腺相关病毒末端反向重复顺序折叠结构在真核细胞染色体基因组上的整合作用

Study on the Integration of the Adeno-associated Virus Inverted Terminal Repeat Fold Structure in Eukaryotic Cell Chromosome in Vitro

腺相关病毒（Ａｄｅｎｏ－ａｓｓｏｃｉａｔｅｄｖｉｒｕｓ，ＡＡＶ）是一类线状单股ＤＮＡ（ＳＳ－ＤＮＡ）的微小病毒。依据Ｃａｖａｌｉｅｒ－Ｓｍｉｔｈ的ＳＳ－ＤＮＡ病毒复制模型原理，将其末端反向重复顺序（ＩＴＲｓ）分离，变为折叠十字架结构，再以其发夹中的３＇－ＯＨ为引物合成另一股姊妹染色体ＤＮＡ，然后共价连接到亲代分子上，与辅助病毒Ａｄ２和ＰＡＡＶ／Ａｄ共转染真核生物细胞。Ｓｏｕｔｈｅｒｎ印迹分子杂交结果表明：插入这一折叠结构中由ＳＶ４０早期启动子控制的报告基因－新霉素抗性基因拷贝已整合到细胞基因组染色体上，这种以无细菌ＤＮＡ分子为支架的折叠结构不仅证明了Ｃａｖａｌｉｅｒ－Ｓｍｉｔｈ的理论，而且可望为外源基因导入真核细胞提供新的又一系统。

Adeno-associated virus is a single-stranded linear DNA parvovirus.Depending on Cavalier-Smith's model theory for the replication of SS-DNA virus,the inverted terminal repeat structure(ITRs)from PAAV/Neo was isolated,the linear fragment was denatured and refolded over,and the daughter DNA was synthesized by using 3'-OH terminus as a primer, then the new strand was covalently linked to the parental strand. After the palindromic structure with resistance neomycin DNA controlled by SV40 early promoter was transferred into eukaryotic cells,southern blot showed that the molecule of harpin structure could facilitate resistance neomycin DNA to integrate into chromosomes of mammalian cells in vitro.The experimental results indicate that the Cavalier-Smith's replicative model is correct and provide other new system for DNA transduction.