非肌肉肌球蛋白Ⅱ型重链A羧基端蛋白的真核表达及在猪繁殖与呼吸综合征病毒感染Marc-145细胞中的作用  

Eukaryotic Expression of Nonmuscle Myosin Heavy Chain II-A C-terminus Protein and Its Roles in Porcine Reproductive and Respiratory Syndrome Virus Infection of Marc-145 Cells

作　　者：吕军华[1] 刘宁宁[1] 赵钦[2] 马玉萍[2] 张冲[2] 王向鹏[2] 胡守彬[1] 赵菲菲[1] 吴海珍[1] 肖一红[1] 周恩民[2] 

机构地区：[1]山东农业大学动物医学院,泰安271018 [2]西北农林科技大学兽医免疫学研究所,西北农林科技大学动物医学院,杨凌712100

出　　处：《畜牧兽医学报》2012年第9期1455-1462,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(30871857;U0931003/L01;30901063);转基因生物新品种培育重大专项(2009ZX08009-147B)

摘　　要：前期研究结果证明抗猪繁殖与呼吸综合征病毒(PRRSV)GP5蛋白的抗独特型抗体特异性结合Marc-145细胞上的非肌肉肌球蛋白Ⅱ型重链A(Nonmuscle myosin heavy chainⅡ-A,NMHCⅡ-A)蛋白,其结合位点位于NMHCⅡ-A的羧基端。本研究通过真核表达NMHCⅡ-A的羧基端(PRA)蛋白,验证其对PRRSV感染Marc-145细胞的作用。通过Bac-to-Bac杆状病毒表达系统表达PRA蛋白。Western blot和间接免疫荧光鉴定PRA蛋白的表达及其与Marc-145细胞的结合。通过病毒中和试验、荧光聚焦中和试验和荧光定量PCR检测PRA蛋白对PRRSV感染Marc-145细胞的作用。Western blot和间接免疫荧光结果表明,PRA蛋白在真核细胞中得到成功表达,其最高表达量为接种1个MOI杆状病毒后96h时。PRA蛋白特异性结合Marc-145细胞,使PRRSV感染Marc-145细胞延迟24h并且感染效率降低60%。真核表达的NMHCⅡ-A羧基端蛋白能特异性结合Marc-145细胞并有效降低PRRSV的感染。这些结果为进一步阐明NMHCⅡ-A在PRRSV感染细胞过程中的作用提供了新的依据。Our previous studies demonstrated that anti idiotypic antibody against porcine repro- ductive and respiratory syndrome virus （PRRSV） GP5 protein specifically binds nonmuscle myo- sin heavy chain II-A （NMHC II-A） on Marc-145 cells and the binding site is in the NMHC II-A C terminus region （named PRA）. In this study, PRA protein was expressed in eukaryotic system and examined for its roles in PRRSV infection of Marc-145 cells. PRA protein containing 310amino acids was expressed in Bac-to-Bac baculovirus expression system and identified by Western blot. Its binding to Marc-145 cells was detected by indirect immunofluorescence assay. The abili- ty of PRA protein to block PRRSV infection of Marc-145 cells was examined by virus neutraliza- tion test, fluorescent focus neutralization test and quantitative RT-PCR. PRA protein was ex pressed in eukaryotic system with the highest expression level at MOI 1 after 96 hours of infec- tion. The results showed that PRA protein specifically bound with Marc-145 cells and inhibited the PRRSV infectivity up to 60‰. Our results indicated that NMHC Ⅱ A C-terminus protein was successfully expressed in baculovirus expression system and could inhibite the PRRSV infec tivity of Marc-145 cells up to 60%. These results provided additional information on the function of NMHC II-A during PRRSV infection.

关 键 词：猪繁殖与呼吸综合征病毒 非肌肉肌球蛋白Ⅱ型重链A 杆状病毒 抑制作用 

分 类 号：S852.659.6[农业科学—基础兽医学]