APOBEC3F的真核表达及其抗猪繁殖与呼吸综合征病毒作用的分析  

Eukaryotic expression of APOBEC3F and its effect on porcine reproductive and respiratory syndrome virus

作　　者：胡璇 田浪 王怡然 温贵兰[1,2] 陈启青 陈佳琪 叶泥[1,2] 龚新勇 HU Xuan;TIAN Lang;WANG Yi-ran;WEN Gui-lan;CHEN Qi-qing;CHEN Jia-qi;YE Ni;GONG Xin-yong(College of Animal Science,Guizhou University,Guiyang 550025,China;Guizhou Engineering Research Center for Animal Biological Products,Guiyang 550016,China;Agriculture and Rural Bureau of Huishui County,Guiyang 550600,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省动物生物制品工程技术研究中心,贵州贵阳550016 [3]贵州省惠水县农业农村局,贵州贵阳550600

出　　处：《中国预防兽医学报》2023年第8期829-836,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(32060794);贵州大学2020年省级教学内容和课程体系改革项目(GZJG20200035)。

摘　　要：为研究载脂蛋白B mRNA编辑酶催化多肽样3F(APOBEC3F)对猪繁殖与呼吸综合征病毒(PRRSV)增殖的影响,本研究根据GenBank中登录的猪源APOBEC3F基因序列(EU871586)设计引物,经PCR从苏太猪外周血单个核淋巴细胞中扩增APOBEC3F基因,经测序后构建系统进化树分析其遗传进化特性。结果显示,猪源APOBEC3F基因编码区(CDS)长1257 bp,编码418个氨基酸,与其他物种该基因的同源性在60%~70%。利用扩增的APOBEC3F基因构建真核表达载体pcDNA3.1(+)5'Flag-APOBEC3F经菌液PCR和测序鉴定正确后,转染Marc-145细胞,采用间接免疫荧光试验(IFA)和western blot鉴定重组APOBEC3F蛋白的表达。IFA结果显示,转染pcDNA3.1(+)5'Flag-APOBEC3F的Marc-145细胞质中出现特异性红色荧光;western blot结果显示,转染pcDNA3.1(+)5'Flag-APOBEC3F的细胞样品在44 ku处有特异性条带,表明猪源APOBEC3F在Marc-145细胞中获得了表达。在Marc-145细胞中转染不同剂量的pcDNA3.1(+)5'Flag-APOBEC3F或不同剂量的针对APOBEC3F的siRNA,过表达或抑制APOBEC3F表达后感染PRRSV,通过RT-qPCR检测PRRSV N基因mRNA的转录水平,采用western blot检测细胞中PRRSV N蛋白的表达水平,采用TCID50测定PRRSV滴度。结果显示,在Marc-145细胞中过表达的APOBEC3F可以显著抑制PRRSV N基因mRNA转录水平(P<0.01)和N蛋白的表达水平,降低PRRSV滴度(P<0.01、P<0.001),表明APOBEC3F具有抑制PRRSV增殖的作用,且抑制效率与重组质粒的转染剂量呈正相关。在Marc-145细胞中抑制APOBEC3F的表达后,PRRSV N基因的mRNA转录水平(P<0.01)、N蛋白的表达水平与PRRSV滴度(P<0.05、P<0.001)均显著升高,表明抑制APOBEC3F的表达具有促进PRRSV增殖的作用,且该促进效率与针对APOBEC3F的siRNA转染剂量呈正相关。本研究首次证明APOBEC3F对PRRSV在Marc-145细胞中的增殖有抑制作用,为研究APOBEC3F功能提供实验依据,也为研究抗PRRSV相关机制的研究提供参考。To investigate the effect of APOBEC3F on PRRSV proliferation,we designed primers based on the GenBank-registered APOBEC3F gene sequence(EU871586).The APOBEC3F gene was amplified by PCR from peripheral blood single nuclei of Sutai pigs,the resulting amplicons were sequenced and the obtained sequence was used to construct a phylogenetic tree for analyzing its genetic evolutionary characteristics.The results showed that the coding region(CDS)of porcine APOBEC3F gene was 1257bp in length,encoding 418 amino acids,and sharing the 60%and 70%of homology with other species.The eukaryotic vector pcDNA3.1(+)5'Flag-APOBEC3F was constructed by inserting the obtained APOBEC3F gene and transfected into Marc-145 cells.The expression of recombinant APOBEC3F protein was identified by IFA and western blot.IFA results showed that the specific red fluorescence was available in the cytoplasm of Marc-145 cells transfected with pcDNA3.1(+)5'Flag-APOBEC3F;western blot results showed that the specific protein band was obtained at 44ku in the cells transfected with pcDNA3.1(+)5'Flag-APOBEC3F,indicating that porcine APOBEC3F was expressed in Marc-145 cells.The experiments were carried out by transfecting different doses of pcDNA3.1(+)5'Flag-APOBEC3F or different doses of siRNA against APOBEC3F into Marc-145 cells as well as infecting with PRRSV on the cells where the APOBEC3F was overexpressed or inhibited.The mRNA transcript levels of the PRRSV N gene were detected by RT-qPCR,and the N protein of PRRSV was detected by western blot,and the titers of PRRSV were detected by TCID50.The results showed that APOBEC3F overexpressed in Marc-145 cells significantly inhibited the mRNA levels of PRRSV N gene(P<0.01)and N protein expression of PRRSV reducing the titers of PRRSV infection,and had the effect of inhibiting PRRSV proliferation.Furthermore the inhibitory efficiency was positively correlated with the transfection dose of recombinant plasmid.Inhibition of APOBEC3F expression in Marc-145 cells significantly increased the mRNA level of PRRSV N

关 键 词：猪繁殖与呼吸综合征病毒 载脂蛋白B mRNA编辑酶催化多肽样3F MARC-145细胞 

分 类 号：S852.65[农业科学—基础兽医学]