作　　者： ; ; ; ; ; ; ;

机构地区： 第三军医大学大坪医院

出　　处： 《中国生物制品学杂志》 2013年第1期17-21,共5页

摘　　要： 目的构建人乳腺癌抗雌激素药物耐药蛋白1(Breast cancer anti-estrogen resistance 1,BCAR1)基因siRNA慢病毒载体,并进行鉴定。方法针对人BCAR1基因靶序列设计并合成4条siRNA序列(PLVT350、PLVT351、PLVT352、PLVT353)及1条阴性对照序列(PLVT4),分别退火后,插入经AgeⅠ和EcoRⅠ双酶切的慢病毒载体pMagic 4.1中,连接产物转化感受态大肠杆菌DH5α,经PCR和DNA测序筛选阳性克隆,提取重组干扰质粒,与辅助质粒共同感染293T细胞,进行慢病毒包装,浓缩后,采用梯度稀释法测定病毒滴度。将重组慢病毒感染A549细胞,荧光显微镜下观察感染效率,实时荧光定量PCR检测干扰效率,筛选干扰效果最好的重组慢病毒感染肺癌A549细胞,Western blot检测BCAR1蛋白的表达水平。结果成功构建了4种人BCAR1基因慢病毒干扰质粒,PLVT350、PLVT351、PLVT352和PLVT353的病毒滴度分别为3.45×108、2.13×108、3.01×108和2.47×108TU/ml;慢病毒感染3 d后,A549细胞的感染效率均达90%以上;除PLVT350外,其他3个均为有效靶点,PLVT351、PLVT352和PLVT353的沉默率分别为82%、73%和82%,BCAR1基因的表达量显著低于未转染和PLVT4组A549细胞(P<0.001);PLVT351感染的A549细胞中BCAR1蛋白的表达水平明显低于未感染和PLVT4组A549细胞(P<0.01)。结论成功构建了人BCAR1基因siRNA慢病毒载体,并建立了稳定表达的A549细胞株,为进一步探索BCAR1基因在肺癌中的相关功能及其分子机制奠定了基础。 Objective To construct and identify the RNAi lentiviral vector for human breast cancer anti-estrogen resistance 1（BCAR1） gene.Methods Four siRNA sequences,i.e.PLVT350,PLVT351,PLVT352 and PLVT353,and a negative control sequence PLVT4 were designed and synthesized according to the target sequence of human BCAR1 gene and,after annealing,inserted respectively into lentiviral vector pMagic 4.1 digested with Age Ⅰ and EcoR Ⅰ.The constructed recombinant plasmids were transformed to competent E.coli DH5α,and positive clones were screened by PCR and DNA sequencing,from which recombinant RNAi plasmids were extracted and co-infected to 293T cells with helper plasmid for packaging.The obtained lentivirus was concentrated and determined for titer by gradient dilution.A549 cells were infected with the recombinant lentivirus particles and observed for infection efficacy by fluorescent microscopy.The recombinant lentivirus particles with satisfactory interfering effect were screened and infected to A549 cells in which the expression level of BCAR1 was determined by Western blot.Results Four RNAi lentiviral vectors were successfully constructed.The titers of PLVT350,PVLT351,PLVT352 and PLVT353 were 3.45 × 108,2.13 ×108,3.01 × 108 and 2.47 × 108 TU / ml,respectively.The infection efficiency of A549 cells was more than 90% 3 d after infection with recombinant lentivirus.Except PLVT350,all the other three siRNA sequences were effective targets.The silencing rates of PLVT351,PLVT352 and PLTV353 were 82%,73% and 82% respectively.The expression levels of BCAR1 gene in A549 cells transfected with recombinant RNAi plasmids containing the three sequences were significantly lower than those untransfected and those transfected with recombinant plasmid containing PLVT4（P 0.001）.However,the expression level of BCAR1 protein in A549 cells infected with recombinant lentivirus containing PLVT351 sequence was significantly lower than those uninfected and those infected with lentivirus containing PLVT4 sequence（P 0.01）.Conclus

关 键 词： 乳腺癌抗雌激素药物耐药蛋白 干扰 慢病毒 肺癌 细胞

领　　域： [生物学]