ALKBH2基因RNA干扰慢病毒载体的构建与鉴定
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摘要
目的 ALKBH2基因高表达于尿路上皮肿瘤,RNA干扰(RNA interference,RNAi)技术难以稳定均一性的下调靶基因。文中旨在构建人源ABH2基因shRNA干扰慢病毒表达载体并进行鉴定。方法选取ABH2基因,分析该基因的编码区序列,进行单链oligo的设计及合成,利用PCR对合成的oligo进行拼接反应,将合成好的序列装入pcDNA3.1(+)载体并转化至感受态细胞DH5α,测序鉴定重组克隆中基因序列后构建表达载体pcDNA3.1(+)/ABH2。根据不同的载体分为4组:siRNA-468组、siRNA-571组、siRNA-662组和对照组。根据靶基因序列设计并合成3对shRNA,构建入U6载体。将shRNA载体分别与表达载体共转染入HEK293细胞,通过QPCR检测筛选出最优干扰序列。将筛选到最有效的shRNA-571干扰序列构建入慢病毒载体pL/shRNA/F载体,用构建的慢病毒干扰载体和包装质粒共转染293T细胞,包装病毒,收集病毒原液,超速离心浓缩,并测定滴度。将LV-shRNA-571与过表达载体pcDNA3.1(+)/ABH2共转染肾癌细胞株ACHN,通过QPCR鉴定干扰效果。结果克隆测序验证无误,慢病毒载体构建成功。转染实验48 h后,收集293细胞分泌的病毒上清测定病毒的滴度为1.1×108TU/mL。对照组ABH2 mRNA相对表达量为0.785±0.082,siRNA-571组为0.078±0.006,RNAi后ABH2基因的mRNA表达水平(0.078±0.006)与对照组(0.785±0.082)相比显著降低(P<0.01),慢病毒PL/shRNA/F-ALKBH2-571对目的基因ABH2的干扰效率为92.2%。结论成功构建ABH2基因RNAi慢病毒载体并得到鉴定。
Objective The ALKBH2 gene is highly expressed in urothelial tumors,and RNA interference( RNAi) technology cannot downregulate the target genes stably. The aim of this study was to construct and identify a lentiviral vector for RNA interference of human ALKBH2( ABH2). Methods The coding region of the ABH2 gene sequences were analyzed for a single-stranded oligo design and synthesis. PCR was performed for splicing reaction of synthetic oligo,which was synthesized into the pcDNA3. 1( +) vector and transformed into the competent cell DH5α. An expression vector pcDNA3. 1( +) / ABH2 was constructed after sequencing and identifying the gene sequence of the recombinant clones. According to the target gene sequence,three pairs of shRNA were designed and synthesized and built into the U6 carrier. The shRNA vectors were co-transfected with expression vectors into the HEK-293 cell line,and the optimal interference sequence was screened by QPCR. The screened shRNA-571,as the most effective interference sequence,was constructed into the lentiviral vector,pL / shRNA / F carrier. The 293T cells were co-transfected with the constructed lentiviral interference vectors and packaging plasmids,the viruses were packed,the virus stock was collected and concentrated by ultracentrifugation,and the titer of the viruses was determined. The LV-shRNA-57 was co-transfected with the over-expression vector pcDNA3. 1( +) /ABH2 into ACHN renal carcinoma,and the optimal interference sequence was screened by QPCR. Results Gene sequencing proved that the recombinant clone gene sequence was correct,and the lentiviral vector was successfully constructed. The titer of the concentrated virus was 1. 1 × 108TU / mL in the viral supernatant of 293T cells collected at 48 hours after co-transfection. The relative expression of ABH2 mRNA was significantly reduced in the siRNA-571 group after RNAi as compared with that in the control group( 0. 078 ± 0. 006 vs 0. 785 ± 0. 082,P < 0. 01). The interference efficiency of the lentiviral vector PL / shRNA / F-ALKBH2-571 in the target gene ABH2 was 92. 2%. Conclusion A lentiviral RNAi expression vector targeting the ABH2 gene was successfully constructed and identified.
