摘要
为构建鸡维甲酸相关孤核受体α(retinoid acid receptor related orphan receptorα,RORα)的真核表达载体,并检测其在MSB1细胞中的表达效果,本研究参照GenBank中鸡RORα基因序列(登录号:NM_001289887.1)设计特异性引物,从鸡肝脏组织中提取总RNA,经反转录合成cDNA,以此为模板进行PCR扩增,将扩增的特异性RORα基因片段连接pMD18-T载体,构建克隆载体pMD18-T-RORα,再用SalⅠ和BamHⅠ双酶切克隆载体pMD18-T-RORα和真核表达载体pEGFP-N1,将酶切回收的RORα与pEGFP-N1片段进行连接,构建重组真核表达载体pEGFP-N1-RORα,经PCR、酶切和测序验证准确性后,将其转染MSB1细胞,于转染后48h荧光显微镜下观察EGFP的表达情况,并采用实时荧光定量PCR检测鸡RORα在MSB1细胞中的表达水平。结果显示,从构建的重组质粒pEGFP-N1-RORα中可扩增出大小约1 600bp的特异性片段,用SalⅠ和BamHⅠ可切出大小约4 700和1 600bp的两条带。实时荧光定量PCR结果显示,转染MSB1细胞48h后,与对照组相比,重组真核表达载体转染组中鸡RORα基因mRNA水平显著增加(P<0.05)。表明本试验成功构建了鸡RORα的真核表达载体,该载体可在MSB1细胞中表达鸡RORα,为进一步研究鸡RORα对MSB1细胞增殖的影响奠定基础。
In order to construct an eukaryotic expression vector of chicken retinoid acid receptor related orphan receptor alpha（RORα）and detect its expression efficiency in MSB1 cell,the specific primers of RORαgene were designed according to the gene sequence of chicken RORαgene from GenBank（accession No.：NM001289887.1）.Total RNA was extracted from chicken liver tissue,the first strand of cDNA was synthesized by reverse transcription,and the chicken RORαgene fragment was amplified from cDNA,then cloning vector pMD18-T-RORαwas constructed.The eukaryotic expression vector pEGFP-N1 and cloning vector pMD18-T-RORαwere digestedby the restriction enzyme SalⅠ and BamH Ⅰ,the RORαand pEGFP-N1 fragments were recycled,then the RORαgene fragment were connected to pEGFP-N1 to construct the recombinant vector pEGFP-N1-RORα,and the recombinant vector was identified by PCR,double enzyme digestion and squencing.The results showed that the 1 600 bp fragment was amplified from the recombinant eukaryotic expression vector,and two segments length about 4 700 and 1 600 bp were cut using restriction endonuclease SalⅠ and BamH Ⅰfrom the recombinant vector pEGFP-N1-RORα.Then the recombinant vector pEGFP-N1-RORαwas transfected into the MSB1 cells,the EGFP-positive cells was observed under the fluorescence microscope,and the expression level of RORαin MSB1 cell was evaluated which measured by Real-time quantitative PCR at 48 hafter transfection.Compared with control group,the expression level of RORα was significantly increased in pEGFP-N1-RORαtransfected MSB1 cell（P0.05）.The result suggested that the eukaryotic expression vector of RORα was successfully constructed,and the recombinant vector could express RORαin MSB1 cell,which laid a foundation to further study the effect of RORαon the biological behavior of MSB1 cell.
作者
尤钊
左珂朦
余祖华
丁轲
王垚垚
刘彤
张梦珂
邱静静
YOU Zhao;ZUO Kemeng;YU Zuhua;DING Ke;WANG Yaoyao;LIU Tong;ZHANG Mengke;QIU Jingjing(Key Laboratory of Animal Disease and Public Health of Henan University of Science and Technology,Luoyang 471003,China;Hongxiang Biological Feed Laboratory of Henan University of Science and Technology,Luoyang 471003,China)
出处
《中国畜牧兽医》
CAS
北大核心
2018年第7期1791-1797,共7页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金项目(U1504308
31702207)
河南科技大学博士科研启动基金项目(13480068)
河南科技大学大学生研究训练计划(SRTP)项目(2017317)
