可调控的鼠白细胞介素-12双亚基共表达质粒的构建及在体外表达  

Construction of Regulatable Murine IL-12 Eukaryotic Expression Plasmid of Single Chain Fusion Gene and Identification of Its Expression in Vitro

作　　者：陈坚[1] 张斌[1] 薛绪潮 方国恩[2] 苏长青[3] 钱其军[3] 

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

出　　处：《中国普外基础与临床杂志》2008年第12期892-897,共6页Chinese Journal of Bases and Clinics In General Surgery

基　　金：国家自然科学基金资助项目(编号:30571830)~~

摘　　要：目的构建可经米非司酮(RU486)调控表达小鼠白细胞介素-12(mIL-12)单链融合基因的真核载体,并鉴定其调控表达效果。方法以GCp35Ep40PN质粒为模板,分别获取mIL-12p40和p35亚基的基因,重叠PCR引入linker后,克隆入pCA14质粒,测序正确之后,装入可调控载体pRS-17而构建成真核表达质粒pRS-RUmIL-12。用Lipofectamine2000将pRS-RUmIL-12质粒转染HEK293细胞,以不同剂量的RU486诱导其表达,然后用ELISA法检测其培养上清液中mIL-12蛋白的含量。结果所得mIL-12单链融合基因序列与设计一致。pRS-RUmIL-12在体外转染HEK293细胞后,ELISA检测结果表明该系统具有良好的调控能力:无诱导剂RU486时,mIL-12蛋白表达量很低,而加入诱导剂RU486后,可以诱导mIL-12的表达,并在一定范围内mIL-12的蛋白表达量与诱导剂的浓度呈正相关。结论成功构建了可经RU486调控的mIL-12双亚基共表达的真核表达质粒,可用于进一步的基因调控和基因治疗研究。Objective To construct a regulatable plasmid containing single chain fusion gene of murine interleukin-12 （mIL-12） which was regulated with mifepristone （RU486） and explore its expression in vitro. Methods The p40 and p35 subunit sequence of mIL-20 were respectively obtained from the plasmid GCp35Ep40PN by polymerase chain reaction （PCR） and they were cloned into pCA14 plasmid after introducing a linker by overlap PCR. The single chain mIL-12 gene was comfirmed by sequencing and subcloned into pRS-17 vector which contains RU486 regulator cassette. The positive clone named pRS-RUmIL-12 was identified by restriction endonuclease digestion and PCR. Lipofectamine 2000 was used to transfect the pRS-RUmIL-12 to HEK293 cells followed by manufacturer＇s recommendations. The protein concentration of mIL-12 induced with RU486 in supernatant of the transfected HEK293 cells was measured by ELISA. Results The sequence of single chain mIL-12 what we obtained was the same as the expected result. The results Of restriction endonuclease digestion and PCR showed that the RU486-inducible regulatory vector （pRS-RUmIL-12） was successfully constructed. No significant raiL-12 protein concentration in supernatant of HEK293 cells activation was measured without the inducer RU486, whereas higher concentration of the mIL-12 protein was observed in the presence of RU486. The relationship of concentration of the mIL-12 protein and RU486 was positive correlated under definite range. Conclusion A regulatable eukaryotie expression plasmid of mlL-12 single chain fusion gene was constructed, which could be used in the further research of gene regulation and gene therapy.

关 键 词：白细胞介素-12 融合基因 真核表达 米非司酮 

分 类 号：Q78[生物学—分子生物学]