[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.

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.