突变型低氧诱导因子-1α腺病毒载体的构建及常氧表达

Construction and normoxic expression of mutant hypoxia inducible factor-1α adenovirus vector

目的为了在常氧条件下观察低氧诱导因子-1α（HIF-1α）在骨缺损部位促新血管生成作用，构建能够同时表达突变型HIF-1仅和人源化绿色荧光蛋白（hrGFP）的腺病毒载体并检测其在兔骨髓间充质干细胞（MSCs）中的表达。方法利用聚合酶链式反应（PCR）定点突变人HIF-1α编码区（CDS）第402、564和803位氨基酸，将突变后HIF-1α和hrGFP重组入pAdEasy-1系统，包装病毒并测定滴度。以感染复数（MOI）=100将腺病毒转染MSCs，逆转录-聚合酶链反应（RT—PCR）和Westernblot方法检测转染细胞中HIF-1αmRNA和蛋白表达。结果（1）第402、564和803位氨基酸均定点突变为丙氨酸。（2）A、B两组HIF-10tmRNA表达量明显高于C、D两组，差异有统计学意义（P〈0．01）；A组HIF-1α蛋白表达量明显高于其他3组，差异有统计学意义（P〈0．01）。结论（1）腺病毒载体Ad—HIF-1α mu-IRES—hrGFP-1构建成功。（2）突变后HIF-1α基因能够在常氧条件下大量且高效表达。

Objective To study the angiogenesis promoting effect of hypoxia inducible factor （HIF）-1α gene at bone detect and loss location under normoxic conditions, construct a new adenovirus vector which can express mutant hypoxia HIF-1α and human renilla reniformis green fluorescent protein （hrGFP） simultaneously and detect the expression of the vector in rabbit marrow stromal cells （MSCs）. Methods Site-directed mutagenesis of the 402, 564 and 803 position amino acids in HIF-1α gene coding sequence （CDS） was clone by using polymerase chain reaction （PCR） technique. The mutant HIF-1α and hrGFP gene were recombined into pAdEasy-1 adenovirus system, the viruses were packed and the titer was assayed. The recombinant adenovirus was transfeeted into MSCs following MOI = 100, and the HIF-1α mRNA and protein expression was detected by using reverse transcription （RT） -PCR and Western blotting. Results （ 1 ） The 402, 564 and 803 amino acids all had site-directed mutagenesis into 2-aminopropionic acid. （2） There was statistically significant difference in the HIF-1α mRNA expression between group A or B and group C or D （P 〈0. 01 ）. The HIF-1α protein expression in group A was significantly higher than other three groups （ P 〈 0. 01 ）. Conclusion （ 1 ） A novel adenovirus vector namely Ad-CMV-HIF-1α mu- IRES-hrGFP-1 was constructed successfully; （2） The expression of HIF-1α gene after mutation is abun- dant and efficient under the normoxie conditions.