Objective To construct lentiviral vector earring point mutant HIF-1a and to detect its anti-degradation effect under normoxic conditions. Methods Based on human HIF-1a mRNA sequences ( NM_001530 ), site-directed mutagenesis of HIF-1 a was made as follows: asparagines 803 to alanine, proline 564 to alanine and proline 402 to alanine. Three pairs of primers were designed and synthesized for introducing mutant sites. Sequence fragment was amplified by PCR and then PCR product of the target gene was ligated with vector pEGFP-N1 to construct eukaryotic expression vector pEGFP- N1-mutant/HIF-1a. After being identified by Pad and AscI, target sequence was recombined into lentiviral vector plenti6. 3 V5-DEST by LR recombination system to construct Lenti-MT ( mutant type HIF-1a) and Lenti-WT ( wild type HIF-1a ). Lenti-MT, Lenti-WT and Lenti-LacZ ( control group ) were transduced into 293T cells and the expression of target gene was detected by qPCR. The transduced cells were cultured for 48 h under hypoxic conditions and for another 48 h under normoxic conditions. The expression level of HIF-1a protein was analyzed by Western blot in different oxygen concentration conditions. Results MT had been completed when compared with the sequence of WT. pEGFP-N1-mutant/HIF-1 a was successfully constructed as confirmed by restriction enzyme digestion. qPCR results indicated that the expression of target gene in the Lenti-MT group was higher than that of the Lenti-WT group with a statistically signicant difference. The mRNA and protein expression of HIF-1 a in the MT and WT groups showed no significant difference under hypoxic conditions. However, HIF-1a protein expression in the WT group decreased remarkably compared with little change in the MT group after 48 h culture under normoxic conditions. These results proved that MT had obvious resistance to degradation. Conclusion The mutant HIF-1a has been successfully constructed and Lenti-MT has notable anti-degradation effect under normoxic conditions.%目的 构建点突变型低氧诱导因子-1α(HIF-1α)慢病毒载体,并检测其在常氧条件下的抗降解作用.方法 根据人的HIF-1α基因(NM_001530),对其全长编码区序列进行如下定点突变:803位天冬酰胺突变成丙氨酸,564位脯氨酸突变成丙氨酸,402位脯氨酸突变成丙氨酸.设计并合成3对引物用以导入变异点.PCR扩增序列片段,然后将目的 基因PCR产物连接到载体pEGFP-N1上,构建真核表达载体pEGFP-N1-mutant/HIF-1α.PacI和AscI酶切鉴定后,用LR重组系统将目的 序列重组到慢病毒载体plenti6.3V5-DEST上,构建慢病毒载体Lenti-MT(突变型HIF-1α)及Lenti-WT(野生型HIF-1α).将Lenti-MT、Lenti-WT及Lenti-LacZ(对照组)转染293T细胞后,采用qPCR检测目的 基因的表达.转染后的细胞在低氧条件下(2% O2)培养48 h,然后常氧条件下培养48 h,采用Western blot检测HIF-1α蛋白在不同氧浓度条件下的表达情况.结果 与WT测序结果对比表明MT已被完成.酶切结果证明已成功构建pEGFP-N1-mutant/HIF-1α.qPCR结果显示Lenti-MT组目的 基因表达高于Lenti-WT组,且差异具有统计学意义.低氧条件下,HIF-1α在WT组和MT组中的蛋白表达差异无统计学意义.但常氧条件下培养48 h后,WT组目的 蛋白的表达显著下降,而MT组变化不大.上述结果表明MT具有明显的抗降解作用.结论 成功构建了突变型HIF-1α,慢病毒载体在常氧条件下,Lenti-MT具有显著的抗降解作用.
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