HPV6b野生型E7基因与优化密码E7基因mRNA表达的分析  

mRNA expression levels of wild type HPV6bE7gene and Codon optimized HPV6bE7 gene

作　　者：郑燕[1] 赵蔚明[1] 周亚滨[1] 王红[1] 栾怡[1] 于修平[1] 

机构地区：[1]山东大学医学院病原生物学研究所

出　　处：《山东大学学报（医学版）》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)

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.

关 键 词：乳头状瘤病毒 质粒 真核细胞 MRNA表达 

分 类 号：Q782[生物学—分子生物学] R373[医药卫生—病原生物学]