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目的人工合成恒河猴粒细胞-巨噬细胞集落刺激因子(Macaca mulatta granulocyte-macrophage colony stimulatingfactor,mGM-CSF)基因,在大肠杆菌中高效表达并纯化。方法根据大肠杆菌遗传密码子偏爱性优化设计并合成mGM-CSF基因,克隆至原核表达载体pET-43.1a(+)中,构建重组表达质粒pET-43.1a-mGM-CSF,转化大肠杆菌BL21-CodonPlus(DE3)-RIPL,IPTG诱导表达。表达的重组mGM-CSF蛋白经Sephacryl S-200分子筛层析纯化,复性后,Western blot检测其反应原性,MTT法检测其生物学活性。结果重组表达质粒pET-43.1a-mGM-CSF经双酶切及测序证实构建正确;表达的重组蛋白相对分子质量约为15 000,表达量约占菌体总蛋白的30%,主要以包涵体形式存在;纯化复性后的重组蛋白纯度可达95%以上,并可与大鼠抗人GM-CSF单克隆抗体特异性结合,比活性为1.2×107IU/mg。结论在大肠杆菌中高效表达了重组mGM-CSF蛋白,纯化复性后的蛋白具有良好的生物学活性。
Objective To synthetize the gene of Macaca mulatta granulocyte-macrophage colony stimulating factor (mGM-CSF) and express it in Escherichia coli. Methods The mGM-CSF gene was designed and synthesized according to the preference of E.coli genetic code and cloned into the prokaryotic expression vector pET-43.1a (+). The recombinant plasmid pET-43.1a-mGM-CSF was constructed and transformed into E.coli BL21- CodonPlus (DE3) -RIPL, IPTG induced expression. The expressed recombinant mGM-CSF protein was purified by Sephacryl S-200 molecular sieve chromatography. After refolding, the recombinant protein was detected by Western blot and its biological activity was detected by MTT assay. Results The recombinant plasmid pET-43.1a-mGM-CSF was correctly constructed by double enzyme digestion and sequencing. The relative molecular mass of the expressed recombinant protein was about 15 000 and the expression level was about 30% of the total bacterial protein. The purity of the purified recombinant protein was more than 95%, and it could specifically bind to the anti-human GM-CSF monoclonal antibody. The specific activity was 1.2 × 107IU / mg. Conclusion The recombinant mGM-CSF protein is highly expressed in E. coli, and the purified recombinant protein has good biological activity.