绵羊c-Myc与EGFP融合蛋白的真核表达及其对细胞增殖的调控作用  

Eukaryotic Expression of Sheep c-Myc and EGFP Fusion Protein and Its Regulation on the Cell Proliferation

作　　者：韩高链 郭丽荣 李孟心 郑腾飞 李俊玲 秦健 杜荣[1] HAN Gaolian;GUO Lirong;LI Mengxin;ZHENG Tengfei;LI Junling;QIN Jian;DU Rong(College of Animal Science and Veterinary Medicine,Shanxi Agricultural University,Taigu 030801,China;Centre of Experiment Teaching,Shanxi Agricultural University,Taigu 030801,China;College of Life Science,Shanxi Agricultural University,Taigu 030801,China)

机构地区：[1]山西农业大学动物科技学院,太谷030801 [2]山西农业大学实验教学中心,太谷030801 [3]山西农业大学生命科学学院,太谷030801

出　　处：《畜牧兽医学报》2020年第5期965-975,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31872438);国家留学基金(201708140150);山西省回国留学人员科研资助项目(2016-067);山西省留学回国人员科技活动择优资助项目;山西省平台基地和人才专项优秀人才科技创新项目(201605D211028);山西省重点研发计划项目(201803D221022-4);山西农业大学中青年拔尖创新人才支持计划(BJRC201204);山西农业大学横向科技项目(2015HX12)。

摘　　要：为了探讨c-Myc基因对细胞增殖及相关基因表达的调控作用,本试验构建了c-Myc-(G4S)3-EGFP融合基因的真核表达载体pc-Myc-EGFP,并利用生物信息学分析了其结构和理化特征。转染后分别通过细胞计数和qRT-PCR检测了其对绵羊胚胎成纤维细胞(sheep embryonic fibroblasts,SEF)增殖及相关基因表达的影响,并利用启动子分析软件解析了相应的表达调控机制。酶切鉴定和测序结果表明,载体构建成功。生物信息学分析表明,绵羊c-Myc蛋白主要定位于细胞核,含有螺旋环螺旋(helix-loop-helix,HLH)结构域,其分子量为48474.78 u。融合蛋白c-Myc-(G4S)3-EGFP有较强的亲水性,且含有31个磷酸化位点。因(G4S)3有较高的灵活度(9.8485),两侧的c-Myc和EGFP都保持各自的空间构象且互不干扰。进一步的细胞试验结果表明,重组质粒pc-Myc-EGFP在SEF中能够表达。过表达c-Myc促进了SEF的增殖,使cyclin D2、Cdk4、Cdk6、cyclin E1、cyclin A2、cdc25A mRNA的表达水平分别升高至5.20、3.10、6.54、6.52、23.37和8.22倍(P<0.01),E2F1 mRNA的表达升高至1.83倍(P<0.05)。启动子分析结果表明,各基因的5′调控区存在c-Myc和E2F1的结合元件。综上所述,绵羊c-Myc通过上调细胞周期相关基因的表达促进SEF细胞增殖,c-Myc的作用机制可能是通过直接作用于靶基因5′调控区的E-box元件,也可能是通过其他转录因子(如E2F1)的间接调控作用而实现。In order to explore the regulatory effects of c-Myc gene on the cell proliferation and the related genes expression,the eukaryotic expression vector pc-Myc-EGFP of the c-Myc-(G 4S)3-EGFP fusion gene was constructed and its structure and physicochemical characteristics were analyzed by bioinformatics in this experiment.After transfecting into sheep embryonic fibroblasts(SEF),the effects of c-Myc on the cell proliferation and the related genes expression were detected by cell counting and qRT-PCR,respectively,and the corresponding expression regulation mechanism was analyzed by the promoter analysis software.The results of restriction enzyme digestion and sequencing indicated that the vector was successfully constructed.The bioinformatics analysis showed that the sheep c-Myc protein,with a molecular weight of 48474.78 u,was mainly located in the nucleus and contained a helix-loop-helix(HLH)domain.The fusion protein c-Myc-(G 4S)3-EGFP was highly hydrophilic and contained 31 phosphorylation sites.(G 4S)3 had a high degree of flexibility(9.8485),c-Myc and EGFP on both sides maintained their respective spatial conformation and did not interfere with each other.The further cellular assay results indicated that the recombinant plasmid pc-Myc-EGFP was expressed successfully in SEF.Overexpression of c-Myc promoted the proliferation of SEF and increased the expression level of cyclin D 2,Cdk 4,Cdk 6,cyclin E 1,cyclin A 2 and cdc 25A mRNA up to 5.20,3.10,6.54,6.52,23.37 and 8.22 times(P<0.01)respectively,while E2 F1 mRNA up to 1.83 times(P<0.05).The results of promoter analysis indicated that there were binding elements for c-Myc and E2F1 on the 5′regulatory region of the above genes.In conclusion,sheep c-Myc promoted SEF proliferation by up-regulating the expression of the cell cycle-related genes,which might be through directly acting on E-box elements of the 5′regulatory region of target genes,or through the indirect regulatory role of other transcription factors such as E2F1.

关 键 词：绵羊 C-MYC基因 EGFP 真核表达 生物信息学 细胞周期相关基因 调控元件 

分 类 号：S826.2[农业科学—畜牧学]