中文摘要：

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pAdeno-EGFP-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-1 α 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5.1 kb 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2×10 10 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。

英文摘要：

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.