摘要
目的:构建人乳头瘤病毒6b型(HPV6b)E7基因重组真核表达质粒,比较优化密码E7基因(hu-E7)与野生型E7基因(wtE7)在mRNA水平的表达差异,初步探讨优化密码增强E7基因表达的机制。方法:EcoRI和KpnI双酶切pUC-wtE7质粒,游离wtE7片段,定向克隆入pcDNA3的EcoRI和KpnI位点,构建含wtE7的重组真核表达质粒pcDNA3-wtE7;与含hu-E7的pcDNA3-huE7质粒同时体外脂质体转染人羊膜细胞(wish细胞),提取细胞总RNA,采用RT-PCR方法,以β-actin做内参照半定量分析比较hu-E7与wtE7在wish细胞内的mRNA表达水平。结果:用EcoRI和KpnI双酶切重组表达质粒pcDNA3-wtE7可释放5.4kb和314bp两片段,说明质粒构建正确。RT-PCR结果显示,HPV6b-huE7与HPV6b-wtE7转染组在预计分子量190bp处均有明显特异性条带,对照组则无。凝胶成像分析仪半定量结果分析,二者无明显差异,无统计学意义P>0.05。结论:成功构建pcDNA3-wtE7真核表达质粒,并在wish细胞中获得特异性E7mRNA表达。HPV6b-hu-E7与HPV6b-wtE7基因在wish细胞内特异性E7mRNA的表达水平没有明显差异。
Objective:To construct a recombinant eukaryotic expression plasmid of HPV6bE7gene,and compare mRNA expression levels of Codon optimized HPV6bE7gene and wild type HPV6bE7gene.Methods:Plasmid pUC-wtE7was digested with EcoRI and KpnI to get wtE7segment,which was cloned into vector pcDNA3.After confirming the correctness of the new constructed recombinant plasmid by restri-cation enzyme analysis,we transfected it into WISH cell instantly with liposome.At the same time plasmid pcDNA3-huE7was transfected into WISH cell by the same method.The mRNA expression levels of the two plasmid were analysed by RT-PCR.Results:The recombinant plasmid pcDNA3-wtE7was digested with restrication enzyme EcoRI and KpnI and two fragments5.4kb and314bp were got,indicating that the re-combinant plasmid was correct.The result of RT-PCR suggested that the cell transfected with recombinant plasmids pcDNA3-wtE7and pcDNA3-huE7got a specific amplified fragment of190bp,but the control group had no this band.Statistical analysis of the mRNA expression levels of the two plasmids showed that there was no evident difference between them(P>0.05).Conclusion:The pcDNA3-wtE7eukaryotic ex-pression plasmid was constructed successfully and the E7mRNA was expressed well in WISH cells.There is no evident difference between Codon optimized HPV6bE7gene and wild type HPV6bE7gene at mRNA expression levels.
出处
《山东大学学报（医学版）》
CAS
2003年第4期355-357,361,共4页
Journal of Shandong University:Health Sciences
基金
国家自然科学基金资助项目(3987065330271193)
