人IL-29的定点突变及抗肿瘤活性初步分析

为探讨人白细胞介素-29(h IL-29)变异体的抗肿瘤活性,根据h IL-29成熟肽的生物信息学分析数据,采用大引物PCR方法对其肽链第33位赖氨酸、35位精氨酸的编码基因进行定点突变,获得的h IL-29变异体基因构建重组真核表达质粒转化毕赤酵母(Pichia pastoris)GS115进行发酵表达,经纯化得到重组人白细胞介素-29变异体蛋白(rh IL-29mut33,35)。经CCK-8法检测抗肿瘤细胞增殖的数据显示,rh IL-29mut33,35对肝癌细胞BEL7402、结肠癌细胞HCT8和胃癌细胞SGC7901的增殖均具有抑制作用,高剂量组对这3种肿瘤细胞的增殖抑制率分别为(30.99±1.58)%、(22.47±1.37)%和(32.05±2.02)%,而且抗增殖作用比野生型rh IL-29的更强(P0.01),表明变异体rh IL-29mut33,35具有潜在的医药开发价值。

Site-directed mutagenesis of human IL-29 and antineoplastic activity of the recombinant human IL-29 variant

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-29mut33,35 was amplified by site-directed mutagenesis and megaprimer PCR. The hIL-29mut33,35 was inserted into an eukaryotic expression plasmid pPIC9K and successfully expressed in Pichia pastoris GS115. A recombinant variant protein (rhIL-29mut33,35) was purified from the ferment supernatant of the engineering GS115. To observe the antineoplastic activity of the variant rhIL-29mut33,35, 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-29mut33,35 was stronger than that of wild type rhIL-29 (P < 0.01). This indicates the variant rhIL-29mut33,35 has potential development value for medicine.