Generation of a series of recombinant herpes simplex viruses which can provide replicating and packaging functions for recombinant adeno-associated virus / 中华实验和临床病毒学杂志
Chinese Journal of Experimental and Clinical Virology
;
(6): 74-78, 2002.
Article
in Chinese
| WPRIM
| ID: wpr-355121
ABSTRACT
<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Physiology
/
Recombination, Genetic
/
Virus Replication
/
Cosmids
/
Transfection
/
Cells, Cultured
/
Herpesvirus 1, Human
/
Dependovirus
/
Genetic Vectors
/
Genetics
Limits:
Animals
Language:
Chinese
Journal:
Chinese Journal of Experimental and Clinical Virology
Year:
2002
Type:
Article
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