Prokaryotic Expression Analysis And Subcellular Localization Of The CIB From Silkworm, Bombyx Mori

Calcium and integrin binding protein(CIB),which contains EF-hand motifs, is one member of the Ca²⁺ binding protein superfamily. It binds many effector proteins including the plateletαⅡbβ3 integrin and potentially regulates their functions in a Ca²⁺ related manner. CIB are widely distributed in a variety of tissues, as a intermediate molecular it plays an important role in many cell possess. To date, most researches of CIB were focused on rather vertebrate than invertebrate.Here we screened a cDNA sequence from a silkworm pupa cDNA database constructed by our laboratory. After blasting, we found that it is Bombyx mori calcium and integrin binding protein gene (BmCIB). The CIB cDNA is 939 bp in size, containing a 558 bp open reading frame that encodes 185 amino acids.We obtained His-tagged fusion protein by transforming pET28a(+)-CIB recombinant plasmid into E.coli BL21 (DE3). The fusion protein which was purified with liquid solution of inclusion bodies and metal-chelating affinity chromatography. The molecular weight of this fusion protein was 24877.0508 Da (His tag was weight of 3.56 kD, and BmCIB was weight of 21.16 kD), characterized by mass-spectrum after dialysis. This result proved that His-tag BmCIB was expressed in the right form. The purified fusion protein was used to immunize a male New Zealand rabbit. Then, antiserum was harvested, and IgG was prepared by affinity chromatography using immobilized protein A. According to real-time Quantitative PCR and Western blotting analyses, we found that BmCIB was widely expressed in a variety of tissues and different developmental stages of silkworm. The transcriptional level of BmCIB mRNA is highest in gonad and pupa. The expression of protein can be detected in all the developmental stages and is highest in gonad among different tissues. Moreover, after being stained with immunofluorescence, the subcellular localization of the BmCIB was analyzed. The result show that BmCIB proteins are all accumulated in cytoplasm. The protein of BmCIB has been cloned and expressed successfully. The specific polyclonal antibody against the protein of BmCIB has been obtained which can be used for further research. Additionally we have completed the related expression and subcellular localization analysis of BmCIB which can be used for further research into the function of BmCIB.