猪肾细胞源三种蛋白质对猪圆环病毒2型复制的调节作用

doi:10.11843/j.issn.0366-6964.2015.11.017

畜牧兽医学报 ›› 2015, Vol. 46 ›› Issue (11): 2040-2049.doi: 10.11843/j.issn.0366-6964.2015.11.017

猪肾细胞源三种蛋白质对猪圆环病毒2型复制的调节作用

张辉¹，唐青海^2*，黄立平¹，危艳武¹，刘长明^1*

（1.中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/猪传染病研究室，哈尔滨 150001；2.南阳师范学院南阳市兽医生物工程技术研究中心/昆虫生物反应器河南省工程实验室，南阳 473061）

收稿日期:2014-12-19 出版日期:2015-11-23 发布日期:2015-11-20
通讯作者: 唐青海，E-mail：qinghaitang109@126.com；刘长明，E-mail：lcm@hvri.ac.cn，Fax:0451-82733132
作者简介:张辉（1987-），女，山东泰安人，博士生，主要从事动物病毒学研究
基金资助:
国家自然基金青年基金（31101837）

Regulation Effect of Three Proteins Derived from Porcine Kidney Cells on Porcine Circovirus Type 2 Replication

ZHANG Hui¹，TANG Qing-hai^2* ， HUANG Li-ping¹，WEI Yan-wu¹，LIU Chang-ming^1*

(1.Division of Swine Infectious Diseases，State Key Laboratory of Veterinary Biotechnology，Harbin Veterinary Research Institute，Chinese Academy of Agricultural Sciences，Harbin 150001，China；2.Center for Nanyang Veterinary Biological Engineering Technology，Henan Provincial Engineering Laboratory of Insect Bio-reactor，Nanyang Normal University，Nanyang 473061，China)

Received:2014-12-19 Online:2015-11-23 Published:2015-11-20

摘要/Abstract

摘要：

为了验证猪肾细胞系（PK-15）源3种蛋白质基因（AP2α2、Elf4和ISCU）表达水平对猪圆环病毒2型（PCV2）复制的调节作用，采用RT-PCR扩增这3种蛋白质编码基因，构建真核表达载体；同时设计这3种蛋白质的RNAi片段，分别插入到pGPU6-GFP载体构建shRNA干扰载体。以荧光定量PCR法检测干扰效率，选取干扰效率较高的干扰载体，利用G418筛选转染真核表达载体或干扰载体后的PK-15细胞。采用酶联免疫吸附试验（ELISA）和免疫过氧化物酶单层细胞试验（IPMA）法检测PCV2 LG毒株在这些细胞中的复制效率。结果表明，Elf4蛋白和ISCU蛋白基因过表达能够显著增强PCV2的复制；而AP2α2蛋白基因过表达对PCV2的复制无显著影响。AP2α2、ISCU和Elf4这3种蛋白质基因被干扰表达后均可降低PCV2复制效率，表明这3种宿主细胞蛋白质对该病毒复制具有调节作用。

关键词: 猪圆环病毒2型, 复制, AP2&alpha, 2, Elf4, ISCU

Abstract:

To investigate the regulative effect of expression level of three proteins gene (AP2α2，Elf4 and ISCU) derived from porcine kidney cell line (PK-15) on porcine circovirus type 2 (PCV2) propagation efficiency,the genes of three proteins were amplified by RT-PCR and then three eukaryotic expression vectors were constructed.The RNAi fragments of these proteins were designed and cloned into pGPU6-GFP vector to construct shRNA vectors respectively.The efficiency of interference was determined by real-time PCR and three shRNA expression plasmids with higher efficiency of interference were determined.The PK-15 cells transfected with eukaryotic expression vectors or shRNA were screened by antibiotics of G418.The propagation efficiency of strains PCV2 LG in the cell lines was investigated using a capture enzyme-linked immunosorbent assay (ELISA) and an immunoperoxidase monolayer assay (IPMA).The results showed that Elf4 and ISCU overexpression increased PCV2 replication.In contrast，overexpression of AP2α2 exhibited no significant influence on PCV2 replication.In contrast，AP2α2，ISCU and Elf4 down-regulation by shRNA resulted in decreased PCV2 propagation，which demonstrated that these three proteins play a regulative role in PCV2 replication.

Key words: porcine circovirus type 2, replication, AP2α2, Elf4, ISCU

中图分类号:

S852.659.2

张辉，唐青海，黄立平，危艳武，刘长明. 猪肾细胞源三种蛋白质对猪圆环病毒2型复制的调节作用[J]. 畜牧兽医学报, 2015, 46(11): 2040-2049.

ZHANG Hui，TANG Qing-hai， HUANG Li-ping，WEI Yan-wu，LIU Chang-ming. Regulation Effect of Three Proteins Derived from Porcine Kidney Cells on Porcine Circovirus Type 2 Replication[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(11): 2040-2049.

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猪肾细胞源三种蛋白质对猪圆环病毒2型复制的调节作用

Regulation Effect of Three Proteins Derived from Porcine Kidney Cells on Porcine Circovirus Type 2 Replication

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