Expression And Functional Analysis Of HMGA From Bombyx Mori

HMGA (High Mobility GroupA) proteins are a kind of nonhistone proteins situated in the chromosome. All of HMGA proteins have nine conservative amino acid sequence and a C terminal with negative charge, the former bind with the minor groove of DNA sequence riched in AT, called AT-hook domain. Now, there are many researches about HMGA, but the only research about HMGA in silkworm was the function of the HMGA about ovicell maturation.A gene sequence named HMGA was screened from NCBI Genbank. By blast contrasting with Bombyx mori genome, it was certeined gene of Bombyx mori. So we named this gene as BmHMGA (Bombyx mori HMGA). The the accession number was DQ402512. The BmHMGA gene contained only one exon. The length of the cDNA is 1 205 bp, it contains an ORF of 345 bp, encoding 114 amino acids with the predicted molecular weight of 12.24 kD and isoelectric point of 9.58.Two primers were designed to amplify the coding region of BmHMGA gene with template of c DNA of pupa. The ORF of this gene was cloned into pET-28a(+)vector with BamH I and Xho I digestion. The recombinant plasmid was transformed into E.coli BL21(DE3) star. PCR and digestion with BamH I/Xho I showed that the designed fragment was inserted correctly. The recombinant plasmid was sequenced and the result indicated that a recombinant expression plasmid was constructed successfully. Recombinant protein was expressed successfully in E.coli BL21 (DE3) star after induced by IPTG with the final concentration of 1 mmol/L. The analysis of SDS-PAGE showed that the fusion protein His-BmHMGA was expressed highly in BL21 with a molecular weight of 22 kD, then characterized by mass-spectrum, the result proved that His-tag BmHMGA was expressed in the right form. Most of recombinant protein was soluble and was purified with metal-chelating affinity chromatograpHy. Polyclonal antibody were generated by immoluning a New Zealand rabbit with recombinant protein and the titer of it was over 1:12 800, measuring by ELISA. The total RNA were extracted from silkworm four different stages and the fifth instar larva tissues.The transcription level of BmHMGA was analyzed by RT-PCR, The results showed that the BmHMGA gene transcripts was higher in egg compared to other stages, and higher in brain compared to other tissues in fifth instar larva Then The total proteins were extracted from silkworm four different stages and the fifth instar larva tissues.The expression level of BmHMGA was analyzed by Western blotting and ELISA semiquantitative analysis. The results of Western blotting and ELISA semiquantitative analysis showed the similar conclusion with the results of RT-PCR. Immolunocytochemistry in BmN cells showed that the protein existed only in cytoplasm. These results laid a good foundation for further studies on BmHMGA gene.