一种携带人α1bIFN的单纯疱疹病毒载体的构建

Construction of Recombinant Herpes Simplex Virus Type 1 with α 1b IFN

为研究用单纯疱疹病毒作为表达细胞因子载体的生物重要性,构建了一种表达人α1b干扰素(IFN)的新型单纯疱疹病毒载体。基于一组含有完整单纯疱疹病毒全基因组的粘粒,α1bIFN表达盒插入到cosmid6上HSV1的UL2基因中,生成cos6-αIFN△UL2。通过将cos6-αIFN/△UL2与cosmid28、cosmid14、cosmid56和cosmid48共转染BHK-21细胞,同源重组产生重组病毒HSV1-αIFN/△UL2。在体外用标准VSV空斑抑制试验检测重组毒感染细胞上清α1bIFN的表达。证实HSV1-αIFN/△UL2感染的细胞中表达的人α1bIFN是有生物活性的。MOI=10感染时,24h后产生2 8×104IU/ml。而对照病毒感染时,这些细胞并不分泌可以检测到的α1bIFN。鉴于HSV1载体的嗜神经性,该载体对于分析体内神经系统局部表达的IFN的抗病毒能力是有用的。

To study the biological r elevance of using herpes simplex vir uses as a vector for expressing cy tokines,a new kind of recombinant herpes simplex virus type 1(HSV1) that expressed human interferon α1 bIFN was constructedBased on a se t of cosmids containing the entire HSV1 genome,the α1bIFN expression bo x (the α1bIFN gene was placed under the control of the HCMV promoter) was inserted into HSV1 UL2 gene on cosmid 6 to generate cos6/α1bIFNBy co-transfecting cos6/α1bIFN with the other cosmids such as cosmid28,cos mid14,cosmid56 and cosmid48,HSV1-α 1bIFN/△UL2 was generated in the cell s by recombinationTo evaluate α1bIFN expression,vector infected cell cu lture media were assayed for α1bIF N releaseIn vitro characterizatio n of the recombinant interferon-α 1bIFN confirmed that the α1bIFN ex pressed in HSV1 infected cells was biologically activeThese cells di d not secrete detectable levels of α1bIFN when infected with control virusesCells infected at a multip licity of infection of 10 with HSV-α 1bIFN/△UL2 produced approximately 2 8×104IU/ml α1bIFN in 24 hrs,as de termined by a standard vecicular s tomatitis virus(VSV) replication inhibition assay. These vectors should be useful for in vivo analysis of the antiviral potential of α1bIFN ex pression from within the nervous system