人IL-29定点突变体抗肿瘤细胞增殖活性分析

Anti-Tumor Proliferation Activity of hIL-29 Variant

DOI：10.3969/j.issn.1673-1689.2017.05.018

中文关键词: 人白细胞介素-29 定点突变 重组蛋白 抗肿瘤活性

英文关键词: human interleukin-29,site-directed mutation,recombinant protein,antineoplastic activity

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中文摘要:

为分析人白细胞介素-29（hIL-29） 变异体抗肿瘤细胞增殖活性，采用大引物PCR方法对人hIL-29基因第104位碱基进行点突变，使hIL-29肽链第35位Arg定点改造为Lys。得到的hIL-29变异体基因经重组后转化毕赤酵母GS115和诱导表达，经纯化获得重组蛋白rhIL-29mut35。SDS-PAGE分析显示rhIL-29mut35的相对分子质量约23×103，Western Blotting鉴定显示其与羊抗人IL-29多克隆抗体发生特异性结合反应。用CCK-8试剂检测rhIL-29mut35对肿瘤细胞增殖的影响，rhIL-29mut35在不同质量浓度时对胃癌细胞SGC7901、肝癌细胞BEL7402、肺腺癌细胞A549和结肠癌细胞HCT8的增殖均有抑制作用，高剂量组（1 000 ng/mL）rhIL-29mut35对胃癌细胞SGC7901、肝癌细胞BEL7402、肺腺癌细胞A549和结肠癌细胞HCT8增值的抑制率分别为（34.20±1.50）%、（27.30±0.31）%、（30.20±0.68）%、（29.13±1.40）%，与阴性对照组相比，差异有统计学意义（P＜0.01）。而且高剂量组的抗增殖效应高于野生型rhIL-29的（P＜0.01），这为进一步研制开发IL-29的抗肿瘤药物奠定基础。

英文摘要:

In order to analyze the antineoplastic activity of human interleukin-29（hIL-29） variant，a mutant gene of hIL-29 was amplified by megaprimer PCR，and the Arg35 of hIL-29 peptide chain was mutated to Lys35. A recombinant plasmid was constructed and transformed into Pichia pastoris GS115，the recombinant protein rhIL-29mut35 was induced and purified. The purified rhIL-29mut35 had a relative molecular mass of about 23×103 on SDS-PAGE profile and showed specific reaction with goat anti-human IL-29 polyclonal antibody in Western blotting. With the Cell Counting Kit-8 reagent，the rhIL-29mut35 was found to has anti-proliferation effect on tumor cells SGC7901，BEL7402，A549 and HCT8 separately with inhibition ratios of （34.20±1.50）%，（27.30±0.31）%，（30.20±0.68）% and（29.13±1.40）% in the high dose group. The anti-proliferation effect of rhIL-29mut35 was stronger than that of wild type rhIL-29（P

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