Abstract
BackgroundLactobacillus casei (L. casei) is well known for its probiotic property in human and animals. Lactoferricin (Lfcin) polypeptide can effectively modulate host immune responses and have antimicrobial activity in vivo and in vitro. In order to develop a food-grade L. casei system constitutively expressing bovine Lfcin, this study constructed a thymidine auxotrophy (ΔthyA) recombinant L. casei.ResultsBased on the thymidylate synthase gene (thyA) insert site, LFEC(Lfcin expression cassette)was inserted into L. casei genome through homologous recombination, successfully expressed and could be stably inherited. The recombinant L. casei, ΔthyA L. casei-LFEC, is sensitive to chloramphenicol and limited when cultured without thymine. Meanwhile, ΔthyA L. casei-LFEC has both good antibacterial activity against Escherichia coli and Staphylococcus aureus and antiviral activity against porcine epidemic diarrhea virus (PEDV).ConclusionsWe successfully constructed a recombinant L. casei strain expressing Lfcin, ΔthyA L. casei-LFEC, which could only survive in the presence of thymine, and had excellent antimicrobial and antiviral activity with good genetic stability and sensitivity. This research provides a cost-effective alternative to the antibiotics with additional biological functions and wider applicability prospect. Using ΔthyA as the selectable mark instead of antibiotic to construct genetic engineering L.casei provides a safe and effective approach of feed additives in livestock raising.
Highlights
Lactobacillus casei (L. casei) is well known for its probiotic property in human and animals
Thymidylate synthase encoded by thymidylate synthase gene is a kind of highly conserved isozyme present in different bacteria, which plays a crucial role in DNA synthesis, and works as the key enzyme in de novo synthesis of phosphorylated deoxythymidine uracil by catalyzing deoxyuridine ribonucleotides into Deoxythymidine uracil (dTMP) via methylation modification [5, 6]
Test of auxotrophy and chloramphenicol-sensitivity of Thymidine auxotrophy (ΔthyA) L.casei-LFEC The results showed that only pGBHCupp-TF-LFEC-TR/ L.casei (PT) could grow on the GM17 plate supplemented with Cmr and thymine (Fig. 3a), indicating no residual antibiotic resistance present in ΔthyA L.casei-LFEC; on the GM17 plate supplemented with thymine and without Cmr, L. casei, pGBHCupp-TF-LFEC-TR /L.casei and ΔthyA L.casei-LFEC could normally grow (Fig. 3b); on the GM17 plate without thymine and Cmr, L. casei and pGBHCupp-TF-LFEC-TR/L.casei could normally grow, but not ΔthyA L.casei-LFEC (Fig. 3c)
Summary
Lactobacillus casei (L. casei) is well known for its probiotic property in human and animals. Lactoferricin (Lfcin) polypeptide can effectively modulate host immune responses and have antimicrobial activity in vivo and in vitro. Lactobacillus casei (L. casei) is well known as a kind of gram positive bacteria with its probiotic property for human and animals, which can maintain microflora homeostasis, inhibit pathogens growing and regulate pH balance in the host gastrointestinal environment [1]. Lactoferricin (Lfcin) polypeptide is dissociated off from lactoferrin under acidic condition, which can effectively modulate host immune responses, such as recruiting and promoting the balance of the production of immune cells [8]. The Lfcin possesses many other probiotic properties, such as antioxidation, antiviral activity, inhibiting tumor cell growth and regulating the immunity of the organism [12,13,14,15,16]
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