携带白细胞介素2和NK4双基因真核共表达质粒的构建及活性观察

目的 构建同时携带白细胞介素2（IL-2）和NK4基因的真核表达载体（pIRES-IL-2-IRES-NK4）,观察其在防治肿瘤中的潜在应用前景.方法 根据Pubmed上公布的IL-2及NK4 mRNA序列,设计扩增IL-2及NK4 cDNA的特异性引物,以PBV220-IL-2质粒和PCAGGS/hNK4质粒为模板,分别扩增IL-2及NK4基因,并将IL-2及NK4基因分别克隆在真核表达载体pIRES-SEQ的XhoI、MLuI位点和SalI、NotⅠ位点.获得同时携带IL-2和NK4基因的重组真核表达质粒pIRES-IL-2-IRES-NK4.该重组质粒转染肝癌细胞HepG2后,检测目的基因的转染表达及表达产物对HepG2细胞增殖的影响.结果 通过对构建质粒克隆进行测序及酶切,证实携带IL-2和NK4双基因的真核表达质粒pIRES-IL-2-IRES-NK4构建成功.用pIRES-IL-2-IRES-NK4转染HepG2细胞后24 h后在荧光显微镜下可观察到细胞有较强的绿色荧光表达,48 h时荧光更强;逆转录聚合酶链反应结果表明转染pIRES-IL-2-IRES-NK4质粒细胞的IL-2及NK4基因表达明显增强.酶联免疫吸附测定检测结果表明,转染组细胞较对照组细胞培养上清中IL-2及NK4蛋白表达水平明显增强.转染组48 h后的IL-2、NK4蛋白表达水平为2.139、1.956 mg/L,明显高于未转染组的0.492、0.620 mg/L.四甲基偶氮唑盐法结果表明转染真核表达质粒pIRES-IL-2-IRES-NK4的细胞表达上清,有明显抑制肝癌细胞HepG2增殖的活性,且有剂量效应关系.结论 本研究成功构建了同时携带IL-2和NK4基因的重组真核表达质粒pIRES-IL-2-IRES-NK4,该质粒可有效转染肝癌细胞,且转染细胞表达上清有明显抑制肝癌细胞HepG2增殖的活性,提示该重组质粒有潜在防治肿瘤的应用前景.

Construction of a plasmid vector containing interleukin-2 gene and NK4 gene

Objective To construct a recombinant plasmid （pIRES-IL-2-IRES-NK4） encoding inteneukin-2（IL-2） gene and NK4 gene and to investigate its expression in liver cancer cells.Methods IL-2 gene and NK4 gene were obtained from plasmid PBV220-IL-2 and PCAGGS/hNK4 by polymerase chain reaction（PCR）,respectively.IL-2 gene was subcloned into plasmid pIRES-SEQ at Xho I and MLu I sites ; NK4 gene was subcloned into plasmid pIRES-IL-2 at Sal I and Not I sites.A recombinant plasmid pIRES-IL-2-IRES-NK4 was obtained.The plasmid was transfected into HepG2 ; the expression level of IL-2 and NK4 protein was evaluated by RT-PCR and ELISA.The biological effects of the IL-2/NK4-expressing product at different doses on liver cancer cells were assessed by MTT.Results The recombinant plasmid vector for IL-2 gene and NK4 gene （pIRES-IL-2-IRES-NK4） was successfully constructed.The green fluorescence protein could be observed under fluorescent microscope in HepG2 at 24 h after transfection and showed a stronger degree after 48 h.The result of RT-PCR revealed that the expression of IL-2 gene and NK4 gene was significantly stronger in pIRES-IL-2-IRES-NK4-transfected cells than that in pIRES-GFP-transfected cells.The expression of IL-2 and NK4 protein was also higher in supernatant of pIRES-IL-2-IRES-NK4-transfected group（2.139,1.956 mg/L） than that of in supernatant of pIRES-GFP-transfected group（0.492,0.620 mg/L）.The IL-2/NK4-expressing product could significantly inhibit proliferation of liver cancer cells in a dose-dependent manner.Conclusions A recombinant plasmid pIRES-IL-2-IRES-NK4,encoding IL-2 gene and NK4 gene,can be constructed in vitro and expressed successfully in HepG2 cells.It provides the material basis for further studying the biologic function and potential application of the recombinant plasmid.