人endostatin基因重组逆转录病毒载体的构建及其在NIH3T3细胞中表达

Construction of Human Endostatin Recombinant RetroviralPlasmid and its Expression in NIH3T3 Cells

为探讨转人血管内皮抑制基因(endostatin)在转染细胞中的表达,利用逆转录病毒载体构建人endostatin基因的重组质粒,通过脂质体(lipofectamine)将重组质粒导入包装细胞PA317,制备重组病毒液.用重组病毒液感染NIH3T3细胞,经G418筛选获得转人endostatin基因细胞株NIH3T3＿endo.同法制备对照细胞株NIH3T3＿pLncx.PCR检测NIH3T3＿endo细胞基因组,在扩增产物中有一550bp人endostatin基因特异性片段,对照组为阴性.免疫组化测定示仅NIH3T3＿endo细胞中有外源性endostatin蛋白的表达.说明人endostatin基因已被成功导入NIH3T3细胞,并获得稳定表达.

To explore the expression of human endostatin in transfected cells mediated by gene transfer, recombinant retroviral plasmid containing human endostatin gene was engineered and transferred into PA317 cells by lipofectamine method to produce retrovirus. The endostatin_transfected NIH3T3_endo cell strain was developed after transfection of mouse NIH3T3 cells with the above retrovirus following the selection of G418. Control NIH3T3_pLncx cell strain was developed in a similar way except that the plasmid pLncx_endo was replaced by empty plasmid pLncx. PCR analysis proved that only the genome of the NIH3T3_endo cells included a 550 bp specific human endostatin fragment. Immunohistochemistry assay confirmed that a lot of brown particles were seen within the endostatin_transfected NIH3T3_endo cells, with the control NIH3T3_pLncx cells showing negative. It was suggested that human endostatin gene could be transferred into and stably expressed in mouse fibroblasts NIH3T3 cells by retroviral virus.