人3突变型低氧诱导因子1α真核表达载体和腺病毒表达载体的构建及鉴定  

Construction and Identification of pShuttle2 and Adenovirus Vetor of Human Hypoxia-inducible Factor-1α of Triple Mutant

作　　者：赖艳娴[1] 刘城[1] 王月刚[1] 谢宜军[1] 童锴[1] 胡英芳[1] 韦莉莉[1] 吴平生[1] 

机构地区：[1]南方医科大学南方医院心内科,广州510515

出　　处：《中国医科大学学报》2008年第1期86-89,共4页Journal of China Medical University

基　　金：国家自然科学基金资助项目(30370587)

摘　　要：目的为了深入研究人低氧诱导因子1α基因对冠心病的血管新生作用,构建人3突变型低氧诱导因子1α腺病毒表达载体(pAdeno-HIF-1α-Ala402-Ala564-Ala803)。方法双酶切pShuttle2-HIF-1α-Ala402-Ala564、pShuttle2-HIF-1α-Ala564-Ala803,凝胶回收前者酶切片段中大小为300bp的小片段及后者酶切片段中大小为6300bp的大片段,并将两者连接,重组成3突变型穿梭质粒pShuttle2-HIF-1α-Ala402-Ala564-Ala803,并进行酶切和PCR鉴定。鉴定正确后,采用分子克隆技术,以PI-SceI和I-CeuI双酶切重组3突变型穿梭载体,获得含有3突变型HIF-1α(pShuttle2-HIF-1α-Ala402-Ala564-Ala803)的表达盒,通过体外连接法与线性化的腺病毒骨架载体Adeno-XTM Viral DNA连接,重组成3突变型腺病毒载体(pAdeno-HIF-1α-Ala402-Ala564-Ala803),再进行酶切及测序鉴定。结果经酶切鉴定及基因测序证实重组3突变型真核表达载体和腺病毒表达载体质粒构建成功。结论成功构建重组人3突变型低氧诱导因子1α真核表达载体(pShuttle2-HIF-1α-Ala402-Ala564-Ala803)和人3突变型低氧诱导因子1α腺病毒表达载体(pAdeno-HIF-1α-Ala402-Ala564-Ala803)。Objective Construct adenovirus vetor of human hypoxia-inducible factor-1α of triple mutant for further study about it＇s effect in therapeutic angiogenesis of coronary heart disease. Methods Double digest pShuttle2-HIF-1α-Ala402-Ala564.pShuttle2-HIF-1α-Ala564- Ala803,gel extraction the fragment of 300bp from the pShuttle2-HIF-1α-Ala402-Ala564 and 6300bp from the pShuttle2-HIF-1α-Ala564- Ala803 ,connect them,so that it＇s the recombinant of shuttle plasmid pShuttle2-HIF-1α-Ala402-Ala564-Ala803.Then assessment it by digestion and polymerase chain reaction （ PCR ） amplification.Digested it by PI-SceI and I-CeuI and then ligated to Adeno-X Viral DNA with in vitro ligafion. The recombinant adenovirus was confirmed by PCR and the sequencing was determined. Results The recombinant was correctly constructed and restriction endonuclease analysis and PCR amplification. Conclusion The recombinant of pShuttle2 and adenovirus vetor of human hypoxia-inducible factor-1α of triple mutant was successfully constructed. It provides further appreciable foundation of HIF-1 alpha gene theraphy for Coronary Athemsclemtic Heart Disease.

关 键 词：低氧诱导因子-1Α 基因治疗 血管新生 载体 

分 类 号：Q78[生物学—分子生物学]