小鼠白介素-10真核表达载体的构建及表达

摘要

目的构建小鼠白介素-10（murine interleukin-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Western blot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 an eukaryotic expression plasmid contained murine IL-10 gene and investigate the expression of the recombinant plasmid in 293T cells. Methods The gene of murine IL-10 was obtained by RT-PCR, and then was integrated into a eukaryotic expression plasmid pcDNA3.0, and finally the recombinant plasmid was confirmed by enzyme digestion analysis and DNA sequencing. The plasmid was transfected into 293T cells by liposome transfecting technique and was identified by Western blot. Results The recombinant plasmid pcDNA3.0-mIL-10 was constructed successfully. Furthermore, the protein was extracted from 293T cells, transfected with the plasmid showed reactivity with antibody against IL-10. Conclusion The recombinant pcDNA3.0-mIL-10 was constructed successfully and was highly expressed in 293T cells, which will lay a foundation for the further study of the biologic activity of IL-10 in vivo.