Effects Of Lactobacilli On The Gene Expression Of Antimicrobial Peptide AvBD9 In Chicken Intestinal Epithelial Cells

Antimicrobial peptides produced by intestinal epithelial cells that serve as the first line of defense for the body are an important component of the intestinal mucosal innate immunity, and show properties such as potent broad-spectrum antimicrobial activity, high stability, and no antimicrobial resistance. The avian beta-defensin 9 (AvBD9), an inducible antimicrobial peptide expressed in the epithelial cells of chicken small intestine, plays an important role in maintaining the homeostasis of gastrointestinal microflora and intestinal immune system. The probiotic Lactobacilli, predominantly clonized in the chicken small intestine, interact with epithelial cells by adhesion to it and can modulate the physiological function of the epithelial cells.In vitro cultured intestinal mucosa epithelial cells were used in this study to investigate the effect of Lactobacilli on the gene expression of antibacterial peptide AvBD9. The isolation methods of the intestinal epithelium were compared and tested, and the optimal culture conditions was determinted in this study, a primary culture model of small intestinal epithelial cells of chicken embryo was then successfully established. The isolated epithelial cells were inoculated into cell culture dishes and attached readily to culture dishes within 12-24 h, forming coalescing colonies of epithelium within 48-72 h. Epithelial cells reached confluence within 6-7 days liking slabstone paving way. Alkaline phosphatase staining and immunohistochemical characterization of the cells showed that>85% of the primary cultured cells were epithelial cell.AvBD9 peptide sequence was cloned into the prokaryotic expression vector pGEX-6P-1. The recombinant expression plasmid pGEX-6P-AvBD9 was constracted and then transformed into E. coli to express fusion protein. The fusion protein was expressed in E. coli as inclusion body. The GST-AvBD9 fusion protein purified from the cutting gel was used to immunize New Zealand white rabbit. As dectected by indirect ELISA and Western blot, high titer of specific polyclonal antibody against AvBD9 was obtained after the rabbit was immunized with purified GST-AvBD9 fusion protein, which will contribute to the further investigation in determining the antimicrobial peptide AvBD-9 expression from protein level.Seven strains of probiotic lactobacilli and one strain of probiotic enterococcus were selected to investigate the effects of lactobacillus stimuli on the AvBD9 mRNA expression in cultured intestinal epithelial cells and to compare the induction differences of AvBD9 expression between different probiotic bacterial strains. The time-and dose-response of AvBD9 gene expression upon stimulation of epithelial cells with defined probiotic lactobacilli was also examined. The AvBD9 mRNA expression was determinted by fluorescence quantitative PCR (FQ-PCR). AvBD9 expression differences were observed among probiotic bacteria and different stimulation concentrations of bacteria. Lactobacillus fermentum F6, Lactobacillus rhamnosus MLGA, Lactobacillus rhamnosus MB 12, Lactobacillus plantarum SJ and Enterococcus faecium DM5 could stimulate AvBD9 mRNA expression in a different extent. From a comparison of all tested lactobacillus strains, the Lactobacillus ramous MLGA has the strongest capability to promote the AvBD9 expression. Heat-inactivated Lactobacillus ramous MLGA also up regulated the expression of AvBD9, and showed stronger induction response than live bacteria (P<0.05). The lactobacilli strains promoted AvBD9 mRNA transcription in time-dependent manner. The AvBD9 mRNA expression peaked at 12 h of incubation upon treatment of epithelial cells with lactobacilli. AvBD9 peptide was detected by Western blot in the supernatants of cultured epithelial cells treated with Lactobacillus ramous MLGA, indicating that AvBD9 peptide releases into extracellular medium.