猪Mx1和牛Mx1蛋白在PK-15细胞中的表达及其对伪狂犬病病毒的抑制

doi:10.13523/j.cb.20150301

中国生物工程杂志

2015, Vol. 35

Issue (3): 1-7 DOI: 10.13523/j.cb.20150301

研究报告

猪Mx1和牛Mx1蛋白在PK-15细胞中的表达及其对伪狂犬病病毒的抑制

王鹏, 郑兆鑫, 刘明秋

复旦大学生命科学学院上海工业菌株工程技术研究中心上海 200433

Expression and Antiviral Effects of Sus Scrofa Mx1 and Bos taurus Mx1 on PRV

WANG Peng, ZHENG Zhao-xin, LIU Ming-qiu

School of Life Sciences, Fudan University, Shanghai Engineering Research Center of Industrial Microorganisms, Shanghai 200433, China

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摘要：

目的: 研究猪Mx1和牛Mx1蛋白在PK-15细胞中的表达并检测其是否对伪狂犬病病毒 (PRV)具有抑制作用.方法:从IBRS-2细胞和MDBK细胞中分别调取猪 Mx1和牛Mx1 基因,并克隆到pcDNA3.1/myc-His(-)B,构建得到真核重组表达质粒,以脂质体转染的方法将其分别导入到PK-15细胞,从mRNA水平和蛋白质水平鉴定重组质粒在细胞内的表达情况,然后用细胞毒性试剂盒检测这两种蛋白是否对PK-15细胞具有毒性.之后,通过荧光定量PCR检测猪 Mx1和牛Mx1 在攻毒后不同时间、不同攻毒剂量的条件下对PRV的抑制情况,并观察100TCID₅₀病毒攻击细胞72h后的病变程度.结果: 成功克隆了猪 Mx1和牛Mx1 基因,经mRNA水平和蛋白质水平证实,两种重组质粒在PK-15细胞内能够正常表达.从荧光定量PCR和细胞病变的角度来看,细胞内表达的Mx1蛋白对PRV具有显著性的抑制(P <0.001).结论: 猪 Mx1和牛Mx1 基因在PK-15细胞中表达的Mx1 蛋白能够抑制PRV在胞内的复制.

关键词： 猪Mx1; 牛Mx1; PK-15细胞; 伪狂犬病病毒

Abstract:

Objective: To study the ability of Sus Mx1 and Bos Mx1 to inhibit the replication of PRV. Methods: Sus scrofa Mx1 gene and Bos taurus Mx1 gene were cloned from IBRS-2 and MDBK cells to pcDNA3.1/myc-His (-) B vector respectively, and transfected into PK-15 cells with Lipofectamine ^TM2000 to detect whether they can express successfully in cells by RT-PCR and Western blot respectively. And then the cytotoxicity to cells WST-1 and the ability of Sus Mx1 and Bos Mx1 to inhibit the replication of PRV were checked. The PK-15 cells were infected with 20TCID₅₀, 100TCID₅₀PRV after transfection 24h. Subsequently, samples were collected after infection 24h, 48h, 72h, and total viral copies were tested by extracting RNAs of frozen and thawed samples repeatedly. Meantime, the cytopathic effect after infecting 100TCID₅₀PRV in 72h was observed. Results: both recombinant eukaryotic plasmids were constructed and expressed successfully. They were able to reduce the amount of RNA of PRV significantly by real time PCR (P <0.01). Conclusion: Sus scrofa Mx1 and Bos taurus Mx1 can inhibit the replication of PRV in PK-15.

Key words: Sus scrofa Mx1 Bos taurus Mx1 PK-15 cells PRV

收稿日期: 2014-12-11 出版日期: 2015-03-25

ZTFLH:

Q812

基金资助:

国家科技重大专项(2013ZX08007-004),教育部留学回国人员科研启动基金,上海市科学技术委员会项目(13DZ2252000)资助项目

通讯作者: 刘明秋 E-mail: liumq@fudan.edu.cn

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引用本文:

王鹏, 郑兆鑫, 刘明秋. 猪Mx1和牛Mx1蛋白在PK-15细胞中的表达及其对伪狂犬病病毒的抑制[J]. 中国生物工程杂志, 2015, 35(3): 1-7.

WANG Peng, ZHENG Zhao-xin, LIU Ming-qiu. Expression and Antiviral Effects of Sus Scrofa Mx1 and Bos taurus Mx1 on PRV. China Biotechnology, 2015, 35(3): 1-7.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150301 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I3/1

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