Prokaryotic Expression Of The Gene Of Bombyx Mori Squid And Positioning Analysis

It is the first time that Bmsqd-1 gene was studied in Bombyx mori. BmSQD-1 protein contains two RRM conserved domains, which included many conserved amino acids residues. RRM RNA binding protein takes part in many post transcription bioprocess including RNA spicing,localization and stability of RNA. It is also related to human genetic diseases and tumor. Squid gene in Drosophila encodes three subtypes,containing 345,321 and 308 amino acids residues, which have the same 1-285 amino acids s sequence. We deduced that BmSQD-1 maybe one of the hnRNPs.A cDNA sequence was screened from NCBI database and cloned from silkworm pupa cDNA library constructed by our labtorary. After blasting, it was found that this was a Bmsqd-1 gene. The ORF of this gene contained 864 bp bases, encoding 286 amino acids with a predicted molecular weight of 30.99 kDa. The ORF of this gene was cloned into the pET-28a(+) vector to construct the procaryotic expression plasmid pET-28a(+)-Bmsqd-1. The recombinant plasmid was transformed into E.coli BL21(DE3). After induced with IPTG, identified by SDS-PAGE the recombinant protein was expressed successfully.The purified fusion protein was used to immunize a male New Zealand rabbit. Then, antisera (polyclonal antibody) was harvested and IgG was prepared by affinity chromatography using immobilized protein A. The titer of the polyclonal antibody reaches 1:12800 measured by ELISA. According to Western blotting analyses, it was showed that the BmSQD-1 expression level was the highest in malpighian tubules of the fifth instar larva, followed by egg, and was the lowest in epidermal. The result of real-time PCR revealed that Bmsqd-1 mRNA was widespread in different tissues and developmental stages of silkworm, with the highest expression level in Ovarian and the lowest one in adult. With immunofluorescence method, the subcellular localization of the BmSQD-1 was analyzed. BmSQD-1 accumulated in nucleus and not in cytoplasm.