基于IRES转录效率不同构建绿色荧光蛋白差异表达载体  

Construction of plasmids for differential expression of green fluorescent protein based on different transcription efficiency of IRES

作　　者：骈亚亚 陶凤蓉[2] 聂晶晶 高振祥[1] 许成山 胡继红[1] Pian Yaya;Tao Fengrong;Nie Jingjing;Gao Zhenxiang;Xu Chengshan;Hu Jihong(National Center for Clinical Laboratories,Beijing Hospital,National Center of Gerontology,Institute of Geriatric Medicine,Chinese Academy of Medical Sciences,Beijing 100730,China; Department of Laboratory Medicine,Beijing Hospital,National Center of Gerontology,Institute of Geriatric Medicine,Chinese Academy of Medical Sciences,Beijing 100730,China)

机构地区：[1]北京医院国家老年医学中心,国家卫生健康委临床检验中心,中国医学科学院老年医学研究院,100730 [2]北京医院检验科,国家老年医学中心,中国医学科学院老年医学研究院,100730

出　　处：《中华微生物学和免疫学杂志》2020年第6期459-464,共6页Chinese Journal of Microbiology and Immunology

基　　金：北京医院博士启动基金项目(bj-2018-027);北京市科协金桥工程种子资金项目(ZZ19059);北京市东城区优秀人才培养资助项目(2019DCT-M-11)。

摘　　要：目的根据IRES末端序列的不同,构建4个GFP表达强度不同的逆转录病毒载体,为后续流式检测或成像提供基础。方法利用脑心肌炎病毒IRES的转录效率依赖于其末端的基因序列,通过重叠PCR方法将IRES与EGFP融合成4个不同连接方式的片段,然后克隆至逆转录病毒载体pMSCV-NGFR中;PCR扩增NGFR片段,克隆至逆转录病毒载体pMSCV-IRES-EGFP前面;逆转录病毒载体pMSCV-NGFR-IRES-EGFP转染293T细胞,分析EGFP的表达比例和平均荧光强度(MFI),同时分析NGFR的表达。结果成功构建4个逆转录病毒载体pMSCV-NGFR-IRES-EGFP;在293T细胞转染4个逆转录病毒载体,24 h和48 h时EGFP的表达效率没有明显差异,但荧光强度却有明显不同,同时NGFR的表达没有明显不同,说明IRES与EGFP之间加入不同的核苷酸序列,会使EGFP的荧光强度呈现明显差异。结论 EGFP的表达强度受IRES与EGFP之间序列的影响,不同强度EGFP的逆转录病毒载体可以适用于不同的实验要求。Objective To construct four retroviral plasmids for differential expression of green fluorescent protein(GFP)based on different terminal sequences of internal ribosome entry site(IRES)and provide reference for subsequent flow analysis or imaging.Methods Based on the fact that the transcription efficiency of encephalomyocarditis virus IRES depends on its terminal sequence,IRES and enhanced GFP(EGFP)were fused into four fragments with different connection modes by overlapping PCR,and then cloned into retroviral plasmid pMSCV-NGFR.NGFR fragment was amplified by PCR and inserted in front of the retroviral plasmids pMSCV-IRES(1-4)-EGFP.These retrovirus plasmids pMSCV-NGFR-IRES(1-4)-EGFP were transfected into 293T cells.The expression ratio and mean fluorescence intensity(MFI)of EGFP were analyzed,and the expression of NGFR was also detected.Results Four retroviral plasmids pMSCV-NGFR-IRES(1-4)-EGFP were successfully constructed.No significant difference in the expression efficiency of EGFP at 24 or 48 h was observed in 293T cells transfected with the four different retroviral plasmids,but there was significant difference in fluorescence intensity.Moreover,the expression of NGFR was not significantly different,indicating that the addition of different nucleotide sequences between IRES and EGFP would make a significant difference in the fluorescence intensity of EGFP.Conclusions The expression intensity of EGFP was affected by the sequence between IRES and EGFP.Retroviral plasmids expressing EGFP of different intensity could meet different experimental requirements.

关 键 词：翻译起始 真核生物mRNA 核糖体内部进入位点 病毒 

分 类 号：Q78[生物学—分子生物学]