[Construction and identification of recombinant adeno-associated virus vector harboring fusion gene NT4-Ant-Shepherdin[79-87]].
OBJECTIVE
To construct a recombinant adeno-associated virus vector harboring fusion gene NT4-Ant-Shepherdin[79-87] and investigate Survivin as a anticancer therapeutic target by use of Shepherdin[79-87].


METHODS
The gene of Ant-Shepherdin[79-87] was obtained by PCR and T-vector method. After inserted in PBV220-NT4 vector and digested with restricted enzyme, The fusion gene of NT4-Ant-Shepherdin[79-87] 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 Dot-blot hybridization. The effect of rAAV-NT4-Ant-Shepherdin[79-87] on A549 cell line was measured by a colorimetric 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay.


RESULTS
DNA sequencing results verified that the sequence of Ant-Shepherdin[79-87] was consistent with that we had designed. After transformed E.coli DH5alpha, a fragment of 321 bp was confirmed. High titer of recombinant adeno-associated virus was obtained by homologous recombination in HEK-293 cell lines (3.4x10(13)pfu/L). rAAV-NT4-Ant-Shepherdin[79-87] had strong induce apoptosis effect on A549 cells.


CONCLUSIONS
The recombinant adeno-associated virus vector encoding fusion gene NT4-Ant-Shepherdin[79-87] is successfully constructed in this experiment by molecular cloning and in vitro recombination techniques, which provided the basis of further research of Survivin for cancer gene therapy.
Tang
Xiao-Jiang
XJ
Department of Oncosurgery, the First Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an 710061, China. txjiang@hotmail.com
Ping
Bao-Hua
BH
Pan
Cheng-En
CE
Yang
Guang-Xiao
GX
Wang
Quan-Ying
QY
D017209:Apoptosis / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D045744:Cell Line, Tumor; D000229:Dependovirus / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D010446:Peptide Fragments / Q000235:genetics / Q000502:physiology*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000502:physiology*
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C503112:shepherdin