猪繁殖与呼吸综合征病毒感染细胞中非结构蛋白Nsp2表达分析

doi:10.11843/j.issn.0366-6964.2013.07.016

畜牧兽医学报 ›› 2013, Vol. 44 ›› Issue (7): 1109-1116.doi: 10.11843/j.issn.0366-6964.2013.07.016

猪繁殖与呼吸综合征病毒感染细胞中非结构蛋白Nsp2表达分析

温贵兰^1，2，张涵淞¹，扈鸿霞¹，章先¹，张毅²，王晓杜¹，李肖梁¹，方维焕^1*

(1. 浙江大学动物预防医学研究所，浙江省动物预防医学重点实验室，杭州 310058；2. 贵州大学动物预防医学实验室，贵阳 550025)

收稿日期:2013-01-22 出版日期:2013-07-23 发布日期:2013-07-23
通讯作者: 方维焕，教授，Tel/Fax：+86-0571-88982242，E-mail: whfang@zju.edu.cn
作者简介:温贵兰（1977-），女，贵州普定人，副教授，博士，主要从事分子微生物学研究，E-mail:guilanwen@hotmail.com, Tel: 0571-88982986
基金资助:
浙江省重大科技专项重点农业项目（2012C12003-2）；宁波市农业攻关项目（2011C10003）；中国博士后基金（2012M521187）

Analysis of Nsp2 Expression in Porcine Reproductive and Respiratory Syndrome Virus Infected Cells

WEN Gui-lan¹^，2, ZHANG Han-song¹, HU Hong-xia¹, ZHANG Xian¹, ZHANG Yi², WANG Xiao-du¹, LI Xiao-liang¹, FANG Wei-huan^1*

（1. Institute of Preventive Veterinary Medicine & Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310058, China; 2. Laboratory of Preventive Veterinary Medicine, Guizhou University, Guiyang 550025, China）

Received:2013-01-22 Online:2013-07-23 Published:2013-07-23

摘要/Abstract

摘要：

猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus，PRRSV) 非结构蛋白Nsp2对病毒复制和致病具有重要作用，本研究旨在分析Nsp2在Marc-145细胞内的分布与表达。构建nsp2含PL2区域基因片段的原核表达载体，重组蛋白纯化后作为免疫抗原，制备Nsp2特异性单克隆抗体。将携带nsp2基因的真核表达载体转染至Marc-145及HEK293细胞，或将PRRSV感染Marc-145细胞后不同时间点收集细胞样品。免疫荧光观察Nsp2在转染细胞中的亚细胞定位，免疫印迹分析Nsp2在真核细胞中的表达。筛选获得了1株稳定分泌特异性抗Nsp2的单克隆杂交瘤细胞株，能用于免疫荧光和印迹分析。重组真核表达质粒转染Marc-145与HEK293细胞后，Nsp2主要定位于细胞质中，免疫印迹检测到约为120与50 ku的蛋白条带。PRRSV感染Marc-145细胞4 h即可检测到Nsp2的表达，主要定位于细胞核周围，随着感染时间延长Nsp2分布范围逐渐扩大至整个细胞质。病毒感染8 h，可检测到120 ku左右的Nsp2蛋白条带，感染后12 h可检测到70和50 ku左右的Nsp2蛋白，且蛋白表达量随感染时间延长显著增多。PRRSV感染细胞中Nsp2的表达与剪切分析，为深入研究该蛋白在病毒复制和致病中的功能奠定了基础。

Abstract:

The Nsp2 protein of porcine reproductive and respiratory syndrome virus (PRRSV) plays an important role in viral replication and pathogenesis. We attempted to analyze the expression and distribution of Nsp2 in Marc-145 cells infected with PRRSV to provide some basis of further investigation of its pathogenesis. The nsp2 gene fragment (covering the PL2 region) was cloned into the prokaryotic expression vector and purified recombinant protein was used as antigen to prepare specific monoclonal antibody. The eukaryotic expression plasmid containing nsp2 gene fragment was transfected into Marc-145 or HEK293 cells. PRRSV infected-Marc-145 cells were collected at different time points. Immunofluoresence and Western blotting were used to analyze the distribution and expression of Nsp2 in cells. A monoclonal hybridoma cell line secreting specific anti-Nsp2 antibody was obtained. The antibody was suitable for immunoblotting and immunofluorescence. Nsp2 was present in the cytosol of Marc-145 or HEK293 cells transfected with the recombinant plasmid. There were two protein bands of about 120 and 50 kDa shown by Western blotting. Expression of Nsp2 in PRRSV-infected Marc-145 cells could be detected initially in the perinuclear cytoplasm at 4 h post-infection, and then throughout the cytoplasm. The major Nsp2 protein at about 120 kDa was seen at 8 h post-infection. Two smaller bands of 70 and 50 kDa were detected at 12 h post-infection. Protein expression increased over time. The expression and cleavage patterns of Nsp2 in PRRSV-infected cells might indicate differential functions of the protein during viral replication and pathogenesis that await further investigation.

中图分类号:

S852.659.6

温贵兰，张涵淞，扈鸿霞，章先，张毅，王晓杜，李肖梁，方维焕. 猪繁殖与呼吸综合征病毒感染细胞中非结构蛋白Nsp2表达分析[J]. 畜牧兽医学报, 2013, 44(7): 1109-1116.

WEN Gui-lan ZHANG Han-song, HU Hong-xia, ZHANG Xian, ZHANG Yi, WANG Xiao-du, LI Xiao-liang, FANG Wei-huan. Analysis of Nsp2 Expression in Porcine Reproductive and Respiratory Syndrome Virus Infected Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(7): 1109-1116.

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猪繁殖与呼吸综合征病毒感染细胞中非结构蛋白Nsp2表达分析

Analysis of Nsp2 Expression in Porcine Reproductive and Respiratory Syndrome Virus Infected Cells

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