Construction Of Recombinant Adenovirus Vector Of Mouse KGF And KGFR

Background: As a classic growth factor, KGF has multiple protective effects on AECs to obviate future harm, which has been proven in the past experiments, from the acute course of Acute Lung Injury/ Acute Respiratory Distress Syndrome ALI/ARDS to the chronic course of PF.However, there are still many problems for its cost in protein therapy, for its only contribution of prevention but not protection which may have something with the decrease of KGFR on epithelial cells after the injuries to the lung, and its effects of a kind of cytokines associated with immune network which has been ignored. Therefore, successful construction of recombinant adenovirus vector of KGF and KGFR may be a better way to cure the pulmonary diseases such as PF, and to discover the roles of KGF and even other growth factor in the immune course of lung injuries.Objective: To construct the recombinant adenovirus vectors expressing mouse KGF or KGFR.Methods: (1)According to the gene order of mouse KGF and KGFR provided by the gene Bank in National Center for Biotechnology Information, we had designed the sense and the antisense primer, with which we amplified the KGF/KGFR gene by PCR, with cDNA acquired from mouse lung RNA using RT-PCR. (2)pShuttle-CMV with the drug resistance of kanamycin to be the carrying plasmid, we inserted the KGF or KGFR gene into it, to construct the shuttle vector pKGF and pKGFR. (3) We then linearized pKGF and pKGFR, transformed them, respectively, into the E.Coli BJ5183 which contain virus backbones, followed by the step transfection of HEK293cells after identification of endonuclease Pac I and amplification. Finally, we harvested the virus after amplification, caesium chloride density gradient centrifugation purification and measurement of virus titers.Results: (1)The results of DNA sequencing suggested the success of construction of shuttle vector pKGF and pKGFR. (2)The bacteria could grow in the culture with kanamycin, as well as the agarose gel electrophoresis showed the proper strap, which had shown the success of construction of virus plasmid expressing KGF or KGFR. (3)HFK293 cells showed the pathologic morphologic changes, under microscope, which suggested the synthesis and duplication of recombinant adenovirus. (4)Finally, we had measured the virus titers, which had achieved the requirement of the future experiments in vivo and in vitro.Conclusion: We have successfully constructed the recombinant adenovirus vector of KGF or KGFR, which had supplied a tool to understand more about the role of KGF in pulmonary disease, especially as a cytokine in immune reaction, and even might be a new, a lower cost way to cure PF.