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  • Title: [Construction and expression of replication-deficient recombinant adenovirus vector with hNET gene by adeasy-1 system].
    Author: Jia ZY, Ou XH, Wei HY, Deng HF, Huang R, Wang JR, Zhang SQ.
    Journal: Sichuan Da Xue Xue Bao Yi Xue Ban; 2008 Jul; 39(4):523-6. PubMed ID: 18798484.
    Abstract:
    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.
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