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目的在大肠杆菌(E.coli)中表达并纯化重组小鼠白细胞介素-17F(rmIL-17F),并鉴定其生物活性。方法经RT-PCR扩增mIL-17f基因片段,定向插入原核表达载体pET28a,构建重组表达质粒pET28a/mIL-17f,转化E.coliBL21(DE3),经IPTG诱导表达,Ni2+-NTA亲和层析纯化rmIL-17F/His融合蛋白,Western blot鉴定其反应原性。以纯化复性的rmIL-17F滴鼻干预小鼠,采用实时荧光定量PCR法检测小鼠肺组织IL-6 mRNA的表达,ELISA法检测小鼠外周血IL-17F、IL-4和IFNγ的水平。结果扩增的mIL-17f基因DNA序列与GenBank中登录的mIL-17f基因序列一致,所构建的重组表达质粒pET28a/mIL-17f构建正确;表达的重组蛋白相对分子质量约为19000,主要以包涵体形式表达,表达量约占菌体总蛋白的30%;纯化的重组蛋白纯度约为95%,具有良好的反应原性,经黏膜给药后,可促进小鼠肺组织中IL-6 mRNA的表达,并提高小鼠血清中IL-4和IFNγ的水平。结论已成功地在大肠杆菌中高效表达并纯化了具有生物学活性的rmIL-17F,为进一步研究IL-17F的功能奠定了基础。
Objective To express and purify recombinant mouse interleukin-17F (rmIL-17F) in E.coli and identify its biological activity. Methods The mIL-17f gene fragment was amplified by RT-PCR and inserted into prokaryotic expression vector pET28a. The recombinant plasmid pET28a / mIL-17f was transformed into E.coli BL21 (DE3) and induced by IPTG. Purified rmIL-17F / His fusion protein, Western blot identification of its reaction. The purified rmIL-17F nasal drops were used to pretreat mice. The expression of IL-6 mRNA was detected by real-time fluorescence quantitative PCR. The level of IL-17F, IL-4 and IFNγ . Results The sequence of mIL-17f gene amplified was consistent with the sequence of mIL-17f in GenBank. The constructed recombinant plasmid pET28a / mIL-17f was constructed correctly. The relative molecular mass of the expressed recombinant protein was about 19000, The expression level of the purified recombinant protein is about 30% of the total bacterial protein. The purified recombinant protein has a purity of about 95% and has good reactionogenicity. After being administered mucosally, IL-6 mRNA can be promoted in the lung tissue of mice And increase the level of IL-4 and IFNγ in mouse serum. Conclusion The rmIL-17F with high biological activity has been successfully expressed and purified in Escherichia coli, which lays the foundation for further study on the function of IL-17F.