单启动子p53/p14ARF非融合表达载体的构建及其在MG-63中的表达鉴定

目的构建单启动子非融合表达载体pIRES-p53-p14~(ARF)并观察其对骨肉瘤细胞增殖生长的影响及其功能初探。方法将p53与p14~(ARF)全长cDNA编码区依次亚克隆至pIRES载体中,并通过PCR、酶切鉴定重组质粒。用Lipofectamin 2000将野生型pIRES-p53-p14~(ARF)真核表达载体转染MG-63细胞,48h后采用axiovent200型倒置显微镜、荧光免疫细胞化学,观察其在细胞内的表达并初步探究其抑瘤功能。结果成功构建出含p53与p14~(ARF)全长基因片段的单启动子非融合表达载体pIRES-p53-p14~(ARF)。RT-PCR,酶切鉴定质粒大小、位点均符合实验设计。荧光免疫细胞化学检测显示p53阳性细胞同时也表现为p14~(ARF)免疫反应阳性,转染后瘤细胞可见散在凋亡现象。结论pIRES-p53-p14~(ARF)非融合表达载体的构建与真核细胞导入、目的基因表达成功。

Construction of Non-fusion Expression Vector of Single Promoter for p53/p14ARF and Assessment of Its Expression in MG-63 Cells Proliferation

Objectives To obtain the non-fusion expression vector pIRES-p53-p14~(ARF) and de- termine its effect on MG-63 cells proliferation.Methods p53 and p14~(ARF) full-length cDNA coding region were subcloned into the internal ribosome entry site (IRES) vector one by one by using gene recombination technology.The constructed plasmids were identified by polymerase chain reaction (PCR) in combination with restriction enzyme digestion and sequence analysis.The recombinat plasmids pIRES-p53-p14~(ARF) was transfected into MG-63 cells by lipofectamin 2000.After 48 hours, its expression in cells was observed by inverted microscopy (Axiovent 2000) and immunohistochem- istry,and its tumor-inhibiting effects were preliminarily examined.Results The recombinant plas- mid pIRES-p53-p14~(ARF) was successfully constructed.The recombinant plasmid was in consistency with the experimental design as confirmed by RT-PCR and restriction enzyme digestion.In the ge- home of these transfected target cells,the expression of p53 and p14~(ARF) and protein were immunohis- tochemically detected in vitro.After the transfection,sporadic apoptosis could be seen in the tumor cells.Conclusions The non-fusion expression vector pIRES-p53-p14~(ARF) was successfully construc- ted and expressed.