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Construction of a recombinant adenovirus containing MMP-9 signal peptide and PEX domain of human MMP-2 and study of its activity
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Abstract:
Objective：To construct a recombinant adenovirus (abbreviated as ET-M9-PEX) containing MMP-9 signal peptide and noncatalytic carboxyl-terminal hemopexin domain of human MMP-2, and to use the constructed adenovirus as a drug bioreactor in vivo to enhance the expression of an anti-angiogenesis factor for treatment of tumor by a gene therapy strategy. Methods: Adenovirus vector containing M9-PEX gene was constructed by PCR and gene recombination, and was packaged and amplified in L293 cells to obtain ET-M9-PEX recombinant adenovirus with infective ability. The expression and secretion of PEX in ET-M9-PEX-infected cells were detected by Western-blotting and immunofluorescent staining. The inhibitory effect of ET-M9-PEX-conditioned medium on EC cells proliferation was detected by growth curve and its inhibitory effects on angiogenesis and tumor growth were determined by chicken chorioallantoic membrane (CAM) assay in vivo. Results:ET-M9-PEX was successfully constructed and the expression and secretion of PEX in ET-M9-PEX-infected cells were verified. The ET-M9-PEX conditioned medium significantly inhibited the proliferating rate of EC cells. The tumor weights from ET-M9-PEX-infected PG cells in CAM and gradeⅠvessel number were reduced by 57.57%（P<0.01）and 33.52%（P<0.01）, respectively, compared with E-T. However, neither the tumor weight nor the vessel number was significantly different between E-T and PBS groups.Conclusion: ET-M9-PEX constructed in the present study shows remarkable inhibitory effects on EC proliferation, tumor growth and angiogenesis on CAM, suggesting that ET-M9-PEX may be a promising candidate for tumor gene therapy.
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