[Construction and expression of recombinant adeno-associated virus vector containing HSV1-TK gene].
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.


CONCLUSIONS
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.
Ding
Zhi-xiang
ZX
Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha 410008, China.
Tan
Qian
Q
Liu
Shuang-zhen
SZ
Liu
Dan
D
Li
Zhong-qing
ZQ
Peng
Jian-qiang
JQ
D000818:Animals; D003001:Cloning, Molecular; D006224:Cricetinae; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism*; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection
D011993:Recombinant Fusion Proteins; D013937:Thymidine Kinase