人白细胞介素32基因的克隆表达及其生物学活性的研究

Cloning and expression of human interleukin-32 and studies on its bioactivity

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。

AIM： To clone human interleukin-32 （ hIL- 32） gene and express it in E. coil efficiently. METHODS： The gene of hIL-32 was amplified by RT-PCR from human peripheral blood mononuclear cells （PBMC） which stimulated with Con A for 60 h. The PCR product was inserted into the pMD18-T vector. The hIL-32 cDNA confirmed by sequencing was inserted into expression vector pET-30a （ ＋ ） and expressed in E. coil BL21 （DE3） strain. The hIL-32 protein expression was induced by IPTG and assayed by SDS- PAGE and coomassie blue stain. The recombination protein was identified by Western blot and its biological activity was analyzed. RESULTS： DNA sequencing confirmed that the cloned cDNA was identical to the published sequence of hIL- 32 that the nucleotide sequence of this gene was 567 bp. The recombinant plasmid pET30a-hIL32 was transformed into E. coil BL21 （DE3） strain for expression. An expected 28 kDa protein of hIL-32 was found mainly in the induced host strains by SDS-PAGE and coomassie blue stain. The 28 kDa protein was recognized by anti-IL-32 antibody in western blot. The purified recombination protein could induce the producing of IL-6 in the human PBMC. CONCLUSION： We have successfully cloned the gene and expressed the protein of hIL-32 and the expressed protein has specific bioactivity.