一种包装AAV2/5的重组单纯疱疹病毒的构建

为了得到一种可以包装AAV2/5和表达绿色荧光蛋白的重组单纯疱疹病毒,设计并构建了一个由AAV2rep基因和AAV5cap基因嵌合而成的rep2cap5基因,然后,利用一套携带HSV1基因组的粘粒系统(cos6、cos28、cos14、cos56、cos48),将rep2cap5基因插入cos6粘粒上HSV1基因组片段的UL2基因中,而将EGFP的表达单位插入cos56粘粒上HSV1基因组片段的UL44基因中,用这2个重组粘粒与其它3个粘粒(cos14、cos28、cos48)共转染BHK-21细胞获得了重组病毒HSV1-r2c5-EGFP并进行了空斑纯化。HSV1-r2c5-EGFP病毒能够在BHK-21细胞连续传代,并且可以观察到几乎所有的感染细胞都能产生绿色荧光。用PCR方法以及Southern杂交方法表明所获得的HSV1-r2c5-EGFP中携带有rep2cap5基因,用HSV1-r2c5-EGFP感染携带报告基因LacZ的AAV载体细胞株,获得了具有感染性的重组AAV2/5-LacZ。结果表明,所获得的重组单纯疱疹病毒HSV1-r2c5-EGFP可提供AAV2/5载体包装所需的全部辅助功能,是一种能简便、高效制备重组AAV2/5病毒的通用性辅助病毒。

A Recombinant Herpes Simplex Virus Type 1 for AAV2/5 Production

In order to get a recombinant HSV1(herpes simplex virus Type 1) which can express enhanced green fluorescent protein and help to produce AAV2/5(adeno-associated virus 2/5),we constructed a pUC18 based plasmid which is combined by the rep gene from AAV2 and the cap gene from AAV5.With the Helper Free system ,we found the plasmid pUC18-rep2cap5 is able to package AAV2/5.Then the rep2cap5 gene was cloned into the UL2 gene of HSV1,which genome was dispersed in five cosmids(cos6,cos28,cos14,cos56,cos48),while the UL2 gene was part of the cos6.Another gene egfp was cloned into the UL44 gene of HSV1.Both of them transfected into the BHK-21 cells with other three cosmids to acquire the recombinant virus HSV1-r2c5-EGFP.The rep2cap5 gene was proved to exist in the HSV1-r2c5-EGFP genome by PCR and Southern blotting.Under fluorescence microscope,we found the cell was green which indicated the egfp gene was correctly expressed.Furthermore,the BHK/AAV2-LacZ cells infected with HSV1-r2c5-EGFP was able to produce the infectious recombinant AAV2/5-LacZ.So the recombinant HSV1-r2c5-EGFP can serve as the helper virus to produce recombinant AAV2/5 and express EGFP correctly.