表达Kallistatin的9型重组腺相关病毒的制备及其体外表达效率

Production and Expression Efficiency in Vitro of Recombinant Adeno-Associated Virus 9 Mediating Kallistatin

采用三质粒磷酸钙共转染生产系统,制备出可以表达内源性抗肿瘤血管生成因子(Kallistatin)的9型重组腺相关病毒(rAAV2/9)和2型重组腺相关病毒(rAAV2/2).rAAV经银染法鉴定其纯度;采用WesternBlotting法比较rAAV2/9和rAAV2/2外壳蛋白的大小;qPCR方法检测rAAV的滴度.使用rAAV2/9-Kal-listatin和rAAV2/2-Kallistatin分别感染人脐静脉血管内皮细胞(HUVEC)后,RT-PCR和ELISA法分别检测Kallistatin的mRNA和蛋白表达水平.结果表明:纯化的病毒纯度达到98%以上;rAAV9外壳蛋白比rAAV2的大.感染HUVEC后,rAAV2/9介导Kallistatin在mRNA和蛋白表达水平都要比rAAV2/2高.

Recombinant adeno-associated virus 9（rAAV2/9） and recombinant adeno-associated virus 2（rAAV2/2） had been produced by three plasmids packaging system.rAAV2/9 and rAAV2/2 could express Kallistatin that had the activity of anti-angiogenesis.The purity of rAAV was detected by Silver stain.The molecular weight of rAAV2/9 and rAAV2/2 were detected by Western Blotting.The titers of rAAV were detected by real-time quantitative PCR（qPCR）.After rAAV2/9-or rAAV2/2-Kallistatin infecting human umbilical vein endothelial cell（HUVEC）,the expression levels of mRNA and protein were detected by reverse transcription PCR（RT-PCR） and enzyme-linked immunosorbnent assay（ELISA） respectively.The results indicated that the purity of rAAV was more than 98% and molecular weight of rAAV2/9 was more than rAAV2/2′s.After infecting HUVEC,the expression levels of mRNA and protein of Kallistatin mediated by rAAV2/9 were higher than rAAV2/2′s.