米非司酮调控的单链鼠白介素12真核表达载体的构建及鉴定  

Construction and identification of mefepristone-regulated eukaryotic expression vector containing single chain fusion murine IL-12 gene

作　　者：陈坚[1] 薛绪潮 方国恩[2] 苏长青 钱其军 

机构地区：[1]解放军第81医院肿瘤外科,江苏南京210002 [2]上海第二军医大学附属长海医院普通外科,上海200433 [3]东方肝胆医院病毒基因治疗实验室,上海200438

出　　处：《生物学杂志》2008年第6期17-21,共5页Journal of Biology

基　　金：国家自然科学基金资助(项目批号:30571830)

摘　　要：构建含有单链鼠白细胞介素12(mIL-12)基因,并可经米非司酮调控表达的真核载体,在体外鉴定其调控表达效果。用PCR技术从GCp35Ep40PN质粒上获取白细胞介素12的p40和p35亚基的基因,通过2次PCR技术引入linker后得到单链白细胞介素12基因,将其与米非司酮诱导调控系统克隆入pDC312质粒,构建成真核表达载体pDC-RUmIL-12,通过限制性内切酶和PCR鉴定其正确性。在体外用脂质体转染法将pDC-RUmIL-12载体转染HEK293细胞株,以不同的剂量米非司酮诱导载体表达,然后用ELISA法检测培养液上清中mIL-12蛋白的含量。测序结果表明所得鼠白细胞介素-12融合单链基因序列与设计相一致。酶切分析和PCR证实载体pDC-RUmIL-12构建正确。将pDC-RUmIL-12转染HEK293细胞并经米非司酮诱导表达后,ELISA检测结果表明该载体有良好的调控能力。没有诱导剂米非司酮时,只有极少量的mIL-12蛋白表达,而加入诱导剂米非司酮后,mIL-12表达量明显增加,并在一定范围内与米非司酮剂量呈正比。成功构建了含有单链鼠白细胞介素12(mIL-12)基因,并可经米非司酮调控表达的真核载体,可用于肿瘤的基因治疗研究。To construct mefepristone-regulated eukaryotic expression vector containing single chain fusion murine IL-12 （miL- 12） gene,and identify its inducilbe expression in vivo, the p40 and p35 subunit were obtained from the plasmid GCp35Ep40PN by PCR technique. Single chain fusion mIL-12 gene was constructed by introducing a linker after twice PCR. The regulatable eukaryotic expression vector was constructed with molecular biological methods including single chain mIL-12 gene and mifepristoneinducible system. The positive clone named pDC-RUmIL-12 was identified by restriction endonuclease digestion and further confirmed by PCR and ONA sequencing. The ELISA method was used to demonstrate the activation of this regulatable vector after transient transfection in HEK293 cells using lipofeetamine2000. Sequencing analysis result showed the single chain fusion murine IL-12 gene was identical to the designed. The regnlatable vector was confirmed by enzyme digestion and PCR assay. The mIL-12 protein concentration in supernatant was very low without the inducer mifepristone, whereas expression of mIL-12 protein was increased significantly in the presence of mifepristone. The relationship between protein concentration and the dose of inducer was positive correlation under definite range. Regulatable eukaryotic expression vector of single chain fusion mIL-12 gene was successfully constructed. This inducible expression vector can be used for the research of gene therapy.

关 键 词：白细胞介素-12 米非司酮 诱导表达 融合基因 

分 类 号：Q781[生物学—分子生物学]