Amplification, Cloning And Expression Of Porcineβ₂-Adrenergic Receptor Gene

There were two cut sites of endonuclease EcoR I located in 5?terminal of iS 2-adrenergic receptor( iS 2-AR) gene in the primary plasmid pMDAR. A recombinant plasmid pMDARIE with single EcoR I site was produced after digestion by EcoR I and ligation by T4 DNA ligase Porcine fi2-AR coding region was cut out from pMDARIE and fused into 3? terminal of thioredoxin (trxA) gene in pET-32C vector. Primers for PCR screening were designed on the basis of T7 promoter of pET-32C vector and porcine iS2-AR eDNA. Five candidates were screened, of which three had right DNA inserts. By restriction enzyme analysis and DNA sequencing, it was demonstrated that the recombinant plasmid pET-32CAR harboring porcine fl,- AR gene was constructed, and the reading frame of the fusion gene was correct. pET-32CAR was transformed into E. coil BL21(DE3), and 1mM IPTG was used to induce the fusion gene to expression. The expression of the fusion protein was monitored at 0, 1, 2, 3, and 4 hour after induction. After preparation of bacterial membrane and electrophoretic analysis, it was indicated that porcine fl2-AR expressed in fusion protein form in vitro which was assembled into bacterial membrane. After scan, it showed that the yield of fusion protein expressed in E. coli reached peak after being induced about 3 hour by LPTG. Another study revealed that IPTG concentration (0.05, 0.1, 0.5, and 1mM) had no significant effect on the output of the fusion protein. The fusion protein produced in 1mM group was about 10.2% of the total bacterial membrane protein, which was relatively higher than that in other groups.