[Construction of recombinant adenovirus vector of mouse keratinocyte growth factor]

Li Chen, Xin-ling Zhang, Yin-ping Li, Jian-li Wang, Tan-shi Li
Zhongguo Wei Zhong Bing Ji Jiu Yi Xue, Chinese Critical Care Medicine, Zhongguo Weizhongbing Jijiuyixue 2007, 19 (10): 610-3

OBJECTIVE: To construct a recombinant adenovirus vector expressing keratinocyte growth factor (KGF) gene in mouse.

METHODS: KGF gene amplified from mouse cDNA by polymerase chain reaction (PCR). It was reverse transcript from RNA that had been harvested from C57BL/6 mouse, and then inserted into the plasmid pShuttle-CMV to construct the shuttle plasmid pShuttle-CMV-KGF (pKGF). After linearized by restriction enzyme, the plasmid was transformed into E.Coli BJ5183 containing adenovirus backbone. The homologous recombinant pAdeasy-1-pShuttle-CMV-KGF (pAd-KGF) was identified, linearized, and then transfected into HEK293 cells using the lipofectamine TM 2000 to package the adenovirus, Adeasy-1-pShuttle-CMV-KGF (Ad-KGF), followed by further amplification, caesium chloride density gradient centrifugation purification and measurement of virus titer. Ad-GFP was used as control, and its transfection efficacy was observed.

RESULTS: (1) The shuttle plasmid pKGF was proved to be successfully constructed by gene sequencing and restriction enzyme, as well as the recombinant adenovirus plasmid. (2) The cytopathic effects of HEK293 cells observed under the microscope suggested that the duplication of the virus was successful. (3) The plague titration of HEK293 cells showed virus titers were 3.0 x 10(10) pfu/ml,the concentration of which was adequate for future test in vivo or in vitro.

CONCLUSION: The harvest of recombinant adenovirus vector of Ad-KGF, is the first step for the future test to investigate the effects of KGF in pulmonary diseases, and the possible gene therapy to treat pulmonary fibrosis.

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