【摘要】 目的:构建携带prohibitin（PHB-1）基因的MiR RNAi真核表达载体pcDNA^TM6.2-GW/EmGFP-MiR,并观察其转染HEK293细胞株前后PHB-1的表达变化。方法:根据GenBank中prohibitin的序列,设计特异的两条互补的单链寡核苷酸退火后形成双链,克隆至pcDNA^TM6.2-GW/EmGFP-MiR质粒缺口末端,连接在质粒上生成含MiR RNAi pcDNA^TM 6.2-GW/EmGFP-MiR-PHB载体,测序鉴定后,用脂质体将重组子转染至HEK293细胞中,用Western blotting检测干扰后HEK293细胞内PHB-1表达的变化。结果:将目的序列成功连接到载体上,并经测序分析证实载体构建成功。Western blotting检测结果证实构建的PHB-1 MiR RNA表达重组体可显著抑制HEK293细胞内PHB-1的表达。结论:成功构建了携带PHB-1基因的MiR RNAi真核表达载体。更多还原

【Abstract】 Aim:To construct the RNAi eukaryotic vector of inhibitory member of the prohibitin(PHB-1)gene and observe the interfering effect in HEK293 cell line after the vector transfection.Methods:The specific Mi RNA sequence was designed according to the PHB-1 sequence in GenBank,complementary single-strand DNA oligonucleotides were designed and synthesized,and annealed the single-stranded oligonucleotides to generate a double strands oligonucleotides,cloned the oligonucleotides into pcDNATM6.2-GW/EmGFP-MiR-PHB to obtain an entry clone and then sequence analysis was performed.The recombinant plasmid was transfected into HEK293 cell by liposome.PHB-1 expression was detected by Western blotting.Results:The DNA sequence of interest clone to the vector was constructed to generate an entry clone and an ex- pression clone successfully,which were proved by sequence determination.Western blotting analysis demonstrated that PHB-1 MiR RNA expression construction could suppress the expression of PHB-1.Conclusion:A RNAi eukaryotic vector containing prohibitin gene was successfully constructed.更多还原