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[Site-directed mutagenesis of human IL-29 and antineoplastic activity of the recombinant human IL-29 variant]
Abstract:
To explore the anti-tumor proliferation activity of human interleukin-29 (hIL-29) variant and based on bioinformatics analyzed data of hIL-29, a mutant gene hIL-29(mut33,35) was amplified by site-directed mutagenesis and megaprimer PCR. The hIL-29(mut33,35) was inserted into an eukaryotic expression plasmid pPIC9K and successfully expressed in Pichia pastoris GS115. A recombinant variant protein (rhIL-29(mut33,35)) was purified from the ferment supernatant of the engineering GS115. To observe the antineoplastic activity of the variant rhIL-29(mut33,35), a CCK-8 reagent was used to detect the anti-proliferation effect. Results show that it has strong anti-proliferation effect when acted on liver cancer cell BEL7402, colon cancer cell HCT8 and gastric cancer cell SGC7901. The inhibition ratios of the three tumor cells were (30.99 ± 1.58)%, (22.47 ± 1.37)% and (32.05 ± 2.02)%, respectively. In high dose group, the anti-proliferation effect of the rhIL-29(mut33,35) was stronger than that of wild type rhIL-29 (P < 0.01). This indicates the variant rhIL-29(mut33,35) has potential development value for medicine.
Insights
A novel variant of human interleukin-29 (hIL-29) demonstrated significant anti-tumor proliferation effects against liver, colon, and gastric cancer cells. This engineered protein shows greater efficacy than the wild type, indicating its potential as a cancer therapeutic.
Area of Science:
- Biotechnology
- Molecular Biology
- Cancer Research
Background:
- Human interleukin-29 (hIL-29) is a cytokine with known biological activities.
- Bioinformatics analysis suggested potential for enhanced therapeutic properties through mutation.
- Developing novel anti-cancer agents is crucial for effective tumor treatment.
Purpose of the Study:
- To engineer and express a mutant form of hIL-29 (hIL-29(mut33,35)).
- To evaluate the anti-tumor proliferation activity of the recombinant variant protein (rhIL-29(mut33,35)).
- To compare the efficacy of the variant protein against the wild-type hIL-29.
Main Methods:
- Site-directed mutagenesis and megaprimer PCR were used to create the hIL-29(mut33,35) gene.
- The gene was cloned into a pPIC9K plasmid for expression in Pichia pastoris GS115.
- Recombinant protein purification and CCK-8 assay were performed to assess anti-proliferation effects.
Main Results:
- The recombinant rhIL-29(mut33,35) was successfully expressed and purified.
- The variant protein exhibited significant anti-proliferation effects on BEL7402 (liver), HCT8 (colon), and SGC7901 (gastric) cancer cell lines.
- Inhibition ratios were 30.99%, 22.47%, and 32.05% for the respective cell lines.
- At high doses, rhIL-29(mut33,35) showed a stronger anti-proliferation effect compared to wild-type rhIL-29 (P < 0.01).
Conclusions:
- The engineered rhIL-29(mut33,35) variant possesses potent anti-cancer properties.
- The variant demonstrates superior anti-proliferation activity compared to the wild-type protein.
- rhIL-29(mut33,35) holds significant potential for development as a novel anti-cancer therapeutic agent.
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