摘要
针对p58IPK基因设计2个靶位点,并根据靶位点合成2对寡聚核苷酸序列,退火后连接到经BglⅡ和HindⅢ双酶切的pSUPER载体上,分别构建抑制p58IPK蛋白基因表达的p58IPK-siRNA1和p58IPK-siRNA2载体并鉴定其功能。重组构建的pSUPER-p58IPK载体经酶切鉴定及基因序列分析,结果表明60个碱基成功插入到预计位点,并且序列完全一致。将构建的2个RNAi载体分别转染到293T细胞中,通过Real-time PCR和Western blotting的方法检测其对p58IPK的抑制效果,结果证明p58IPK-siRNA2可显著抑制细胞中p58IPK基因的表达,可为进一步研究p58IPK在禽流感病毒感染中的作用机制奠定基础。
The aim of this study is to construct RNAi vector which can inhibit the expression of p58IPK gene .Based on the analysis of p58IPK mRNA sequence,two siRNA binding sites were designed and se-lected .Two pairs of oligo nucleotides for targeting sites were synthesized,annealed and inserted into pSUPER vector digested with BglⅡand HindⅢ,respectively.The two recombinant p58IPK siRNA plas-mids were identified by restriction enzyme digestion and nucleotide sequencing .In order to test RNA in-terference efficiency,p58IPK-siRNA 1 and 2 were transfected into 293T cells,respectively .Real-time PCR and western blotting were used to detect p58IPK gene expression on mRNA and protein level .The re-sults showed that p58IPK-siRNA 2 significantly inhibited the expression of p58IPK,which could be used for exploring the role of p58IPK in influenza virus infection .
出处
《吉林农业大学学报》
CAS
CSCD
北大核心
2014年第1期102-105,共4页
Journal of Jilin Agricultural University
基金
国家重点基础研究发展计划项目("973"计划)(2012CB518900)
关键词
pSUPER载体
RNA干扰
p58IPK
Real-time PCR
p58IPK
pSUPER vector
RNA interference
Real time-PCR
