T7 RNA聚合酶的克隆及其介导基因转录功能的鉴定

The clone of T7 RNA polymerase and its function direct gene transcription

目的构建含T7RNA聚合酶(T7RNA Polymerase,T7RNP)的真核表达载体,同时构建含T7启动子、增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein,EGFP)和T7RNP转录终止信号的载体,并通过观察EGFP的表达推测T7RNP表达和T7表达系统介导基因转录的功能。方法根据编码T7RNP的基因序列,应用PCR技术从含有T7RNP基因的E.coli BL21(DE3)基因组中扩增编码T7RNP的基因片段,克隆至载体pcDNA3·1(+)。同时从px8δt载体和pGEM-Teasy/EGFP上切下T7RNP识别的终止信号(terminator,TER)和编码EGFP的基因,克隆至载体pcD-NAII。将获得的两个重组质粒共转染BHK细胞。48h后,通过观察EGFP基因在真核细胞内的表达情况,推测T7RNP基因的表达及表达后识别T7启动子介导基因转录功能。结果成功构建了含T7RNP编码基因的真核表达载体pcDNA3·1(+)/T7RNP和原核表达载体pcDNAII/EG-FP/TER,共转染BHK细胞后,可观察到EGFP表达的绿色荧光。结论T7RNP能顺利在真核细胞内表达,并通过与T7启动子和TER的相互作用,成功实现了EGFP基因的转录和表达。

Objective To detect the expression of enhanced green fluorescent protein （EGFP） that in prokaryotic expressive vector pcDNAⅡ in Baby Hamster Kidney （BHK） cells, so that we can speculate the expression of T7 RNP and the effect of T7 RNP with T7 promoter and T7 RNP terminate signal （TER） in eukaryotic cells. Methods According to the full sequence of T7 RNP, a pair of primers that containing EcoRⅤ and XhoⅠ were designed and synthesized. A specific gene fragment of T7 RNP was obtained by PCR amplification, then the PCR product was subcloned into the eukaryotic vector pcDNA3. 1 （ ＋ ）. Meanwhile, TER was cut from vector px8δt and was cloned into prokaryotic vector pcDNAⅡ, and EGFP was chipped off from recombinant vector pGEM-T easy/EGFP and was cloned into pcDNAⅡ/TER between T7 promoter and TER. Its transient expression was observed in 48 hours after the two recombinant vectors co-transfected into BHK cells. Results The eukaryotic vector pcDNA3.1 （ ＋ ）/T7 RNP and the prokaryotic vector pcDNAⅡ/EGFP/TER were successfully constructed and the fluorescent of EGFP could be observed in BHK cells with fluorescent microscopy. Conclusion The eukaryotic vector pcDNA3.1 （ ＋ ） / T7 RNP can express T7 RNP, and T7 RNP can lead EGFP that in the prokaryotic vector pcDNAⅡ transcribed successfully. So, it provides a solid basis for the application of T7 RNP on reverse genetics of negative RNA virus in the future.