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

doi:10.16431/j.cnki.1671-7236.2017.11.003

Abstract

Abstract:

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.

Key words: pig; β₂AR gene; eukaryotic expression vector; mutant; Western blotting

CLC Number:

S859.1

ZHANG Hai-jie, XIE Hua-jie, JIANG Li-jie, LIAN Jun-wei, LIU Min, Min Tian-qi, WANG Zhi-qiang. Construction and Expression of the Eukaryotic Expression Plasmid and Mutant of β₂ Adrenergic Receptor of Pig[J]. , 2017, 44(11): 3130-3136.

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

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

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