猪β2肾上腺素能受体基因真核表达载体及突变体的构建及表达  

Construction and Expression of the Eukaryotic Expression Plasmid and Mutant ofβ_2Adrenergic Receptor of Pig

作　　者：张海洁 谢华杰 江礼捷 连俊伟 刘敏[1,2] 闵天奇 王志强 ZHANG Hai jie;XIE Hua jie;JIANG Li jie;LIAN Jun wei;LIU Min;Min Tian qi;WANG Zhi qiang(Laboratory of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009,China;Jiangsu Co innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009,China)

机构地区：[1]扬州大学兽医学院,兽医药理毒理研究室,扬州225009 [2]江苏高校动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《中国畜牧兽医》2017年第11期3130-3136,共7页China Animal Husbandry & Veterinary Medicine

基　　金：江苏高校优势学科建设工程项目(PAPD)

摘　　要：试验旨在构建猪β_2肾上腺素能受体(β_2adrenergic receptor,β_2AR)基因及其突变体真核表达载体,并鉴定其在HEK293T细胞中的表达。通过猪基因组DNA克隆猪β_2AR基因,利用同源重组技术将其连接至真核表达载体pcDNA3.1(+),构建真核表达载体pcDNA3.1(+)-β_2AR,加入c-myc标签后命名为myc-pcDNA3.1(+)-β_2AR。以真核表达载体为模板构建突变体myc-pcDNA3.1(+)-β_2AR-D130N、myc-pcDNA3.1(+)-β_2AR-C285S和mycpcDNA3.1(+)-β_2AR-D130N/C285S,并将构建的真核表达载体和突变体转染HEK293T细胞,利用Western blotting技术验证其表达。结果显示,猪β_2AR基因已正确重组入pcDNA3.1(+)载体中;经测序鉴定,猪β_2AR的第130位天冬氨酸成功突变为天冬酰胺,第285位半胱氨酸成功突变为丝氨酸。Western blotting检测结果证明所构建的表达载体均可在HEK293T细胞中表达。本研究成功构建了猪β_2AR野生型的真核表达载体及2个单点突变和1个双点突变的突变体,并验证其在HEK293T细胞中正常表达,为进一步研究猪β_2AR蛋白表达及其药理学活性奠定基础。This study was aimed to construct the eukaryotic expression plasmid ofβ2adrenergic receptor(β2AR)and mutants including D130N,C285S and D130N/C285S and demonstrate the plasmids could be expressed in HEK293T successfully.Theβ2AR gene was amplified by PCR from pig genome DNA,then subcloned into pcDNA3.1(+)vector by homologous recombination to construct the eukaryotic expression plasmid,added the c myc tag in the plasmid which named myc pcDNA3.1(+)β2AR.We made the eukaryotic expression vector as the template to construct the mutant,transfected the plasmids into HEK293T,extracted the protein and demonstrated the expression by Western blotting.The results of sequencing showed that the130th amino acid had been mutate from aspartic acid to asparagine,the285th amino acid had been mutate from cysteine to serine.Also,we confirmed that the plasmids could be expressed in HEK293T successfully.The results showed that the recombinant plasmid myc pcDNA3.1(+)β2AR,myc pcDNA3.1(+)β2AR D130N,myc pcDNA3.1(+)β2AR C285S and myc pcDNA3.1(+)β2AR D130N/C285S were constructed correctly and could be expressed in HEK293T,which laid the foundation for further studies on the function and the pharmacology activity ofβ2AR.

关 键 词：猪 β2AR基因 真核表达载体 突变体 WESTERN BLOTTING 

分 类 号：S859.1[农业科学—临床兽医学]