TPP1基因慢病毒干扰载体的构建及鉴定

目的:构建抑制TPP1基因的短发夹RNA(sh RNA)干扰载体。方法:以人的TPP1基因为靶序列,设计并合成sh RNA序列TPP1-si1和TPP1-si2,分别与RNA干扰慢病毒载体pll3.7连接,双酶切鉴定质粒得到阳性克隆pll-TPP1-si1和pll-TPP1-si2,经测序正确后将其与慢病毒包装载体(RRE、REV、VSVG)共转染293T细胞,进行病毒的包装,将得到的病毒感染稳定高表达外源TPP1蛋白的HT1080细胞,通过Western印迹检测其对TPP1蛋白表达的抑制效果,并进行比较;将抑制效果好的病毒感染高表达外源Pot1蛋白的Hep G2细胞,检测Pot1蛋白在内源TPP1被敲低的情况下能否在端粒定位。结果:经双酶切验证,外源片段成功插入载体pll-TPP1-si1和pll-TPP1-si2中;pll-TPP1-si1和pll-TPP1-si2均能明显抑制TPP1的表达,其中pll-TPP1-si1的抑制效果最好;pll-TPP1-si1病毒感染高表达外源Pot1蛋白的Hep G2细胞,经免疫荧光鉴定能够有效抑制内源TPP1的表达,使Pot1不再端粒定位。结论:构建的抑制TPP1基因的sh RNA干扰载体能有效抑制TPP1的表达,为端粒蛋白TPP1功能的研究奠定了实验基础。

Construction and Identification of Lentiviral Interference Vector of TPP1 Gene

Objective： To construct a shRNA interference vector of TPP1 gene. Methods： The shRNA oligonucle？otide sequences TPP1-si1 and TPP1-si2 were designed and synthesized according to human TPP1 gene sequence and the synthesized sequences were annealed to form double-strand oligonucleotide and cloned into interference vector plasmid pll3.7. The plasmids were restriction enzyme digested and sequenced to confirm the shRNA oligonu？cleotide sequences have successfully cloned into interference vector plasmid pll3.7, and called pll-TPP1-si1 and pll-TPP1-si2 for short. The shRNA interference vectors were cotransfected into 293T cells with the lentivirus pack？ing vectors（RRE, REV, VSVG）, and the HT1080 cells stable high expressing heterologous protein TPP1 were in？fected with the virus. The shRNA interference effects of these vectors were detected by Western blot, and the bet？ter virus were chosen to infect the HepG2 cells high expressing heterologous protein Pot1 to detect its suppressive function Results： TPP1 gene specific oligonucleotide sequences were successfully cloned into shRNA interference vectors pll3.7 through the restriction enzyme digestion and sequencing. It was found that TPPl-si1 and TPPl-si2 can obviously inhibit the expression of the TPP1 protein, and TPPl-si1 had the highest interference effect on TPP1 compared with other vectors. After packaging into lentivirus, TPP1-si1 was infected into HepG2 cells high expressing heterologous protein Pot1. It was shown that TPP1-si1 also could also significantly suppress TPP1 ex？pression through immunofluorescence, which made Pot1 no longer telomere location. Conclusion： The constructed shRNA interference vector of TPP1 gene can effectively inhibit the expression of TPP1, which provided a powerful method for studying TPP1 function.