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目的构建小鼠白介素-10(murineinterleukin-10,mIL-10)的真核表达载体,并研究其在293T细胞中的表达。方法 RT-PCR扩增小鼠脾脏IL-10基因后,克隆到真核表达载体pcDNA3.0上,酶切和测序鉴定重组质粒的大小、序列。采用脂质体转染法将重组质粒pcDNA3.0-mIL-10瞬时转染293T细胞,用Westernblot法检测IL-10表达。结果重组质粒pcDNA3.0-mIL-10构建成功,而且转染了293T细胞中提取的蛋白可检测到活性蛋白。结论经酶切和测序鉴定重组质粒pcDNA3.0-mIL-10构建成功,并在293T细胞中成功表达活性蛋白,为进一步研究其生物学功能奠定了基础。
Objective To construct a murine interleukin-10 (mIL-10) eukaryotic expression vector and study its expression in 293T cells. Methods The spleen IL-10 gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.0. The size and sequence of recombinant plasmid were identified by restriction enzyme digestion and sequencing. The recombinant plasmid pcDNA3.0-mIL-10 was transiently transfected into 293T cells by lipofection method, and the expression of IL-10 was detected by Western blot. Results The recombinant plasmid pcDNA3.0-mIL-10 was successfully constructed and the protein extracted from 293T cells transfected with the recombinant protein could detect the active protein. Conclusion The recombinant plasmid pcDNA3.0-mIL-10 was successfully constructed and successfully expressed in 293T cells by enzyme digestion and sequencing, which laid the foundation for further study of its biological functions.