鼠抑铁素2蛋白的原核表达及鉴定

Prokaryotic Expression and Identification of Murine Lipocalin 2

目的表达鼠抑铁素2(lcn2)蛋白,并检测表达产物的生物学活性。方法从小鼠RAW264.7巨噬细胞提取总RNA,逆转录合成cDNA,以cDNA为模板,PCR扩增lcn2基因片段,插入原核表达质粒pET-32a(+),转化大肠杆菌BL21(DE3)plysS,用IPTG诱导表达。表达产物经His-tagin-gel stain鉴定后,采用Ni2+-NTA亲和层析纯化,并检测其生物学活性。结果经核苷酸序列测定和酶切鉴定,表明重组质粒pET-32a(+)-lcn2构建正确。表达的重组蛋白经SDS-PAGE分析,相对分子质量约为21000,表达量占菌体总蛋白的35%,主要以包涵体形式存在。纯化后蛋白浓度为1.0g/L,并对大肠杆菌和乙型链球菌生长有一定的抑制作用。结论成功地在原核细胞中表达了重组lcn2蛋白,且表达的蛋白具有一定的抑菌作用。

Objective To express murine lipocalin 2 （lcn2） and study its biological activity. Methods Extract total RNA from murine macrophage RAW264.7 strain for amplification of lcn2 gene by RT-PCR. Insert the amplified lcn2 gene into prokaryotic expression vector pET-32a （＋）, and transform the constructed recombinant plasmid pET-32a （＋）-lcn2 to E. coli BL21 （DE3） plysS for expression under induction of IPTG. The expressed product was identified by His-tag in-gel staining, purified by Ni^2＋-NTA affinity chromatography and determined for biological activity. Results Both nucleotide sequencing and restriction analysis proved that recombinant plasmid pET-32a （＋）-lcn2 was constructed correctly. SDS-PAGE showed that expressed recombinant lcn2 protein, with a relative molecular mass of about 21 000, mainly existed in a form of inclusion body and contained 35% of total somatic protein. The expressed product reached a protein concentration of 1. 0 g/L after purification and showed a certain inhibitory effect on both E.coli and β-streptococcus. Conclusion Recombinant lcn2 protein was successfully expressed in prokaryotic cells and showed a certain bacleriostatic effect.