家蚕脂蛋白的克隆表达、组织定位与亚细胞定位

he sequences are blasted on NCBI ,we can find one cDNA sequence that encode silkworm lipoprotein in our laboratory’s silkworm pupae cDNA database. We can know that contains 917 bp. This gene has an open reading frame of 786 bp, encoding 261 amino acid. The predicted molecular weight of 30.013 kDa and isoelectric point of 7.193. We nominated this novel gene as BmLp-c23 (Bombyx mori lipoprotein). To characterize the BmLp-c23 from silkworm pupae, The recombinant plasmid pET-28a- BmLp-c23 was transformed into E.coli BL21 (DE3) and the recombinant protein was expressed. The protein formed the inclusion bodies. After purification with liquid solution, we have used this purified fusion protein to produce polyclonal antibodies against BmLp-c23 for ELISA analyse. This analyse has revealed that the titer of the polyclonal antibodies reaches 1:8000. Western blotting analyses and real-time reverse transcription/polymerase chain reaction have shown that BmLp-c23 is expressed in larval, pupa and moth, but we can not find the BmLp-c23 proteins in egg. We also compared the quantity of BmLp-c23 mRNA in different tissues of the fifth instar larva with Real-time Quantitative PCR. These results suggested that BmLp-c23 expressed in silkworm’s all tissues except silk glands. The results of the subcellular localization showed that BmLp-c23 proteins are all in the nucleus, membrane and cytoplasm.