的病毒滴度,证实该细胞对DEN2感染有一定的敏感性。机械刮取或胰蛋白酶消化法收集ECV304细胞分离膜蛋白,SDS-PAGE见胰酶处理样品缺失一43 kDa的膜蛋白。将ECV304细胞膜蛋白与-^(35)S-Met标记的DEN2进行病毒重叠蛋白结合试验(VOPBA),有29、34和43 kDa的3种膜蛋白可与DV结合,其中29 kDa的蛋白对胰酶耐受。培养的ECV304细胞中加入重组E蛋白(rEgp)对DEN2吸附进行阻断试验,微量蚀斑法与间接免疫荧光表明rEgp抑制DEN2感染该细胞。VOPBA中rEgp可阻断病毒与细胞膜蛋白的结合。结果表明ECV304细胞表面可能存在29、34、43 kDa的3种与DEN2结合的相关蛋白,DEN2 E蛋白可直接介导DV感染血管内皮细胞。

A human endothelial cell line (ECV304) derived from umbilical vein was used to study the mechanism of Dengue virus type 2 (DEN2) infection on endothelial cells. Virus growth curve on the ECV304 cells showed that the cells were sensitive to DEN2 infection. Membrane preparations from ECV304 cells were collected either by mechanical scraping or by trypsin digestion. SDS-PAGE analysis showed that there lacks of a protein with a molecular size 43 kDa in the membrane preparation treated by trypsin. Virus overlay protein binding assays (VOPBA) were performed by binding ^(35)S-Met labeled DEN2 with separated membrane proteins of ECV304 cells. Three proteins each of 29, 34 and 43 kDa located on the surface of ECV304 were found to bind with the labeled virus. The virus-binding effects of the 34 and 43 kDa proteins could be inhibited when the ECV304 cells were treated with trypsin. Preincubation of ECV304 cells with rEgp could inhibit DEN2-binding and block virus infection. Preincubation of the protein electro-transferred nitrocellulose membrane with the rEgp could also block DEN2 binding with the three identified proteins on the surface of ECV304 cells in VOPBA. The result suggests that three proteins each of 29, 34 and 43 kDa on the surface of ECV304 cell might associate with the receptor complex for DEN2 binding, and envelope E glycoprotein could mediate initial binding of DEN2 to human vascular endothelial cells.