pcDNA3.1/hIL-18真核表达载体的构建及表达

目的 :构建重组真核表达质粒pcDNA3.1/IL 18,并在哺乳动物细胞COS 7和Rlc310中进行瞬时和稳定性表达。方法 :从含hIL 18基因的中介载体 pGEM TEasy( pGEM T/hIL 18)中 ,以限制性内切酶酶切方法获得目的片段 ,克隆入真核表达质粒 pcDNA3.1( )中。以脂质体法转染COS 7和Rlc310细胞 ,用RT PCR检测IL 18mRNA的水平 ,免疫组化染色法检测蛋白表达。结果 :构建了hIL 18基因的重组真核表达质粒pcDNA3.1/IL 18,并可在哺乳动物细胞中瞬时、稳定表达 ,获得了可稳定表达hIL 18基因的Rlc310细胞株。结论 :pcDNA3.1/IL 18的构建及表达 ,为IL 18抗肿瘤作用的研究奠定了基础

Construction and expression of eukaryotic expression plasmid pcDNA3.1/hIL-18

AIM: To construct an eukaryotic expression plasmid pcDNA3.1/hIL 18 and express it in mammalian cells. METHODS: cDNA encoding mature hIL 18 was cleavaged by enzyme digestion from mesomeric clone vector pGEM T/hIL 18 and inserted into an eukaryotic expression plasmid pcDNA3.1 to construct a recombinant expression plasmid pcDNA3.1/hIL 18. Then the constructed plasmid was transfected into COS 7 and Rlc310 cells by liposome mediated gene transfer method. hIL 18 expressed in transfected COS 7 and Rlc310 cells was detected by immunohistochemical staining and level of hIL 18 mRNA in transfected Rlc310 cells was assayed by RT PCR. RESULTS: A recombinant eukaryotic expression plasmid pcDNA3.1/hIL 18 was successfully constructed and expressed transiently in COS 7 cells and stably in Rlc310 cells. CONCLUSION: The construction and expression of pcDNA3.1/hIL 18 have been achieved successfully, which lays a foundation for further research on anti tumor effect of IL 18.