真核表达载体pcDNA3.1(-)-IL-3-PE38KDEL的构建及其在CIK细胞中的表达

Construction of eukaryotic expression plasmid pcDNA3.1(-)-IL-3-PE38KDEL and its expression in cytokine-induced killer cells

目的:构建人白细胞介素-3(interleukin-3,IL-3)和铜绿假单胞菌外毒素(pseudomonas exotoxin,PE)衍生物(PE38KDEL)基因的真核融合表达载体,研究其在细胞因子诱导的杀伤(cytokine induced killer,CIK)细胞中的表达情况。方法:通过PCR获得实验需要的IL-3-PE38KDEL基因片段,构建IL-3-PE38KDEL融合基因的真核表达载体pcDNA3.1(-)-IL-3-PE38KDEL。重组质粒经限制性内切酶及DNA序列测定证实构建成功后,用Liprofectamine2000脂质体转染CIK细胞,然后用RT-PCR及Western blot检测IL-3-PE38KDEL融合蛋白在CIK细胞的表达,用MTS检测细胞上清中融合蛋白的细胞毒作用。结果:酶切分析及DNA序列测定证实,IL-3-PE38KDEL融合基因被成功克隆入真核表达质粒载体pcDNA3.1(-);RT-PCR及Western blot法检测证实重组基因可在CIK细胞中表达,且表达的融合蛋白对HL60细胞有杀伤活性。结论:IL-3-PE38KDEL融合基因可在CIK细胞中表达,为进一步研究融合毒素IL-3-PE38KDEL联合CIK细胞过继免疫治疗对肿瘤细胞的体内外杀伤作用奠定了基础。

Objective：To construct an eukaryotic expressive vector of interleukin-3（IL-3）-PE38KDEL and to study the expressions of this gene in cytokine-induced killer（CIK） cells.Methods：IL-3-PE38KDEL fusion gene was cloned into the eukaryotic expression vector to get the recombinant plasmid pcDNA3.1（-）-IL-3-PE38KDEL by molecular cloning technique.The fusion gene was transfected into CIK cells by Liprofectamine2000 immediately after it was confirmed by restrictive enzyme analysis and sequencing.RTPCR and Western blot were used to confirm expressions of the fusion gene in the CIK cells.Prohibitory effect of fusion protein IL-3PE38KDEL on HL60 cells was assessed by MTS assay.Results：Restrictive endonuclease digestion and sequence analysis revealed that IL-3-PE38KDEL fusion gene was cloned into the eukaryotic expression plasmid vector pcDNA3.1（-）.IL-3-PE38KDEL fusion gene could express in the CIK cells and the fusion protein could inhibit HL60 cell growth.Conclusions：IL-3-PE38KDEL fusion gene eukaryotic expression vector is constructed successfully and can be expressed in CIK cells,which provide the basis for the further experiment in vitro and in vivo.