Study On The Expression And Functional Analysis Of Bmphb Gene From Silkworm Pupae(Bombyx Mori)

Prohibitin(PHB) is an evolutionarily conserved protein which was originally thought to have an important role in the inhibition of cell proliferation, ubiquitously distributed in many species such as mammals, plants and bacteria. Many researches reveal that Prohibitin(PHB) is a novel chaperone protein, and has involved in diverse cellular processes such as cell proliferation and apoptosis. However, its mechanism of action is unknown as yet.According to the large-scale cDNA sequencing of silkworm pupae, a novel cDNA sequence was identified from the cDNA library. The cDNA sequence is 1145 bp in length, which has an ORF of 825 bp, encoding a predicted 274 amino acids, molecular weight 30.08 kD, isoelectric point 6.79. Via bioinformation analyses, the novel gene contains a Band_7prohibitin domain, so we nominate it as Bmphb(Bombyx mori prohibitin), and the GenBank accession number is DQ311431. An alignment of the Bmphb cDNA sequence with the silkworm genome sequences reveals that there are 3 introns and 4 exons in it, and all of them conformed the canonical GT-AG rules. According to the ORF of Bmphb, two primers were designed to obtain the coding region of Bmphb gene. The ORF of the gene was cloned into pET-28a(+) vector digested with BamH I and Xho I. PCR, double digestion and sequencing indicated that the recombinant expression plasmid pET-28a(+)-Bmphb was constructed successfully. Screening positive clones and then transformed into E.coli Rosetta(DE3), and the expression of the His-tagged fusion protein was induced with 1 mM IPTG. The analyses of SDS-PAGE showed that the fusion protein His-BmPHB was expressed highly in Rosetta(DE3) with 35 kD that was accorded with the theory value. The recombinant protein was insoluble inclusion body and purified with affinity and reversed-phase chromatographies. The eluted His-BmPHB was then identified with mass spectrometer. Polyclonal antibody was generated by immunization of New Zealand rabbit with purified recombinant protein His-BmPHB. Polyclonal antibody was prepared by affinity chromatography using immobilized protein A and the titer was larger than 1:12800, measuring by ELISA. Purified anti-BmPHB polyclonal antibody was used to determine the subcellular localization. Immunostaining indicated that BmPHB can be found in both nucleus and cytoplasm but is located primarily in cytoplasm. Western blotting analyses indicated that, in the fifth instar larva, BmPHB was expressed descendingly in gonad, malpighian tubule, trachea, fatty body, intestine and head. However, none was detected in larva's silk gland and epidermis. Additionally, BmPHB was expressed in the nascent egg, larva and pupa, but none was detected in the moth. Therefore, we hypothesized that BmPHB may play an important role on silkworm development. To date, we have not found any report about the prohibitin protein from silkworm pupae, B.mori, so these results lay a foundation to further researches on BmPHB protein.