CAJ | 학술논문

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。
침대p58IPK기인설계2개파위점，병근거파위점합성2대과취핵감산서렬，퇴화후련접도경 BglⅡ화HindⅢ쌍매절적pSUPER재체상，분별구건억제p58IPK단백기인표체적p58IPK-siRNA1화p58IPK-siRNA2재체병감정기공능。중조구건적pSUPER-p58IPK재체경매절감정급기인서렬분석，결과표명60개감기성공삽입도예계위점，병차서렬완전일치。장구건적2개RNAi재체분별전염도293T세포중，통과Real-time PCR화Western blotting적방법검측기대p58IPK적억제효과，결과증명p58IPK-siRNA2가현저억제세포중p58IPK기인적표체，가위진일보연구p58IPK재금류감병독감염중적작용궤제전정기출。
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 .