携带人分泌型内皮抑素基因重组腺病毒的制备和鉴定

目的制备包含人分泌型内皮抑素基因的重组腺病毒,为下一步探讨其在血管生成依赖性疾病的治疗研究中的应用提供基础。方法以T-Endostatin质粒为模板,通过PCR扩增回收hEndostatin,将hEndostatin连接到pShuttle2中,构建pShuttle2-hEndostatin表达质粒,将此表达质粒连接到缺陷型腺病毒载体,构建Ad-hEndo。其线性化后转染HEK293T细胞,纯化后的重组腺病毒Ad-hEndo在HEK293T细胞中大量扩增并通过氯化铯密度梯度离心法纯化,并测定其滴度。结果利用PCR反应进行鉴定,扩增得到的hEndostatin经酶切鉴定,DNA测序证实为重组腺病毒,测定病毒滴度为5.2×109PFU/ml。结论本实验成功构建了人分泌型内皮抑素重组腺病毒Ad-hEndo,可直接用于血管生成依赖性疾病基因治疗的实验研究。

Construction of a recombinant adenoviral vector expressing human endostatin

OBJECTIVE: To construct the recombinant adenovirus vector expressing human endostatin. METHODS: Human endostatin gene extracted from pGEM-T Easy vector containing the target gene fragment was successfully amplified using PCR and cloned into pShuttle2 vector. The target gene was subcloned into an adenovirus vector and the resulted recombinant adenovirus (Ad-hEndo) was linearized before transfected into HEK 293 packaging cells. The Ad-hEndo recombinant adenovirus was efficiently amplified in 293T cells and purified by CsCl density centrifugation, and the titer of the virus was determined. RESULTS: The amplified hEndostatin cDNA was verified by PCR and sequencing, and the resulted virus titer reached 5.2 x 10(9) pfu/ml. CONCLUSION: The recombinant adenovirus containing human endostatin gene has been successfully constructed, which may provide important basis for gene therapy research for angiogenesis-dependent diseases.