Skp2-siRNA重组腺病毒载体构建及鉴定

Construction and Identification of Recombinant Adenovirus Vector of siRNA Targeting Skp2

目的:构建针对Skp2的siRNA腺病毒载体。方法:合成针对Skp2的siRNA靶DNA序列及相应阴性对照序列,退火成DNA双链,然后亚克隆穿梭质粒pShuttle-H1,Pme I线性化后,与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中进行同源重组,转染AD293细胞,包装得到pAd-Skp2-siRNA和pAd-Skp2-siRNA-NC重组腺病毒。用病毒体外感染结肠癌SW480细胞,Western blot检测Skp2蛋白表达水平。结果:重组腺病毒载体经酶切、鉴定正确,制备的病毒感染效率高,能显著抑制Skp2蛋白表达。结论:细菌内同源重组成功构建了Ad-Skp2-siRNA重组腺病毒及阴性对照病毒。

Construc tion and Iden tifica t ion of Recomb ina nt Adenoviru s Vector of siRNA T ar geting Skp2 WE I Gang1, 2, SUN Ze- qun2, KONG X ia3, HUANG Yong- zhang3, WANG Bin2, TANG Jun-m ing3, XU Shao- yong2* ( 1Second Clinica l College, Wuha n University, Wuha n, H ubei 430072; 2Depa rtment of Ga stroen terology; 3Institute of Clinica lMed icine, Renmin H ospita l, Hubei Univer sity of Med icine, Shiyan, H ubei 442000, China ) Abstr ac t: Ob je c tive To construct an adenovirus vector express ing sma ll interfer ing RNA( siRNA) targe ting to Skp2. Me thods The siRNA conta in ing DNA sequence ta rgeting to Skp2 and its nega tive control sequence were designed, synthesized, annea led and subcloned into pShuttle-H 1 conta in ing the green fluorescen t protein. The linearized shu ttle p lasm id by Pme I was recombined with back- bone pAdEasy- 1 in BJ5183 bacteria. The recomb inan t pAd- Skp2- s iRNA adenovirus partic les were produced by transfection ofAD293 ce lls and subsequently infected colon ic cancer SW480 ce lls. The Skp2 prote in leve ls were detected byWeste rn b lot. Re s u lts The adenovirus vectors were ver ified by enzyme d igestion and DNA sequencing, the in fection effic iency of constructed adenovirus was high, and cou ld obviously inh ibit the expression of Skp2. Conc lus ion The recomb inant pAd- Skp2- siRNA and negative control bacteria lwere successfully constructed by bacte rial homologous recomb ination.