| 摘要: |
| 目的 构建并鉴定人神经源性分化蛋白基因(NeuroD)RNA干扰慢病毒表达载体。方法 根据已公布的NeuroD基因序列(GenBank:NM_002500),设计并合成4 对shRNA(NeuroD1~D4),与载体pcDNATM 6.2-GW/EmGFP-miR连接,构建pcDNATM 6.2-GW/EmGFP-miR-NeuroD 表达载体,转化入感受态细胞DH5α; real-time PCR 技术检测pcDNATM 6.2-GW/EmGFP-miR-NeuroD表达载体对293T细胞内靶基因的干扰效果。将筛选出的干扰载体pcDNATM6.2-GW/EmGFP-miR-NeuroD1与慢病毒载体pLenti6.3/V5-DEST经酶切后连接,构建慢病毒表达载体pLenti6.3-EGFP-NeuroD1-miR。用构建的慢病毒表达载体和包装质粒(Packaging Mix)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。采用PCR法对重组载体进行鉴定,利用绿色荧光蛋白作为报告基因,对病毒滴度和感染效率进行检测。结果 成功构建针对靶基因的4个干扰质粒,测序结果表明,4个pcDNATM 6.2-GW/EmGFP-miR-NeuroD表达载体序列与参考序列一致,real-time PCR 检测显示以pcDNATM 6.2-GW/EmGFP-miR-NeuroD1的沉默效应最佳(P<0.01)。将重组获得的慢病毒表达载体pLenti6.3-EGFP-NeuroD1-miR转染至293T细胞株,酶切鉴定及PCR结果与病毒载体的预期一致,病毒滴度达1.18×108 ifu/mL。结论 成功构建、筛选了针对人NeuroD基因的RNA干扰慢病毒表达载体。 |
| 关键词: 慢病毒 NeuroD基因 RNA干扰 胰腺肿瘤 |
| DOI： |
| 投稿时间：2013-05-01修订日期：2013-07-09 |
| 基金项目:国家自然科学基金(30770996,81172310),长海医院“1255学科建设计划”(CH125521106). |
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| Construction and identification of RNAi leniviral vector targeting NeuroD |
| WANG Yang,GAO Li,BU Hai ji,CHEN Ying,ZHU Ming hua* |
(Department of Pathology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
*Corresponding author.) |
| Abstract: |
| Objective To construct and identify RNAi lentiviral expression vectors targeting human neurogenic differentiation gene (NeuroD). Methods The oligo DNA sequences of 4 pairs of shRNA, named as NeuroD1, NeuroD2, NeuroD3, and NeuroD4, were designed according to NeuroD gene sequence (GenBank:NM_002500). The single strand of oligo DNA was annealed to form double strand DNA, and then was cloned into the empty plasmid pcDNATM 6.2-GW/EmGFP-miR. Four interference plasmids were constructed and transformed into competent cells DH5α. Interference carrier was transiently transfected into target cells and the interference effect against target genes was detected by real-time PCR．The interference plasmid pcDNATM 6.2-GW/EmGFP-miR-NeuroD1 was linked to lentiviral destination vector pLenti6.3/V5-DEST to form the lentiviral expression vector pLenti6.3-EGFP-NeuroD1-miR. Constructed lentiviral vector carrier and packaging plasmids (Packaging Mix) were cotransfected into 293T cells, and followed by packaging virus, collecting the virus stock solution, ultra-centrifugating, condensing, and detecting the titer．PCR method was used to identify the recombinant vector; enhanced green fluorescent protein (EGFP) expression was used to determine the titer and the infection rate of the recombinant lentivirus under fluorescent microscope. Results Four interference plasmids for target gene were successfully constructed. The sequences of expression vector pcDNATM 6.2-GW/EmGFP-miR-NeuroD1/2/3/4 were proven correct using sequencing method. miR-NeuroD1 sequence showed the best silencing effect after transfected into 293T cells (P<0.01). Restriction endonuclease and PCR analysis confirmed that the pcDNATM 6.2-GW/EmGFP-miR-NeuroD1 was successfully inserted into the lentivirus vector. The titer of the recombinant lentivirus harboring NeuroD1 shRNA was 1.18×108 ifu/mL. Conclusion The recombinant lentivirus pLenti6.3-EGFP-NeuroD1-miR has been constructed successfully, which lays a foundation for future study of NeuroD function. |
| Key words: lentivirus NeuroD gene RNA interference pancreatic neoplasms |
