摘要
目的构建短发夹RNA（shRNA）／mdr1的质粒表达载体并验证其体外表达效率。方法按照pSUPER的设计要求合成mdr1基因RNAi靶序列的64bp寡核苷酸序列，退火后双酶切法（BglⅡ和HindⅢ）克隆，得到质粒pSUPER-shRNA／mdr1，转染感受态大肠杆菌，筛选阳性克隆，测序证实后扩增培养并提取，然后转染融合达90％以上的HepG2／mdr1细胞，阴性载体为对照组，实时聚合酶链反应（Real-timePCR）、流式细胞术检测mdr1基因mRNA、P-gp表达、细胞耐药性等功能变化。结果成功构建质粒载体pSUPER-shRNA／mdr1，基因测序证实靶序列插入正确；HepG2／mdr1-si组mdr1基因的mRNA表达水平是耐药株HepG2／mdr1组的56分之一；HepG2／mdr1-si组细胞和阴性对照组HepG2／mdr1细胞的P-gP表达率分别为11.0％和98．6％，P〈0．05；HepG2／mdr1组细胞耐药倍数从62．5下降到1．4，相对逆转效率99．34％；细胞内DNR累积量也明显增加，与对照组比较（79．32％比37．96％），P〈0．05。结论多药耐药基因mdr1质粒表达载体pSU-PER-shRNA／mdr1能在HepG2／mdr1内稳定表达，并逆转其耐药性。
Objective To construct the expressing vector of shRNA/mdr1 and to study its reversed effect in vitro. Methods 64 bp oligonucleotides of pSUPER and the targeted sequence of siRNA/mdr1 were synthesized and annealed to form duplex strand, then were cloned into pSUPER to construct pSUPER-shRNA/mdr1 vector. Competent E. coli was transfected by vector of pSUPER-shRNA/mdr1 to screen the positive clones for sequencing and extracting plasmid. The plasmids extracted were used to transfect HepG2/mdr1 cells with control groups by negative vectors. The expression of mRNA was measured by real-time PCR, and P-glycoprotein and resistance of HepG2/mdr1 by flow cytometry. Results pSUPER-shRNA/mdr1 was established successfully and was sequenced to test its accuracy. The expression of mRNA in HepG2/mdr1-si was lower than that in HepG2/mdr1 （1-fold vs 56.30-fold,P 〈0.01 ）. Compared to HepG2/mdr1, the expression of P-gp in HepG2/mdr1-si was lower （11% vs 98.6% ,P 〈 0.05 ）. The sensitivity of HepG2/mdr1-si to adiramycin was higher than that of HepG2/mdr1 （62.5-fold vs 1,4-fold,P 〈0.01 ）. Also, compared to controls, the accumulation of DNR in HepG2/mdr1-si was increased significandy （79.32% vs 37.96%, P 〈 0.05）. Conclusion Vector of pSUPER-shRNA/mdr1 can be constructed by technique of enzymatic incision. The multidrug resistance of HepG2/mdr1 can be reversed.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2007年第2期169-172,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（30170925）
关键词
多药耐药基因
质粒
表达
Multidyry gene
Plasmid
Expression
作者简介
潘光栋（现在广西医科大学第五附院肝胆外科，545001）
