Cloning And Expression Of β₂ Adrenergic Receptor Gene

The safety of products from livestock has been given more attention by the public. As hormone drugs, clenbuterol and Ractopamine (β -agonists) were abused during the feeding for livestock, and resulted in their accumulation in edible tissues and muscle. The foods from these products directly affect the health of human . Therefore, the establishment of rapid detecting multi-residues in feed and animal products is a very important pathway for preventing the harmful substance from into the products from livestock.β₂ Adrenergic receptor belongs to the family of seven thansmembrane domains proteins. β ₂ Agonists, whether synthetic (e.g.,clenbuterol) or natural (e.g.,adrenaline),bring about physiological responses through a series of biochemical reactions initiated at the β₂AR on the cell membrane. The object for this study is to isolate the β₂ Adrenergic receptor gene, and identify the expression of this gene in E.Coli. in vitro, and purify the encoded protein by this gene, provide the support of technique and material guarantee for detection in hormone drugs by the property of binding between the receptor and hormone substance.In this study, the β₂ AR gene was isolated using the total RNA from the lung cells of greater long-tailed hamster as template by RT-PCR with a pair of specific primers, The sequencing data showed that this gene has 87.8%⁹6% identity with the genes from other animals registered in the NCBI GenBank, suggesting that this gene belongs to the member of β₂ Adrenergic receptor family, and carries the 1254 bp nucleotides and encodes 417 amino acids.In this study, The full length, four truncated, and two 6His tag of β₂AR were inserted into the pGEX-4T-1 vectors, and the recombinants were transformed into the E.Coil. strain Origami? B. SDS-PAGE assay showed that the fusion protein from the pGEX-4T-1-AR62-6His was expressed in the E.coli. in vitro after induction by IPTG. Western blot revealed that the fusion protein of interest bound specifically with GST antibody.Based on the analysis of E.Coli., the gene of β₂AR was inserted into the expression pPIC9 vector to establish yeast (Pichia pastoris) expression systerm. Four different recombinants pPIC9-β₂AR, pPIC9-β₂ARl-307, pPIC9-β₂AR-6His, pPIC9- β₂AR62-6His have been constructed. DNA sequencing showed the fragments of pPIC9- β₂AR have already directly inserted into the pPIC9 vector. This expression system will provide convenience for expression of β₂AR protein in yeast strain Pichia pastoris.