Protective effects of a food-grade recombinant Lactobacillus plantarum with surface displayed AMA1 and EtMIC2 proteins of Eimeria tenella in broiler chickens
Protective effects of a food-grade recombinant Lactobacillus plantarum with surface displayed AMA1 and EtMIC2 proteins of Eimeria tenella in broiler chickens
复制标题
表面展示柔嫩艾美耳球虫 AMA1 和 EtMIC2 蛋白的食品级重组植物乳杆菌对肉鸡的保护作用
DOI：
10.1186/s12934-020-1297-4
复制
发表时间：
2020-02-11
影响因子：
6.4
通讯作者：
Wang, Chunfeng
中科院分区：
文献类型：
--
作者：
Liu, Qiong;Jiang, Yanlong;Wang, Chunfeng
背景
禽球虫病对家禽生产构成严重威胁。除了商业减毒疫苗外，迫切需要其他防治球虫病的策略。植物乳杆菌作为一种口服疫苗递送系统，常使用传统的红霉素抗性基因（erm）来表达外源蛋白。然而，在蛋白表达过程中经常使用抗生素选择标记，它们存在将抗生素抗性基因转移到环境中的风险，并极大地限制了在田间生产中的应用。因此，一种食品级重组植物乳杆菌候选疫苗将极大地提高其在家禽业中的应用潜力。
结果
在本研究中，我们首先用非抗生素的asd - alr融合基因替换了pLp_1261Inv衍生表达载体的红霉素抗性基因（erm），产生了一系列无抗生素且可靠的食品级表达载体。此外，我们设计了一种双表达载体，它利用来自枯草芽孢杆菌的截短的多聚 -γ - 谷氨酸合成酶A（pgsA'）或嗜酸乳杆菌表层蛋白（SlpA）的锚定序列，在植物乳杆菌表面展示两种外源蛋白。绿色荧光蛋白（EGFP）和红色荧光蛋白（mCherry）被用作标记蛋白来评估重组植物乳杆菌菌株的表面展示特性，并通过蛋白质印迹、流式细胞术和荧光显微镜进行检测。为了进一步确定其作为口服疫苗候选物的应用，将柔嫩艾美耳球虫的AMA1和EtMIC2基因锚定在植物乳杆菌菌株表面。在鸡口服免疫后，重组植物乳杆菌菌株能够诱导抗原特异性的体液免疫、黏膜免疫和T细胞介导的免疫反应，为抵抗球虫病攻击提供有效保护。
结论
新构建的具有双表面展示抗原的食品级重组植物乳杆菌菌株为球虫病提供了一种潜在的高效口服疫苗候选物。
Background
Avian coccidiosis posts a severe threat to poultry production. In addition to commercial attenuated vaccines, other strategies to combat coccidiosis are urgently needed. Lactobacillus plantarum has been frequently used for expression of foreign proteins as an oral vaccine delivery system using traditional erythromycin resistance gene (erm). However, antibiotic selection markers were often used during protein expression and they pose a risk of transferring antibiotic resistance genes to the environment, and significantly restricting the application in field production. Therefore, a food-grade recombinant L. plantarum vaccine candidate would dramatically improve its application potential in the poultry industry.
Results
In this study, we firstly replaced the erythromycin resistance gene (erm) of the pLp_1261Inv-derived expression vector with a non-antibiotic, asd-alr fusion gene, yielding a series of non-antibiotic and reliable, food grade expression vectors. In addition, we designed a dual-expression vector that displayed two foreign proteins on the surface of L. plantarum using the anchoring sequences from either a truncated poly-γ-glutamic acid synthetase A (pgsA′) from Bacillus subtilis or the L. acidophilus surface layer protein (SlpA). EGFP and mCherry were used as marker proteins to evaluate the surface displayed properties of recombinant L. plantarum strains and were inspected by western blot, flow cytometry and fluorescence microscopy. To further determine its application as oral vaccine candidate, the AMA1 and EtMIC2 genes of E. tenella were anchored on the surface of L. plantarum strain. After oral immunization in chickens, the recombinant L. plantarum strain was able to induce antigen specific humoral, mucosal, and T cell-mediated immune responses, providing efficient protection against coccidiosis challenge.
Conclusions
The novel constructed food grade recombinant L. plantarum strain with double surface displayed antigens provides a potential efficient oral vaccine candidate for coccidiosis.