ABSTRACT
Integrins are transmembrane glycoproteins, closely related to many physiological and pathological processes. In order to explore its role in silkworm, by PCR and Rapid-amplification of cDNA ends (RACE) technology, the full-length cDNA of Bmintegrin β1 in silkworm was acquired. The domain was predicted by domain prediction website. Phylogenetic tree was constructed to analyze its evolutionary relationship. By prokaryotic expression system, protein purification method and immunizing mouse, the antibody against Bmintegrin β1 recombinant protein was obtained. The spatial-temporal expression profile of Bmintegrin β1 was investigated by semi quantitative PCR and Western blotting. Then we identified all 3 different spliceosomes, and they shared a common open reading frame of 2 502 bp, encoding 833 amino acids. Bmintegrin β1 contained all the classic domains of the integrin family, such as Integrin-B-tail, transmembrane domain etc. Phylogenetic analysis indicated that Bmintegrin β1 was close to the homologous proteins from Heliothis assulta and Danaus plexippus. In order to understand the function of Bmintegrin β1 further, we generated the antibody. In addition, Western blotting demonstrated that the antibody recognized the Bmintegrin β1 recombinant protein. Then, semi quantitative PCR and Western blotting results showed that Bmintegrin β1 was widely expressed in most of tissues, among of them, it's exhibited the highest expression level in hemacyte. Overall, this study provides a foundation for the study of silkworm integrin family.
ABSTRACT
Scavenger receptor class B is involved in various indispensable physiological processes, like the formation and inhibition of atherosclerosis or other cardiovascular diseases, innate immune defense and the removal of apoptotic cells. Here, we cloned BmSCRB8, a member of scavenger receptor class B in silkworm. We obtained the full-length cDNA sequence of BmSCRB8 by rapid amplification of cDNA ends (RACE), including 2 668 bp. The ORF of BmSCRB8 is 1 704 bp, encoding 567 amino acids. Online software prediction indicated that the molecular weight of BmSCRB8 is 63.87 kDa and the isoelectric point (pI) is 6.06. The space-time expression profile of BmSCRB8 was detected by reverse transcription PCR (RT-PCR), which implicated that BmSCRB8 is extensively expressed in each tissue and at each stage of blood. In addition, BmSCRB8 is highest expressed in fat body of silkworm, and is highly expressed in metamorphosis periods. Anti-BmSCRB8 polyclonal antibody was generated through prokaryotic expression, protein purification and mice immunization. Simultaneously, we constructed BmSCRB8 eukaryotic vector and then transfected embryonic cell line of silkworm. Immunofluorescence and overexpression showed that BmSCRB8 expressed specifically in membrane. Western blotting demonstrated that BmSCRB8 protein can be specifically recognized by anti-serum generated after mice immunization.