Construction of the plasmid coding for the expression of the EGFP-M-IL-2(88Arg 125Ala) fusion protein and the anti-tumor effects exerted by the fusion protein in HeLa-60 cells

摘要

Gene therapy is a promising therapeutic option for the treatment of various cancers, and tumor-targeted plasmids encoding toxic protein genes are potential tools for gene therapy. In the present study, a recombinant plasmid containing the genes for the toxic protein melittin and interleukin-2 (IL-2) was constructed. Melittin and IL-2 are known to play key roles in immunoregulation and cancer therapy, but they each possess defects that limit the clinical application of these proteins. The present study aimed to construct a novel recombinant expression plasmid, pLEGFP-C1-M-IL-2(⁸⁸Arg, ¹²⁵Ala), and to improve the biological activity of IL-2 and melittin. The M-IL-2(⁸⁸Arg, ¹²⁵Ala) gene was excised from the pPICZαA/M-IL-2(⁸⁸Arg, ¹²⁵Ala) plasmid by polymerase chain reaction (PCR). The pLEGFP-C1 plasmid carrying the enhanced green fluorescent protein (EGFP) gene was used as a shuttle plasmid. Subsequent to digestion, the M-IL-2(⁸⁸Arg, ¹²⁵Ala) gene was subcloned into the pLEGFP-C1 vector to build the pLEGFP-C1-M-IL-2(⁸⁸Arg, ¹²⁵Ala) eukaryotic expression plasmid, which was identified by restriction enzyme digestion and gene sequencing. Confocal microscopy was used to determine the transfection efficiency subsequent to the plasmid being transfected into the cervical cancer HeLa cell line. The cells transfected with the pLEGFP-C1-M-IL-2(⁸⁸Arg, ¹²⁵Ala) plasmid demonstrated a decreased transfection efficiency compared with the cells transfected with the pLEGFP-C1 plasmid. The cellular expression of M-IL-2(⁸⁸Arg, ¹²⁵Ala) was detected by reverse transcription PCR and western blot analysis. Finally, cell counting kit-8 and apoptosis assays were performed to investigate the effects of the expression of the M-IL-2(⁸⁸Arg, ¹²⁵Ala) fusion protein on HeLa cells and to analyze the antitumor activity of the protein. In conclusion, a recombinant eukaryotic pLEGFP-C1-M-IL-2(⁸⁸Arg, ¹²⁵Ala) expression plasmid containing the M-IL-2(⁸⁸Arg, ¹²⁵Ala) fusion gene was constructed and the M-IL-2(⁸⁸Arg, ¹²⁵Ala) fusion protein was successfully expressed in HeLa cells. Furthermore, the M-IL-2(⁸⁸Arg, ¹²⁵Ala) fusion protein was able to inhibit HeLa cell proliferation and induce apoptosis in the tumor cells. These findings may offer an alternative method for anticancer therapy. The present study has provided a basis for future studies into the M-IL-2(⁸⁸Arg, ¹²⁵Ala) fusion gene.