人低氧诱导因子1α腺病毒载体的构建及鉴定

Construction and identification of human hypoxia inducible factor-1αadenovirus vector

目的构建人HIF1α腺病毒表达载体,研究人低氧诱导因子1α基因对冠心病的血管新生作用。方法采用分子克隆技术,从pcDNA3.1/V5-HisA-HIF1α质粒获得HIF1αcDNA,经pcDNA3.1(+)克隆到穿梭质粒pShuttle2,以PI-SceI和I-CeuI双酶切重组穿梭质粒,含有HIF1αcDNA的表达盒通过体外连接法与线性化的腺病毒骨架质粒Adeno-XViralDNA连接,重组成pAdeno-HIF1α腺病毒质粒,经酶切及测序鉴定正确后,在HEK293细胞中包装成为重组Adeno-HIF1α腺病毒,并进行PCR鉴定及滴度测定。结果经酶切鉴定及基因测序证实重组腺病毒质粒构建成功,包装后冻融细胞的上清PCR检测重组腺病毒包装成功,病毒滴度为2×109pfu/mL。结论成功构建重组腺病毒Adeno-HIF1α,为冠心病的基因治疗研究奠定基础。

[Objective] To construct adenovirus vector containing the HIF1α gene for studying therapeutic angiogenesis of coronary heart disease. [Methods] Human HIF1α cDNA obtained from the plasmid pcDNA3.1/V5HisA-HIF1α was cloned into plasmid pcDNA3.1（＋） and further cloned into plasmid pShuttle2. The expression cas- sette containing HIF1α cDNA was obtained from the recombinant pShuttle2 with double digestion of PⅠ- Sce Ⅰ and Ⅰ- Ceu Ⅰ, then ligated to Adeno-X Viral DNA with in vitro ligation. The recombinant adenoviral plasmid was identified and transfected into the adenoviral packaging cell HEK293 by lipofectamine 2000 mediated gene transfer method to pack the virus. The recombinant adenovius was confirmed by polymerase chain reaction （PCR） and the titer was determined. [Results] The recombinant pAdeno-HIF1α was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. The transfected HEK293 cells were lysed by freezethawing to obtain the recombinant adenovirus in the lysate. The PCR product of the lysate confirmed the presence of recombinant adenovirus. The viral titer was 3×10^9 pfu/mL. [Conclusion] The recombinant adenovirus containing the HIF1α gene was successfully constructed. It provided the further foundation of HIF1α gene therapy for coronary heart disease.