HIF-1α和EGFP双基因共表达重组腺病毒载体的构建及鉴定  

Construction and identifi cation of recombinant adenovirus vector co-expressing HIF-1α and EGFP

作　　者：李永明[1] 吕刚[1] 范仲凯[1] 曹阳[1] 王岩松[1] 庄培袁 张玉强[1] 李刚[1] 

机构地区：[1]辽宁医学院附属第一医院,辽宁锦州121000

出　　处：《解放军医学院学报》2014年第2期166-169,177,共5页Academic Journal of Chinese PLA Medical School

基　　金：国家自然科学基金项目(81101421)~~

摘　　要：目的构建pAdeno-EGFP-HIF-1α重组腺病毒载体,探讨其在脊髓损伤中的作用。方法采用聚合酶链反应(polymerase chain reaction,PCR)法扩增目的基因HIF-1α并亚克隆到含有报告基因EGFP的pShuttle-CMV-EGFP穿梭载体中,得到pShuttle-EGFP-HIF-1α重组穿梭质粒。将已构建的重组穿梭质粒转移至pAdeno骨架载体上,获得pAdenoEGFP-HIF-1α重组腺病毒质粒,继而在H293细胞中扩增,纯化后进行PCR鉴定并测定病毒滴度。结果 pShuttle-EGFPHIF-1α经KpnI/BamHI双酶切后琼脂糖凝胶电泳可见阳性克隆组在2.5 kb和5.1 kb处出现两条带,而阴性克隆组只有5.1kb处一条带,证明pShuttle-EGFP-HIF-1α重组穿梭质粒构建成功;pShuttle-EGFP-HIF-1α重组穿梭质粒与pAdeno载体重组后,经XhoI单酶切后琼脂糖凝胶电泳可见阳性克隆组出现14.5 kb,11.7 kb,2.66 kb,2.6 kb,2.48 kb,2.47 kb,1.45 kb,0.6 kb八条带,而阴性克隆的腺病毒空载组有14 kb,11.8 kb,4.0 kb,2.47 kb,1.45 kb,0.6 kb六条带,证明重组pAdeno-EGFP-HIF1α腺病毒质粒构建成功;pAdeno-EGFP-HIF-1α重组腺病毒成功包装纯化后经PCR鉴定,实验组和阳性对照组中均扩增到2.5 kb片段,证明目的病毒中成功整合了HIF-1α基因;TCID50法测定纯化后的病毒滴度为2×1010 PUF/ml。结论成功构建了HIF-1α和EGFP双基因共表达重组腺病毒载体,为进一步研究其在脊髓损伤中的作用奠定了基础。Objective To study how to construct the recombinant adenovirus vector of pAdeno-EGFP-HIF-1 α . Methods The recombinant pShuttle-EGFP-HIF-1 α vector was constructed by amplifying HIF-1 α in PCR and cloned into the pShuttle-EGFPHIF- 1 α vector containing EGFP. The recombinant pAdeno-EGFP-HIF-1 α adenoviral vector was constructed by transferring the constructed recombinant pShuttle-EGFP-HIF-1 α vector to the pAdeno skeleton vector, amplified in H293 cells and purified. Their viral titer was measured and identified by PCR. Results Agarose gel electrophoresis displayed 2 fragments at 2.5 kb and 5.1 kb in positive clones and 1 fragment at 5.1 kb in negative clones of pShuttle-EGFP-HIF-1 α after KpnI/BamHI enzyme digestion, indicating that the recombinant pShuttle-EGFP-HIF-1 α vector was successfully constructed. Agarose gel electrophoresis showed 8 fragments at 4.5 kb, 11.7 kb, 2.66 kb, 2.6 kb, 2.48 kb, 2.47 kb, 1.45 kb, 0.6 kb in positive clones and 6 fragments at 14 kb, 11.8 kb, 4.0 kb, 2.47 kb, 1.45 kb, 0.6 kb in negative clones of recombinant pShuttle-EGFP-HIF-1 α vector and pAdeno vector, indicating that the recombinant pAdeno-EGFP-HIF-1 α adenovirus vector was successfully constructed. PCR showed that the recombinant pShuttle-EGFP-HIF-1 α adenovirus vector was amplified to a 2.5 kb fragment after package and purification, suggesting that the HIF- 1 α was integrated in the adenovirus with a titer of 2 × 10 10 pfu/ml as measured by TCID50. Conclusion The successful construction of recombinant pAdeno-EGFP-HIF-1 α adenoviral vector lays a foundation for the further study of its role in spinal cord injury.

关 键 词：低氧诱导因子-l α 增强型绿色荧光蛋白 腺病毒载体 基因重组 

分 类 号：R683.2[医药卫生—骨科学]