摘要
本研究通过慢病毒载体(pRNAT-U6.2/Lenti)构建靶向Beclin1基因的RNA干扰(RNAi)重组体,用以建立Beclin1基因稳定沉默的非小细胞肺癌(NSCLC)A549细胞株。PCR鉴定结果显示3个扩增的阳性片段均已插入pRNAT-U6.2/Lenti载体;测序结果证明3个重组慢病毒载体pRNAT-U6.2/Lenti-si356、pRNAT-U6.2/Lenti-si423和pRNAT-U6.2/Lenti-si684的插入序列完全正确;重组慢病毒载体感染A549细胞后,RT-PCR和Westernblot检测结果均证实3组重组体感染的细胞内Beclin1mRNA和蛋白表达都受到不同程度的抑制,Beclin1mRNA的沉默效率分别为35.56%、89.22%和66.78%。结果显示成功构建了Beclin1基因的RNAi病毒重组载体,并建立了其稳定表达的NSCLC A549细胞株,为探讨Beclin1基因对NSCLCA549细胞生物学行为的影响提供了新的细胞模型。
The lentiviral vector was used for construction of a recombinant mediating RNA interference （RNAi） a- gainst Beclinl gene in this study. Recombinant vector plasmid was transfected into non small cell lung cancer （NSCLC） A549 cells by liposome. PCR results showed that three amplified positive fragments were inserted into pRNAT-U6.2/Lenti vectors. DNA sequencing results showed that the three recombinant lentivirus plasmids, pR- NAT-U6.2/Lenti-si356, oRNAT-U6.2/Lenti-si423 and pRNAT-U6.2/ Lenti-si684 were constructed successfully. After transfection with liposome, RT-PCR and Western blot analysis confirmed that the expression of Beclinl mRNA and protein was inhibited in the three recombinant lentivirus plasmids transfected groups, and gene silencing efficacy was 35.56 %, 89.22 %0 and 66.78%, respectively. The results demonstrated that the lentiviral vectors of RNAi tar- geting Beclinl gene were successfully constructed, and NSCLC A549 stable cell line with Beclinl gene knockdown was established. This study finally provided a new cell model to explore the biological behavior of the Beclinl gene in NSCLC A549 cells.
出处
《生物医学工程学杂志》
EI
CAS
CSCD
北大核心
2013年第1期131-135,共5页
Journal of Biomedical Engineering
基金
河南省科技创新人才工程项目资助(0623060900)
