针对核因子-κBP65基因的短发夹干扰RNA逆转录病毒载体构建与鉴定

Construction and identification of human Nuclear factor kappa B P65 siRNA retrovirus expression vector in vitro

目的:构建针对人核因子κB亚基P65基因mRNA的短发夹干扰RNA(shRNA)逆转录病毒表达载体,并探讨小干扰RNA(siRNA)靶向抑制NF-κBP65基因表达的作用。方法:根据shRNA设计原则,在人NF-κBP65全长序列中选取含19个核苷酸靶序列,设计形成siRNA的DNA模板并克隆到shRNA表达载体pSUPER.retro.neo中,构建针对NF-κBP65基因的shRNA表达载体。经293A细胞包装,并感染NIH3T3细胞进行病毒滴度测定后,感染THP-1细胞。分别采用RT-PCR和Westernblot从mRNA和蛋白水平检测干扰效果。结果:限制性酶切和基因测序证实针对人NF-κBP65亚基的shRNA表达逆转录病毒载体成功构建;其感染THP-1细胞后,NF-κBP65的mRNA和蛋白表达明显抑制。结论:成功构建了NF-κBP65shRNA逆转录病毒表达载体,该载体能高效感染THP-1并明显抑制NF-κBP65的表达。

Objective： To construct human NF-κB P65 siRNA retrovirus expression vector and investigate its inhibitory effect in vitro. Methods： The Oligonucleotides of NF-κB P65 shRNA were synthesized and inserted into the retrovirus expression vector pSUPER. retro.neo and transfected into the packaging cells 293A. The retrovirus supernatant infected NIH3T3 in vitro to determine the virus titer. Then we used THP-1 as a cell model to test the silencing effect on NF-κB P65 by RT-PCR and Western blot after it was infected with P65 siRNA retrovirus expression vector. Results： The results of restrictive enzyme and gene sequence confirmed that the NF-κB P65 siRNA retrovirus expression vector was constructed successfully. The level of NF-κB P65 in mRNA and protein were dramatically decreased, compared with normal cells. Conclusions： The P65 siRNA retrovirus expression vector was successfully constructed, which could inhibit the activation of NF-κB through intervention of the expression of NF-κB P65.