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目的 在大肠杆菌中表达重组人白细胞介素10(Human Interleukin,hIL-10)。方法 用RT-PCR自白血病细胞株K562 RNA扩增hIL-10 cDNA片段(约500bp),克隆至质粒载体pSK(+),并对克隆的DNA片段进行序列分析。用限制酶EcoRI和 BamHI消化pSK/IL-10重组质粒,分离hIL-10片段,并插入原核表达载体pBV220的相应限制酶位点,酶谱分析鉴定重组表达载体pBV/IL-10。转化菌株经42℃诱导,用SDS-PAGE和Westem blot鉴定表达的重组蛋白。表达的重组蛋白经谷胱甘肽复性缓冲液复性,用RT-PCR检测其对外周血单核细胞(PBMC)合成IFN-γ的抑制作用。结果 RT-PCR扩增的DNA片段与hIL-10 cDNA大小一致。重组质粒pSK/IL-10的DNA序列分析显示,克隆的DNA序列与文献报道的hIL-10 cDNA序列一致。SDS-PAGE表明,重组蛋白相对分子质量为18000,其表达量达菌体总蛋白的20％左右。Westem blot分析显示,重组蛋白能特异性地与抗hIL-10抗体结合,复性的重组蛋白能明显抑制PBMC合成IFN-γ。结论 已成功地构建了表达具有生物学活性的重组hIL-10(Recombinant hIL-10,rhIL-10)的工程菌株。
Objective To express recombinant human interleukin (hIL-10) in E. coli. Methods The cDNA fragment of hIL-10 (about 500 bp) was amplified by RT-PCR from leukemia cell line K562 RNA and cloned into the plasmid vector pSK (+). The cloned DNA fragment was sequenced. The recombinant plasmid pSK / IL-10 was digested with restriction enzymes EcoRI and BamHI. The hIL-10 fragment was isolated and inserted into the corresponding restriction site of prokaryotic expression vector pBV220. The recombinant expression vector pBV / IL-10 was identified by zymography. The transformed strain was induced at 42 ° C and the expressed recombinant protein was identified by SDS-PAGE and Western blot. The expressed recombinant protein was refolded by glutathione refolding buffer, and its inhibitory effect on IFN-γ production by peripheral blood mononuclear cells (PBMC) was detected by RT-PCR. Results The DNA fragment amplified by RT-PCR was consistent with the hIL-10 cDNA. DNA sequence analysis of the recombinant plasmid pSK / IL-10 showed that the cloned DNA sequence was consistent with the reported hIL-10 cDNA sequence. SDS-PAGE showed that the recombinant protein relative molecular weight of 18000, the expression of the total amount of bacterial protein about 20%. Westem blot analysis showed that the recombinant protein can specifically bind to the anti-hIL-10 antibody, and renaturation of the recombinant protein can significantly inhibit PBMC synthesis of IFN-γ. Conclusion The engineered strains expressing recombinant hIL-10 (rhIL-10) with biological activity have been successfully constructed.