Construction,expression,purification and quality identification of kanamycin resistant strain of recombinant human interleukin-1 receptor antagonist / 中国生物制品学杂志

ABSTRACT

@#Objective To construct a recombinant human interleukin-1 receptor antagonist(rhIL-1Ra)strain of kanamycin resistance,express,purify and identify rhIL-1Ra protein in order to reduce the risks of β-lactam antibiotics.Methods The ampicillin-resistant rhIL-1Ra(A-rhIL-1Ra)plasmid and PET-28a plasmid were used as templates to prepare the linearized vector and kanamycin gene fragment by PCR.The kanamycin resistant rhIL-1Ra plasmid(K-rhIL-1Ra)was constructed by homologous recombination,which was transformed into E.coli BL21(DE3)to construct the recombinant engineered bacteria after correct sequencing.The engineered bacteria were induced by IPTG and then purified by CM Bestarose Fast Flow and DEAE Bestarose Fast Flow column chromatography.The purified products were collected,and then detected for the purity by 15% SDS-PAGE,size-exclusion high-performance liquid chromatography(SEC-HPLC),and reversed-phase high-performance liquid chromatography(RP-HPLC),determined for the relative molecular mass by tandem mass spectrometry,identified for the specificity by Western blot,measured for the biological activity by reporter gene assay,and compared for the related impurities by liquid chromatography-mass spectrometry(LC-MS)analysis.Results Colony PCR and sequencing results showed that K-rhIL-1Ra plasmid was constructed correctly.The expressed K-rhIL-1Ra protein had a relative molecular mass of about 17 000,mainly existing in soluble form,and the expression amount accounted for more than 30% of the total bacterial proteins.The purity of K-rhIL-1Ra protein purified by two steps was 97% and 99%,the monomer content was 99.33%and 100%,and the chromatographic purity was 91.86% and 96.96%,respectively.The mass spectral molecular mass was consistent with that of the standard(A-rhIL-1Ra protein),and the protein reacted specifically relative with mouse antihuman IL-1Ra monoclonal antibody.The biological activity of the purified K-rhIL-1Ra protein was 1.29 × 105U/mg,and the chemical modification types of related proteins were the same as those of the standard(A-rhIL-1Ra protein).Conclusion The K-rhIL-1Ra strain was successfully constructed,and the expressed and purified protein was in line with the characteristics and quality standards of rhIL-1Ra,which lays a foundation for the study of rhIL-1Ra strain change and comparability