CD163片段在PRRSV感染过程中的作用

Function of CD163 fragments in porcine reproductive and respiratory syndrome virus infection

使用Scanprosite软件对猪肺泡巨噬细胞的CD163蛋白结构和功能域进行预测,对其进行分段克隆,构建原核表达载体p ET-28a（Y-P1,Y-P2,Y-P3）,片段位置Y-P1（160-798bp）,Y-P2（790-20146bp）,Y-P3（2143-3084bp）。利用镍柱纯化重组蛋白,免疫小鼠获得特异性抗体。通过病毒阻断试验,验证Y-P1,Y-P2,Y-P3的PRRSV阻断情况;构建真核表达载体p CD163,P1（1-798bp）,P2（790-2046bp）,P3（2023-3345bp）分别转染PRRSV非易感细胞系BHK-21,验证其PRRSV感染情况。结果显示,经PCR、双酶切及测序鉴定构建的原核和真核表达载体是正确的,SDS-PAGE检验Y-P1,Y-P2,Y-P3蛋白大小分别为27000,46000,39000。Y-P2蛋白,抗Y-P2的小鼠血清能够阻断PRRSV感染Marc-145细胞;转染p CD163的细胞系能够感染PRRSV而P1,P2,P3片段不感染PRRSV。CD163其790-2046bp可能是PRRSV感染细胞与CD163受体作用位点。

CD163 fragment P1（1-798 bp） ,P2（790-2 046 bp） ,P3（2 023-3 345 bp） ,Y-P1（160- 798 bp） ,Y-P2（790-2 046 bp） ,Y-P3（2 143-3 084 bp）were expressed by eukaryotic and prokary- otic expression systems, respectively. The infection experiments revealed that non-permissive BHK-21 cells transfected with pCD163 could be infected by PRRSV. However, cells with truncated CD163 （P1,P2, P3） were not susceptible to PRRSV. Meanwhile, Y-P1, Y-P2 and Y-P3 were ex- pressed in E. coil and antisera to these peptides were prepared in mice. A virus blocking test showed that Y-P2 protein and anti-Y-P2 mouse serum could block PRRSV infection in a dose-de- pendent manner,while Y-P3 protein could improve virus infection. The results suggested that the CD163 （790-2 046 bp） was involved in PRRSV infection of cells.