人白细胞介素32基因的克隆表达及其生物学活性的研究

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.

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

AIM: To clone human interleukin-32(hIL-32) gene and express it in E.coli 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.coli BL21(DE3) strain.The hIL-32 protein expression was induced by IPTG and assayed by SDS-PAGE and coomassie blue stain.The re...