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  

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

作　　者：王鹏[1] 郑兆鑫[1] 刘明秋[1] 

机构地区：[1]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

出　　处：《中国生物工程杂志》2015年第3期1-7,共7页China Biotechnology

基　　金：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

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pc DNA3.1/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。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.

关 键 词：猪Mx1 牛Mx1 PK-15细胞 伪狂犬病病毒 

分 类 号：Q812[生物学—生物工程]