摘要
目的 构建大鼠β防御素2（rBD2）基因RNAi慢病毒重组载体,转染培养细胞,检测其沉默效应,筛选出最佳的RNAi慢病毒载体.方法 序列软件设计3条针对rBD2基因CDS区的siRNA序列,合成单链后退火形成双链DNA,分别与酶切处理的慢病毒载体lentivirus连接构成3个RNAi慢病毒重组载体,再转化细菌,测序鉴定.脂质体法转染细胞,荧光实时定量PCR（RT-PCR）及Western免疫印迹测rBD-2 mRNA及蛋白表达,筛选沉默效果最佳的为LV-shrBD2载体.用慢病毒包装系统对LV-shrBD2进行慢病毒颗粒包装并梯度稀释法测定病毒滴度.结果 凝胶电泳显示3个RNAi慢病毒重组载体的PCR产物为316 bp,测序结果表明序列正确.转染细胞后RT-PCR及Western免疫印迹检测,siRNA序列1构建的重组载体mRNA抑制率达82%,干扰效率最高,为所需的rBD2基因RNAi慢病毒载体LV-shrBD2.包装慢病毒颗粒并调整病毒滴度至1×105ifu/μl.结论 成功构建并筛选出沉默效应最佳的rBD2基因RNAi慢病毒表达载体LV-sh1rBD2,为进一步开展rBD2研究提供了依据.
Objective To construct RNAi lentiviral recombinant vector of rat β-defensin-2 （rBD2） gene, detect its silencing effect by transfecting cultured cells, and to identify the RNAi lentiviral vector with best silencing effects. Methods Three siRNA sequences binding to CDS region of rBD2 gene were designed with software and used to generate single stands which formed double-stranded DNA by annealing. Three RNAi lentiviral recombinant vectors were constructed by connecting these DNAs to enzyme- digested lentivirus vectors and were identified by sequencing after transfecting into bacteria. Recombinant vector was transfected into cells by liposome. Expressions of rBD2 mRNA and protein were tested with fluorescence RTPCR and Western blotting. Recombinant vector with best silencing effect was screened as LV-shrBD2. The virus- like particles of LV- shrBD2 was packed with lentiviral packaging system and the viral titer was determined by slow-gradient dilution. Results Gel electrophoresis showed that the PCR products of three siRNAs were 316 bp in size with correct sequences. RT-PCR and Western blotting indicated that the recombinant vector with siRNA sequence 1 exhibited the best interference effect with an inhibition rate of 82%, and was therefore identified as rBD2 gene RNAi lentiviral vector LV-shrBD2. The lentiviral vector particle packaging was complete, and the virus titer was adjusted to 1×105 ifu/μl. Conclusion The RNAi lentiviral expression vector of rBD2 gene LV-shrBD2 that exhibits best gene-silencing effect is successfully constructed, which may provide evidences for further research of rBD2.
出处
《中华生物医学工程杂志》
CAS
2010年第5期453-456,共4页
Chinese Journal of Biomedical Engineering
基金
国家自然科学基金（30670932）
上海浦东新区社会发展局基金（pw2005A-10）
上海浦东新区医学领先人才培养计划（PwRd2007-17）
关键词
β防御素2
转染
慢病毒属
RNA干扰
沉默效应
β-defensin-2 （rBD2） Transfection Lentivirus RNA interferenc Silencing effect
