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

试验旨在构建猪β₂肾上腺素能受体（β₂ adrenergic receptor，β₂AR）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β₂AR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（+）-β₂AR，加入c-myc标签后命名为myc-pcDNA3.1（+）-β₂AR。以真核表达载体为模板构建突变体myc-pcDNA3.1（+）-β₂AR-D130N、myc-pcDNA3.1（+）-β₂AR-C285S和myc-pcDNA3.1（+）-β₂AR-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β₂AR基因已正确重组入pcDNA3.1（+）载体中；经测序鉴定，猪β₂AR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β₂AR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β₂AR蛋白表达及其药理学活性奠定基础。

Construction and Expression of the Eukaryotic Expression Plasmid and Mutant of β2 Adrenergic Receptor of Pig

This study was aimed to construct the eukaryotic expression plasmid of β₂ adrenergic receptor (β₂AR) and mutants including D130N, C285S and D130N/C285S and demonstrate the plasmids could be expressed in HEK293T successfully. The β₂AR 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 the 130th amino acid had been mutate from aspartic acid to asparagine, the 285th 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 of β₂AR.