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目的构建人低氧诱导因子-1α(HIF-1α)真核表达载体pcDNA3.1+-HIF-1α的两种突变体pcDNA3.1+-HIF-1α-564Ala和pcDNA3.1+-HIF-1α-564Ala-803Ala,并检测它们在人肺微血管内皮细胞(HMVECs)中的表达。方法用定点突变的方法将pcDNA3.1+-HIF-1α的HIF-1α第564位脯氨酸(Pro)密码子CCC突变为丙氨酸(Ala)密码子GCC,构建成单个突变HIF-1α真核表达载体pcDNA3.1+-HIF-1α-564Ala。在第一次突变的基础上,仍然用定点突变的方法将pcDNA3.1+-HIF-1α-564Ala的HIF-1α-564Ala第803位天冬酰胺(Asn)密码子ATT突变为丙氨酸(Ala)密码子GCT,构建成双个点突变HIF-1α真核表达载体pcDNA3.1+-HIF-1α-564Ala-803Ala。将3种HIF-1α表达载体用脂质体法分别转入HMVECs,以RT-PCR、免疫荧光法和Westernblotting法分别对转染细胞进行表达分析。结果测序证实,定点突变成功,构建成重组真核表达载体pcDNA3.1+-HIF-1α-564Ala和pcDNA3.1+-HIF-1α-564Ala-803Ala;3种载体均能表达相应的蛋白和mRNA,但转化突变HIF-1α表达载体的细胞蛋白量比空白细胞和转化无突变HIF-1α载体的细胞显著增加。结论成功构建能够在HMVECs中表达的单个和双个点突变的HIF-1α基因的真核表达载体。
Objective To construct two kinds of pcDNA3.1 + -HIF-1α-564Ala and pcDNA3.1 + -HIF-1α eukaryotic expression vector of human hypoxia inducible factor-1α (HIF-1α) -564Ala-803Ala and tested for their expression in human pulmonary microvascular endothelial cells (HMVECs). Methods Site-directed mutagenesis was used to mutate the proline (Pro) codon CCC at position 564 of HIF-1α of pcDNA3.1 + -HIF-1α into alanine (Ala) codon GCC to construct a single mutant HIF-1α Eukaryotic expression vector pcDNA3.1 + -HIF-1α-564Ala. Based on the first mutation, site-directed mutagenesis was used to mutate ATT of Asn at codon 803 of HIF-1α-564Ala in pcDNA3.1 + -HIF-1α-564Ala to alanine Ala) codon GCT was constructed to construct a double point mutant HIF-1α eukaryotic expression vector pcDNA3.1 + -HIF-1α-564Ala-803Ala. Three HIF-1α expression vectors were transformed into HMVECs by lipofectamine respectively. The expression of HIF-1α was analyzed by RT-PCR, immunofluorescence and Western blotting. Results Sequencing confirmed that the site-directed mutagenesis was successful and was constructed into recombinant eukaryotic expression vector pcDNA3.1 + -HIF-1α-564Ala and pcDNA3.1 + -HIF-1α-564Ala-803Ala. All three vectors expressed the corresponding proteins and mRNA , But the amount of cellular protein of the transformed mutant HIF-1α expression vector was significantly higher than that of the blank cells and the cells transformed with the non-mutant HIF-1α vector. Conclusion The eukaryotic expression vector of single and double point mutated HIF-1α gene that can be expressed in HMVECs was successfully constructed.