Zhou Han, Li Xuechun, Wang Zongying, Yin Jiyuan, Tan Hongchao, Wang Li, Qiao Xinyuan, Jiang Yanping, Cui Wen, Liu Min, Li Yijing, Xu Yigang, Tang Lijie
College of Veterinary Medicine, Northeast Agricultural University, Chang Jiang road No. 600, Xiang Fang District, Harbin, China.
Heilongjiang Key Laboratory for Animal Disease Control and Pharmaceutical Development, Harbin, China.
BMC Vet Res. 2018 Jun 27;14(1):206. doi: 10.1186/s12917-018-1516-y.
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. In order to develop a food-grade L. casei system constitutively expressing bovine Lfcin, this study constructed a thymidine auxotrophy (ΔthyA) recombinant L. casei.
Based 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).
We 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.
干酪乳杆菌（L. casei）因其在人和动物体内的益生菌特性而闻名。乳铁蛋白素（Lfcin）多肽可有效调节宿主免疫反应，并在体内和体外具有抗菌活性。为了开发一种组成型表达牛Lfcin的食品级L. casei系统，本研究构建了胸苷营养缺陷型（ΔthyA）重组L. casei。
基于胸苷酸合成酶基因（thyA）插入位点，通过同源重组将Lfcin表达盒（LFEC）插入L. casei基因组，成功表达并可稳定遗传。重组L. casei，即ΔthyA L. casei-LFEC，对氯霉素敏感，在无胸腺嘧啶培养时生长受限。同时，ΔthyA L. casei-LFEC对大肠杆菌和金黄色葡萄球菌均具有良好的抗菌活性，对猪流行性腹泻病毒（PEDV）具有抗病毒活性。
我们成功构建了表达Lfcin的重组L. casei菌株ΔthyA L. casei-LFEC，其仅在胸腺嘧啶存在下存活，具有优异的抗菌和抗病毒活性，遗传稳定性良好且具有敏感性。本研究为具有额外生物学功能和更广泛应用前景的抗生素提供了一种经济高效的替代品。使用ΔthyA作为选择标记而非抗生素来构建基因工程L. casei为家畜饲养中的饲料添加剂提供了一种安全有效的方法。
