人IL-18cDNA克隆及原核表达

Gene cloning and prokaryotic expression of recombinant human interleukin-18

目的 从外周血单核细胞中克隆人IL 1 8成熟编码序列cDNA ,对其进行测序并构建原核表达载体 ,进行原核表达。方法 从人外周血单核细胞中分离总RNA ,进行RT PCR获得人IL 1 8cDNA。测序证实其结果正确后 ,构建原核表达载体。E .coli的表达产物通过SDS PAGE、WesternBlot证实。结果 所克隆的人IL 1 8cDNA编码序列经测序证实与GenBank所报道的序列完全一致 ,细菌表达产物经SDS PAGE证实其大小约为 1 8.3KDa,WesternBlot进一步证实了所表达产物的正确性。结论 本研究结果为进一步探讨IL 1 8的生物学活性和特性奠定了基础 ,同时亦为利用人IL 1 8进行免疫基因治疗的研究打下了良好的基础。

ObjectiveTo clone human interleukin18 cod ing cDNA from monocytes of PBMC,sequence the cDNA and construct the recombinant hIL 18 prokaryotic expression plasmids to express hIL18 in E.coli.Met hodsTotal RNA from monocytes of PBMC was used to perform RTPCR to acquire hIL18 cDNA.After the sequence was confirme d by sequencing,the prokaryotic expression vectors are constructed.The expressio n product of E.coli was confirmed by SDSPAGE and Western Blot.Results The sequence of the cloned human open reading frame of hIL18 was exa ctly the same as that reported in GenBank.(Accession Number:E17135).SDSPAGE di splayed that the size of the bacterial expression product was approximately 18.3 KDa. The expression protein was further confirmed by Western Blot.ConclusionThe work has settled the foundation for further biol ogical research on hIL18,including immunogene therapy through hIL18.