自分泌运动因子受体基因及其干扰RNA表达载体的构建

Construction of expression vector of autocrine motility factor receptor and its interference RNA

目的构建AMFR基因的表达载体和AMFRsiRNA表达载体,观察AMFRsiRNA对AMFR基因表达的影响。方法采用PCR技术扩增AMFR基因,并将其插入到带FLAG标签的真核表达载体上;利用RNAi技术,设计并合成了两条针对AMFR基因的siRNA,将其克隆到pSliencer 2.1-U6 neo表达载体上。将构建的AMFR基因表达载体和AMFRsiRNA表达载体共转染人胚肾细胞293T,通过Western blot实验了解AMFRsiRNA对AMFR基因表达的影响。结果通过双酶切和测序鉴定表明,构建的AMFR基因表达载体和AMFRsiRNA表达载体序列正确;通过West-ern blot实验证明,构建的AMFRsiRNA能有效抑制AMFR基因的表达。结论成功构建了AMFR基因表达载体和AMFRsiRNA表达载体,且表达的AMFRsiRNA能有效地抑制AMFR基因的表达。

Objective To construct the expression vector of AMFR gene and AMFR siRNA,and to investigate the effect of AMFR siRNA on the expression of AMFR gene.Methods AMFR gene was amplified by PCR technique and the product of PCR was inserted into the pcDNA3-FLAGC eukaryotic expression vector.Two AMFR siRNAs were developed by RNAi technique and the AMFR siRNAs were inserted into the pSliencer 2.1-U6 neo expression vector.Then human embryo kidney 293T cells were co-transfected with the AMFR siRNA expression vectors and FLAG-tagged AMFR expression vector.The effect of AMFR siRNAs on the expression of AMFR gene was identified by Western blot.Results The expression vectors of AMFR gene and AMFR siRNAs were constructed and confirmed by restriction enzyme digestion and DNA sequence analysis.Western blot showed that AMFR siRNAs could effectively inhibit the expression of AMFR gene.Conclusion The expression vectors of AMFR gene and AMFR siRNAs were successfully constructed,and the AMFR siRNAs effectively inhibited the expression of AMFR gene.