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为获得人白介素-29(h IL-29)变异体并考察其对肿瘤细胞增殖的影响,基于对h IL-29成熟肽生物信息学的分析结果,通过大引物PCR方法将其肽链第43位赖氨酸(Lys)编码基因进行定点突变。将得到的h IL-29变异体基因插入质粒p PIC9KM,构建重组真核表达质粒p PIC9KM-h IL-29~(mut43),导入毕赤酵母(Pichia pastoris)GS115,经G418筛选得到高产菌株p PIC9KM-h IL-29~(mut43)/GS115,用甲醇诱导表达重组蛋白rh IL-29~(mut43)。检测rh IL-29~(mut43)对肿瘤细胞增殖的影响,结果显示该重组蛋白对结肠癌细胞HCT8、肺腺癌细胞A549、胃癌细胞SGC7901和肝癌细胞BEL7402的增殖均有抑制作用,且高浓度(1 000 ng/ml)下对上述4种肿瘤细胞的增殖抑制作用更强,增殖抑制率分别为(18.45±0.83)%、(27.14±2.99)%、(29.94±1.95)%和(25.22±0.42)%。与阴性对照组相比,差异有统计学意义(P<0.01)。且其抑制增殖效应强于野生型h IL-29和阳性对照药人干扰素(IFN)-a2b。
In order to obtain the human interleukin-29 (hIL-29) variant and investigate its effect on tumor cell proliferation, based on the bioinformatics analysis of hIL-29 mature peptide, Lys (Lys) encoding gene for site-directed mutagenesis. The resulting hIL-29 variant gene was inserted into the plasmid p PIC9KM to construct a recombinant eukaryotic expression plasmid p PIC9KM-h IL-29 ~ (mut43) which was introduced into Pichia pastoris GS115 and screened by G418 to obtain a high-yield strain p PIC9KM-h IL-29 ~ (mut43) / GS115 was induced with methanol to express the recombinant protein rh IL-29 ~ (mut43). The effect of rhIL-29 ~ (mut43) on the proliferation of tumor cells was examined. The results showed that the recombinant protein could inhibit the proliferation of colon cancer cell line HCT8, lung adenocarcinoma cell line A549, gastric cancer cell line SGC7901 and liver cancer cell line BEL7402, (1 000 ng / ml), the proliferation inhibition rate of the above four kinds of tumor cells were stronger than that of the control group (18.45 ± 0.83)%, (27.14 ± 2.99)%, (29.94 ± 1.95)% and (25.22 ± 0.42)%. Compared with the negative control group, the difference was statistically significant (P <0.01). And its inhibitory effect on proliferation was stronger than that of wild-type hIL-29 and positive control human interferon (IFN) -a2b.