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目的:构建人细胞周期素依赖蛋白激酶2(CDK2)基因RNA干扰慢病毒载体。方法:利用Invitro-gen公司在线软件设计人CDK2(NM001798)shRNA序列,退火形成dsoligo后克隆到pENTRTM/U6载体的黏性末端,测序,再与慢病毒载体重组,测序鉴定,在脂质体的介导下将慢病毒的包装混合物和CDK2基因重组慢病毒载体转染293FT细胞,包装成病毒后,收集细胞培养上清液,测定病毒滴度。结果:测序证实pENTRTM/U6-CDK2-shRNA为阳性克隆,与慢病毒载体重组后测序结果显示也为阳性克隆,CDK2基因重组慢病毒载体传染293FT细胞后48h,细胞培养上清液,病毒的滴度为6×108TU/L。结论:成功构建人CDK2基因RNAi慢病毒载体,为研究CDK2在自身免疫病中的应用提供了稳定的转染细胞载体。
OBJECTIVE: To construct RNA interference lentiviral vector of human cyclin dependent protein kinase 2 (CDK2) gene. Methods: Human CDK2 (NM001798) shRNA sequence was designed and synthesized by Invitro-gen software. After annealing, dsoligo was cloned into the cohesive end of pENTRTM / U6 vector, sequenced, recombinated with lentiviral vector, sequenced and identified. Under the guidance of the lentivirus packaging mixture and CDK2 gene recombinant lentiviral vector transfected 293FT cells, packaged into a virus, the cell culture supernatants were collected, the virus titer was measured. Results: Sequencing confirmed that pENTRTM / U6-CDK2-shRNA was positive and recombined with lentiviral vector. The result of sequencing also showed that the positive clones were positive. The cell culture supernatant and virus droplet 48 hours after infected with CDK2 gene recombinant lentiviral vector Degree of 6 × 108TU / L. Conclusion: The RNAi lentiviral vector of human CDK2 gene was successfully constructed, which provided a stable transfected cell vector for studying the application of CDK2 in autoimmune diseases.