生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2014年
6期
760-764
,共5页
张金丁%金蕊%杨丹丹%王亚楠%黄君健%苏金为
張金丁%金蕊%楊丹丹%王亞楠%黃君健%囌金為
장금정%금예%양단단%왕아남%황군건%소금위
TPP1基因%shRNA干扰%Western印迹%免疫荧光
TPP1基因%shRNA榦擾%Western印跡%免疫熒光
TPP1기인%shRNA간우%Western인적%면역형광
TPP1 gene%shRNA interference%Western blot%immunofluorescence
目的：构建抑制TPP1基因的短发夹RNA（shRNA）干扰载体。方法：以人的TPP1基因为靶序列，设计并合成shRNA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HepG2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HepG2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shRNA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。
目的：構建抑製TPP1基因的短髮夾RNA（shRNA）榦擾載體。方法：以人的TPP1基因為靶序列，設計併閤成shRNA序列TPP1-si1和TPP1-si2，分彆與RNA榦擾慢病毒載體pll3.7連接，雙酶切鑒定質粒得到暘性剋隆pll-TPP1-si1和pll-TPP1-si2，經測序正確後將其與慢病毒包裝載體（RRE、REV、VSVG）共轉染293T細胞，進行病毒的包裝，將得到的病毒感染穩定高錶達外源TPP1蛋白的HT1080細胞，通過Western印跡檢測其對TPP1蛋白錶達的抑製效果，併進行比較；將抑製效果好的病毒感染高錶達外源Pot1蛋白的HepG2細胞，檢測Pot1蛋白在內源TPP1被敲低的情況下能否在耑粒定位。結果：經雙酶切驗證，外源片段成功插入載體pll-TPP1-si1和pll-TPP1-si2中；pll-TPP1-si1和pll-TPP1-si2均能明顯抑製TPP1的錶達，其中pll-TPP1-si1的抑製效果最好；pll-TPP1-si1病毒感染高錶達外源Pot1蛋白的HepG2細胞，經免疫熒光鑒定能夠有效抑製內源TPP1的錶達，使Pot1不再耑粒定位。結論：構建的抑製TPP1基因的shRNA榦擾載體能有效抑製TPP1的錶達，為耑粒蛋白TPP1功能的研究奠定瞭實驗基礎。
목적：구건억제TPP1기인적단발협RNA（shRNA）간우재체。방법：이인적TPP1기인위파서렬，설계병합성shRNA서렬TPP1-si1화TPP1-si2，분별여RNA간우만병독재체pll3.7련접，쌍매절감정질립득도양성극륭pll-TPP1-si1화pll-TPP1-si2，경측서정학후장기여만병독포장재체（RRE、REV、VSVG）공전염293T세포，진행병독적포장，장득도적병독감염은정고표체외원TPP1단백적HT1080세포，통과Western인적검측기대TPP1단백표체적억제효과，병진행비교；장억제효과호적병독감염고표체외원Pot1단백적HepG2세포，검측Pot1단백재내원TPP1피고저적정황하능부재단립정위。결과：경쌍매절험증，외원편단성공삽입재체pll-TPP1-si1화pll-TPP1-si2중；pll-TPP1-si1화pll-TPP1-si2균능명현억제TPP1적표체，기중pll-TPP1-si1적억제효과최호；pll-TPP1-si1병독감염고표체외원Pot1단백적HepG2세포，경면역형광감정능구유효억제내원TPP1적표체，사Pot1불재단립정위。결론：구건적억제TPP1기인적shRNA간우재체능유효억제TPP1적표체，위단립단백TPP1공능적연구전정료실험기출。
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.
