【摘要】 目的:外源RNA导入细胞特异性上调或下调基因表达,目前外源RNA的制备方法主要有化学合成、体外转录、细胞提取。Alu DNA和Alu RNA是人基因组和转录组中最重要的成分,参与基因表达调节。建立工程菌制备基因工程人源Alu RNA(Alu RNA)的技术,所提取的RNA满足一般生物学实验要求。方法和结果:将人Alu序列插入pET-28α质粒(pET),转化BL-21菌,探讨不同条件对Alu RNA产生的影响。用pET-Alu×8质粒转化BMBL-21(DE3)感受态细胞(简称DE3),异丙基-β-D-硫代半乳糖苷(IPTG)诱导减弱细菌生长;用IPTG诱导2h、4h、6h、8h、10h、12h、14h和16h,用Northern杂交检测Alu RNA的量,发现诱导4小时RNA产量最高;1、2、4、8、14拷贝的Alu序列插入pET,转化DE3菌,随拷贝数增加Alu RNA产量上升;pET-Alu×8 DE3菌液,不加IPTG诱导,没有Alu RNA产生,0.1~0.4mg/ml IPTG诱导时,Alu RNA产量没有区别,偏离该浓度时,RNA产量略下降;34℃、37℃和40℃培养pET-Alu×8 DE3菌液,IPTG诱导4h,在37℃培养条件下,RNA产量最高;将pET-Alu×8质粒转化3种BL-21感受态细胞,包括DE3、BMBL21-DE3-pLysS(简称pLysS)和Trans BL 21(简称TransBL),发现转化DE3感受态细胞后Alu RNA产量最高。结论:建立了基因工程制备Alu RNA的技术:pET-Alu×14质粒转化DE3菌,37℃培养至600nm OD为1.0时,加入终浓度为0.2mg/ml的IPTG诱导4h,获得最高Alu RNA产量,纯Alu RNA在提取的RNA中的含量达15.8%,每100ml菌液纯Alu RNA产量平均为0.46mg。更多还原

【Abstract】 Objective: Exogenous RNAs can specifically up-regulate or down-regulate gene expression after they enter into cells. The preparation methods of exogenous RNAs mainly include chemical synthesis,in vitro transcription and extraction from cells. Alu DNA and Alu RNA are the main constituents of human genome and transcriptome and participate in gene expression regulation. The technology method of preparing genetically engineered humanized Alu RNA( Alu RNA) from engineering bacteria was established. The extracted RNAs using this technology method can satisfy the requirement of general biology experiments. Methods and results:Different copies of human Alu elements were inserted into pET-28α plasmid( pET) to construct pET-Alu plasmids that were transformed into BL-21 bacteria. The effects of different conditions on the Alu RNA production were explored. Isopropylthio-β-d-galactoside( IPTG) induction inhibited transformed bacterial growth after BMBL-21( DE3) competent cells( DE3 cells) were transformed by pET-Alu × 8 plasmid( 8 copies of Alus were inserted into pET); Northern blotting was used to detect the amount of Alu RNA after 2,4,6,8,10,12,14 and 16 hours inducing with IPTG. The results showed that the amount of Alu RNA was highest at 4 hours; 1,2,4,8 or 14 copies of Alu elements were inserted into the pET to construct pET-Alu plasmids that were transformed into DE3 bacteria,the Northern blotting results showed that Alu RNA production amount increased with the increase of Alu copy number; pET-Alu × 8 DE3 bacteria did not produce Alu RNA without IPTG induction,Alu RNA production kept similar when inducing by 0. 1mg/ml to 0. 4mg/ml IPTG induction,however,Alu RNA production slightly decreased if deviating from the above concentration range; pET-Alu × 8 DE3 bacteria were cultured at 34℃,37℃ or 40℃ and then were induced by IPTG for 4 hours,the results showed that,under the condition of 37℃ cultivation,Alu RNA production was the highest; pET-Alu × 8 plasmid was transformed into three kinds of BL-21 cells,including DE3,BMBL21-DE3-pLysS( pLys S) and Trans BL 21( TransBL),the results showed that Alu RNA production was the highest when using pET-Alu × 8 DE3. Conclusion: The preparation technology of genetically engineered humanized Alu RNA was established. The highest production of Alu RNA can be obtained based on the following conditions: pET-Alu × 14 plasmid was transformed into DE3 bacteria; the transformed bacteria were cultured at 37℃ to OD 1. 0 at 600 nm,and then were induced by 0. 2mg/ml IPTG for 4 hours. Pure Alu RNA occupies 15. 8% of extractive RNA and the mean yield of pure Alu RNA in 100 ml bacteria solution is 0. 46 mg.更多还原