一组可提供AAV载体复制和包装功能的重组HSV-1

目的 构建一组具有AAV载体复制和包装功能的重组HSV-1，从中选出功能较强者用于重组AAV的生产。方法 采用一套含有HSV-1基因组的粘粒系统构建重组HSV-1。首先将2型腺病毒伴随病毒（AAV-2）rep基因的起始密码子ATG人工定点突变成ACG；然后将自身启动子控制下的AAV-2rep(起始密码子突变或未突变）和cap基因分别插入粘粒上HSV-1的UL2基因或UL44基因，构建成重组粘粒cos6-rmc/△UL2,cos56-rc/△UL44cos56-rmc/△UL44；将重组粘粒上的HSV-1片段分别与HSV-1的其余片段进行同源重组，得到3株重组HSV-1，连同过去报道的一株重组HSV-1一起，分别命名为HSV1-rc/△UL2,HSV1-rmc/△UL2,HSV1-rc/△UL44,HSV1-rmc/△UL44。结果 4株重组HSV-1经PCR鉴定均含有rep基因，分别感染携带绿色荧光蛋白（GFP）基因的AAV载体细胞株后均能产生重组AAV，重组AAV可在BHK-21细胞中表达GFP，HSV1-rc/△UL2和HSV1-rmc/△UL2对AAV载体的包装能力明显强于HSV1-rc/△UL44和HSV1-rmc/△UL44。结论 构建的4株重组HSV-1均具有复制和包装重组AAV的能力，其中HSV1-rc/△UL2和HSV1-rmc/△UL2功能较强，有望用于重组AAV的大规模生产。

Generation of a series of recombinant herpes simplex viruses which can provide replicating and packaging functions for recombinant adeno-associated virus

BACKGROUND: To construct a series of recombinant herpes simplex viruses that can provide replicating and packaging functions for recombinant adeno-associated virus (rAAV), and to select the strains possessing stronger functions for large-scale production of rAAV. METHODS: A set of cosmids that represents the whole genome of HSV-1 was used to generate recombinant HSV-1 expressing rep and cap proteins of AAV-2. An ATG-to-ACG mutation in the start codon of AAV-2 rep protein was generated by site-directed mutagenesis. rep and cap genes, under control of their native promoters, with or without the ATG-to-ACG mutation in the start codon of rep, were inserted into the Xba site of UL2 or UL44 genes, respectively, resulting in the recombinant cosmids cos6-rmc/ UL2, cos56-rc/ UL44 and cos56-rmc/ UL44. Homologous recombination among the HSV-1 fragment on the recombinant cosmids and the rest fragments of HSV-1 genome resulted in three strains of recombinant HSV-1. Together with the one was constructed previously, there were four strains of recombinant HSV-1named HSV1-rc/ UL2, HSV1-rmc/ UL2, HSV1-rc/ UL44 and HSV1-rmc/ UL44 respectively. RESULTS: PCR detection confirmed the existence of rep- gene in the genomes of all four strains of the recombinant HSV-1. Recombinant AAV was produced after infecting the AAV vector cell line that carrying the GFP expression cassette with the four strains of recombinant HSV-1 respectively. However, HSV1-rc/ UL2 and HSV1-rmc/ UL2 produced much more rAAV than HSV1-rc/ UL44 and HSV1c/ UL44 did. CONCLUSIONS: All the four strains of recombinant HSV-1 support rAAV replication and packaging. HSV1 UL2 and HSV1-rmc/ UL2 that provide much stronger functions may be useful for large-scale production of rAAV.