摘要
目的:克隆人全长Notch1基因,构建人全长Notch1/p CMV6-Entry真核重组质粒并在HEK293 T细胞中进行瞬时表达.方法:采用PCR公知不认方法扩增人全长Notch1基因并克隆到真核表达载体p CMV6-Entry中并用限制性内切酶分析和DNA测序鉴定重组质粒.将该重组质粒转染HEK293 T细胞24h后,通过实时荧光定量PCR(q-PCR)和Western blot检测鉴定人全长Notch1的表达.结果:成功构建了人Notch1/p CMV6-Entry重组真核表达质粒,人全长Notch1基因在mRNA和蛋白水平上的表达显著高于p CMV6-Entry载体对照组和HEK293 T细胞对照组.结论:克隆获得人全长Notch1基因并验证在HEK293 T细胞中表达.
Aim： To construct recombinant eukaryotic expression plasmid Notch1/p CMV6-Entry and express it in HEK293 T cells. Methods： Human Notch1 gene was amplified by use of Polymerase Chain Reaction（ PCR） and cloned into eukaryotic expression vector p CMV6-Entry with flag tag to construct recombinant Notch1/p CMV6-Entry. Enzyme restriction and sequencing were used to identify the recombinant plasmid,which then was transiently transinfected into HEK293 T cells for 24 h. Real-time quantitative PCR and Western blot were performed to confirm the expression. Results： The recombinant eukaryotic expression plasmid was successfully constructed. Expression of Notch1 mRNA and the protein was both higher that the blank control and negative control, which demonstrated that Notch1 was transiently expressed in HEK293 T cells. Conclusion： Human Notch1 was successfully cloned and expressed in HEK293 T cells.
出处
《暨南大学学报（自然科学与医学版）》
CAS
CSCD
北大核心
2017年第5期373-379,共7页
Journal of Jinan University(Natural Science & Medicine Edition)
基金
国家自然科学基金项目(81470831)
国家自然科学基金-广东省联合基金重点项目(U1401223)
国家高技术研究发展计划(863计划)项目(2014AA020909)
广东省科技计划-创新载体建设项目(2013B090800036)
广东省科技计划项目(2015A040404021
2016B090919019)
广东省大学生创新创业项目(201512121122)
广东省大学生创新训练项目(201612121100
201612121243
201612121034
201612121246)
