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首页> 外文期刊>Virus Research: An International Journal of Molecular and Cellular Virology >Genetically modified VSVNJ vector is capable of accommodating a large foreign gene insert and allows high level gene expression
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Genetically modified VSVNJ vector is capable of accommodating a large foreign gene insert and allows high level gene expression

机译：转基因的VSVNJ载体能够容纳较大的外源基因插入片段并允许高水平的基因表达

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摘要

It is desirable to develop a RNA virus vector capable of accommodating large foreign genes for high level gene expression. Vesicular stomatitis virus (VSV) has been used as a gene expression vector, especially Indiana serotype (VSVInd), but less with New Jersey serotype (VSVNJ). Here, we report constructions of genetically modified rVSVNJ vector carrying various lengths of human hepatitis C virus (HCV) non-structural (NS) protein genes, level of inserted gene expression and characterization of rVSVNJ. We modified the M gene of VSVNJ by changing methionine to arginine at positions 48 and 51 (rVSVNJ-M) (Kim and Kang, 2007) for construction of rVSVNJ with various lengths of HCV non-structural genes. The NS polyprotein genes of HCV were inserted between the G and L genes of the rVSVNJ-M vector, and recombinant VSVNJ-M viruses with HCV gene inserts were recovered by the reverse genetics. The recombinant VSVNJ-M vector with the HCV NS genes express high levels of all different forms of the NS proteins. The electron microscopic examination showed that lengths of recombinant VSVNJ-M without gene of interests, VSVNJ-M with a gene of HCV NS3 and NS4A (VSVNJ-M-NS3/4A), VSVNJ-M with a gene of HCV NS4AB plus NS5AB (VSVNJ-M-NS4AB/5AB), and VSVNJ-M carrying a gene of HCV NS3, NS4AB, and NS5AB (VSVNJ-M-NS3/4AB/5AB) were 172??10.5nm, 201??12.5nm, 226??12.9nm, and 247??18.2nm, respectively. The lengths of recombinant VSVs increased approximately 10nm by insertion of 1kb of foreign genes. The diameter of these recombinant viruses also increased slightly by longer HCV gene inserts. Our results showed that the recombinant VSVNJ-M vector can accommodate as much as 6000 bases of the foreign gene. We compared the magnitude of the IFN induction in mouse fibroblast L(Y) cells infected with rVSVNJ wild type and rVSVNJ M mutant viruses and show that the rVSVNJ M mutant virus infection induced a higher level of the IFN-?? compare to the wild type virus. In addition, we showed that the NS protein expression level in IFN-incompetent cells (Mouse-L) infected with rVSVNJ-M viruses was higher than in IFN-competent L(Y) cells. In addition, we confirmed that HCV NS protein genes were expressed and properly processed. We also confirmed that NS3 protein expressed from the rVSVNJ-M cleaves NS polyprotein at junctions and that NS4A plays an important role as a co-factor for NS3 protease to cleave at the NS4B/5A site and at the NS5A/5B site. ? 2012 Elsevier B.V.
机译：期望开发一种能够容纳大型外源基因用于高水平基因表达的RNA病毒载体。水泡性口炎病毒（VSV）已被用作基因表达载体，尤其是印第安纳州血清型（VSVInd），但新泽西州血清型（VSVNJ）较少。在这里，我们报道了携带各种长度的人类丙型肝炎病毒（HCV）非结构性（NS）蛋白基因，转基因表达水平和rVSVNJ的特征的转基因rVSVNJ载体的构建。我们通过将第48和51位的蛋氨酸改为精氨酸来修饰VSVNJ的M基因（rVSVNJ-M）（Kim和Kang，2007），以构建具有各种长度的HCV非结构基因的rVSVNJ。将HCV的NS多蛋白基因插入rVSVNJ-M载体的G和L基因之间，并通过反向遗传学回收具有HCV基因插入物的重组VSVNJ-M病毒。具有HCV NS基因的重组VSVNJ-M载体可高水平表达所有不同形式的NS蛋白。电子显微镜检查显示，没有目的基因的重组VSVNJ-M，具有HCV NS3和NS4A基因的VSVNJ-M（VSVNJ-M-NS3 / 4A），具有HCV NS4AB基因加上NS5AB（带有HCV NS3，NS4AB和NS5AB（VSVNJ-M-NS3 / 4AB / 5AB）基因的VSVNJ-M-NS4AB / 5AB）和VSHCNJ-M分别为172？10.5nm，201？12.5nm，226？分别为λ12.9nm和247λ18.2nm。通过插入1kb的外源基因，重组VSV的长度增加了约10nm。更长的HCV基因插入片段也会使这些重组病毒的直径略有增加。我们的结果表明，重组VSVNJ-M载体可容纳多达6000个外源基因碱基。我们比较了用rVSVNJ野生型和rVSVNJ M突变病毒感染的小鼠成纤维细胞L（Y）细胞中IFN诱导的幅度，并表明rVSVNJ M突变病毒感染引起了更高水平的IFN-γ。与野生型病毒相比。此外，我们表明感染rVSVNJ-M病毒的无IFN能力的细胞（小鼠L）的NS蛋白表达水平高于有IFN的L（Y）细胞。另外，我们证实了HCV NS蛋白基因被表达并被正确加工。我们还证实了从rVSVNJ-M表达的NS3蛋白在连接处裂解NS多蛋白，并且NS4A作为NS3蛋白酶在NS4B / 5A和NS5A / 5B裂解的辅助因子起着重要作用。 ？ 2012年Elsevier B.V.

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