白细胞介素-24基因重组表达载体的构建及原核表达

Construction of recombinant expression vector of IL-24 gene and prokaryotic expression

目的:构建人白细胞介素-24(hIL-24)基因的表达载体并在大肠杆菌中表达。方法:用PCR从质粒TRAP-IL-24中扩增hIL-24cDNA片段,并将该片段插入pGEX-KG原核表达载体中,实现插入基因的融合表达。用SDS-PAGE和West-ernblot对表达产物进行鉴定。采用MTT法检测GST-IL-24融合蛋白的生物学活性。结果:酶切结果证实,成功地构建了pGEX-KG IL24原核表达载体,并在大肠杆菌中获得稳定的表达,表达产物的相对分子质量(Mr)同预期值相一致。GST-IL24融合蛋白能够抑制THP1细胞的生长。结论:成功地构建了重组表达载体pGEX-KG-IL24,并在E.coliBL21中表达了具有生物学活性的GST-IL-24融合蛋白,为下一步研究人IL24的功能奠定了基础。

AIM: To construct a recombinant expression vector of human IL-24 gene and express it in E.coli. METHODS: The hIL-24 cDNA fragment was amplified from plasmid TRAP-hIL-24 by PCR, then cloned into the prokaryotic vector pGEX-KG, and expressed as a fusion protein in E.coli. The expressed IL-24-GST fusion protein was purified via GST-Sepharose 4B Column and identified by SDS-PAGE and Western blot. The bioactivity of GST-IL-24 fusion protein was measured by MTT assay. RESULTS: Restriction enzyme digestion analysis showed that the recombinant prokaryotic expression vector pGEX-KG-IL-24 was successfully constructed and expressed in E.coli. The relative molecular mass(M_r) of the expression product was identical with the prediced value. The proliferation of THP-1 cells was inhibited by GST-IL-24 fusion protein. CONCLUSION: The recombinant expression vector pGEX-KG-IL-24 has been constructed successfully and expressed as a bioactive fusion protein in E.coli BL21 (BlysS), which is helpful for the further study of the biological function of IL-24.