构建双突变型低氧诱导因子1α腺病毒载体的实验  

Construction of the adenovirus vector containing double mutant human hypoxia-inducible factor-1 alpha

作　　者：胡英芳[1] 王月刚[1] 谢宜军[1] 赖文岩[1] 赖艳娴[1] 吴平生[1] 

机构地区：[1]南方医科大学南方医院心内科,广东省广州市510515

出　　处：《中国组织工程研究与临床康复》2007年第33期6565-6568,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金(30370987)~~

摘　　要：目的:前期实验已成功构建无突变及564位是单突变腺病毒载体。在此基础上构建人双突变型低氧诱导因子1α腺病毒表达载体,为进一步研究低氧诱导因子1α基因及其突变体的临床应用奠定基础。方法:实验于2005-12/2006-12在南方医院心内科重点实验室完成。①实验材料:限制性内切酶PacI(NEB),PI-SceI、I-CeuI(Clontech);IPTG、X-Gal(Takara),转染试剂Lipofectamine2000(Invitrogen),凝胶回收试剂盒(Omega);DMEM(Omega);小牛血清(PAA);北京天为时代公司病毒基因组提取试剂盒;HIF-1α单克隆抗体(Chemicon International公司);羊抗鼠辣根过氧化物酶标记二抗(北京鼎国生物公司)。质粒、菌珠、细胞系等:Adeno-XTM-Adenoviral Expression Systems(BD.CLONTECH):包括有pShuttle2、pShuttle2-lacZ、Adeno-XTMViralDNA等;重组穿梭质粒pShuttle2-HIF-1α-Ala564-Ala803及pShuttle2-lacZ(本室已构建成功);大肠杆菌DH5α(本室保存);HEK293A细胞(中科院上海细胞所)。②实验方法:采用分子克隆技术,以PI-SceI和I-CeuI双酶切重组穿梭质粒pShuttle2-HIF-1α-Ala564-Ala803,然后切下含有低氧诱导因子1αcDNA表达的盒片段,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-HIF-1α腺病毒质粒,经酶切及测序鉴定正确后,在HEK293A细胞中包装成为重组腺病毒Adeno-HIF-1α-Ala564-Ala803。③实验评估:进行PCR鉴定及滴度测定。用重组腺病毒转染293A,采用Westernblot鉴定低氧诱导因子1α蛋白表达。结果:提取重组腺病毒质粒pAdeno-HIF-1α-Ala564-Ala803以引物A、B和引物1、2、3、4进行PCR鉴定,分别扩增出287,460,214bp的3种引物片段,与预期一致,经酶切鉴定及基因测序证实重组腺病毒质粒构建成功。pAdeno-lacZ转染293A细胞后X-Gal原位染色,显示约有近20%左右细胞染成蓝色,病毒滴度为6.8×107pfu/mL。Adeno-HIF-1α-Ala564-Ala803转染293A后稳定表达低氧诱导因子1α蛋白。结论:成功构建了重组腺病毒�In the previous experiments, the adenovirus vector with no mutant and single mutant at 564 site has been constructed successfully. In this study, the adenovirus vector containing double mutant hypoxia inducible factor-lα （HIF-1α） gene was constructed, so as to provide the basis for the further study on the effects of HIF-1α and its mutant on angiogenesis in coronary heart disease and the clinical practice in future. METHODS： The experiment was carried out in the Key Laboratory of Department of Cardiology, Nanfang Hospital from December 2005 to December 2006. ①The materials included restriction enzyme Pacl （NEB）, PI-Scel, I-Ceul （Clontech）; IPTG, X-Gal （Takara）, transfection agent Lipofectamine2000 （Invitrogen）, gel retrieval kit （Omega）; DMEM （Omega）; fetal bovine serum （PAA）; extract kit （Beijing Tiangen Biotech Co., Ltd）; HIF-1α monoclonal antibody （Chemicon International Company）; plasmids, bacterium, and cell line： Adeno-XTM-Adenoviral Expression Systems （BD.CLONTECH） including pShuttle2, pShuttle2-1acZ, Adeno-XTM Viral DNA; recombinant shuttle plasmids pShuttle2-HIF-1α-Ala564-Ala803 and pShuttle2-1acZ; E. coil DH5α （Key Laboratory of Department of Cardiology, Nanfang Hospital）; HEK293A cells （Shanghai Biochemistry and Cell Biology, Chinese Academy of Sciences）. ②The expression cassette containing HIF-Iα cDNA was obtained from the recombinant pShuttle2- H IF-1α-Ala564-Ala803 with double digestion of PI-Sce I and I- Ceul, then ligated to Adeno-X Viral DNA by in vitro ligation. The recombinant adenoviral plasmid was identified and transfected into the adenovira.I packaging cell HEK293A by Lipofectamine 2000 mediated gene transfer method to pack the virus. ③The recombinant adenovirus was confirmed by polymerase chain reaction （PCR） and the titer was determined. The protein of HIF was detected by Westem blot. RF^ULTS： The recombinant pAdeno-HIF1α-Ala564-Ala803 was extracted and identified by primers A, B and primers 1, 2, 3, and 4. Three

关 键 词：低氧诱导因子 冠状动脉粥样硬化性心脏病 基因治疗 腺病毒载体 

分 类 号：R541.4[医药卫生—心血管疾病]