[Construction and identification of recombinant adeno-associated virus vector harboring fusion gene NT4-Apoptin-HA2-TAT].
OBJECTIVE
To construct a recombinant adeno-associated virus vector harboring fusion gene NT4-Apoptin-HA2-TAT, laying a foundation for gene therapy research of malignant tumors.


METHODS
The Apoptin and HA2-TAT gene were inserted in pUC19/NT4 vector after digested with restriction enzyme. The fusion gene of NT4-Apoptin-HA2-TAT was sub-cloned into the shuttle plasmid of adeno-associated virus; the products were co-transferred into HEK-293 cell line with helper plasmid pAAV/Ad and adeno-plasmid pFG140.The recombinant adeno-associated virus was produced by homologous recombination of above 3 plasmids in HEK-293 cells and its titer was measured by quantitative dot blot hybridization. The effect of AAV-NT4-Apoptin -HA2-TAT on HepG2 cell line was measured by a colorimetric 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay.


RESULTS
The NT4-Apoptin-HA2-TAT was confirmed by restriction enzyme digestion and DNA sequencing. High titer of recombinant adeno-associated virus was obtained by homologous recombination in HEK-293 cells (3.14 x 10(15) pfu/L). AAV-NT4-Apoptin-HA2-TAT had strong deduce apoptosis effect on HepG2 cells. Compared with Adeno-associated mock virus and in normal cell line NIH3T3, Aden-associated virus NT4-Apoptin-HA2-TAT significantly decreased the survival rate of HepG2 cells.


CONCLUSIONS
The recombinant adeno-associated virus vector encoding gene NT4-Apoptin-HA2-TAT has been successfully constructed in this experiment by molecular cloning and in vitro recombination techniques, laying a foundation for further research of gene therapy of cancer.
Wang
Jian-Sheng
JS
Department of Oncology Surgery, the First Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi'an 710061, China. wangjsh@mail.xjtu.edu.cn
Zhang
Ming-Xin
MX
Liu
De-Chun
DC
Duan
Xiao-Yi
XY
Zhou
Su-Na
SN
Zhang
Guang-Jian
GJ
Yang
Guang-Xiao
GX
Wang
Quan-Ying
QY
D000818:Animals; D036022:Capsid Proteins / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D000229:Dependovirus / Q000235:genetics*; D015696:Gene Products, tat / Q000235:genetics / Q000502:physiology*; D005822:Genetic Vectors / Q000235:genetics*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D041681:NIH 3T3 Cells; D009414:Nerve Growth Factors / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000502:physiology*
D036022:Capsid Proteins; D015696:Gene Products, tat; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D009414:Nerve Growth Factors; D011993:Recombinant Fusion Proteins