家蚕BmLp-c21蛋白的表达分析及其亚细胞定位

cDNA sequence which encodes lipoprotein was found from the pupa cDNA library, the gene was named BmLp-c21 (Bombyx mori lipoprotein pBmHPC-21). The length of the cDNA is 2803 bp, the open reading frame(ORF) is 792 bp and encodes a protein of 263 amino acid residues with the predicted molecular weight of 30.241 kD and isoelectric point of 7.23. BmLp-c21 was inserted into the expression vector pET-28a (+) to construct the recombinant plasmid. The fusion protein was expressed in the E.coli Rosetta (DE3) induced by IPTG, and then was purified by affinity chromatograph. Polyclonal antibody was generated by immunizating New Zealand rabbit with the purified fusion protein .The titer of the polyclonal antibody was larger than 1:6400, measured by ELISA. Immunofluorescence method was employed to detect the subcellular localization of BmLp-c21 in BmN cells. We found that BmLp-c21 was distributed mainly in the cytoplasm. We found that the transcription and expression level of BmLp-c21 were different in the different tissues of the fifth instar larva and developmental stages of Bombyx mori by real-time PCR and Western blotting. The result revealed that: BmLp-c21 gene transcripts and expresses higher in pupa than other stages and higher in blood compared to other tissues of the fifth instar larva. It laid a foundation for further Studies on BmLp-c21 gene.