兔膜联蛋白A1基因短发夹RNA慢病毒载体构建与RNA干扰效率的鉴定

背景:膜联蛋白A1参与细胞凋亡的调控。目的:针对兔膜联蛋白A1基因构建短发夹RNA慢病毒载体,并检测其对成骨细胞的沉默效率。方法:针对兔膜联蛋白A1基因设计RNA干扰靶序列,合成Oligo DNA,退火形成双链DNA,与BamHI和EcoRI双酶切后的pGLV/H1/GFP载体连接成pGLV-shANXA1,PCR筛选阳性克隆,测序鉴定。pGLV-shANXA1和pGLV/helper-1、pGLV/helper-2、pGLV/helper-3共转染293FT细胞包装产生慢病毒颗粒LV-shANXA1,并测定其滴度,随后将LV-shANXA1感染兔成骨细胞。结果与结论:经PCR和测序证实构建片段大小及DNA序列与目标序列一致,shANXA1片段完全正确插入慢病毒载体pGLV/H1/GFP中。包装浓缩慢病毒颗粒LV-shANXA1的滴度为3.8×108TU/L。经嘌呤霉素筛选后,感染复数为50时,LV-shANXA1对成骨细胞感染效率为80%;感染复数为100时,感染效率为95%。Real-time PCR和Western blot检测显示,LV-shANXA1-901对膜联蛋白A1基因的沉默效率最高,在感染复数为50时,沉默效率可达71.2%。表明实验成功构建的兔膜联蛋白A1基因短发夹RNA慢病毒载体能够在细胞水平有效沉默靶基因。

Construction of short hairpin RNA lentiviral vector targeting rabbit Annexin A1 gene and identification of RNA interference efficiency

BACKGROUND: Annexin A1 (ANXA1) protein is involved in the regulation of cell apoptosis. OBJECTIVE: To construct a short hairpin RNA (shRNA) lentiviral vector targeting rabbit ANXA1 gene and to detect its efficiency of gene silence in osteoblasts. METHODS: Targeting rabbit ANXA1 gene sequences, a pair of complementary small hairpin RNA (shRNA) oligonucleotides were designed, synthesized, annealed and cloned into pGLV/H1/GFP plasmid digested by BamHI and EcoRI to construct a vector named pGLV-shANXA1. The recombinant plasmid was identified by PCR and DNA sequencing. 293FT cells were co-transfected with pGLV-shANXA1, pGLV/helper-1,pGLV/helper-2 and pGLV/helper-3 to package into lentivirus particles named LV-shANXA1.The titer of virus was tested according to the expression level of GFP. The lentivirus particles were then transmitted into rabbit osteoblasts and its infection efficiency was assessed by flow cytometry. 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 pGLV/H1/GFP vector. The titer of concentrated LV-shANXA1 was 3.8×108 TU/L. The infection efficiency of lentiviral particles was 80% at multiplicity of infection (MOI) of 50 and 95% at MOI of 100 in osteoblasts after puromycin selection. Real-time PCR and Western blot results showed that the expression of ANXA1 in LV-shANXA1-901 infected osteoblasts were lower than that in the blank control, negative control, and other LV-shANXA1 infected cells (P < 0.05), it could achieve 71.2% interference efficiency at MOI of 50. The results demonstrate a lentiviral shRNA expression vector targeting the ANXA1 gene is successfully constructed and it can knockdown the expression of ANXA1 in osteoblasts.