摘要
目的设计并构建针对ECEL1基因的RNA干扰慢病毒载体。方法依照ECEL1基因为模板,设计RNA干扰靶点,根据选定的靶点序列,设计短发夹RNA(shRNA)干扰序列,在两端添加相应的限制性内切酶酶切位点,合成单链DNA oligo,退火缓冲液中配对形成双链DNA oligo。利用Age I和EcoR I双酶线性化GV115载体。把载体和DNA oligo相连接,其连接产物转化大肠杆菌感受态细胞,经PCR扩增并测序鉴定。再通过质粒抽提,转染,浓缩与纯化后获得重组的ECEL1基因RNAi慢病毒,用"HIV-1p24抗原ELISA法"测定样品滴度。选取检测合格的慢病毒感染人肝癌细胞(BEL-7404),并设置对照组,荧光观察感染率。并使用Real-time PCR法及Western blot检测人肝癌细胞(BEL-7404)中ECEL1基因敲减后mRNA和蛋白质的表达量。2组间比较采用两独立样本t检验。结果(1)根据ECEL1基因模板设计后,选定psc48784片段作为RNA干扰靶点,制备双链DNA oligo,PCR鉴定阳性重组子并测序,验证psc48784为正确的克隆。经过质粒抽提,转染以及浓缩纯化后,成功构建ECEL1基因RNAi慢病毒。物理检测与无菌检测合格。(2)通过HIV-1 p24抗原ELISA法测定ECEL1基因RNAi慢病毒样品病毒滴,测定样品病毒滴度为3×108TU/ml;表明已成功构建高滴度且合格的ECEL1基因RNA干扰慢病毒。(3)用ECEL1基因RNAi慢病毒感染人肝癌细胞(BEL-7404),荧光观察结果显示细胞感染效率达到80%,细胞状态稳定;使用Real-time PCR的方法及Western blot检测实验组人肝癌细胞(BEL-7404)中ECEL1基因在mRNA和蛋白质水平的表达量受到抑制(P值均<0.05),敲减效率达到70.5%。结论成功构建ECEL1基因PSCSI-GFP慢病毒载体并获得稳定高滴度的病毒样品,并获得稳定的ECEL1基因敲减的人肝癌细胞(BEL-7404)。
Objective To design and construct the RNA interference(RNAi) lentiviral vector targeting the endothelin-converting enzyme-like 1(ECEL1) gene.Methods The RNA interference target was designed with the ECEL1 gene as the template,and the short-hairpin RNA(shRNA) interference sequence was designed based on the selected target sequence.Enzyme cut sites for restriction endonuclease were added to both ends of this sequence to synthesize single-stranded DNA oligo,which was then paired in annealing buffer to form double-stranded DNA oligo.GV115 vectors were linearized by double digestion with Age I and EcoR I enzymes,and then they were connected with the DNA oligo;the grafted product transformed Escherichia coli receptor cells and was amplified,sequenced,and identified by PCR.The recombinant ECEL1 gene RNAi lentivirus was obtained by plasmid extraction,transfection,concentration,and purification,and HIV-1 p24 antigen ELISA was used to determine the titer of the recombinant RNAi lentivirus.Human hepatoma BEL-7404 cells were transfected with the qualified lentivirus,and a control group was set up.Fluorescence observation was performed to determine transfection rate.Real-time PCR and Western blot were used to measure the mRNA and protein expression of target gene after ECEL1 gene knockdown in human hepatoma BEL-7404 cells.The two-independent-samples t test was used for comparison between two groups.Results After the design with the ECEL1 gene as the template,the psc48784 fragment was selected as the target for RNAi;the double-stranded DNA oligo was prepared and the positive recons were identified and sequenced by PCR,and the results showed that psc48784 was the correct clone.The ECEL1 gene RNAi lentivirus was successfully constructed after plasmid extraction,transfection,concentration,and purification and was qualified for physical test and sterility test.HIV-1 p24 antigen ELISA showed that the ECEL1 gene RNAi lentivirus had a virus titer of 3 E+ 8 TU/ml,suggesting that the qualified ECEL1 gene RNAi lentivirus with a high tit
作者
何剑
廖红伍
阳学风
HE Jian;LIAO Hongwu;YANG Xuefeng(Department of Gastroenterology, Nanhua Hospital Affiliated to Nanhua University, Hengyang, Hunan 421001, China)
出处
《临床肝胆病杂志》
CAS
北大核心
2019年第6期1286-1292,共7页
Journal of Clinical Hepatology
基金
国家自然科学基金面上项目(81373465)
湖南省自然科学省市联合基金(2016JJ5010)
湖南省卫生计生委科研课题(A2017015)
