The Silkworm Bmmp54 Gene With The Polyhedrin Gene Fusion Expression Of Its Functions Preliminary Study

We selected a novel gene with an open reading frame(ORF) of 312 bp and encoding 103 amino acid residues from the silkworm pupae cDNA library constructed by our laboratory. The length of the gene is 1086 bp. The predicted molecular weight is 65 kD and the isoelectric point is 9.90. By bioinformatics analysis, two transmembrane domains were identified in this transmembrane protein. After blasting the sequence homology against the NCBI database, we identified it as an unknow gene in the silkworm pupae and nominated the gene as BmMP54, and the GenBank accession number is DY231399.1。We cloned the fusion gene of BmMP54 and gene Polh (polyhedrin, Polh) into the vector pGEX-4T-1 and pFastBacHT B to construct rocombinant plasmid, and the fusion protein was expressed by E.coli system and Bombyx mori NPV expressing system, respectively. According to the ORF of BmMP54 and Polh, two primers were designed to obtain the coding region of BmMP54 gene and Polh gene from silkworm pupae EST database. The fusion gene was cloned into pGEX-4T-1 vector digested with BamH I/EcoR I and EcoR I/Xho I. The recombinant plasmid was transformed into E.coli BL21(DE3). PCR and digestion with BamH I and Xho I showed that the designed fragment was inserted correctly. The recombinant plasmid was sequenced and the sequence map indicated that a recombinant expression plasmid was constructed successfully. Recombinant protein was expressed successfully in E.coli BL21 (DE3) induced by IPTG with the final concentration of 1mM. The analysis of SDS-PAGE and Western blotting showed that the fusion protein GST-Polh-BmMP54 was expressed highly in BL21 with 65 kD that was accorded with the theory value. The fusion protein expressed in the E.coli system remained as inclusion body. According to the principle of the alkali fusion of Polh protein, we can initial purify the fusion protein and use ion-exchange chromagraph to purify it furthery. we use Baculoviral Polyhedrin as a Novel Fusion Partner for facilitating expression of BmMP54 in Bac-to-Bac baculovirus expression system. We constructed the baculovirus transfer vector pFastBac HTb-Polh-BmMP54. The recombinant transfer plasmid was transformed into the BmDH10Bac competent cells, and inserted into the Bacmid DNA by transposition. We constructed the recombinant virus vBmPolh--BmMP54. was infected by this kind of recombinant viruses. The BmN insect cells was infected with the recombinant virus vBmPolh-BmMP54, and the aimed 65 kD protein expressed in the BmN insect cells was identified with Western blotting.Real-time fluorescent quantificational PCR was conducted to investigate the BmMP54 transcription at different tissues of the fifth instar larva. The results indicated that BmMP54 was highly expressed in silkworm's stigma and epiploon, resepectively. In addition, we knokdown the gene BmMP54 by RNAi and observe the cell cycle by flow cytometry. The result showed that the cells would pass the G1/S and S/G2 faster, showing some contributions of gene BmMP54 to blot cells to enter the checkpoints of G1/S and S/G2. These results laid a good foundation for further researching the biological function of BmMP54 gene in the growth and development process of silkworm.