大鼠ATPase a3基因siRNA筛选及慢病毒载体构建

摘要

[目的]设计、筛选大鼠破骨细胞(osteoclast,OC)骨吸收关键基因ATPase a3小干扰RNA(small interfering RNA,siRNA)并构建其慢病毒表达载体,为研究以OC亢进的骨代谢疾病莫定基础.[方法]设计ATPase a3基因shRNA序列并合成小发卡RNA(short hairpin RNA,shRNA)结构的DNA,退火形成双链DNA,与经Hpa I和Xho I酶切慢病毒pGCL-GFP载体连接,构建成携带ATPase a3-shRNA慢病毒质粒载体(pGCL-ATPase a3-shRNA),PCR及测序鉴定后,Western blot筛选出携带最佳siRNA的慢病毒质粒载体,并利用慢病毒包装质粒(pHelper 1.0和pHelper 2.0)将其制备成ATPase a3-shRNA慢病毒,并测定病毒滴度.[结果]成功构建出ATPase a3-shRNA慢病毒载体,Western blot筛选出携带最佳siRNA片段,并将其成功包装成5×108Tu.mL-1滴度的ATPase a3-shRNA慢病毒.[结论]成功构建大鼠ATPase a3基因RNAi慢病毒.%[Objective] To design and sieve small interfering RNA sequence targeting rat ATPase a3, the key gene of bone resorption of osteoclast, and to construct recombinant lentivirus for research on diseases of bone metabolism by OC accentuation. [Method] It was to design the effective sequence of small interfering RNA targeting ATPase a3 gene and to synthesis the DNA of its short hairpin RNA(shRNA) struction. The complementary DNA sequence was synthesized and cloned into the pGCL-GFP vector, which was cut by Hpa Ⅰ and Xho Ⅰ. It was named ATPase a3-shRNA lentiviral vector(pGCL-ATPase a3-shRNA), the resulting lentiviral vector containing ATPase a3 shRNA, and it was confirmed by PCR and sequencing. By western blot, the best small interfering RNA sequence was certificated and it's best pGCL-ATPase a3-shRNA. With best ATPase a3-shRNA lentiviral vector, pHelper 1.0 and pHelper 2. 0, ATPase a3-shRNA Lentivirus was produced, and the titer of virus was tested. [Result]It was demonstrated that the lentivirus vector targeting rat ATPase a3 was successfully constructed, and best small interfering RNA was perfectly selected by western blot, and ATPase a3-shRNA Lentivirus was successfully produced, its titer was 5 × l08 Tu. ml1. [Conclusion]The lentivirus RNAi of ATPase a3 was constructed successfully.