重组人白细胞介素-1受体拮抗剂卡那霉素抗性菌种的构建、表达、纯化及质量鉴定

Construction,expression,purification and quality identification of kanamycin resistant strain of recombinant human interleukin-1 receptor antagonist

目的构建卡那霉素抗性的重组人白细胞介素-1受体拮抗剂(recombinant human interleukin-1 receptor antagonist,rhIL-1Ra)菌种,并进行rhIL-1Ra蛋白的表达、纯化及质量鉴定,以降低β-内酰胺抗生素带来的风险。方法分别以氨苄抗性的质粒rhIL-1Ra(A-rhIL-1Ra)、pET-28a为模板,进行PCR扩增,获得线性化载体和卡那霉素基因片段,经同源重组构建卡那霉素抗性的质粒rhIL-1Ra(K-rhIL-1Ra),测序正确后转化E.coli BL21(DE3),构建重组工程菌,经IPTG诱导表达后,进行CM Bestarose Fast Flow、DEAE Bestarose Fast Flow两步柱层析纯化。收集纯化产物,15%SDSPAGE、分子排阻高效液相色谱法(size-exclusion high-performance liquid chromatography,SEC-HPLC)、反相高效液相色谱法(reversed-phase high-performance liquid chromatography,RP-HPLC)检测纯度,串联质谱法检测质谱相对分子质量,Western blot法鉴定特异性,报告基因法检测生物学活性,液相色谱-质谱(liquid chromatography-mass spectrometry,LC-MS)法分析比对产品相关杂质。结果经菌落PCR及测序鉴定证明质粒K-rhIL-1Ra构建正确。表达的K-rhIL-1Ra蛋白相对分子质量约17000,主要以可溶性形式存在,表达量占菌体总蛋白的30%以上;两步纯化的K-rhIL-1Ra蛋白纯度分别为97%和99%,单体含量分别为99.33%和100%,色谱纯度分别为91.86%和96.96%,质谱相对分子质量与标准品(A-rhIL-1Ra蛋白)一致,可与小鼠抗人IL-1Ra单抗发生特异性反应;纯化的K-rhIL-1Ra蛋白生物学活性为1.29×10^(5)U/mg,相关蛋白化学修饰类型与标准品(A-rhIL-1Ra蛋白)一致。结论成功构建了K-rhIL-1Ra菌种,表达并纯化后的蛋白符合rhIL-1Ra的特征和质量标准,为rhIL-1Ra菌种变更及可比性研究奠定了基础。

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 ofβ-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 17000,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 1.29×10^(5)U/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.