Construction and characterization of thymidine auxotrophic (ΔthyA) recombinant Lactobacillus casei expressing bovine lactoferricin.
Construction and characterization of thymidine auxotrophic (ΔthyA) recombinant Lactobacillus casei expressing bovine lactoferricin.
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表达牛乳铁素的胸苷营养缺陷型（Delta thyA）重组干酪乳杆菌的构建和表征
DOI：
10.1186/s12917-018-1516-y
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发表时间：
2018-06-27
影响因子：
2.6
通讯作者：
Tang L
中科院分区：
文献类型：
--
作者：
Zhou H;Li X;Wang Z;Yin J;Tan H;Wang L;Qiao X;Jiang Y;Cui W;Liu M;Li Y;Xu Y;Tang L
干酪乳杆菌（Lactobacillus casei， L. casei）以其对人类和动物的益生菌特性而闻名。乳铁蛋白（Lfcin）多肽能有效调节宿主免疫反应，在体内和体外均具有抗菌活性。为了构建可组成性表达牛Lfcin的食品级干酪乳杆菌体系，本研究构建了胸苷缺乏症重组干酪乳杆菌（ΔthyA）。基于胸腺苷酸合成酶基因（thyA）插入位点，通过同源重组将LFEC（Lfcin表达盒）插入干酪乳杆菌基因组，成功表达并稳定遗传。重组干酪乳杆菌（ΔthyA L. casei- lfec）对氯霉素敏感，在没有胸腺嘧啶的情况下培养受限。同时，ΔthyA L. casei-LFEC对大肠杆菌和金黄色葡萄球菌具有良好的抑菌活性，对猪流行性腹泻病毒（PEDV）具有抗病毒活性。我们成功构建了一株表达Lfcin的重组干酪乳杆菌（ΔthyA L. casei- lfec），该菌株只能在胸腺嘧啶存在下存活，具有良好的抗菌和抗病毒活性，遗传稳定性和敏感性良好。本研究提供了一种具有额外生物学功能的低成本抗生素替代品，具有广阔的应用前景。利用ΔthyA作为替代抗生素构建基因工程干酪乳杆菌的选择性标记，为畜禽养殖饲料添加剂提供了一条安全有效的途径。
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.
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影响因子：
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作者：
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通讯作者：
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作者：
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