Construction of double-gene adenovirus vector carrying vascular endothelial growth factor and hepatocyte growth factor

Abstract

The invention provides construction of a double-gene adenovirus vector carrying a Vascular Endothelial Growth Factor (VEGF) 165 and a Hepatocyte Growth Factor (HGF). The adenovirus vector expressing the VEGF and the HGF is constructed by utilizing a shuttle vector carrying an endogenous ribosome entry site (IRES) element, and a new choice is provided for biological treatment of CLI patients. A method comprises the following steps: obtaining a target fragment by utilizing PCR amplification, and performing directional cloning for two times to obtain a recombinant shuttle vector; performing homologous recombination on linearized shuttle plasmids and framework plasmids, performing digestion on screened positive recombinant PacI, transfecting AD293 cells by virtue of lipidosome, and performingadenovirus packaging and amplification. Recombinant adenovirus simultaneously and efficiently expressing hVEGF165 and hHGF proteins is successfully constructed by utilizing the shuttle vector pShuttle-IRES, and is efficiently expressed in hVSMCs, and a powerful tool and method are provided for double-gene treatment.

Description

technical field [0001] The invention belongs to the construction field of adenovirus vectors, and relates to the construction of adenovirus vectors carrying vascular endothelial growth factor and hepatocyte growth factor double genes. Background technique [0002] At present, atherosclerosis is one of the main causes of arterial diseases; with the economic development and the significant improvement of people's living standards, the incidence of atherosclerotic diseases is gradually increasing, and the patients are showing a younger trend. The prevalence of peripheral arterial disease (PAD) is high, reaching 15% of the general population in developed countries and as high as 30% in the annual population, and even higher in developing countries. PAD patients generally have critical limb ischemia (CLI), and some patients even face amputation. The more severe the PAD, the greater the risk of death from myocardial infarction and stroke. Severe symptomatic large-vessel PAD, vascu...

AI Technical Summary

Problems solved by technology

[0006] In order to increase the probability of homologous recombination of the adenovirus backbone plasmid carrying two target genes, and to solve the problem of low expression level of the target gene due to mutual interference between promoters of conventional expression vectors, the present invention proposes to carry vascular endothelial growth factor and Construction of Hepatocyte Growth Factor Double-gene Adenoviral Vector

Images