| Lactobacillus plantarum is a common lactic acid bacteria that own a wide range of applications in the field of industrial lactic acid fermentation and healthcare, especially it has immunomodulatory activity, which can effectively prevent some diseases. The strain has been largely utilized as vehicles for oral pharmaceutical proteins to solve the hydrolysis in the gastrointestinal tract(GIT) in recent years, but expression products are secreted in its cytoplasm mostly. To a great extent, the cell wall of this bacteria is the biggest obstacle for exogenous proteins to play a role in extracellular. As a constituent protein of Bacillus subtilis, poly-γ-Glutamic Acid Synthetase A(pgsA) can display exogenous proteins on the surface of host strains. PgsA has been mainly applied to Lactobacillus casei or Lactococcus lactis, laying the foundation of solving the extracellular transport problem of heterogenous proteins expressed by Lb. plantarum after using it as a surface display component. However, the construction of recombinant bacteria and expression of exogenous proteins would be affected because of its longer genes(1143 bp). Therefore, we also intended to increase the ability of carrying genes by plasmids and expression efficiency of recombinant bacteria by truncating pgsA.The study is divided into three parts, including the construction of recombinant Lb. plantarum, the identification of physiological properties of recombinant Lb. plantarum and expression functions of them in vitro.(1) pgsA’, the shortened anchored gene, obtained according to the prediction of protein structure of pgsA by DNAstar software firstly, then Lb. plantarum-EGFP,Lb. plantarum-pgsA-EGFP, Lb. plantarum-pgsA’-EGFP,Lb. plantarum-pgsA were constructed.(2) The physiological properties of recombinant Lb. plantarum were detected, including optimal induced conditions, growth rate, the ability of surface expression of EGFP, the stability of EGFP expression, the relative copy number of plasmids as well as the stability of plasmids.(3) Lb. plantarum-IL10, Lb. plantarum-pgsA-IL10, Lb. plantarum-pgsA’-IL10 were constructed and surface expression of IL10 on bacteria was measured by Western blot. Subsequently, RAW264.7 cells were co-cultured with these bacteria before the stimulation of Poly(I: C) or lipopolysaccharide(LPS), then the expression of inflammatory cytokines such as TNF-α, IFN-γ, IL-1β, IL-6 and nuclear transcription factor NF-κB p65 were detected by qRT-PCR, ELISA, and immunofluorescence.The results indicated that both EGFP and IL10 could be expressed on the surface of recombinant strains with anchorin as well as higher expression in Lb. plantarum-pgsA’-IL10 were verified by Western Blot. The optimal induction condition:the strains were inoculated with addition of SppIP simultaneously and cultured under the condition of 30 ℃ for 12 h. The growth rates of recombinant bacteria containing pgsA and pgsA’ were obviously decreased, but the two strains had a lower loss rate of plasmids and could effectively maintain EGFP expression for 48 h or more. The synergistic effects of rLAB and two TLR ligands in RAW264.7 cells elucidated that the upregulation of TNF-α, IFN-γ, IL-1β, IL-6 and NF-κB p65 by Poly(I:C) or LPS was exacerbated after co-culture with Lb.plantarum-pgsA, but the IL10 of recombinant bacteria could inhibit these factors, and that transcription of IL-1β, IL-6 as well as expression of TNF-α, IFN-γ, IL-1β, IL-6, NF-κB p65 arised from recombinant strains with anchorin(especially in Lb. plantarum-pgsA’-IL10) were significantly lower than the anchored proteins-free ones.This study not only confirmed both pgsA and pgsA’ could display exogenous proteins on the surface of Lb. plantarum, but also found pgsA’ had a higher efficiency of surface-displayed, providing a reference for subsequent studies of heterogeneous proteins expression on the surface of recombinant latic acid bacteria. |
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