Expression And Subcellular Localization Of A Novel Gene Bm29 From Bombyx Mori

A new gene was screened from Bombyx mori pupa cDNA library constructed by our labtorary. The cDNA is 819 bp in length and contained an open reading frame encoding a protein of 272 amino acid residues, with the predicted molecular mass of 28.44 kD and pI of 4.1. Then the sequence of this new gene was analysed in NCBI datebase, there is 57% similarity of the sequence with a predicted protein 29, which is identified in Lonomia obliqua caterpillars. So this gene was named as Bm29. GenBank accession number is DQ985599. The gene Bm29 was cloned by PCR from Bombyx mori pupa cDNA library, then the PCR product was digested by BamHⅠand XhoⅠand inserted into pET-28a(+) to construct the recombinant plasmid pET-28a(+)–Bm29. The recombinant plasmid was transformed into E. coli Rosetta and was induced by IPTG to express the fusion protein His-Bm29. The results of SDS-PAGE showed that the fusion protein His-Bm29 was in supernatant with a molecular weight of 32 kD which was consistent with the theory value. Then the fusion protein was purified with ion exchang chromatography and Ni2+ affinity chromatography, Polyclonal antibodies were generated from a New Zealand rabbit after immunization with affinity-purified fusion protein protein His-Bm29. The titer of Polyclonal antibodies was over 1:12800 measured by ELISA.Total protein of each of the developmental stages of silkworm and eight tissues of the fifth instar lavae were extracted and analyzed by Western blotting. Total RNA from each of the developmental stages of silkworm and eight tissues of the fifth instar lavae were extracted and analyzed by RT-PCR. The results showed that the levels of transcription and expression of Bm29 were different obviously in the different developmengtal stages and tissues of Bombyx mori. The real-time RT-PCR results showed that Bm29 gene transcription level was higher in pupa stage than other developmental stages. Western blotting results showed that the expression levels of Bm29 were high in malpighian tubule and ovary, followed by head, silk gland, and low in the midgut and fat body. With immunocytochemistry method, the subcellular location of Bm29 protein showed that Bm29 protein existed in cytoplasm close to cell membrane. The above results will be the foundation for the deeply functional studies on the new gene Bm29.