特异性针对大鼠甲状旁腺激素受体1基因siRNA表达载体构建及鉴定

Construction and identification of siRNA recombinant expression vector targeting parathyroid hormone 1 receptor gene

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 the expression vector of siRNA specific for parathyroid hormone 1 receptor gene.Methods Genome sequences of parathyroid hormone 1 receptor gene was retrieved from gene bank,and 4 pairs of oligonucleotides were synthesized and inserted into pSUPER.retro RNAi,which was identified by RT-PCR and sequence analysis,and then was transfected into INS-1 cells.The expression of parathyroid hormone 1 receptor gene was observed with Western blot and the transfection efficiency was identified.Results It was confirmed by RT-PCR and sequence analysis that siRNA recombinant expression vector targeting parathyroid hormone 1 receptor gene was coincided with the designs completely,then transfected into INS-1 cell successfully.The best gene was picked up by Western blot.Conclusion The successful construction of recombinant parathyroid hormone 1 receptor-siRNA vectors establishes a favorable foundation for further study on anti-apoptosis of the parathyroid hormone 1 receptor gene.