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目的:利用siRNA表达载体构建抑制人类心脏发育候选基因KLHL31表达的pSUPER RNAi载体(pSUPER-KLHL31)。方法:化学合成一对编码短发夹RNA序列的、靶向心脏发育候选基因KLHL31的寡核苷酸链60个碱基,退火,克隆到经BglⅡ、Xho Ⅰ双酶切的pSUPER质粒上,构建重组RNAi质粒(pSUPER-KLHL31),通过酶切鉴定及测序分析验证构建效果,将正确构建的质粒转染大鼠心肌细胞H9c2,建立产生逆转录病毒的细胞克隆。通过Western blotting检测转染pSUPER-KLHL31后细胞中KLHL31蛋白的表达量。结果:pSUPER-KLHL31载体经酶切鉴定及测序分析,结果表明60个碱基成功插入到预计位点,且序列完全一致;重组载体转染包装细胞,可表达绿色荧光蛋白,表明包装成功;Western blotting检测结果显示转染pSUPER-KLHL31的细胞中KLHL31蛋白表达量明显降低。结论:靶向KLHL31的pSUPER RNAi载体构建成功,为进一步从分子水平探讨KLHL31在心脏发育中的功能奠定了基础。
OBJECTIVE: To construct the pSUPER RNAi vector (pSUPER-KLHL31) that inhibits the expression of human cardiac development candidate KLHL31 by siRNA expression vector. METHODS: A pair of oligonucleotide strands targeting KLHL31 encoding short hairpin RNA sequence was chemically synthesized and annealed. The oligonucleotides were annealed and cloned into pSUPER plasmid digested with BglII and XhoI to construct Recombinant RNAi plasmid (pSUPER-KLHL31) was constructed and verified by restriction enzyme analysis and sequencing analysis. The constructed plasmid was transfected into rat cardiomyocytes H9c2 to construct a retrovirus-producing cell clone. The expression of KLHL31 protein in cells transfected with pSUPER-KLHL31 was detected by Western blotting. Results: The pSUPER-KLHL31 vector was identified by restriction enzyme digestion and sequencing analysis. The results showed that 60 bases were successfully inserted into the predicted site and the sequence was identical. The recombinant vector transfected into packaging cells expressed green fluorescent protein, indicating that the packaging was successful. The results of blotting showed that KLHL31 protein expression in transfected pSUPER-KLHL31 cells was significantly decreased. CONCLUSION: The pSUPER RNAi vector targeting KLHL31 is successfully constructed, which lays the foundation for further exploring the function of KLHL31 in cardiac development at the molecular level.