关键词: 大肠杆菌, 人白介素24, 原核表达, 二次PCR, 融合蛋白

Abstract: Taking the interleukin (IL-24) in the lab as the template, recombinant prokaryotic vector of expressing pET28a-IL-24 was constructed, the vector was transferred into E.coli DH5α and the monoclones was picked out. The mutant(G→A in IL-24 cDNA)resulted in the change of IL-24 protein(A412T), The mutants(A→G, C→T, in IL-24 cDNA)did not change the amino acids on the bit. By designing mutation primers, correction of mutation was performed by a two-step PCR reaction, the correct recombinant vector was transformed into E.coli. BL21. The optimal condition was confirmed by changing the concentration of IPTG and induction time and temperature, the fusion protein 6his-IL-24 was detected by SDS-PAGE and Western blotting. As a result, by two-step PCR, the prokaryotic expression vector was constructed, the expression product was existed in the form of inclusion body, the best induction temperature was 28 ℃, the optimal IPTG concentration was 1 mmol/L and the best induction time was 8 h.

Key words: Escherichia coli, interleukin-24(IL-24), prokaryotic expression, two-step PCR, fusion protein