Expression Of Bombyx Mori Cecropin B In Silkworm Larvae By Using BmNPV/Bac-to-Bac Expression System With Double Promoters

Antimicrobial peptides Cecropin B is a kind of small molecular weight protein produced by immune organisms of silkworm, molecular mass of about4kDa, it cannot form intramolecular disulfide bonds, N-terminal with strong alkaline, C-terminal with strong hydrophobicity and have a broad range of antimicrobial activity against bacteria, viruses, and fungi. In addition, antibacterial peptide is the biggest role as green feed additive to improve animal immunity, promote animal health growth in animal husbandry. Antibacterial peptide has become a hot spot in the study of life science. It is difficult to separation of a large number of antibacterial peptides from the biological tissue, the cost is high. AMPs are usually expressed as fusion proteins in E. coli, but the products are difficult to purify and the posttranslational modification is not complete, consequently, the bioactivity of expressed products is quite low. BmNPV/Bac-to-Bac Expression System, the recombinant baculoviruses, without the protection of the polyhedrin protein, is needed to inoculate silkworm larvae through subcutaneous injection, which results in low efficiency. Therefore, the expression of both polh gene and foreign gene in one eukaryotic expression system has become a hot topic in engineering research of for antibacterial peptide.In this study, we successfully expressed Bm cecropin B (CecB2) and polh genes in the pFastBacDual vector, The recombinant CecB2was confirmed to have antimicrobial activity, and we measured the concentration of recombinant CecB2after infection using ELIS A assay, the results of the study are as follows:1. Recombinant baculovirus BmNPV-CecB2/polh The fragments of Bm CecB2and polh genes were amplified from the plasmids PET-32a-Bm-CecB2and wild BmNPV, respectively. Two gene cloning to pFastBacDual transfer vector. Construction of recombinant plasmid pFastBacDual-CecB2/polh, recombinant plasmid transformd into DH10Bac E.coli cells, construction of recombinant Bacmid-CecB2/polh, Bacmid DNA through the transfection BmN cells get recombinant baculovirus BmNPV-CecB2/polh.2. The expression and appraisal of foreign gene in silkworm larvaThe recombinant Bacmid-CecB2/polh was transfected into BmN cells using lipofectamine.72h after transfection, BmN cells showed obvious symptom of infection, most of cells were in round shape and lots of polyhedrins were observed under microscope144h after transfection, the silkworm larvae fed with recombinant virus have obvious signs of infection, silkworm blood is ivory, and moreover, we analyzed the expression of CecB2and Polh proteins in the blood cells by Tricine-SDS-PAGE. polh protein (29KDa) was observed in the blood of silkworm larvae after infection with pED-CecB2/polh, while the predicted band for CecB2protein (4.9KDa) was only observed in the blood cells from pFD-CecB2/polh baculoviruses infected silkworm larvae. The supernatant from silkworm larvae blood cells after ultrasonic disruption was used for the detection of antimicrobial activity. Our result showed the recombinant CecB2in silkworm larvae blood cells had obvious antimicrobial activity to E. Coli, the strength of antimicrobial activity was up to10mm in diameter.and ELISA quantative assay showed average concentration to0.115μg/mL CecB2was detected in the body (crude extract) and0.109μg/mL in the hemolopha, which provide the basis for further study in the future.The studies show that we successfully expressed Bm cecropin B (CecB2) and polh genes in the pFastBac DUAL vector, the recombinant baculoviruses were generated by Bm cell line, BmN and used to orally inoculate silkworm larvae, which provide the theoretical basis for the application of large-scale production and animal husbandry.