正义、反义MCP-1基因逆转录病毒载体重组质粒的构建和包装

Construction and Packaging of Recombinant Retroviral Vector Containing Sense-and Antisense-Rat Monocyte Chemoattractant Protein-1 Gene

为构建含单核细胞趋化蛋白-1(Monocytechemoattractantprotein-1,MCP-1)基因的重组逆转录病毒pLXSN/MCP-1质粒.用RT-PCR技术从大鼠系膜细胞中扩增出MCP-1全长DNA,将其与Pgem-TE连接,用限制性内切酶EcoRⅠ对pTE-MCP-1和逆转录病毒质粒pLXSN分别进行酶切.在T4连接酶的作用下,构建重组逆转录病毒质粒pLXSN/MCP-1.经BglⅡ,XhoⅠ酶切鉴定MCP-1在质粒中的方向,用脂质体介导的方法把重组质粒DNA转染进入包装细胞PA317中,经过G418筛选出抗性克隆.通过NIH3T3细胞测定病毒液的病毒滴度.结果证实经过RT-PCR技术从大鼠系膜细胞中扩增出MCP-1全长DNA与所需的大小一致.重组质粒经酶切分析与预期的结果一致.G418筛选出抗性克隆,能稳定合成并分泌重组逆转录病毒颗粒.NIH3T3细4胞测定病毒滴度为17×10cfu/mL.

In order to construct and package for recombinant retroviral vector containing antisense rat monocyte chemoattractant protein 1 gene,MCP 1 cDNA was amplified by reverse transcription and polymerase chain reaction from total RNA of rats mesangial cell.The PCR fragment was cloned to the cloning vector pGEM T Easy and confirmed by the DNA sequence analysis.Digested by enzyme EcoR I,MCP 1 cDNA was cloned into the EcoR I site of the retroviral vector pLXSN verified by restriction endonuclease maping.Thus pLXSN en coding sense MCP 1 pLXSN MCP 1 and pLXSN encoding antisense MCP 1 pLXSN MCP 1 recombinant eukaryotic expression vectors were constructed.Moreover,antisense pLXSN MCP 1 was transfected into PA317 package cell line with lipofectin and selected with G418.The virus in the supernatant was titrated by transfecting NIH 3T3 cells.The plasmids from the positive clones were cleaved by BglⅡ/XhoⅠ.Tow target fragments at expected positions,respectively were detected by agarose gel electrophoresis.The fragments were confirmed by DNA sequencing and proved identical to the reported cDNA sequence.The presence of MCP 1 cDNA in PA317 genome was detected by PCR.The titer of virus titrated by NIH 3T3 cells was 17×104 cfu/mL.