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目的:克隆人白细胞介素32(hIL-32)基因,构建高效稳定的hIL-32大肠杆菌表达菌株,并对纯化的目的蛋白进行生物学活性的初步测定。方法:将人外周血单个核细胞(PBMC)经刀豆素A(Con A)刺激60 h后提取细胞的总RNA,通过逆转录聚合酶链式反应(RT-PCR)从刺激的PBMC中扩增出hIL-32的基因,通过基因克隆技术,构建hIL-32在pET-30 a(+)中的重组质粒pET30a-hIL32,用IPTG进行诱导,得到hIL-32的原核表达蛋白;采用Ni2+-NTA亲和层析方法纯化目的蛋白;应用ELISA方法检测纯化蛋白诱导人PBMC产生IL-6的情况。结果:序列测定表明,hIL-32基因核苷酸长度为567 bp,编码189个氨基酸。重组质粒pET30-hIL32转化至大肠杆菌BL21(DE3),重组菌菌体裂解物SDS-PAGE可检测到相对分子质量(Mr)为28 000的重组蛋白,表达的重组蛋白IL-32可促进人PBMC产生IL-6,浓度达到(127±4.8)ng/L,而对照组IL-6的浓度仅为(25±2.3)ng/L。结论:成功克隆了hIL-32基因并构建了高效且稳定表达hIL-32基因的大肠杆菌菌株,且表达的目的蛋白能诱导IL-6细胞因子的产生。
OBJECTIVE: To clone human interleukin 32 (hIL-32) gene and construct a highly efficient and stable hIL-32 E. coli expression strain. The purified recombinant protein was preliminarily assayed for its biological activity. Methods: Total RNA was extracted from human peripheral blood mononuclear cells (PBMCs) stimulated by Con A for 60 h. The total RNA was extracted from the stimulated PBMCs by reverse transcription-polymerase chain reaction (RT-PCR) The recombinant plasmid pET30a-hIL32 of hIL-32 in pET-30 a (+) was constructed by gene cloning technique and induced by IPTG to obtain the prokaryotic expression product of hIL-32. NTA affinity chromatography purification of the target protein; ELISA method was used to detect the purified protein induced IL-6 production of human PBMC. Results: Sequence analysis showed that the nucleotide length of hIL-32 gene was 567 bp, encoding 189 amino acids. The recombinant plasmid pET30-hIL32 was transformed into E.coli BL21 (DE3). Recombinant bacterial lysate SDS-PAGE detected a recombinant protein with a molecular weight of 28 000 (Mr). The expressed recombinant protein IL-32 can promote human PBMC The concentration of IL-6 was (127 ± 4.8) ng / L, while that of control group was only (25 ± 2.3) ng / L. CONCLUSION: The hIL-32 gene was cloned successfully and the E. coli strain highly efficient and stably expressing hIL-32 gene was constructed. The expressed protein can induce the production of IL-6 cytokines.