作　　者： ; ; ; ; ; ; ; ;

机构地区： 广州军区广州总医院

出　　处： 《中国微侵袭神经外科杂志》 2007年第7期321-324,共4页

摘　　要： 目的构建大鼠白细胞介素12(rIL-12)基因真核表达载体质粒,建立rIL-12基因修饰并稳定表达的大鼠胶质瘤9L/rIL-12细胞。方法用Trizol提取大鼠腹腔巨噬细胞总RNA,RT-PCR方法分别获取rp40和rp35cDNA,依次克隆入pcDNA3.1(-)/myc-HisA质粒中,以IRES连接双亚基,构建成双顺反子真核表达载体质粒pcDNA3.1/rIL-12。将构建的重组质粒转染大鼠胶质瘤9L细胞,G418筛选单克隆细胞株,ELISA法检测其培养上清rIL-12(p70)蛋白含量,同时抽提细胞RNA,RT-PCR方法检测rp40、rp35基因在9L/rIL-12细胞中的表达。结果所得rp40cDNA序列与GeneBankNM022611及AF133197、rp35cDNA序列与GeneBankNM053390及AF177031均一致,构建的质粒经PCR、酶切及测序鉴定正确。质粒转染9L细胞后,获得2个rIL-12基因稳定、高效表达的9L/rIL-12单克隆细胞株,其培养上清rIL-12(p70)蛋白含量分别为139.0、162.1pg/ml,RT-PCR结果显示细胞中rIL-12基因表达呈阳性。结论成功构建了rIL-12真核表达质粒pcDNA3.1/rIL-12,并建立了9L/rIL-12细胞株,为rIL-12基因修饰的胶质瘤疫苗研究打下了基础。 Objective To construct eukaryotic expression plasmid containing rat interleuldn 12 （rIL-12） genes and establish 9L/rIL-12 strains stably expressing rIL-12. Methods Both rp40 and rp35 cDNA of rIL-12 were amplified by reverse transcription-polymerase chain reaction （RT-PCR） from total RNA of rat peritoneal macrophages stimulated by lipopolysaccharide （LPS）. The amplified fragments were inserted into enkaryotic expression plasmid pcDNA3.1 （-）/myc-His A respectively. Both p40 and p35 cDNA were linked by internal ribosome entry site （IRES）. The constructed plasmid pcDNA3.1/rIL-12 was identified by PCR, restriction enzyme digestion, and DNA sequence analysis. Then the constructed plasmid was transfected into rat glioma 9L cells. The 9L/rIL-12 monoclonal cell sWains were selected with G418 subsequently. The expression of rIL-12 gene in 9L/rIL-12 cells was detected by ELISA and RT-PCR. Results The sequences oflM0 and p35 cDNA were consistent with GenBank NM022611, AF133197 and GenBank NM053390, AF177031 respectively. The constructed plasmid was confirmed by PCR, restriction enzyme digestion and sequencing. Two monoclonal sWains with stably and highly expressed rIL-12 genes were gained, and the concentration of rIL-12 p70 protein in culture supematant of 9L/rIL-12 strains was 139.0 and 162.1 pg/ml respectively. RT-PCR demonstrated expression of both rp40 and rp35 subunit genes in 9L/rIL-12 cells. Conclusion The enkaryotic expression plasmid pcDNA3.1/rIL-12 has been constructed successfully and 9L/rIL-12 strains established, which provides a foundation for studying glioma vaccine modified by IL-12 gene.

关 键 词： 大鼠 白细胞介素 质粒 神经胶质瘤 细胞

领　　域： [生物学]