MoMLVgag-pol基因在NIH3T3细胞中的表达和鉴定

The Expression of gag-pol Fusion Gene in NIH3T3 and Its ldentification

目的:构建含MoMLVgag-pol基因的重组表达载体,实现其在NIH3T3细胞中稳定表达。方法:应用RT-PCR方法反转录并扩增gag-pol基因,克隆入真核表达载体pcDNA4/HisMaxA上,构建重组表达载体pcDNA4/HisMaxA-gag-pol,用脂质体法转染NIH3T3细胞,Zeocin筛选稳定表达细胞株,通过SDS-PAGE分析检测表明,gag-pol基因在NIH3T3细胞实现了表达,产物相对分子质量(kDa)为194.78×103。然后,将逆转录病毒载体导入此细胞系,包装逆转录病毒。用PCR与标记基因补救分析法检测野生型辅助病毒。结果:酶切鉴定的片段大小分别为5.2kb,与预期大小一致,经Zeocin筛选后获得稳定表达细胞株,SDS-PAGE实验表明产物融合蛋白相对分子质量(kDa)为194.78×103,与预期相符。脂质体转染包装细胞,嘌呤霉素加压筛选出高病毒滴度(4.0×106CFU/ml)的细胞克隆,且未检测到辅助病毒。结论:构建的融合表达载体pcDNA4/HisMaxA-gag-pol及其在NIH3T3细胞中的表达,构建成功具有靶向性的逆转录病毒包装细胞系,该细胞系能够包装出高滴度的逆转录病毒,为肝细胞的基因治疗提供了一种新的基因转移系统。

Objective Construction of a fusion expression vector of MoMLV gag-pol and eukaryotic expression of GAG-POL protein. Methods The gag-pol gene of Moloney murine leukemin virus （MoMLV） were amplified by RT-PCR,they were subcloned into eukaryotic expression vector pcDNA4/HisMaxA. The recombinant plasmid pcDNA4/HisMaxA-gag-pol was thus constructed and identified by sequencing and digestion with restriction enzymes. The recombinant plasmid pcDNA4/HisMaxA-gag-pol was transfected into NIH3T3 cells by lipofecamine2000. 72 hours after tranfection, passage the cells 1：10 into selective medium containing Zeocin for stable expression. The fusion protein gene was identified by RT-PCR and SDS-PAGE. A retroviral vector pMSCV/u6-HDVRZ, carrying HDV ribozyme gene, was transferred into the packaging cell line. The titer of retrovirus was assayed on NIH3T3 cells. The helper virus was tested by both PCR and marker rescue assay. Results A recombinant fusion expression plasmid was successfully constructed. The plasmid expressed GAG-POL protein. The fusion expression vector pcDNA4/HisMaxA-gag-pol can construct targeting retrovirus packaging cell lines. The efficiency and safety of this system in gene transfer might provide an optimal system for hepatocyte gene therpy.