[Construction and expression of replication-deficient recombinant adenovirus vector with hNET gene by adeasy-1 system].
OBJECTIVE
To construct and identify the recombinant replication deficient adenovirus vector which codes for human Norepinephrine Transporter (hNET) gene by using the method of homogenous recombination in bacteria.


METHODS
hNET gene was obtained from the recombinant plasmid pCMV5 via Kpn I + Xba I digestion, and subcloned into E1 deleted expression plasmid pAdtrack-CMV shuttle vector, forming transfer vector pAdtrack-CMV-hNET. Then it was linearized with Pme I followed by homologous recombination with bone plasmid pAdEasy-1 in BJ5183 cells to generate recombinant plasmid Ad-hNET. The DNA of identified Ad-hNET was digested with Pac I and transfected to HEK293 cells by liposome-mediated method to package recombinant adenovirus. The PCR technique was applied to detect the target gene and Western Blotting to verify the expression of hNET. The titre of the Ad-hNET was measured with the aid of green fluorescence protein (GFP) expression after multiplication and purification.


RESULTS
By sequencing, it was confirmed that the product was the gene of hNET. PCR test, restriction endonuclease digestion and Western Blotting confirmed the successful construction of the recombinants Ad-hNET. The titre of purified recombinant adenovirus Ad-hNET was 1.2 X 10(10) pfu/mL.


CONCLUSIONS
The recombinant adenovirus with the hNET gene was constructed successfully. It will be helpful for the further investigation of its potentiality to be applied in the tumors targeted therapeutic strategy.
Jia
Zhi-yun
ZY
Department of Nuclear Medicine, West China Hospital, Sichuan University, Chengdu 610041, China.
Ou
Xiao-hong
XH
Wei
Hai-yan
HY
Deng
Hou-fu
HF
Huang
Rui
R
Wang
Jin-rong
JR
Zhang
Shi-qiong
SQ
D000256:Adenoviridae / Q000235:genetics*; D015153:Blotting, Western; D002460:Cell Line; D003673:Defective Viruses / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D050484:Norepinephrine Plasma Membrane Transport Proteins / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D050484:Norepinephrine Plasma Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins