摘要
构建了由RSV-LTR启动子带动并能在细胞内稳定复制的Ribozyme的自身修剪表达质粒pRSV-Rz523、Ribozyme反义对照质粒pRSV-AE7及人增殖细胞核抗原基因(PCNA)启动子带动的HPV16 E7片段(+554～+686)的真核表达质粒pPCNA-E7。经G418抗性筛选获得了稳定表达Ribozyme的CV-1细胞克隆,其表达水平约为9.0pmol/10~6个细胞,其中活性Ribozyme的量大于50fmol/10~6个细胞,分离得到的Ribozyme可在体外特异切割E7靶RNA片段。通过共转染Ribozyme(或反义对照)和底物表达质粒并筛选出细胞克隆,研究了Ribozyme在细胞中对底物表达水平的影响。初步结果显示,Ribozyme的导入可使细胞内底物E7的RNA表达水平降低了90％(反义对照使E7 RNA表达降低20％)。上述结果提示:在CV-1细胞中表达的Ribozyme不仅在体外,同时在细胞内具有一定的生物学活性,有可能应用于逆转宫颈癌细胞的恶性表型。
HPV16 (Human papilloma virus type 16) E7 gene product, an oncoprotein, has been considered to be casually involved in the pathogenesis of anogenital cancer, particularly in cervical cancer. In order to evaluate the effect of suppressing the expression of E7 gene in CV-1 cells by ribozyme,Rz523, with a rans-acting ribozyme targeted to E7 RNA and 5', 3' two processing ribozymes was cloned into the eukaryotic expression plasmid pREP9 under the control of RSV-LTR promoter. The resultant plasmid pRSV-Rz523 was transfected into CV-1 cells by the method of calcium phosphate coprecipita-tion. The expression of the ribozyme in G418-resistant cells was detected by dot-blot hybridization. Ribozymes stablely expressed in CV-1 cells at a level of 9. 0 pmol per 106 cells, in which the active ribozyme molecules were more than 50 fmol per 106 cells. The result of RNase protection assay showed that steady state level of E7 RNA fragment in CV-1 cell lines was significantly reduced by about 90% in ribozyme-expressing cells. In contrast, the antisense control plasmid pRSV-AE7 yielded only a small reduction (about 20%). This research implicated the possibility to reverse the malignant phenotype of cervical cancer by means of suppressing the expression of E7 gene by ribozyme.
出处
《生物工程学报》
CAS
CSCD
北大核心
1996年第4期385-389,共5页
Chinese Journal of Biotechnology
基金
国家863高技术生物领域(863-102-19-06)资助项目
