一组可提供AAV载体复制和包装功能的重组HSV-1

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

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 ΔUL2、cos5 6 rc ΔUL44cos5 6 rmc ΔUL44 ;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 ΔUL2、HSV1 rmc ΔUL2、HSV1 rc ΔUL44、HSV1 rmc ΔUL44。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 2 1细胞中表达GFP。HSV1 rc ΔUL2和HSV1 rmc ΔUL2对AAV载体的包装能力明显强于HSV1 rc ΔUL44和HSV1 rmc ΔUL44。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 ΔUL2和HSV1 rmc ΔUL2功能较强 。

Objective 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 Ⅰ site of UL2 or UL44 genes, respectively, resulting in the recombinant cosmids cos6-rmc/ΔUL2, cos56-rc/ΔUL44 and cos56-rmc/ΔUL44. Homologous recombination among the HSV-1 fragment on the recombinant cosmids and the rest fragments of HSV-1genome resulted in three strains of recombinant HSV-1. Together with the one was constructed previously, there were four strains of recombinant HSV-1 named HSV1-rc/ΔUL2,HSV1-rmc/ΔUL2,HSV1-rc/ΔUL44 and HSV1-rmc/ΔUL44 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/ΔUL2 and HSV1-rmc/ΔUL2 produced much more rAAV than HSV1-rc/ΔUL44 and HSV1-rmc/ΔUL44 did. Conclusion All the four strains of recombinant HSV-1 support rAAV replication and packaging. HSV1-rc/ΔUL2 and HSV1-rmc/ΔUL2 that provide much stronger functions may be useful for large-scale production of rAAV.