摘要
克隆小鼠白细胞介素12(IL-12)p40及p35cDNA,并构建同时含mIL-12p40和p35cDNA的双顺反子真核表达载体及其在哺乳动物细胞中的表达.白细胞介素12是由巨噬细胞,树突状细胞等抗原提呈细胞产生的一种异二聚体细胞因子,对机体的细胞免疫功能起着重要的调节作用.利用脂多糖(100pg/ml)和小鼠重组干扰素(IFN-γ500U/ml)体外联合刺激小鼠腹腔巨噬细胞,从中提取总RNA,经RT-PCR扩增出含信号肽的小鼠白细胞介素12(mIL-12)p40及p35全长cD-NA.PCR产物经酶切后,分别克隆至pBluescriptⅡSK载体中,序列测定结果与文献报道序列一致.然后利用脊髓灰质炎(Polio)病毒内核糖体进入位点(IRES)连接mIL-12p40及p35cDNA,亚克隆至pcDNA3载体中,构建成含mIL-12p40及p35cDNA双顺反子真核表达载体,即pcDNA3/mIL-12,p40及p35cDNA同时受pcDNA3中hCMV启动子驱动,将p40及p35转录至同一mR-NA上.通过LipofectAMINE将pcDNA3/mIL-12转染COS-7细胞,72h收集培养上清,测定m?
Murine interleukin 12 p40 and p35 cDNA,were cloned and bicistronic eukaryotic expression vector containing murine IL 12 p40 and p35 cDNA was constructed.Both p40 and p35 cDNAs of murine interleukin 12 were amplified by means of reverse transcription and polymerase chain reaction from total RNA of murine peritoneal macrophage stimulated synergistically by 500 U/ml IFN γ and 100 pg/ml LPS.The amplified cDNA fragments were inserted into pBluescriptSK plasmid respectively.The nucleotide sequence of both p40 and p35 cDNAs was determined by dideoxy termination and showed that it was the same sequence as that reported previously.Both p40 and p35 cDNAs of mIL 12 were linked by internal ribosome entry site (IRES) of 5′ nontranslation region from poliovirus and then cloned into the eukaryotic expression vector pcDNA3,thus constructing the eukaryotic expression vector pcDNA3/mIL 12 containing both p40 and p35 cDNAs.pcDNA3/mIL 12 was transfected into COS 7 cells by LipofectAMINE.RT PCR and murine IL 12 ELISA demonstrated expression of mIL 12 in COS 7 cells and the cultural supernatant of the COS 7 cells transduced with pcDNA3/mIL 12 could induce proliferation of murine spleen cells activated by ConA.Results suggest that cloning of mIL 12 can provide basis for further study of its immunoregulatory mechanism and antitumor immune response.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
1999年第1期48-53,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家"863"计划基金
国家自然科学基金