巨噬细胞抑制因子在毕赤酵母中的表达及鉴定

Recombinant and Identification Macrophage Inhibitory Cytokine 1 in Pichia Pastries

目的:在毕赤酵母表达系统中获得巨噬细胞抑制因子(MIC-1)高效分泌表达,并鉴定其免疫活性。方法:从BeWo细胞系中提取总RNA,通过反转录PCR获得目的片段,然后将基因亚克隆入pPIC9k表达载体,线性化后转化GS115酵母细胞,经G418抗性筛选获多拷贝重组子并诱导表达,PCR鉴定目的基因的整合。表达产物用Westernblot鉴定生物活性。结果:经过G418筛选到34株阳性克隆,MIC-1蛋白最高表达量为6.332mg/L。结论:成功构建了MIC-1表达载体,在酵母中获得多拷贝高效表达,并且形成正确的蛋白结构。

Objective: To obtain macrophage inhibitory cytokine 1 in pichia pastories, and to identify bioactivity of this protein. Methods: To Extract total RNA from BeWo cell line, and to attain gene of interest by RT-PCR, then to sub-clone this gene into the expression vector-pPIC9k, to transform into yeast GS115 after linearization, to screen multiple-copy transfomants by G418 and identify MIC-1 bioactivity by Western Blotting. Results: 34 strains of multicopy transforms were identified,the highest yield was 6.332mg/L.Conclusion: The protein is secreted in its corrected folded dimeric form at milligram per litre quanitities.