摘要
目的构建双亚基共表达的大鼠白细胞介素12(rIL-12)基因真核表达载体质粒。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rp35两个亚基的cDNA,依次克隆入pcDNA3.1(-)/myc-His A质粒中,以脊髓灰质炎病毒的内核糖体进入位点连接双亚基,构建成双顺反子真核表达载体质粒pcDNA3.1/rIL-12。用电转染法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rp40 cDNA序列与GenBank NM022611和AF133197、rp35 cDNA序列与GenBank NM053390和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,获得9L/rIL-12单克隆细胞株,其培养上清rIL-J2(p70)蛋白含量达139.0～162.1 PG/mL,RT-PCR结果显示细胞中rIL-12基因表达阳性。结论成功构建并鉴定了大鼠IL-12真核表达质粒pcDNA3.1/rIL-12。
Objective To construct eukaryotic expression plasmid containing two subunits of rat interleukin 12 gene. Methods Both p40 and p35 cDNAs of rat interleukin 12 were amplified by means of reverse transcription and polymerase chain reaction（RT-PCR） from total RNA of rat peritoneal macrophages stimulated by lipopolysaccharide （LPS）. The amplified fragments were inserted into eukaryotic expression plasmid pcDNA3.1 （-）/myc-His A respectively. Both p40 and p35 cDNA were linked by internal ribosom entry site （1RES）. The constructed plasmind pcDNA3, 1/rIL-12 was identified by PCR, restricted enzymes and DNA sequence analysis. Then the plasmind was tansfected into rat glioma cells 9L by electroperation. G418 resistant monoclonal cells 9L/IL-12 were selected and rat IL-12 （p70） protein in culture medium was evaluated by enzyme linked immunosorbent assay （ELISA）. Results The sequences of p40 and p35 cDNA were same as GenBank NM022611 AF133197 and NM053390 AF177031 respectively. The electrophoresis strips of constructed plasmid by PCR and digestion of restriction enzymes were coincident with expectation. The concentrations of rIL- 12 （p70） protein in culture supematant of 9L/IL- 12 monoclonal cells were 139.0-162.1 PG/mL. Conclusion The eukaryotic expression plasmid pcDNA3.1/rlL-12 was successfully constructed.
出处
《中华神经医学杂志》
CAS
CSCD
2007年第7期653-656,共4页
Chinese Journal of Neuromedicine
基金
全军医学科研"十五"计划项日(01MA038)
广东省自然科学基金(001122)
