Study On Bombyx Mori BmLp-c21 Protein

The lipoprotein is the particle that richly contains lipid, which contains proteins (apolipoprotein) and polarity lipid (phospholipid and nullilipidic cholesterol) in the ecderon, and the nullipolarity core contains triglyceride and cholesterl ester. In our laboratory's silkworm pupal cDNA library, we find a cDNA sequence that encodes silkworm lipoprotein. We can know that it contains 2803 bp. This gene has an open reading frame of 792 bp, encoding 263 amino acid residues with the predicted molecular weight of 30.24 kD and isoelectric point of 7.23. We nominated this novel gene as BmLp-c21. Two primers were designed to amplify the coding region of BmLp-c21 gene. The ORF of this gene was cloned into pET-28a(+) vector. The recombinant plasmid was transformed into E.coli Rosetta (DE3). Recombinant protein was expressed successfully in E.coli Rosetta (DE3) after induced by IPTG. The analysis of SDS-PAGE showed that the fusion protein was expressed highly in Rosetta with the molecular weight of 34 kD which was consistent with the theory value (His tag was weight of 3.56 kD, and BmLp-c21 was weight of 30.24 kD). The recombinant protein was insoluble, so it was washed, dissolved and centrifuged. The supernatant was purified with metal-chelating affinity chromatography. Polyclonal antibody was generated by immuning a New Zealand rabbit with recombinant protein and the titer of it was over 1:6400, measuring by ELISA. We compared the quantity of BmLp-c21 mRNA in different stages and different tissues of silkworm by RT-PCR. The transcriptional level of BmLp-c21 gene was found in developmengtal stages following from high to low: pupa, larvae, moth and egg. The transcriptional level of BmLp-c21 gene was found differently in fifth instar larva's tissues following from high to low: blood, fat body, trachea, testis, ovary, midgut, silk glands, head, malpighian tubule and epidermis. The total proteins were extracted from silkworm four different stages and the tissues of fifth instar larva. The expression level of BmLp-c21 was analyzed by Western blotting and the results showed that the BmLp-c21 markedly existed in all development stages and genitalia, trachea, fat body ,blood, but we can not find the BmLp-c21 in silk glands, midgut,epidermis ect. Immunocytochemistry in BmN cells showed that the protein mainly existed in cytoplasm.