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目的:设计靶向人端粒酶反转录酶的RNAi序列,构建shRNA表达载体质粒和hTERT真核表达质粒,采用共转染工具细胞筛选出有效靶点。方法:首先以EASYTMsiRNA软件针对hTERT设计5个靶点siRNA序列和1条通用阴性对照序列,合成shRNA oligo表达框架,将其分别连接至经酶切消化的载体质粒、pGCsi-U6/Neo/GFP、pGCL-GFP,重组质粒转化DH5α,阳性克隆进行PCR与测序鉴定;然后从cDNA文库中利用PCR钓取hTERT基因片段,与表达载体pEGFP-C1分别进行双酶切、纯化及定向连接、重组、转化,阳性克隆行PCR与测序鉴定,荧光镜检GFP表达;最后上述2个质粒系统共转染293T细胞,Western印迹检测hTERT蛋白表达水平,筛选最有效序列。应用SPSS16.0软件包对所得数据进行单因素方差分析。结果:BLAST检索5条hTERT siRNA序列均能100%命中,且不与其他基因同源;PCR鉴定及阳性克隆测序验证shRNA oligo与载体质粒成功连接、重组。hTERT基因PCR钓取片段产物大小1.4 Kp,经酶切与质粒连接,形成pEGFP-C1-hTERT表达载体,阳性克隆PCR、测序鉴定确定成功重组;转染293T细胞48h时荧光镜下可观察到GFP标记的hTERT融合蛋白。hTERT表达质粒和shRNA载体质粒共转染293T细胞,48h时Western印迹检测结果显示:实验各组hTERT蛋白量均有不同程度减少,而内参对照β-actin和GAPDH无明显变化。经β-actin校正,得到各组hTERT蛋白相对表达量:KD No.1-5实验组与NC组相比,hTERT基因表达均得到不同程度的敲减(54.61%～76.84%,P<0.05),其中No.4敲减效能最高。结论:5条RNAi设计序列均可有效、特异性地沉默hTERT基因的转录和表达水平,其中以5’-GCAAGTTGCAAAGCATTGGAA-3’敲减效能最优;构建外源基因的过表达载体质粒转染工具细胞,可作为RNAi敲减效率筛选的验绩标靶。
OBJECTIVE: To design the RNAi sequence targeting human telomerase reverse transcriptase, construct the shRNA expression plasmid and hTERT eukaryotic expression plasmid, and use the cotransfection kit to screen the effective target. Methods: Five target siRNA sequences and one universal negative control sequence were designed for hTERT by EASYTM siRNA software. The shRNA oligo expression vector was synthesized and ligated into the vector pGCsi-U6 / Neo / GFP, pGCL-GFP, the recombinant plasmid was transformed into DH5α, and the positive clones were identified by PCR and sequencing. The hTERT gene fragment was then obtained from the cDNA library by PCR, double digested with pEGFP-C1, purified and ligated, recombined and transformed , Positive clones were identified by PCR and sequencing, and GFP expression was detected by fluorescence microscopy. Finally, 293T cells were co-transfected with the two plasmids. The expression of hTERT protein was detected by Western blot and the most effective sequence was screened. The SPSS 16.0 software package was used to perform one-way analysis of variance on the data obtained. Results: All 5 hTERT siRNA sequences were able to hit 100% by BLAST and did not show any homology with other genes. PCR and positive clones were used to confirm that the shRNA oligo was successfully ligated with the vector and recombined. hTERT gene was amplified by PCR. The size of the product was 1.4 Kp. After digested with restriction endonuclease, the plasmid was ligated to form pEGFP-C1-hTERT expression vector. Positive clones were identified by PCR. The recombinant plasmids were identified by sequencing. Labeled hTERT fusion protein. hTERT expression plasmid and shRNA vector plasmid co-transfected 293T cells, 48h Western blot results showed that: the experimental group hTERT protein levels were reduced to varying degrees, while the internal reference control β-actin and GAPDH no significant change. The relative expression of hTERT protein in each group was obtained by β-actin calibration. The expression of hTERT gene in KD No.1-5 experimental group was reduced to some extent (54.61% -76.84%, P <0.05) , Of which No.4 knockdown the highest efficiency. CONCLUSION: All five RNAi designed sequences can effectively and specifically silence the transcription and expression of hTERT gene, and the knockdown efficiency of 5’-GCAAGTTGCAAAGCATTGGAA-3 ’is the best. The constructed plasmid vector for transfection of overexpression vector Cells can be used as a screening target for RNAi knockdown efficiency.