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

畜牧兽医学报 ›› 2012, Vol. 43 ›› Issue (9): 1455-1462.doi:

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

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*

（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）

收稿日期:2011-12-13 出版日期:2012-09-25 发布日期:2012-09-25
通讯作者: 肖一红，副教授，E-mail: xiaoyihong01@163.com；周恩民，教授，E-mail: zhouem@nwsuaf.edu.cn
作者简介:吕军华（1986-），男，山东潍坊人，硕士生，主要从事免疫生物学研究，E-mail: huashuo001@126.com
基金资助:
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）

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

LV Jun-hua¹，LIU Ning-ning¹，ZHAO Qin²， MA Yu-ping²,
ZHANG Chong², WANG Xiang-peng², HU Shou-bin¹，ZHAO Fei-fei¹，WU Hai-zhen¹，
XIAO Yi-hong^1*， ZHOU En-min^2*

(1. College of Veterinary Medicine, Shandong Agricultural University, Taian 271018, China;
2. Immunology Institute of Northwest A & F University, College of Veterinary Medicine,
Northwest A & F University, Yangling 712100, China)

Received:2011-12-13 Online:2012-09-25 Published:2012-09-25

摘要/Abstract

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96 h时。PRA蛋白特异性结合Marc-145细胞，使PRRSV感染Marc-145细胞延迟24 h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。

Abstract: Our previous studies demonstrated that anti-idiotypic antibody against porcine reproductive and respiratory syndrome virus (PRRSV) GP5 protein specifically binds nonmuscle myosin 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 310 amino 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 ability of PRA protein to block PRRSV infection of Marc-145 cells was examined by virus neutralization test, fluorescent focus neutralization test and quantitative RT-PCR. PRA protein was expressed in eukaryotic system with the highest expression level at MOI 1 after 96 hours of infection. 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 infectivity of Marc-145 cells up to 60%. These results provided additional information on the function of NMHC II-A during PRRSV infection.

中图分类号:

S852.659.6

吕军华, 刘宁宁, 赵钦, 马玉萍, 张冲, 王向鹏. 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[J]. 畜牧兽医学报, 2012, 43(9): 1455-1462.

吕Jun-Hua,LIU Ning-Ning, ZHAO Qin, MA Yu-Ping, ZHANG Chong, WANG Xiang-Peng, . 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[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2012, 43(9): 1455-1462.

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