Drebrin蛋白对PEDV在Vero细胞中增殖影响的研究

Effect of Drebrin protein on the proliferation of PEDV in Vero cells

已有研究发现发育调节脑蛋白(Drebrin)参与细胞间信息通讯、神经传递、肿瘤转移、精子发生等生命活动,但其对病毒的作用研究却很少。为研究Drebrin对猪流行性腹泻病毒(PEDV)增殖的影响,本研究根据GenBank登录的非洲绿猴Drebrin基因序列(XM_008015438.2)设计引物,经PCR从Vero细胞中扩增得到Drebrin基因,经测序后构建系统进化树分析其遗传进化关系。结果显示,Vero细胞Drebrin基因编码区(CDS)长2088 bp,编码695个氨基酸,且该基因相对保守,与其他物种Drebrin基因的同源性均较高。利用扩增的Drebrin基因构建真核表达载体pcDNA3.1-Drebrin-Flag,并以不同浓度转染Vero细胞后感染PEDV,利用RT-qPCR检测Vero细胞中PEDV mRNA的转录水平,采用western blot与间接免疫荧光试验(IFA)检测细胞中PEDV N蛋白的表达量,采用TCID50检测病毒滴度。结果显示,Vero细胞中过表达的Drebrin可显著抑制细胞内PEDV mRNA水平(P<0.01)和N蛋白表达量,降低细胞外病毒滴度,具有抑制PEDV增殖的作用,且抑制效率与转染质粒的浓度呈正相关。进一步利用激光共聚焦技术检测Drebrin与PEDV S蛋白的亚细胞定位,结果显示,Drebrin与S蛋白在细胞质中存在共定位现象,提示两种蛋白可能存在互作。本研究首次证明Drebrin对PEDV在Vero细胞中的增殖有抑制作用,为Drebrin的功能研究提供实验基础,也为抗PEDV机制相关研究提供参考。

Previous studies have revealed that developmental regulated brain protein(Drebrin)is involved in cell-to-cell communication,nerve transmission,tumor metastasis,spermatogenesis and other life activities,but there are few studies on viruses.The aim of the current research was therefore,to study the function of Drebrin and its effect on the proliferation of porcine epidemic diarrhea virus(PEDV).The Drebrin gene was cloned according to the Drebrin gene sequence(XM_008015438.2)of Chlorocebus sabaeus registered by Gen Bank,and the phylogenetic tree was constructed to analyze its homology.The results showed that the CDS region of Vero cells Drebrin gene was 2088bp long,encoding 695 amino acids,and was relatively conserved and had high homology with all species.To investigate the effect of Drebrin on the proliferation of PEDV in Vero cells,the eukaryotic expression vector pc DNA3.1-Drebrin-Flag was constructed.After transfection of Vero cells with different concentrations of pc DNA3.1-Drebrin-Flag,cells were infected with PEDV.Our results showed that overexpression of Drebrin in Vero cells could significantly inhibit the intracellular PEDV m RNA level and N protein expression,reduce the extracellular virus titer and inhibit the proliferation of PEDV.Further study on the interaction between Drebrin and PEDV S proteins by laser confocal technique was also performed.The results showed that Drebrin and S protein were co-located in the cytoplasm,suggesting that the two proteins may interact with each other.This study demonstrated for the first time that Drebrin can inhibit PEDV proliferation in Vero cells,laying a foundation for further research in to Drebrin function and provides a valuable information for anti-PEDV research.