人双突变型低氧诱导因子1α腺病毒载体的构建及鉴定

Construction and identification of adenovirus vector for double-mutant human hypoxia-inducible factor-1α gene

目的构建人双突变型HIF-1α-Ala402-Ala564腺病毒表达载体,研究人双突变型低氧诱导因子1α基因对冠心病的血管新生作用。方法在业已完成的pShuttle2-HIF-1α-Ala564的基础上,用PCR定点突变的方法将其第402位脯氨酸密码子CCA突变为丙氨酸密码子GCA,构建成双突变HIF-1α真核表达载体pShuttle2-HIF-1α-Ala402-Ala564,通过体外连接法与线性化的腺病毒骨架质粒连接,重组成pAdeno-HIF-1α-Ala402-Ala564腺病毒质粒,经酶切及测序鉴定正确后,包装成为重组Adeno-HIF-1α-Ala402-Ala564腺病毒。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。结论成功构建重组腺病毒Adeno-HIF-1α-Ala402-Ala564(双突变型),为冠心病的突变型HIF-1α基因治疗研究奠定基础。

Objective To construct an adenovirus vector containing the double-mutant hypoxia-inducible factor-1α （HIF-1α） gene for exploring the therapeutic angiogenesis for coronary heart disease, Methods Human double-mutant HIF-1α cDNA was obtained from PCR of pShuttle-2-HIF-1α containing the mutant HIF-1α gene （564）. The recombinant adenoviral plasmid containing mutant HIF-1α cDNA was constructed by ligation of recombinant pShuttle2 containing double-mutant HIF-1α cDNA and Adeno-X viral DNA, followed by its identification and transfection into adenoviral packaging cells HEK293 via lipofectamine 2000, Result and Conclusion The recombinant pAdeno-HIF-1α was correctly constructed and verified by restriction endonuclease and DNA sequencing analyseis. This recombinant adenovirus containing the double-mutant HIF-1α gene may facilitate further investigation of mutant HIF-1α gene therapy for coronary heart disease.