摘要
目的:构建带有Myc标签的赖氨酸乙酰基转移酶(KAT7)的真核表达载体,获得Myc-KAT7融合蛋白,并检测其对雌激素受体α(ERα)表达的影响。方法:以本实验室保存的乳腺文库为模板,采用PCR技术从中扩增出人KAT7基因的编码序列,插入p XJ-40-Myc载体;将重组质粒与空载体分别转染人胚肾293T细胞,通过Western印迹检测转染细胞的表达情况,并提取总RNA采用q RT-PCR检测ERα的表达。结果:双酶切和测序结果表明Myc-KAT7真核表达质粒构建成功,转染293T细胞后Western印迹鉴定表明融合蛋白得到表达,q RT-PCR表明KAT7在转录水平能够促进ERα表达。结论:构建了带Myc标签的人KAT7真核表达载体,并发现KAT7对ERα的表达具有促进作用。
Objective： To construct the eukaryotic expression vector of human lysine acetyhransferase 7（KAT7） labeled with Myc tag and detect its biological activity preliminarily. Methods： Human KAT7 gene was obtained from human mammary gland cDNA by PCR and cloned into the eukaryotic expression vector pXJ-40. Human 293T cells were transfected with the recombinant plasmid of Myc-KAT7, and the expression was detected by West- ern blot. Real-time quantitative PCR（qRT-PCR） was performed to test the effect of KAT7 on estrogen receptor （ERos） expression. Results： KAT7 eukaryotic expression vector labeled with Myc tag was successfully constructed as demonstrated by double enzyme digestion and gene sequencing. Western blotting showed that KAT7 protein was expressed in 293T cells transfected with Myc-KAT7 eukaryotic expression vector, and qRT-PCR revealed that over- expression of KAT7 caused an increase in ERa mRNA level. Conclusion： The eukaryotie expression vector of Myc-KAT7 was successfully constructed, and KAT7 can modulate the ERα mRNA level, which will contribute to the further study on the function of KAT7.
出处
《生物技术通讯》
CAS
2016年第1期79-82,共4页
Letters in Biotechnology
基金
国家自然科学基金(81472589
31100604)
北京市科技新星计划(Z141102001814055)
北京市自然科学基金(7132155)
全军医学科技"十二五"项目(CWS12J120)
军事医学科学院创新基金转化医学项目(ZHYX003)
关键词
赖氨酸乙酰基转移酶7
克隆
真核表达
雌激素受体Α
lysine acetyltransferase 7（KAT7）
cloning
eukaryotic expression
estrogen receptor α
作者简介
董倩（1989－），女，硕士研究生；
李玲（1991－），女，博士研究生；
宋烨琼（1991－），女，硕士研究生；
徐小洁，（E-mail）miraclexxj@126．com
叶棋浓，（E-mail）yeqn66@yahoo．com：
胡毅，（E-mail）huyi040l@aliyun．com；
