Abstract:Aim To construct an adenovirus expression vector which can express hepatocyte growth factor (HGF) in vascular smooth muscle cells (SMC). Methods The plasmid containing HGF fragment was cleaved by restriction enzyme digestion, and the resultant fragment was inserted directionally into adenoviral shuttle plasmid. The linearized recombinant adeno viral shuttle plasmid and adenovirus expression vector were cotransformed into Escherichia coli BJ5183 cells for homologous recombination . The resultant recombinant plasmid, pAd-HGF, then was transfected into HEK293 cells with liposome for packaging. The recombinant adenoviral shuttle plasmid and pAd-HGF were identified by enzyme digestion and sequencing. The package of pAd-HGF in HEK293 cells was tracked by fluorescent microscope, and was observed by electronic microscope. The expression of packaged pAd-HGF in abdominal aortic SMCs of rat was identified by RT-PCR and Western blotting. Results HGF fragment was inserted correctly into the adenoviral shuttle plasmid and adenovirus expression vector. High-liter packaged adenovirus vector was produced and expressed in abdominal aortic SMCs of rat. Conclusions A recombinant adenovirus expression vector of HGF was constructed successfully. This study suggested that HGF may be a potential target for the gene therapy of vascular diseases and established a foundation for further study.

Abstract:Aim To prepare catalase-contained recombinant adenovirus. Methods Catalase gene was digested from plasmid of pZeoSV2-Cat, subcloned into plasmid of pBluescript Ⅱ sk( + ) and formed plasmid of pBluescript Ⅱ sk( + )-Cat. Then Catalase gene was digested from plasmid of pBluescript Ⅱ sk( + ) -Cat, subcloned into shuttle plasmid of pShuttle-CMV and formed transfer plasmid of pShuttle-CMV-Cat. Adenovirus genomic plasmid of pAdEasy-1 was transformed into BJ5183 bacteria and prepared ultracompletent BJ5183 containing pAdEasy-1. pShuttle-CMV-Cat was linealinzed with Pme I and transformed into ultracompletent BJ5183 containing pAdEasy-1. The identified recombinant adenovirus plasmid of pAdEasy-1-Cat was linealinzed with Pac I and transfected into Ad-293 cells to package recombinant adenovirus particles. The target gene was decteted by PCR. Results The hneahnzed pShuttle-CMV-Cat was transformed into ultracompletent BJ5183 containing pAdEasy-1 .There were 30% positive recombinant plasmid. After pAdEasy-1-Cat was transfected into Ad-293 cells, recombinant adenovirus particles were produced and further amplified. PCR test indicated that the recombinant adenovirus AdCat contained Catalase gene. The liter of the purified AdCat was 4.12 × 1015 OPU/L. Conclusion The homologous recombination in bacteria is a convenient and high efficient method to prepare AdCat. This affords a good gene transfer vector for the gene therapy in restenosis.