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目的:构建抑癌基因PTENshRNA腺病毒载体,探讨PTEN在哮喘患者气道重塑的作用机制。方法:设计形成短发夹序列(shRNA)的PTEN-shRNA引物对应的模板DNA序列,亚克隆至pShuttle-GFP-U6重组穿梭载体,构建穿梭质粒pShuttle-GFP-U6-PTEN-shRNA。鉴定正确后,然后与腺病毒质粒pAdxsi在化学感受态细胞DH5α菌株内同源重组,得到pAdxsi-GFP-U6-PTEN-shRNA病毒质粒,将pAdxsi-GFP-U6-PTEN-shRNA质粒经PacI线性化后转染HEK293细胞,包装重组、并大量扩增pAdxsi-GFP-U6-PTEN-shRNA腺病毒质粒,氯化铯梯度离心纯化,测病毒滴度,Western blot检测PTEN蛋白水平的表达。结果:结果证实pShuttle-GFP-U6-PTEN-shRNA及pAdxsi-GFP-U6-PTEN-shRNA质粒构建正确,收获病毒后的PCR及Western blot检测结果均证明pAdxsi-GFP-U6-PTEN-shRNA包被成功。结论:抑癌基因PTENshRNA腺病毒载体成功构建,为进一步研究PTEN基因应用于哮喘治疗提供了相关的实验基础。
Objective: To construct adenovirus vector of tumor suppressor gene PTEN shRNA to investigate the mechanism of PTEN in airway remodeling in asthmatic patients. Methods: The template DNA sequence of PTEN-shRNA primer was designed and subcloned into pShuttle-GFP-U6 recombinant shuttle vector to construct shuttle plasmid pShuttle-GFP-U6-PTEN-shRNA. After identified correctly, pAdxsi-GFP-U6-PTEN-shRNA plasmid was homologously recombined with the adenovirus plasmid pAdxsi in the chemically competent cell DH5α strain, and the pAdxsi-GFP-U6-PTEN-shRNA plasmid was linearized by PacI HEK293 cells were transfected with HEK293 cells and packaged. Recombinant plasmids were transfected into pAdxsi-GFP-U6-PTEN-shRNA adenovirus vector. The cetyltrimethylammonium chloride gradient centrifugation and virus titer were used to detect the expression of PTEN protein. Results: The pShuttle-GFP-U6-PTEN-shRNA and pAdxsi-GFP-U6-PTEN-shRNA plasmids were constructed correctly. PCR and Western blot results showed that pAdxsi-GFP-U6- success. CONCLUSION: PTEN shRNA Adenovirus vector is successfully constructed and provides the experimental basis for further study on the application of PTEN gene in the treatment of asthma.