Preparation of human interleukin-6 recombinant protein
Objective To express and purify human interleukin-6(IL-6)recombinant protein via prokaryotic expression system,which provides a candidate material for the preparation of IL-6 reference and quality control materials at a later stage.Methods The target gene segment was amplified by PCR using the existing IL-6 prokaryotic expression plasmid 6His-IL-6-pET-28a(+)as a template and a His-tag was added to its N-terminus.The synthetic segment was cloned into the pRSFDuet prokaryotic expression vector by enzyme digestion and ligation to construct a 6His-IL-6-pRSFDuet recombinant plasmid,and then the plasmid was transformed into E.coli BL21 expressing strain.The expression of recombinant IL-6 protein was induced by isopropyl-β-d-thiogalactoside(IPTG),and the protein was purified by nickel affinity chromatography.The protein expression levels of the two strains were compared,and the one with the higher expression was selected for the subsequent experiments.Results The 6His-IL-6-pRSFDuet prokaryotic expression plasmid was successfully constructed,and the 6His-IL-6-pET-28a(+)plasmid was selected for the subsequent experiments according to the protein expression comparison.After optimizing the expression conditions,it was found that the highest expression of IL-6 recombinant protein could be obtained after 20 h induction with IPTG at a final concentration of 0.2 mmol/L at 20℃.The purified IL-6 protein was detectable in the clinical laboratory,and there was a linear relationship between the dilution ratio and the detection results.Conclusion The low cost,high concentration and high purity of IL-6 recombinant proteins are successfully obtained,which lays the foundation for the subsequent preparation of reference and quality control materials.
interleukin-6recombinant proteinprokaryotic expressionreference materialquality control product
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