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Construction of recombinant eukaryotic expression vectors carrying genes encoding attenuated Shiga-like toxin Ⅰ mutant and their anti oophoroma effect
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Abstract:
To construct recombinant eukaryotic expression vectors carrying genes encoding attenuated Shiga-like toxin 1 mutant and to study their anti-oophoroma effect in vitro and in vivo. Methods: The genes encoding attenuated Shiga-like toxin 1 mutant were amplified by overlap PCR and then cloned into eukaryotic expression plasmid pcDNA31. The recombinant plasmids were transfected into SKOV3 cells and RT-PCR was used determine the expression of Stx l mRNA in SKOV3 cells. The influence of attenuated Stx 1 on the cell cycle of SKOV3 cells and the mechanism by which Stx 1 induces apoptosis of SKOV3 cells were observed. Then Stx 1 with different cyotoxicites (after packed by liposome) were intratumorally injected into immunodeficient mice harboring SKOV3 to assess their anti-oophoroma effect in vivo. Results: It was showed that the genes encoding attenuated Stx 1 mutant were successfully cloned into pcDNA3.1 and their mRNA expression in transfected SKOV3 cells was verified by RT-PCR. In vitro study showed that the constructed plasmid arrested SKOV3cells at G 2 /M stage. The post-transfection death of SKOV3 cells was mainly through necrosis as assessed by flow cytometry. In vivo study showed that the constructed plasmid had a significant anti-oophoroma effect on the growth of SKOV3 tumor in immunodefficient mice. Conclusion: Two sequences encoding attenuated Stx 1 mutant have been successfully constructed and expressed in eukaryotic expression system, which have been confirmed to have obvious anti-oophoroma effect in vitro and in vivo .
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