人白细胞介素-29基因的克隆及真核表达

Cloning and eukaryotic expression of human interleukin-29 gene

目的克隆人白细胞介素-29(Interleukin-29,IL-29)基因并进行真核表达。方法提取人外周血单个核细胞(PBMC)总RNA,经逆转录合成cDNA,以其为模板,PCR扩增IL-29基因,插入真核表达载体pPIC9K,构建重组真核表达质粒pPIC9K-29,转化毕赤酵母GS115,甲醇诱导表达,经SDS-PAGE和Western blot鉴定表达产物。结果扩增的IL-29基因序列与GenBank中登录的序列(NM_172140)相同;重组表达质粒pPIC9K-29经双酶切及测序证实构建正确;表达产物经SDS-PAGE分析,可见相对分子质量分别约30 000和27 000的特异蛋白条带,经Western blot分析,均可与羊抗人IL-29多抗发生特异性反应。结论已克隆并在毕赤酵母GS115中表达了人IL-29,为进一步研究其生物学活性及其应用奠定了基础。

Objective To clone human interleukin-29 （IL-29） gene and express in eukaryotic cells. Methods Total RNA was extracted from human peripheral blood mononuclear cells （PBMCs） and reversely transcribed into cDNA as a template with which IL-29 gene was amplified by PCR and inserted into eukaryotic expression vector pPIC9K. The constructed recombinant plasmid pPIC9K-29 was transformed to Pichia pastoris GSll5 and induced with methanol, and the expressed product was identified by SDS- PAGE and Western blot. Results The sequence of amplified IL-29 gene was identical to that reported in GenBank （NM_172140）. Restriction analysis and sequencing proved that recombinant plasmid pPIC9K-29 was constructed correctly. Specific protein bands with relative molecular masses of about 30 000 and about 27 000 respectively were observed on SDS-PAGE profile of expressed product, both of which showed specific reactions with goat anti-human IL-29 polyclonal antibody. Conclusion Human IL-29 gene was cloned and expressed in P. pastoris GS115, which laid a foundation of further study on biological activity and application of IL- 29.