多药耐药基因RNAi重组腺病毒构建  

Construction of Recombinant Adenovirus Vector for RNA Interference with Multidrug Resistance MDR1 Gene

作　　者：陈蕾[1] 冯培民[2] 杨天华[1] 田林郁[1] 程新旺[1] 周东[1] 

机构地区：[1]四川大学华西医院神经内科,成都610041 [2]成都中医药大学消化内科

出　　处：《四川大学学报（医学版）》2007年第6期913-917,共5页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(批准号0040205401094)资助

摘　　要：目的构建抑制多药耐药基因(MDR1)表达的RNAi腺病毒载体,探讨基因治疗改善癫痫多重耐药现象的可行性。方法根据大鼠MDR1基因序列,选择3个19nt的靶序列,设计并合成3对66nt含编码短发夹RNA(shRNA)序列的寡核苷酸,构建pSIREN-shuttle-MDR1重组质粒,测序分析正确后转染通过马桑内酯诱导的已表达多重耐药蛋白的大鼠星形胶质细胞,通过RT-PCR法测定多药耐药蛋白(P-gp)表达量,判断所设计的3条DNA序列对于P-gp表达的抑制作用。选择抑制效率最高的1个重组质粒,将其中的MDR1 shRNA表达结构酶切后插入腺病毒载体pAdeno-X,构建的pAdeno-MDR1经Pac1酶切后与脂质体共转染HEK293细胞进行病毒包装扩增纯化,所得病毒液作酶切电泳及测序分析正确后再转染大鼠星形胶质细胞模型。RT-PCR及免疫组织化学法分别测转染前后星形胶质细胞模型的MDR1及P-gp表达量。结果重组质粒及pAdeno-MDR1病毒经PCR、酶切、测序分析证实构建正确。病毒滴度为6×109pfu/mL。重组腺病毒转染星形胶质细胞后MDR1及P-gp表达量减少,干扰效率接近100%。结论成功构建针对大鼠MDR1基因的RNAi腺病毒载体,并通过体外实验证实其对大鼠MDR1基因的高效抑制作用。为进一步探索难治性癫痫的多药耐药机制和基因治疗奠定了基础。Objective To construct the recombinant adenoviral RNA interference （RNAi） vector in order to inhibit the expression of multidrug resistance MDR1 gene, and probe whether gene therapy for multidrug resistance of epilepsy is feasibility. Methods Three target sequences for short hairpin RNA （shRNA） expression were selected and designed according to MDR1 gene sequence of rat. Annealed oligos were inserted into the downstream of treated pSIREN-shuttle U6 promoter to construct RNAi plasmid pSIREN-shuttle-MDR1. Next, MDR1 shRNA sequence was cloned to pAdeno-X, a transfer vector of adenovirus, to produce the pAdeno-MDR1, which was then packed and amplified in HEK293 cells. Further the recombinant adenovirus was purified by CsC1 and used to infect the rat astrocytes with P170-glucoprotein （P-gp） over-expression which have been induced by coriaria lactone （CL）. Results It was confirmed by restriction digestion, PCR and sequencing that MDR1 shRNA expression structure was correctly cloned to pSIREN-shuttle and pAdeno-X vector respectively. Virus titer was 5 ×10^9 pfu/mL. The interference efficiency of pAdeno-MDR1 to the expression drop of multidrug resistance gene in astrocyte model neared to 100%. Conclusion RNAi adenovirus vector of rat MDR1 gene has been constructed and found its high interference efficiency. It is the essential building required for the remedy of refractory epilepsy and the research on mechanism of multidrug resistance.

关 键 词：多药耐药基因 RNA干扰 腺病毒 星形胶质细胞模型 

分 类 号：R742.1[医药卫生—神经病学与精神病学]