Expression And Purified Analysis Of Ocular Alb Protein From Silkworm (Bombyx Mori)

Ocular_alb(ocular albinism) superfamily,which can bind hetero-trimeric Gi, Go and Gq proteins, is similar to G protein-coupled receptor(GPCR). Unlike anonical GPCRs, the protein is not localized in the plasma membrane, and is exclusively detectable on the membrane of intracellular organelles.The BmOcular_alb(BmOA) cDNA is 645 bp in length, containing a 453 bp ORF, which encodes a polypeptide of 150 amino acids; the molecular weight of this protein is estimated to be 17.7 kD. The ORF of the BmOA was obtained from the total cDNA of silkworm pupa by PCR. The ORF of this gene was cloned into the pBacPAK8 vector to construct the recombinant plasmid pBacPAK8-Ph-tev-BmOA, then the recombinant gene Ph-tev-BmOA. Ph-tev-BmOA was cloned into the pET-28a vector; the prokaryotic expression plasmid pET-28a-Ph-tev-BmOA was constructed. The recombinant plasmid was transformed into E.coli BL21 (DE3). After inducing with IPTG, the E.coli was lysised and the analysis of SDS-PAGE showed that the recombinant protein was expressed compared to control, produced a protein of 50 kD, and the protein formed the inclusion bodies. The recombinant protein with Polh shows the same characteristics to the Native Polh (Ph), the membrane protein with the characteristics was purified easily. The recombinant plasmid pFastBacHTB-Ph-tev-BmOA was constructed. Then, the Bacmid-Ph-tev-BmOA was transected into the BmN cells of Bombyx mori, and the invasion cell was obtained. The expression of BmOA has been detected in mRNA levels. The results showed the expression level was highest in trachea of the tissues which were tested. What’s more, RNA interference was applied to silence the target mRNA followed by detecting the cell condition with flow cytometer.