大鼠硫酸软骨素蛋白多糖基因shRNA慢病毒载体的构建及干扰效率鉴定

背景：最近研究发现硫酸软骨素蛋白多糖（NG2）在中枢神经系统中参与多种生理病理功能,慢病毒载体可感染分裂期细胞或非分裂期的细胞,并能在细胞内高效稳定的表达。目的：构建大鼠源性NG2基因shRNA慢病毒载体并检测其干扰效率。方法：选择大鼠NG2基因RNA干扰的靶序列,合成OligoDNA,退火形成双链DNA,与HpaⅠ和XhoⅠ双酶切后的pFU-GW-RNAi载体连接产生pLV-NG2-RNAi,PCR筛选阳性克隆,测序鉴定。将重组载体与pHelper1.0载体、pHelper2.0载体通过lipofectamineTM2000共转染293T细胞包装产生慢病毒LV-NG2-RNAi,收集病毒上清并浓缩。采用孔稀释滴度测定法计算病毒滴度。将LV-NG2-RNAi慢病毒感染C6细胞,于感染后96h提取细胞总蛋白,采用Westernblot检测NG2的表达。结果与结论：经PCR和测序证实构建片段大小及DNA序列与目的序列一致,实验成功构建大鼠NG2基因shRNA慢病毒载体LV-NG2-RNAi。包装浓缩慢病毒的滴度为8×1011TU/L。Westernblot检测显示在感染复数为50时,感染LV-NG2-RNAi慢病毒的C6细胞较感染对照慢病毒及未感染细胞NG2的表达明显降低,干扰效率可达100%（P〈0.05）。结果证实了实验成功构建大鼠NG2基因shRNA慢病毒载体,且该载体能够在细胞水平有效沉默靶基因。

Construction and identification of a lentiviral vector for RNA interference of rat chondroitin sulfate peoteoglycan gene

BACKGROUND：Recent studies suggest that the chondroitin sulfate peoteoglycan（NG2） is involved in physiological and pathological function in the central nervous system.Lentiviral vector can infect cells in both dividing phase and non-dividing phase,and can be stable expressed in cells with high efficiency.OBJECTIVE：To construct a lentiviral vector for RNA interference（RNAi） of rat NG2 gene and to detect its effect of gene silence in C6 cells.METHODS：Towards rat NG2 gene sequences,a pair of complementary small hairpin RNA（shRNA） oligonucleotides were designed,synthesized,annealed and inserted into pFU-GW-RNAi vector digested by Hpa Ⅰ and Xho Ⅰ.The recombinant plasmid was identified by PCR and DNA sequencing.The recombinant plasmid,pHelper 1.0 and pHelper 2.0 were co-transfected into 293T cells to obtain lentivirus particles.Viral titer was then determined.The lentivirus particles were transmitted into C6 cells.Then,western blot was performed to determine the expression level of the NG2 protein.RESULTS AND CONCLUSION：The PCR identification and DNA sequencing showed that the fragment and nucleotides were in accordance with the target sequence and the shRNA sequence was successfully inserted into the pFU-GW-RNAi vector.The titer of concentrated virus was 8×1011 TU/L.Compared with the C6 cells infected with LV-Con-RNAi or uninfected,the cells infected with LV-NG2-RNAi at MOI of 50 showed significant suppression in the expression of NG2,it could achieve 100% interference efficiency（P〈0.05）.The results demonstrate a lentiviral shRNA expression vector targeting the NG2 gene is successfully constructed and it can knockdown the expression of NG2 in C6 cells.