APP695-siRNA慢病毒载体构建及其对SH-SY5Y细胞中APP695基因的沉默作用

摘要

目的：构建针对APP6995的siRNA慢病毒载体，并检测其对人神经纤维母细胞瘤SH-SY5Y细胞株中APP695基因表达的沉默作用。方法：设计并合成针对筛选确定的APP695基因寡核苷酸序列特异性siRNA有效靶序列，同时合成1对阴性对照寡核苷酸序列，将以上寡核苷酸序列退火后连人pFU-GW-jRNA质粒，酶切和测序鉴定后，将它们分别与包装质粒混合物共感染293T细胞，产生具有感染能力的慢病毒颗粒，并感染SH-SY5Y细胞，分未经病毒感染(CON)组、阴性对照病毒感染(NC)组和慢病毒介导阳性干扰(APP695-RNAi)组；应用实时荧光定量PCR(FQ-RT-PCR)检测各组细胞APP695 mRNA的表达水平。结果：酶切和测序证实目的寡核苷酸片段已被准确克隆到pFU-GW-iRNA质粒；各质粒与包装质粒共感染293T细胞后收获慢病毒颗粒；感染SH-SY5Y细胞72 h后，不同组别APP695 mRNA表达水平不同(F=554.442，P＜0.001)，APP695-siRNA组表达水平低于CON组和NC组。结论：成功构建了APP695-siRNA慢病毒载体，该载体可有效沉默SH-SY5Y细胞中APP695 mRNA的表达。%Aim:To construct a lentiviral vector of RNA interference( RNAi) of APP695 gene and detect the effect of the vector in human neuroblastoma SH-SY5Y cell line(SH-SY5Y cells). Methods:The target sequence of APP695 gene which can be effectively silenced in RNA interfence was confirmed in our previous study. The cDNA encoding for sense and antisense OligoDNA fragments of the targeting sequence was designed, synthesized and cloned into the pFU-GW-iRNA vector, and the vector then the results confirmed by PCR and DNA sequencing. 392T cells were cotransfectd with lentiviral vector pFU-GW-iRNA and packing plasmids, to produce lentiviral vector which can transfect other cells. The transfected SH-SY5Y cells were divided into APP695-siRNA group, control group and negative control group. The changes of APP695 mRNA was detected by fluorescence quantitative Real-time polymerase chain reaetion( FQ-RT-PCR). Results: The positive clones of recombinants were identified by PCR and DNA sequencing, the result showed that the target oligonucleotide was ac-curatly cloned into pFU-GW-iRNA plasmid. The recombinant plasmids and packaged plasmids co-transfected the 293T cells and lentiviral particles were harvested. The SH-SY5Y cells were infected by lentivius vectors after 72 h, the APP695mRNA expression was different in the three groups( F =554.442,P <0.001). Comparing with the control group and negative control group, the APP695mRNA expression of APP695-siRNA group was lower. Conclusion: APP695-siRNA lentivirus vector has been constructed successfully,and it can effectively silence the APP695mRNA expression in SH-SY5Y cells.