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| Molecular Cloning of Sheep β2-adrenergic Receptor Gene (β2-AR) and Expression of Its Second Extracellular Loop in Escherichia coli |
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Abstract The β2-adrenergic receptor (β2-AR) genes of three breeds of sheep (Ovis aries) were cloned for the first time from Suffolk, Small Tail Han and Chinese Merino (Xinjiang type) with a pair of PCR primer corresponding to the consensus sequence of mammalian β2-AR(GenBank accession No.EU707939, EU707940 and EU707941). The nucleotide sequence data showed that the coding region of the gene consisted of 1257 bp open reading frame which encoded 418 amino acid residues. The sheep β2-AR coding region nucleotide sequence was 98.4%, 88.5%, 84.2% and 80.5% identical to those of bovine, porcine, rat and human, respectively. Two copies of the nucleotide sequence coding for the second extracellular loop of sheep β2-AR were synthesized and then cloned into pET-32C (+) expression vector. After being transformed into Escherichia coli BL21 (DE3) and induced by 1 mmol/L IPTG, the recombinant fusion protein expressed in cytoplasm. SDS-PAGE and Western blotting results revealed that the molecular weight of the protein was about 26 kD, and the expression level was 40.3% of total bacterial protein.
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Received: 03 May 2009
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