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[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亢进的骨代谢疾病奠定基础。[方法]设计ATPase a3基因siRNA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5×108 Tu.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 screen small interfering RNA (siRNA) of bone resorption key gene of osteoclast (OC) and construct its lentiviral expression vector. basis. [Method] The siRNA sequence of ATPase a3 gene was designed and synthesized. DNA of small hairpin RNA (shRNA) was synthesized and annealed to form double-stranded DNA. The double-stranded DNA was ligated with the lentiviral vector pGCL-GFP by Hpa I and Xho I, Carrying the ATPase a3-shRNA lentiviral plasmid vector (pGCL-ATPase a3-shRNA), identified by PCR and sequencing, the lentiviral plasmid vector carrying the best siRNA was screened by Western blot and the lentiviral packaging plasmid (pHelper 1.0 and pHelper 2.0 ) Was prepared into ATPase a3-shRNA lentivirus, and the virus titer was determined. [Results] ATPase a3-shRNA lentiviral vector was successfully constructed. The best siRNA fragment carrying the best siRNA fragment was screened by Western blot and successfully packaged into ATPase a3-shRNA lentivirus with 5 × 108 Tu.mL-1 titer. [Conclusion] The rat ATPase a3 gene RNAi lentivirus was successfully constructed.