EIAV Rev核输出活性检测系统的建立

Establishment of nuclear export-activity detection method for EIAV Rev

Rev蛋白是所有慢病毒属成员均具有的附属蛋白,它与病毒mRNA的特定序列结合,将编码病毒结构蛋白的mRNA运输至胞浆,从而促进病毒结构蛋白的表达,将Rev介导病毒mRNA核质运输的生物学功能简称为核输出活性。为建立马传染性贫血病毒(EIAV)附属蛋白Rev的核输出活性检测方法,本研究通过PCR方法克隆了EIAV的结构蛋白Gag-Pol编码区序列及Rev反应元件(RRE)序列,构建pGagpol-RRE表达载体,将其单独或与Rev表达载体(pcDNA-Rev _(FDDV)-HA)共转染HEK293T细胞后,通过western blot分析显示,pGagpol-RRE单独转染HEK293T细胞不表达,须在Rev的作用下才能在HEK293T细胞中表达,而且Gag的表达量随着Rev剂量(0.05μg、0.1μg、0.2μg)的增加而增加。在pcDNA-Rev _(FDDV)-HA的基础上,通过PCR方法将Rev核输出活性的关键位点L^(36)突变成A,构建突变的Rev表达载体pcDNA-Rev _(L36A)-HA,将其与pGagpol-RRE共转染HEK293T细胞后,经western blot检测结果显示,未检测到Gag的表达。上述结果表明表达载体pGagpol-RRE可以用于评价EIAV Rev的核输出活性。在此基础上,本研究将不同EIAV的Rev表达载体与pGagPol-RRE共转染HEK293T细胞,经western blot鉴定结果显示,不同EIAV的Rev均可以介导Gag-Pol的表达。上述结果表明,表达载体pGagpol-RRE转染细胞后经western blot检测Gag表达量的变化可以广泛用于评价不同EIAV Rev的核输出活性。本研究利用Rev与RRE结合介导Gag-Pol表达的原理首次建立了EIAV Rev核输出活性检测系统,为进一步研究Rev的生物学活性奠定了基础。

The Rev protein is an accessory protein shared by all members of the lentivirus genus.It binds to the specific sequence of viral mRNA and transports the mRNA encoding viral structural protein to the cytoplasm to promote the expression of viral structural protein.The biological function of Rev mediated nucleoplasmic transport of viral mRNA is referred to as nuclear export activity.To establish a method for detecting the nuclear export-activity of equine infectious anemia virus (EIAV) accessory protein Rev,in this study,the Gag-Pol coding region sequence and RRE sequence of EIAV were cloned into an expression vector,named pGagpol-RRE,which was transfected into HEK293T cells with or without the Rev expression vector pcDNA-Rev_(FDDV)-HA.Western blot analysis showed that pGagpol-RRE was not expressed alone and could only be expressed under the action of Rev,and the expression level of Gag55 increased with the presence of the Rev in a dose-dependent manner (0.05μg,0.1μg,0.2μg).On the basis of pcDNA-REV_(FDDV)-HA,the key site 36 of Rev nuclear export activity L was mutated into A by PCR method,and the Rev expression vector pcDNA-REV_(L36A)-HA was constructed and co-transfected with pGagpol-RRE in HEK293T cells.Western blot results showed that the expression of Gag55 could not be detected.These results indicate that the expression vector pGagpol-RRE can be used to evaluate the nuclear export activity of EIAV Rev.On this basis,we further identified that Rev derived from different EIAV strains could all mediate the expression of Gag-pol.These results indicated that expression vector p Gagpol-RRE could be used to evaluate the nuclear export-activity of EIAV Rev.