陈凌,郭盛,郝春莉,吴良霞,张建华
CHEN Ling,GUO Sheng,HAO Chun-li,WU Liang-xia,ZHANG Jian-hua(Department of Pediatrics

• 1:上海交通大学附属第六人民医院儿科

摘要(Abstract)：

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 mouse interleukin 17F(mIL-17)/His fusion protein in E.coli,purified the expressed product and analyze its bioactivity.Methods The mIL-17f gene was amplified by RT-PCR and inserted into prokaryotic expression vector pET28a.The constructed recombinant plasmid pET28a /mIL-17f was transformed to E.coli BL21(DE3)was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed rmIL-17F /His fusion protein was purified by Ni2+-NTA affinity chromatography and identified for reactogenicity.BALB/c mice were immunized with the purified and re-naturalized rmIL-17 /His fusion protein by nasal drip,then determined for expression of IL-6 mRNA in lung tissue by real-time fluorescent quantitative PCR,and for IL-17F,IL-4 and IFNγ levels in peripheral blood by ELISA.Results The DNA sequence of amplified mIL-17f gene was consistent with that reported in GenBank.Recombinant plasmid pET28a /mIL-17f was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 19 000,mainly existed in a form of inclusion body and contained about 30% of total somatic protein.The purified fusion protein reached a purity of about 95% and showed good reactogenicity.The mucosal immunization with the purified fusion protein promoted the expression of IL-6 mRNA in lung tissues and increased the IL-4 and IFNγ levels in sera of mice.Conclusion The rmIL-17F with bioactivity was highly expressed in E.coli and purified,which laid a foundation of further study on the function of IL-17F.

关键词(KeyWords)： 白细胞介素-17;原核细胞;基因表达;蛋白纯化;生物活性
Interleukin-17;Prokaryotic cells;Gene expression;Protein purification;Bioactivity

Abstract:

Keywords:

基金项目(Foundation): 上海市自然科学基金资助项目(10ZR1423000)

作者(Author): 陈凌,郭盛,郝春莉,吴良霞,张建华
CHEN Ling,GUO Sheng,HAO Chun-li,WU Liang-xia,ZHANG Jian-hua(Department of Pediatrics

DOI: 10.13200/j.cjb.2010.07.6.chenl.008

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