Expression And Subcelluar Localization Of BmCH Gene From Silkworm Pupae (Bombyx Mori)

A cDNA sequence was screened from a Bombyx mori cDNA library constructed by our laboratory,which contains a CHCH domain identified after blast based on NCBI database. We preliminary nominated this novel gene as BmCH (Bombyx mori BmCH). (GenBank accession number is ABF51514.1). It contains an ORF of 435 bp and encodes 144 amino acids with the predicted molecular weight of 14.82 kDa and isoelectric point of 8.549.According to the ORF of BmCH, two primers were designed to obtain the coding region of BmCH gene. The ORF of the gene was cloned into pET-28a(+) vector with EcoRâ… and Xhoâ… digest. The recombinant plasmid was transformed into E.coli Rosetta (DE3). PCR and digestion with EcoRâ… /Xhoâ… showed that the designed fragment was inserted correctly. Recombinant protein was expressed and successfully induced by IPTG. The analysis of SDS-PAGE showed that the fusion protein His-BmCH was expressed highly in Rosetta with about 20 kDa. The fusion protein formed the inclusion bodies and then was purified with metal-chelating affinity chromatography. Polyclonal antibody was generated by immunization of New Zealand rabbit with fusion protein. The titer of the antiserum can reach 1:12800 with a high specificity by experiment of ELISA. We use Western blot and real-time fluorescent quantitative PCR to analyze the transcriptional level and the expression level of BmCH in developmental stages and different tissues of the fifth instar larva of Bombyx mori. Our results suggested that BmCH was expressed in the four stages, with highest in larva; in fifth instar larva, the positive signal of BmCH was detected obviously in epidermis, gut, testis, Malpighian tubule, ovary, the signal in blood, silk glands, fat body, but not detected in trachea and head. The results were similar to real-time fluorescent quantitative PCR. Finally, we carried out the subcellular localization about BmCH in BmN cell and positive signal of BmCH was mostly found in the cytoplasm. These results laid a good foundation of further studies on BmCH.