Construction and identification of recombinant adenovirus vector Ad5-hBDNF-EGFP

Changsheng WANG; Jianhua LIN; Zhaoyang WU

ABSTRACT

BACKGROUND:

Gene therapy is the direction of spinal cord injury(SCI) therapy,the key of which is construction of targeting gene and vector.

OBJECTIVE:

To construct the recombinant adenovirus vector carrying human brain-derived neurotrophic factor(hBDNF) marked enhanced green fluorescent protein(EGFP). DESIGN,TIME AND

SETTING:

A single sample observation was completed in the First Affiliated Hospital of Fujian Medical University from September 2007 to June 2008. MATERIALSCompetent E. coli DH-5? was obtained from the American Stratagene Company. Plasmid pDC316-hBDNF,pDC316-mCMV-EGFP,pBHGlox_E1,3Cre and package system AdMax and 293 package cell strain were purchased from the Canadian Mixcrobix-Biosystems Company.

METHODS:

The hBDNF gene was constructed by PCR with plasmid pDC316-BDNF as template. With enzyme digestion,the hBDNF gene was inserted into the vector pDC316-mCMV-EGFP and the shuttle plasmid pDC316-hBDNF-mCMV-EGFP was constructed,which was cotransfected with the adenovirus skeleton plasmid pBHGlox_E1,3Cre into 293 cells to obtain the produced replication defective recombinant adenovirus vector Ad-hBDNF-EGFP. The recombinant adenovirus was propagated by repeat infection of 293 cells and purified by ion exchange method,then the virus particles were counted and the purity and titer were determined. MAIN OUTCOME

MEASURES:

①PCR identification of plasmid pDC316-hBDNF. ②Construction and identification of the shuttle plasmid pDC316-hBDNF-mCMV-EGFP. ③Packing,amplification and purification of recombinant adenovirus vector Ad-hBDNF-EGFP. ④PCR identification of the recombinant adenovirus. ⑤Titer of recombinant adenovirus.

RESULTS:

PCR amplification,restriction analysis and sequencing identified that both recombinant shuttle plasmid pDC316-hBDNF-mCMV-EGFP and recombinant adenovirus vector Ad-hBDNF-EGFP were correctly constructed. After amplification and purification,the virus particle count,A260/A280 and titer of recombinant adenovirus were 2.4?1011 VP/mL,2.0 and 0.8?1010 CCID50/mL,respectively.

CONCLUSION:

Recombinant adenovirus vector Ad-hBDNF-EGFP is successfully constructed,which laid a foundation for further study regarding gene function and therapy.