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目的:构建Visfatin原核表达质粒,诱导表达并纯化重组人内脂素(Visfatin)。方法:从人LO2细胞中提取总RNA后,经RT-PCR法得到人Visfatin的c DNA序列,用以构建带HIS标签的Visfatin重组质粒。然后将重组质粒转化到大肠杆菌BL21中,用异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达,用Ni柱亲和层析纯化融合蛋白。结果:PCR扩增出的目的基因片段为1600 bp左右,与理论大小一致。SDS-PAGE、Western blot、质谱分析数据证明了蛋白是Visfatin融合蛋白。结论:成功的表达并纯化得到带HIS标签的Visfatin融合蛋白。
OBJECTIVE: To construct prokaryotic expression plasmid of Visfatin, induce expression and purify recombinant human visfatin. Methods: After total RNA was extracted from human LO2 cells, the c-DNA sequence of human Visfatin was obtained by RT-PCR to construct the Visfatin recombinant plasmid with HIS tag. The recombinant plasmid was then transformed into E. coli BL21 and induced with isopropyl thio-β-D-galactoside (IPTG). The fusion protein was purified by Ni column affinity chromatography. Results: PCR amplification of the target gene fragment of about 1600 bp, consistent with the theoretical size. SDS-PAGE, Western blot, mass spectrometry data show that the protein is Visfatin fusion protein. Conclusion: The HIS-tagged Visfatin fusion protein was successfully expressed and purified.