[Construction and expression of recombinant adeno-associated virus vector containing HSV1-TK gene].
Zhong Nan Da Xue Xue Bao Yi Xue Ban
; 33(3): 210-5, 2008 Mar.
Article
em Zh
| MEDLINE
| ID: mdl-18382054
ABSTRACT
OBJECTIVE:
To construct the recombinant adeno-associated virus(rAAV) vector plasmid pSNAV2.0-TK containing HSV1-TK gene, to produce recombinant adeno-associated virus rAAV2/HSV1-TK, and to detect the integration and expression of HSV1-TK gene in lens epithelial cells transfected by rAAV2/HSV1-TK, and to provide foundation for gene therapy of posterior capsular opacification.METHODS:
The recombinant vector plasmid constructed by gene recombinant technology was analyzed by PCR and restriction enzyme digestion. The cell strain BHK-21/TK was screened by G418 after the plasmid was transfected into BHK-21 cells,with the helper virus HSV1-rc/UL2 to produce the recombinant virus rAAV2/HSV1-TK. The purity of rAAV2/HSV1-TK was detected by SDS-PAGE and HPLC, and the titre of rAAV2/HSV1-TK was observed by dot blot hybridization. The HSV1-TK gene in lens epithelial cells transfected by rAAV2/HSV-TK was investigated by PCR and RT-PCR.RESULTS:
The recombinant plasmid proved successful by PCR and restriction enzyme digestion. The recombinant virus rAAV2/HSV1-TK was produced successfully and its titre was 1 x 10(12) v.g./mL by dot blot hybridization. The HSV1-TK gene was integrated and expressed in lens epithelial cells.CONCLUSION:
The recombinant adeno-associated virus vector plasmid containing HSV1-TK gene is successfully constructed, and high titre recombinant adeno-associated virus (rAAV2/HSV1-TK) is obtained. The HSV1-TK gene in lens epithelial cells is expressed after being transfected by rAAV2/HSV1-TK.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Timidina Quinase
/
Herpesvirus Humano 1
/
Dependovirus
/
Epitélio Corneano
Tipo de estudo:
Risk_factors_studies
Limite:
Animals
Idioma:
Zh
Ano de publicação:
2008
Tipo de documento:
Article