摘要
目的构建特异性强,转染率高,针对人Survivin进行RNA干扰的肿瘤增殖型腺病毒载体(Ad-delE1b55kD-shRNA/Survivin-EGFP)。方法将针对人Survivin的shRNA的基因序列及EGFP基因序列克隆至肿瘤增殖型腺病毒穿梭质粒pAd-delE1b55kD;HEK-293细胞中包装出毒得到重组腺病毒Ad-delE1b55kD-shRNA/Survivin-EGFP并测序鉴定。用重组腺病毒感染结肠癌细胞HT-29并检测转染率,用RT-PCR和Westernblot分别检测转染后的HT-29细胞中SurvivinmRNA和蛋白表达的变化。结果序列测定证明针对人Survivin的shRNA序列及EGFP序列被成功构建到肿瘤增殖型腺病毒载体中;Ad-delE1b55kD-shRNA/Survivin-EGFP对结肠癌细胞HT-29的转染效率显著高于增殖缺陷型腺病毒载体和脂质体载体(P<0.01);而对肝细胞的转染率则显著低于对结肠癌细胞株HT-29的转染(P<0.01)。与复制缺陷型腺病毒载体和脂质体载体比较,转染Ad-delE1b55kD-shRNA/Survivin-EGFP后,HT-29细胞中SurvivinmRNA拷贝数及蛋白表达显著降低(P<0.01)。结论构建成功携带针对人Survivin的shRNA的肿瘤增殖型腺病毒载体Ad-delE1b55kD-shRNA/Sur-vivin-EGFP,其能选择性地高效转染结肠癌细胞株HT-29并有效干扰Survivin,且较之增殖缺陷型腺病毒载体和脂质体载体具有更高的干扰效率。
Objective To construct the conditionally replicating adenovirus vector Ad-delE1b55kD-shRNA/Survivin-EGFP that can transfects into HT-29 effectually and selectively and contains shRNA targeting to human Survivin gene. Methods EGFP gene and shRNA gene were inserted into pAd-delE1b55kD, then pAd-delE1b55kD-shRNA/Survivin-EGFP was obtained. The plasmid and pBHGE3 were cotransfected into HEK-293 cell to obtain the conditionally replicating adenovirus vector Ad-delE1b55kD-shRNA/Survivin-EGFP. Then DNA of the adenovirus vector was extracted and identified. The transfection efficiency of the vector to HT-29 and LO2 was detected. The expression of Survivin mRNA and protein was detected by RT-PCR and Western blot. Results EGFP gene and shRNA gene targeting to human Survivin mRNA were inserted into conditionally replicating adenovirus vector successfully, proven by sequencing. The transfection efficiency to HT-29 of Ad- elE1b55kD-shRNA/Survivin-EGFP was significantly higher than replication defective adenovirus and liposome vector （P 〈 0.01 ）. After transfection of Ad-delE1b55kD-shRNA/Survivin-EGFP, mRNA and protein of Survivin gene in HT-29 cells were obviously reduced. Conclusion A conditionally replicating adenovirus vector was constructed and identified successfully. It can transfect to HT-29 and silence Survivin effectually and selectively.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2007年第13期1293-1297,共5页
Journal of Third Military Medical University
基金
重庆市自然科学基金(2005BB5225)~~
关键词
肿瘤增殖型腺病毒
RNA干扰
存活素
结肠癌
conditionally replicating adenovirus
RNA interference
Survivin
colon carcinoma
