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目的探索构建能表达人B型利钠肽(hBNP)的pLenti6/V5-D-TOPO-hBNP载体的方法,以实现人B型利钠肽基因在骨骼肌成肌细胞中长久、稳定的表达。方法将目的基因人B型利钠肽(hBNP)亚克隆到真核细胞表达载体pLenti6/V5-D-TOPO载体上,构建重组质粒pLenti6/V5-D-TOPO-hBNP。将pLenti6/V5-D-TOPO-hBNP及阳性对照质粒pLenti6/V5-D-TOPO-GFP分别用lipo-fectamin2000介导转染293FF细胞,获得病毒颗粒;用病毒颗粒转染体外培养的SD乳鼠骨骼肌成肌细胞。荧光显微镜下计数确定阳性对照质粒的瞬时转染数,从而估计该基因的瞬时转染效率。加入筛选试剂以获得稳定表达异源B型利钠肽(BNP)的成肌细胞。收集瞬时转染及筛选后的细胞培养基,用酶连结免疫吸附分析(ELISA)检测试剂盒检测人B型利钠肽(hBNP)的表达水平。结果聚合酶链式反应法及DNA测序显示人B型利钠肽基因成功地插入到pLenti6/V5-D-TOPO载体中;阳性对照质粒转染细胞12h后,在荧光显微镜下观察,其转染效率达60%以上。检测收集的上清,结果与阴性对照有显著差别(P<0.01),观察至第4周,人B型利钠肽(hBNP)仍持续稳定表达。结论本实验成功构建了能在真核细胞内表达人B型钠尿肽的重组质粒pLenti6/V5-D-TOPO-hBNP,并且取得了较高的转染效率,因此慢病毒载体介导的基因治疗在心血管疾病中有乐观的应用前景。
Objective To explore a method for constructing pLenti6 / V5-D-TOPO-hBNP vector expressing human B-type natriuretic peptide (hBNP) in order to achieve long-term and stable expression of human B-type natriuretic peptide gene in skeletal myoblasts. Methods The recombinant plasmid pLenti6 / V5-D-TOPO-hBNP was subcloned into eukaryotic cell expression vector pLenti6 / V5-D-TOPO. 293T cells were transfected with pLenti6 / V5-D-TOPO-hBNP and positive control plasmid pLenti6 / V5-D-TOPO-GFP respectively with lipo-fectamin 2000 to obtain virus particles; Skeletal muscle myoblasts. The number of transient transfection of the positive control plasmid was counted under a fluorescence microscope to estimate the transient transfection efficiency of the gene. Screening reagents are added to obtain myoblasts stably expressing heterologous B-type natriuretic peptide (BNP). Collect transiently transfected and cell culture media after screening, and detect the expression level of human B-type natriuretic peptide (hBNP) by enzyme-linked immunosorbent assay (ELISA) kit. Results Polymerase chain reaction (PCR) and DNA sequencing showed that the human B-type natriuretic peptide gene was successfully inserted into pLenti6 / V5-D-TOPO vector. The positive control plasmid was transfected into cells for 12 hours and observed under a fluorescence microscope. Efficiency of more than 60%. The supernatant was collected and the result was significantly different from that of the negative control (P <0.01). At the 4th week, the human B-type natriuretic peptide (hBNP) remained stable and stable. Conclusions The recombinant plasmid pLenti6 / V5-D-TOPO-hBNP expressing human B-type natriuretic peptide in eukaryotic cells has been successfully constructed and a high transfection efficiency has been achieved. Therefore, lentiviral vector-mediated gene Treatment has an optimistic outlook for cardiovascular disease.