牛c-Myc/TAT/9R融合表达载体构建及其原核表达研究  

The Construction and Prokaryotic Expression of TAT-bc-Myc-9R Fusion Vector in Bovines

作　　者：甘鹏[1,2] 熊亮[1] 贾文超[1] 潘传英[1,3] 蓝贤勇[3] 陈宏[3] 

机构地区：[1]西北农林科技大学生命科学学院,陕西杨凌712100 [2]中山大学生命科学学院,广东广州510275 [3]西北农林科技大学动物科技学院,陕西杨凌712100

出　　处：《家畜生态学报》2013年第10期10-15,共6页Journal of Domestic Animal Ecology

基　　金：国家自然科学基金项目(No.31000655);西北农林科技大学博士启动基金(BSJJ2010049)

摘　　要：原癌基因c-Myc虽然在许多肿瘤组织中特异性高表达,但它也是机体正常细胞生长与增殖所必需的因子,然而目前家畜诱导多潜能干细胞(iPS)研究中关于多功能转录因子cMyc的报道很少。为此,研究拟利用蛋白转导区域(PTD)的跨膜作用,构建TAT-bc-Myc-9R融合表达载体,并对其进行原核表达,旨在为转录因子蛋白重编程牛体细胞奠定基础。试验以牛c-Myc基因编码区序列为参考,设计并合成含酶切位点、跨膜转导肽TAT和9R的引物,利用PCR等基因工程方法获得包括牛c-Myc、TAT和9R的重组原核表达载体(pTAT-HA-bcMyc-9R);酶切鉴定和DNA测序结果表明,试验成功获得bc-Myc-9R重组序列,成功构建重组质粒pTAT-bc-Myc-9R;不同IPTG浓度和诱导时间条件对该重组载体的诱导表达和SDSPAGE分析表明,TAT-bc-Myc-9R在0.6mmol/L IPTG和37℃诱导6h条件下表达约57KDa的目的蛋白。这些结果为PTD介导的转录因子蛋白直接重编程牛体细胞以获得iPS研究积累了科学资料。Although c-Myc was originally found to express as a proto-oncogene and active in many human tumors,it is a critical transporter factor which is responsible for normal cell growth and proliferation.However,there is little information about pluripotent stem cell research in livestocks relevant to multifunctional transcription factor c-Myc.Therefore,this study used the transmembrane property of TAT and poly（Arg） to construct TAT-bc-Myc-9R fusion expression vector expressing in prokaryotic cells,which will lay the foundation for TAT-9R-mediated transcription factor trans-inducing into bovine somatic cells for iPS cells.Based on the coding region of bovine c-Myc gene,the artificial primer including enzyme recognizing site,TAT and 9R sequences were synthesized to amplify and obtain the recombinant DNA sequence （c-Myc gene,TAT and 9R）.Then,the recombinant DNA sequence was cloned into pTAT-HA vector,which was called as recombinant vector pTAT-HA-bc-Myc-9R.After identified by enzyme digestion analysis and DNA sequencing,the correct bovine c-Myc gene and TAT-bc-Myc-9R fusion expression vector was successfully obtained.Under the different concentration of IPTG and different stages,TAT-bcMyc expressed an about 57 kDa target protein using SDS-PAGE analysis.The optimized condition was 0.6 mmol/L of IPTG inducing at 37 ℃ for 6 h.These findings will provide a reference for reprogramming somatic cell into iPS.

关 键 词：牛 诱导多潜能干细胞(iPS) C-MYC基因 TAT 多聚精氨酸(9R) 原核表达 

分 类 号：S811.6[农业科学—畜牧学]