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目的:构建并筛选携带针对大鼠甲状旁腺激素受体1基因的pSUPERretro-GFP/Neo干扰逆转录病毒载体及鉴定。方法:根据Genbank筛选甲状旁腺激素受体1基因3个阳性克隆及1个阴性克隆序列,定向克隆入逆转录病毒载体pSUPERretro-GFP/Neo,并用RT-PCR和测序鉴定,将其转染至胰岛素-1细胞中,Westernblot观察甲状旁腺激素受体1基因表达,鉴定其转染效率。结果:菌落RT-PCR及基因测序鉴定结果表明序列正确,并能成功转染到胰岛素-1细胞株中,Western blot筛选最佳抑制基因。结论:成功构建并筛选出特异性沉默大鼠甲状旁腺激素受体1基因的pSUPERretro-GFP/Neo干扰逆转录病毒载体,为深入探讨大鼠甲状旁腺激素受体1基因的抗凋亡作用奠定了试验基础。
OBJECTIVE: To construct and screen pSUPERretro-GFP / Neo interfering retroviral vector carrying rat parathyroid hormone receptor 1 gene. Methods: Three positive and one negative clones of parathyroid hormone receptor 1 gene were screened by GenBank and cloned into the retroviral vector pSUPERretro-GFP / Neo. The recombinant plasmids were identified by RT-PCR and sequencing and transfected into Insulin-1 cells, Western blot analysis of parathyroid hormone receptor 1 gene expression, identification of its transfection efficiency. Results: The results of colony RT-PCR and gene sequencing showed that the sequence was correct and successfully transfected into insulin-1 cell line. The best suppressor gene was screened by Western blot. CONCLUSION: The pSUPERretro-GFP / Neo interfering retrovirus vector of pSPHR1 gene was specifically constructed and screened in order to further investigate the anti-apoptotic effect of PTHR1 gene in rat Lay the foundation for the experiment.