JOURNAL ARTICLE

[Construction and expression of replication-deficient recombinant adenovirus vector with hNET gene by adeasy-1 system]

Zhi-yun Jia, Xiao-hong Ou, Hai-yan Wei, Hou-fu Deng, Rui Huang, Jin-rong Wang, Shi-qiong Zhang
Sichuan da Xue Xue Bao. Yi Xue Ban, Journal of Sichuan University. Medical Science Edition 2008, 39 (4): 523-6
18798484

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.

CONCLUSION: 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.

Full Text Links

Find Full Text Links for this Article

Discussion

You are not logged in. Sign Up or Log In to join the discussion.

Related Papers

Remove bar
Read by QxMD icon Read
18798484
×

Save your favorite articles in one place with a free QxMD account.

×

Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"