CD40-siRNA慢病毒载体的构建及其对小鼠巨噬细胞的感染

Construction of siRNA Lentiviral Vector against CD40 and Measurement of Its Titer and Infection Efficiency on Mouse Macrophages Cells

目的:构建CD40-siRNA慢病毒载体,检测其对小鼠巨噬细胞J774.1的感染效率及测定J774.1细胞的CD40 mRNA水平。方法:设计1段CD40的siRNA序列,构建慢病毒载体p LL3.7-CD40-siRNA,与p MD2.G、p MDLg/pRRE和pRSV-REV按比例采用Lipofectamine LTX&Plus共转染293T细胞制备病毒,感染J774.1细胞后第4、6和8天时用流式细胞仪检测其感染效率,实时荧光定量RT-PCR(qRT-PCR)检测CD40mRNA的表达水平。结果:成功构建CD40-siRNA慢病毒载体,当p LL3.7-CD40-siRNA、p MD2.G、p MDL g/p RRE和与pRSV-REV的比例为2∶1∶1∶1时,制备的慢病毒滴度最高;慢病毒感染J774.1细胞的效率呈时间依赖性,感染第8天时的感染效率最高;同时,p LL3.7-CD40-siRNA组CD40 mRNA的表达明显低于p LL3.7空载体组,差异有统计学意义(P<0.05)。结论:慢病毒感染J774.1细胞的效率可以满足后续实验的要求,CD40-siRNA慢病毒载体可以抑制CD40mRNA的表达。

Objective：To construct CD40-siRNA lentiviral vector and detect its infection efficiency on mouse macrophages cells lines（J774. 1）and the level of CD40 mRNA. Methods：An effective tar-get sequence against CD40 was designed,then CD40-siRNA lentiviral vector was constructed,identi-fied by DNA sequencing and named pLL3. 7-CD40-siRNA. pLL3. 7-CD40-siRNA were co-transfected with pMD2. G,pMDL g/p RRE and pRSV-REV into 293T cells to produce lentivirus on ratio 1：1：1：1,2：1：1：1,3：1：1：1 and 4：1：1：1 respectively by Lipofectamine LTX＆Plus. Lentivirus was infected into mouse J774 . 1 macrophages cells lines and infection efficiency was measured by flow cytometry on 4^th,6^th and 8^th day respectively after infection. The expression level of CD40 mRNA was measured by real-time quantitative RT-PCR methods. Results：The CD40 siRNA lentiviral vector was successfully constructed. The result of flow cytometry showed the lentivirus titer was highest on ratio 2：1：1：1（3. 5 × 10^8 TU/mL）. The infection efficiency of J774. 1 cells was increased on a time-dependent manner and the infection efficiency was highest on 8^th day. Meanwhile,the expression level of CD40 mRNA was significantly lower in pLL3. 7-CD40-siRNA group than in control group. Conclusion：The lentivi-ral vector is able to infect mouse macrophages cells lines（J774. 1）and the infection efficiency can reached the need of subsequent experiments. The CD40 siRNA lentiviral vector can suppress the ex-pression of CD40 mRNA.