摘要
目的:构建人突变型低氧诱导因子1(H IF-1)α腺病毒表达载体,研究人突变型HIF-1α基因对冠心病的血管新生作用.方法:采用分子克隆技术,由pcDNA3.1(+)-HIF1α(突变型)质粒获得突变型HIF1αcDNA,克隆到穿梭质粒pShuttle2,以PI-SceI和I-CeuI双酶切重组穿梭质粒,获得含有突变型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-X Viral DNA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2×1012pfu/L.结论:成功构建重组腺病毒Adeno-HIF1α(突变型),为冠心病的突变型HIF1α基因治疗研究奠定基础.
AIM： To construct adenovirus vector containing the mutant HIF1α gene and to study the effect of mutant hypoxia inducible factor-1α on the angiogenesis of coronary heart disease. METHODS： Human mutant HIF1α cDNA obtained from the plasmid pcDNA3. 1 （ ＋ ）-HIF1α was cloned into plasmid pShuttle2. The expression cassette containing mutant HIFla cDNA was obtained from the recombinant pShuttle2 with double digestion of PI-Sce Ⅰ and Ⅰ- Ceu Ⅰ and 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 lipofectamine2000 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 freeze-thawing to obtain the recombinant adenovirus in the lysate. The PCR product of the lysate confirmed the presence of recombinant adenovirus. The viral titer was 2 × 10^12 pfu/L. CONCLUSION： The recombinant adenovirus containing the mutant HIF1α gene has been successfully constructed, which paves the way for mutant HIF1α gene therapy of coronary heart disease.
出处
《第四军医大学学报》
北大核心
2005年第17期1614-1617,共4页
Journal of the Fourth Military Medical University
基金
国家自然科学基金(30370587)
关键词
低氧诱导因子1Α
突变
腺病毒
基因治疗
hypoxia inducible factor-1α
mutant
adenovirus vector
gene therapy
