三种IL-15基因转染NCI-H446细胞模型的建立与鉴定

Establishment and identification of NCI-H446 cell models transfected by three different IL-15 genes

目的构建并鉴定三种IL-15基因转染的人小细胞肺癌(NCI-H446)细胞模型。方法PCR法扩增得原型IL-15基因片段(FhIL-15)I、L-15成熟肽基因片段(FhIL-15mp)和IL-2信号肽基因片段(FhIL-2sp);利用重叠延伸基因拼接法将FhIL-15mp和FhIL-2sp拼接成改型IL-15基因片段(FhIL-2sp-hIL-15mp)。将三种IL-15基因片段(FhIL-15、FhIL-15mp和FhIL-2sp-hIL-15mp)分别插入真核表达载体pEGFP-N1构建成相应的重组质粒(PhIL-15、PhIL-15mp和PhIL-2sp-hIL-15mp),用脂质体法分别转染NCI-H446、G418筛选得三种IL-15基因转染的NCI-H446细胞(C-hIL-15、C-hIL-15mp、C-hIL-2sp-hIL-15mp)。对所得的各基因片段和重组质粒,用琼脂糖凝胶电泳和测序鉴定;对所得的各转染细胞,用RT-PCR法检测hIL-15mRNA的表达,ELISA法检测hIL-15蛋白的分泌。结果琼脂糖凝胶电泳和测序证明,FhIL-15、FhIL-15mp、FhIL-2sp-hIL-15mp电泳条带位置和基因序列正确。三种转染细胞均有IL-15mRNA表达,但仅C-hIL-2sp-hIL-15mp可测到22pg/mL的IL-15蛋白分泌。初步应用表明,三种转染细胞确有不同的免疫生物学特性。结论正确构建了三种IL-15基因转染的NCI-H446细胞模型,可在进一步研究IL-15基因与肿瘤细胞免疫生物学特性的关系及至机制中应用。

Objective To construct and identify the NCI-H446 cell models transfected by three IL-15 genes of different structure. Methods The prototypic IL-15 gene (F_(hIL-15)), IL-2 signal peptide gene (F_(hIL-2) sp), and IL-15 mature peptide gene (F_(hIL-15mp)) were cloned by PCR, and then the modified IL-15 gene (F_(hIL-2sp-hIL-15mp)) was constructed by fusing F_(hIL-2sp) and F_(hIL-15mp) using the gene splicing by overlap extension (SOEing). FhIL-15, FhIL-15mp, and F_(hIL-2sp-hIL-15mp) were inserted into the vector pEGFP-N1 to construct their recombinant eukaryotic expression vectors (P_(hIL-15), P_(hIL-15mp), and P_(hIL-2sp-hIL-15mp)), and then transfected into NCI-H446 cells by Lipofectamine 2000, respectively. The transfected cells (C-_(hIL-15), C-_(hIL-15mp) and C-_(hIL-2sp-hIL-15mp)) were selected by G418. All the obtained genes and the expression vectors were identified by gel electrophoresis and sequencing. For all transfected cells, the mRNA expression and protein secretion of IL-15 were analyzed by RT-PCR and ELISA, respectively. Results The gel electrophoresis and gene sequencing revealed that the F_(hIL-2sp-hIL-15mp), F_(hIL-15), and F_(hIL-15mp) were coincidence with the Gene Bank and their corresponding recombinant plasmids were constructed successfully. The IL-15 mRNA was detected in all the three kinds of transfected cells, but the hIL-15 protein secretion was only detected in the supernatants of the C-_(hIL-2sp-hIL-15mp). The initial experiments suggested that the three kinds of transfected cells exhibited significant difference in the immunobiological characteristics. Conclusion The obtained model cells are qualified and can be used to investigate the relationship between the IL-15 gene and the tumor cells.