Objective The ALKBH2 gene is highly expressed in urothelial tumors,and RNA interference( RNAi) technology cannot downregulate the target genes stably. The aim of this study was to construct and identify a lentiviral vector for RNA interference of human ALKBH2( ABH2). Methods The coding region of the ABH2 gene sequences were analyzed for a single-stranded oligo design and synthesis. PCR was performed for splicing reaction of synthetic oligo,which was synthesized into the pcDNA3. 1( +) vector and transformed into the competent cell DH5α. An expression vector pcDNA3. 1( +) / ABH2 was constructed after sequencing and identifying the gene sequence of the recombinant clones. According to the target gene sequence,three pairs of shRNA were designed and synthesized and built into the U6 carrier. The shRNA vectors were co-transfected with expression vectors into the HEK-293 cell line,and the optimal interference sequence was screened by QPCR. The screened shRNA-571,as the most effective interference sequence,was constructed into the lentiviral vector,pL / shRNA / F carrier. The 293T cells were co-transfected with the constructed lentiviral interference vectors and packaging plasmids,the viruses were packed,the virus stock was collected and concentrated by ultracentrifugation,and the titer of the viruses was determined. The LV-shRNA-57 was co-transfected with the over-expression vector pcDNA3. 1( +) /ABH2 into ACHN renal carcinoma,and the optimal interference sequence was screened by QPCR. Results Gene sequencing proved that the recombinant clone gene sequence was correct,and the lentiviral vector was successfully constructed. The titer of the concentrated virus was 1. 1 × 108TU / mL in the viral supernatant of 293T cells collected at 48 hours after co-transfection. The relative expression of ABH2 mRNA was significantly reduced in the siRNA-571 group after RNAi as compared with that in the control group( 0. 078 ± 0. 006 vs 0. 785 ± 0. 082,P < 0. 01). The interference efficiency of the lentiviral vector PL / shRNA / F-ALKBH2-571 in the target gene ABH2 was 92. 2%. Conclusion A lentiviral RNAi expression vector targeting the ABH2 gene was successfully constructed and identified.
引文
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[13]姚伟,李凯,徐挺,等.慢病毒介导的shRNA靶向干扰β-catenin神经母细胞瘤稳定细胞株的建立[J].中华小儿外科杂志,2013,34(4):290-294.
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[15]陈立,钟国强,涂荣会,等.Cx43基因shRNA慢病毒载体的构建及其对大鼠心肌细胞Cx43基因的作用[J].山东医药,2013,53(21):1-3.
[2]Chen B,Liu H,Sun X,et al.Mechanistic insight into the recognition of single-stranded and double-stranded DNA substrates by ABH2 and ABH3[J].Mol BioSyst,2010,6(11):2143-2149.
[3]Nay SL,Lee DH,Bates SE,et al.Alkbh2 protects against lethality and mutation in primary mouse embryonic fibroblasts[J].DNA repair,2012,11(5):502-510.
[4]Gilljam KM,Feyzi E,Aas PA,et al.Identification of a novel,widespread,and functionally important PCNA-binding motif[J].J Cell Biol,2009,186(5):645-654.
[5]Gao W,Li L,Xu P,et al.Frequent down-regulation of hABH2in gastric cancer and its involvement in growth of cancer cells[J].J Gastroenterol Hepatol,2011,26(3):577-584.
[6]Wu S,Xu W,Liu S,et al.Down-regulation of ALKBH2 increases cisplatin sensitivity in H1299 lung cancer cells[J].Acta Pharmacol Sin,2011,32(3):393-398.
[7]Johannessen TC,Prestegarden L,Grudic A,et al.The DNA repair protein ALKBH2 mediates temozolomide resistance in human glioblastoma cells[J].Neuro Oncol,2013,15(3):269-278.
[8]Fujii T,Shimada K,Anai S,et al.ALKBH2,a novel AlkB homologue,contributes to human bladder cancer progression by regulating MUC1 expression[J].Cancer Sci,2013,104(3):321.
[9]Wang Q,LüY,Gong Y,et al.Double-stranded Let-7 mimics,potential candidates for cancer gene therapy[J].J Physiol Biochem,2012,68(1):107-119.
[10]胡波,吴苏稼.微小RNA在骨肉瘤中的研究进展[J].医学研究生学报,2013,26(5):524-527.
[11]陈帅,李浩博,李牧,等.靶向树突状细胞的肿瘤疫苗OVA慢病毒载体构建及其体外抗肿瘤效应[J].同济大学学报(医学版),2012,33(1):13-18,23.
[12]陈伟,曹罡,董震,等.人Notch4基因RNAi慢病毒载体的构建及鉴定[J].医学研究生学报,2013,26(2):116-121.
[13]姚伟,李凯,徐挺,等.慢病毒介导的shRNA靶向干扰β-catenin神经母细胞瘤稳定细胞株的建立[J].中华小儿外科杂志,2013,34(4):290-294.
[14]孙懿,曾思聪,卢光琇,等.利用慢病毒载体构建稳定干扰β-catenin的人胚胎干细胞系[J].南方医科大学学报,2012,32(8):1088-1092.
[15]陈立,钟国强,涂荣会,等.Cx43基因shRNA慢病毒载体的构建及其对大鼠心肌细胞Cx43基因的作用[J].山东医药,2013,53(21):1-3.