Abstract: 【Objective】 Receptor assay is a new method suitble for rapid multianalysis for detection of β-agonists compared to traditional immunoassay. The key problem of this method is acquiring pure recombinant receptor with high affinity and selectivity. To provide the basic material for screening of optimal expression system and expression condition, the specific recombinant shuttle expression plasmid was constructed with pig β₂AR gene. 【Method】 Total RNA was extracted from fresh pig liver and a pair of primers was designed and synthesized according to the published pig β₂AR gene sequence in Genbank(AF000134). The recovery of RT-PCR product from agarose gel was connected with cloning vector pMD-18T by T4 DNA ligase at 4℃ overnight. The ligation product was then transformed into competent cell DH5α and the plasmid was extracted after blue and white spot selection. And that the plasmid was confirmed by PCR, double enzyme digestion and sequencing analysis. The DNA sequence and deduced amino acid sequence were firstly analyzed by BLAST, and then phylogenetic tree construction and hydrophobicity were performed, respectively. In order to enhance the expression and binding affinity of receptor protein, N-terminal 186 bp of the cloned gene were truncated, and 6×His-tag was added at C-terminal. Finally, the genetically modified genes were respectively cloned to pTriEx-1.1 Hygro vector, and the ligation products were transformed into competent cell NovaBlue. The recombinant plasmids were extracted from single colonies and identified after Amp resistance screening. 【Result】 The purity, concentration and integrity of the extracted total RNA could meet the requirements of successive test by UV spectrophotometer testing and agarose gel electrophoresis. The RT-PCR product was 1 257 bp by sequencing, which encods 418 amino acids. The sequence has been submitted in Genbank as accession number KF023571.1. The deduced protein was predicted to have a computed molecular mass of 46.73 kD by Compute pI/Mw. Compared with the published pig β₂AR gene sequence (AF000134), the identity of nucleotide between them was 99.68%, and the rate of deduced amino acid was 99.28%. Furthermore, all of the amino acids at the ligand binding sites were cloned correctly. BLAST analysis indicated that there was a high homology of β₂AR between pig and some species. From phylogenetic tree analysis based on β₂AR gene, Sus scrofa is more close to Pecan tajacu and not closely related to Tscherskia triton and Microtus ochrogaster. Hydrophobicity analysis illustrated that the N-terminal and C-terminal of the receptor were dominated by hydrophobic amino acids and hydrophilic amino acids, respectively. Recombinant plasmid named pTriEx-1.1Hygro-β₂AR1-418 and pTriEx-1.1Hygro- β₂AR63-418 were constructed successfully after verification of PCR, double enzyme digestion and sequencing. 【Conclusion】 The β₂AR gene and the amino acids at active sites were highly consistent with the published records. Besides, pTriEx-1.1 Hygro vector contains promoters suitble for Escherichia coli, insect cells and mammalian cells, respectively. It is a good material to explore the expression effect of target gene in different expression systems. Consequently, both of the recombinant shuttle expression plasmid pTriEx-1.1Hygro-β₂AR1-418 and pTriEx-1.1Hygro-β₂AR63-418 can be applied to the further studies on β₂AR expression and purification in all three of expression systems.

Key words: pig, β₂ adrenergic receptor, gene cloning, sequence analysis, shuttle expression plasmid