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Pro apoptotic effect of recombinant anti HER2 fusion protein ScFv/tBid gene on osteosarcoma E10 cells
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Abstract:
Abstract Objective： To construct a fusion gene ScFv/tBid against HER2 and investigate its pro popotic effect on osteosarcoma cell line E10. Methods: HER2 expression on the surface of E10 cells was detected by immunofluorescent staining and flow cytometry (FCM)， then e23sFv fragment, a single chain HER2 antibody, was linked with a PE translocation domain (PE aa253-364) and tBid . The recombinant tBid gene was cloned into a pCMV plasmid to obtain pCMV ScFv/tBid, which was then transfected into E10 cells. Immunofluorescent staining was used to examine the expression of target protein and morphological changes of cells. Meanwhile, the pro apoptotic effect of ScFv/tBid gene was analyzed by Annexin V FITC staining and TUNEL staining. Results: Flow cytometry showed HER2 expression on cell surface, and the recombinant plasmid, pCMV ScFv/tBid, was successfully constructed and transfected into E10 cells. Overexpression of tBid protein was detected in E10 cells as revealed by immunofluorescent staining; and shrinkage and nuclear condensation were also noticed in E10 cells. Annexin V FITC staining and FCM revealed that the apoptosis rate of E10 cells was 161% after transfection with pCMV ScFv/tBid; the apoptosis rate in the control cells was 4.5%. TUNEL staining showed typical apoptosis characteristics of E10 cells after transfection. Conclusion: The recombinant anti HER2 fusion gene, ScFv/tBid, can be expressed in E10 cells transfected with pCMV ScFv/tBid, and subsequently induce apoptosis.
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Project Supported:
Supported by the National Science Foundation for Distinguished Young Scholars of China (No.39925036); the Major National Natural Science Fondation of China (No.30330610); the National Natural Science Foundation of China (No.30471988); the National Postdoctoral Research Foundution of China (No.2005038259)
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