【摘要】 目的构建并产生肿瘤血管抑制肽alphastatin(Al)重组腺伴随病毒(rAAV)载体。方法将目的基因alphastatin插入载体质粒pSSHG-巨细胞病毒(CMV)的EcoRI和BamHI位点,构建重组质粒pSSHG-CMV/NT4-Al。用腺病毒辅助质粒pFG140代替野生型腺病毒,包装质粒pAAV/Ad及已构建的重组腺伴随病毒载体质粒,使用三质粒共转染法转染293细胞,包装得到腺伴随病毒(AAV)-Al。采用氯仿抽提、聚乙二醇沉淀回收纯化,斑点杂交方法测定重组病毒滴度。结果重组病毒rAAV-Al滴度约为2×1012颗粒/ml。结论成功制备了重组病毒rAAV-Al,提供了一种生产足量安全的rAAV-Al简单易行的方法,为肿瘤的基因治疗实验奠定了基础。更多还原

【Abstract】 Objective To construct and produce the vector of antiangiogenic peptide alphastatin (Al) recombinant adeno-associated virus (rAAV).Methods By inserting the resulting gene of Al into the EcoR I and BamH I sites of vector plasmid pSSHG- cytomegalovirus (CMV), the recombinant plasmid vector pSSHG-CMV/NT4-Al was constructed. The 293 cells were co-transfected with the recombinant plasmid, packaging plasmid pAAV/Ad and helper adenovirus plasmid pFG140 instead of wild type adenovirus. The rAAV-A1 viral stock was purified by chloroform and polyethylene glycol precipitation and titrated by dot blot.Results The titer of rAAV-A1 vector was approximately 2×10 12 particles/ml.Conclusion The viral stock of rAAV-Al is successfully prepared, which can lay a basis for the experimental studies of gene therapy of tumors.更多还原