The Fusion Expression Of Bovine Lactoferricin(LfcinB) In Bacillus Thuringiensis

Lactoferricin is a small piece of peptide decomposed by digestive enzyme after taken in by animals. This 25-residue peptide has the specific antibacterial and immunologic function,and can stabilize intestinal flora’s normal ecological environment. Bovine Lactoferricin( LfcinB) is a peptide with molecular weight of 3.1kDa protein, which was derived from the residues 17-41 of bovine lactoferrin. It was reported that Lfcin B has most of the biological activity of 1actoferrin, and its ts antibacterial activity is 400 times greater than 1actoferrin and stand its leading place among all kinds of lactoferricin,which make it receive much attention from researchers. Preliminary research showed that, activated LfcinB can be expressed through E.coli expression system and Pichia pastoris expression system, but with limited yield and difficulty in purification. So building a new LfcinB expression system is significative for raising production and optimizing production process.Baciullus thuringiensis can form ICPs consisting of plenty of δ-endotoxins during spore formation.It was subjected to lysis after fermentation.,releasing spore and crystal into culture medium.This feature makes it easy to pure crystal and industrial fermentation production.Based on the above advantages of Bt, we plan to construct a fusion protein of LfcinB and Cry60 Ba about 35 kDa from Bt subsp. Jegathesan.The fusion gene was constructed by fusing the gene encoding LfcinB to the 3’ terminus of the intact cry60 Ba gene. The recombinant vector containing the fusion gene was transformed into acrystalliferous Bt strain 4Q7 and recombinant strain 4Q7/pPFT60Ba-LfcinB was obtianed. After cultivated in GYS medium for 48 h, the recombinant strain produced parasporal crystal, which was collected and subjected to SDS-PAGE analysis. The result showed that the expected protein band about 38 kDa was obtained.The purified fusion protein was hydrolyzed with 90 mM HCl for 3-7 h, then adjusting pH value to neutral with same volume of 90 mM NaOH. SDS-PAGE analysis showed that 3-kDa target protein was obtained, which was then excised and was subjected to LC-MS/MS analysis. The result showeds that Cry60 Ba and LfcinB from fusion protein was separated after hydrolysis and single Lfcin B was obtained.In summary, we considered that expressing fusion protein of crystal protein and LfcinB in Baciullus thuringiensis may be a efficient expression system of LfcinB, which laid the foundation for massively producting active LfcinB through genetic engineering methods.