Construction and expression of recombinant adeno-associated virus vector containing HSV1-TK gene.
- Author:
Zhi-xiang DING
1
;
Qian TAN
;
Shuang-zhen LIU
;
Dan LIU
;
Zhong-qing LI
;
Jian-qiang PENG
Author Information
1. Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha 410008, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Cloning, Molecular;
Cricetinae;
Dependovirus;
genetics;
metabolism;
Epithelium, Corneal;
cytology;
metabolism;
Genetic Vectors;
Herpesvirus 1, Human;
enzymology;
genetics;
Rabbits;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
Thymidine Kinase;
biosynthesis;
genetics;
Transfection
- From:
Journal of Central South University(Medical Sciences)
2008;33(3):210-215
- CountryChina
- Language:Chinese
-
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.
