Receptor assay is a new method suitble for rapid multianalysis for detection ofβ-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 pigβ2AR gene. [Method] Total RNA was extracted from fresh pig liver and a pair of primers was designed and synthesized according to the published pigβ2AR 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 DH5αand 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 ofβ2AR between pig and some species. From phylogenetic tree analysis based onβ2AR 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] Theβ2AR 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.

<正>苏丹红是一种人工合成的偶氮类染色剂,有Ⅰ、Ⅱ、Ⅲ、Ⅳ号[1]。苏丹红不是一种食品添加剂,但是由于其着色效果好、颜色比较鲜艳,被许多食品厂商作为食品添加剂加入食品中来增加食品的颜色。国际癌症研究机构将苏丹红归为三类致癌物质,但苏丹红的代谢产物却属于二级致癌物质。还有研究表明,苏丹红能诱发癌细胞的产生,可能会使肝部