外源性tRNAser对人乳头瘤病毒E7基因表达的影响

目的:探讨哺乳动物细胞中特定外源性tRNAser(CGA)的表达对HPV6bE7蛋白转译的影响,为研究HPV抗原表达的机制提供实验依据。方法:提取重组真核表达质粒pSVtRNAser(含人tR-NAsercDNA的全部序列)、pcDNA3-WtE7(含野生型HPV6bE7ORF)、pcDNA3-HuE7(含优化密码人源化HPV6bE7ORF),EcoRI酶切鉴定pSVtRNAserKpnI和EcoRI双酶切鉴定pcDNA3-HuE7和pcDNA3-WtE7。将质粒DNApSVtRNAser分别与pcDNA3-WtE7或pcDNA3-HuE7混合,脂质体介导共转染COS-1细胞。转染后48h,做免疫荧光染色。结果:重组质粒酶切鉴定结果正确。免疫荧光检测显示:与野生型E7基因单独转染组相比,pSVtRNAser与pcDNA3-WtE7共转染的COS-1细胞中,E7蛋白的表达增强。pcDNA3-HuE7质粒单独转染的COS-1细胞中优化密码E7基因可获得有效表达,共转染后,荧光阳性细胞未见明显增多。Southernblot检测结果显示,在400bp处可见特异DNA条带,说明质粒pSV2tRNAser已转入COS-1细胞。结论:补充外源性pSVtRNAser对COS-1细胞中野生型HPV6bE7基因的表达有一定程度的增强作用。

The influence of exogenous tRNAser(CGA) on the expression of human papillomavirus E7 gene

Objective: To investigate whether the expression of exogenous tRNAser(CGA) can alter the translation efficiency of the HPV 6bE7 gene in mammalian Cos-1 cell. Methods: The recombinant eukaryotic expressing plasmids encoding tRNAser (CGA) (pSV-tRNAser ) and wild-type (pcDNA3-WtE7) or codon modified (pcDNA3-HuE7) HPV 6b E7 DNA were extracted and identificated by gel analysis of digested products. Cos-1 cells were co-transfected with pSV tRNAser (CGA) and pcDNA3-WtE7 or pcDNA3-HuE7. The expression of the E7 protein were analyzed by immunoflurecence at 48h after transfection. Results: Cos-1 cells co-transfected with pSV-tRNAser and pcDNA3-WtE7 expressed higher levels of E7 protein than similar cells transfected with pcDNA3-WtE7 only, and the cells transfected only by wild-type E7 plasmids were occasionally positive. The expression of codon modified E7 gene were much higher than wild-type E7 gene in transfected Cos-1 cells, but no difference observed between tranfected and co-transfeced groups. The specific tRNAser(CGA) DNA fragment was obtained from the transfected cell by Southern blot analysis. Conclusion: The data indicate that supplement of exogenous tRNAser(CGA)can partly enhance the expression of HPV 6b wild-type E7 gene in mammalian Cos-1 cells.