重组AAV1-Luc病毒制备及体外转导培养细胞的特性

Preparation of rAAV1-Luc and Study on Transduction of Mammalian Cells in vitro

目的制备携带萤火虫荧光素酶基因的重组AAV1病毒(rAAV1-Luc),研究该病毒体外转导培养细胞的特性。方法通过"1株载体细胞/1株辅助病毒"的双因素包装策略制备出rAAV1-Luc,研究该重组病毒体外转导哺乳动物细胞的量效关系,肝素对转导的拮抗作用、rAAV1的竞争抑制作用及丁酸钠对表达水平的增强作用。结果在一定范围内,随着rAAV1-Luc感染细胞的感染复数(MOI,即每个细胞感染的病毒基因组数)值增高,荧光素酶的表达水平也增高;但更高的MOI(>107)反而使荧光素酶的表达水平下降。肝素不能特异性阻断rAAV1介导的荧光素酶表达。携带不同基因的2种rAAV1病毒相互具有明显的竞争抑制作用。丁酸钠可显著增强rAAV1介导的荧光素酶表达水平。结论该研究揭示了rAAV1载体一定的细胞转导特性,对应用rAAV1载体介导基因转移研究具有指导意义。

Objective To prepare the rAAV1-Luc and study on transduction of mammalian cells with in vitro. Methods The rAAV1-Luc was prepared by ＂one helper virus-he vector cell line＂ strategy. The dose-effect relationship,competitive inhibition of rAAV1, the antagonistic effect of .heparin, the effect of butyrate on Luciferase expression levels with rAAV1-Luc in vitro were studied. Results The results showed that,in a certain extent,the higher the MOI ,the higher the LUCiferase expression levels. But the expression levels were decreased when the MOI was higher than 1×10^7. Butyrate could remarkably intensify the Luciferase expression levels by rAAV1. The Luciferase expression was not specially inhibited by heparin.The two kinds of rAAV1 containing different genes had remarkable competitive inhibition effect. Conclusion The study reveals that some transduction characteristics of rAAV1 vector which can be used as guidance in some extend on study of gene transfer by rAAV 1 vector.