凡纳滨对虾肠道中乳酸菌表面粘附相关蛋白及其受体的鉴定与功能研究

Lactobacillus as probiotics is drawing more and more attention in the prevention and control of aquaculture diseases, adhesion is the first step for Lactobacillus colonizing animal intestinal tract to perform beneficial function, and the .adhesion-related components on the surface of the bacteria body is the material foundation of mediated adhesion. Our previous studies have found that the probiotics effect of Lactobacillus pentosus (HC-2) is associated with the adhesion of the bacteria in the Litopenaeus vannamei intestine, and the surface proteins played vital role in those process, but the key proteins at work are unclear. The preliminary experiments showed that HC-2 surface protein molecular weight were 15kDa, 17kDa, 33kDa, 44kDa ect 4 proteins could interact with the intestinal protein of shrimp. Accordingly, in order to further explore the adhesion mechanism of lactobacillus, this project intends to use bioinformatics, antibody probe and mass spectrometry to identify the adhesion proteins and their adhesion receptors in L. vannamei intestine; Exploring and validating the interaction relationship between adhesion protein and their receptors in vivo and in vitro by Yeast double hybridization, cell transfection, co-immunoprecipitation and colloidal gold co-localization; Through the means of SiRNA technology interfering and recombinant homologous proteins simulating adhesion proteins, and blacking adhesion receptors by antibodies to study the function of adhesion protein and its receptors in the bacteria adhesion colonization and pathogenic bacteria inhibition in L. vannamei and Caco-2 cells. This project will provide an important theoretical basis for elucidation of the adhesion mechanism of Lactobacillus and its application in L. vannamei intestine.

乳酸菌作为益生菌在水产病害防治的作用越来越受重视，粘附是乳酸菌定植肠道发挥益生功能的第一步，而菌体表面的各种粘附成分是介导定植的物质基础。前期发现戊糖乳杆菌(HC-2)对凡纳滨对虾的益生效果跟菌体在对虾肠道定植有关，其表面蛋白在菌体粘附过程发挥了重要作用，但具体的关键蛋白尚不清楚。预实验发现其表面分子量为15kDa,17kDa,33kDa,44kDa等4种蛋白可与对虾肠道蛋白发生相互作用。据此，为深入探究乳酸菌粘附机理，本项目拟以生物素标记、抗体探针和质谱技术，鉴定HC-2表面粘附蛋白及其在对虾肠道的粘附受体；通过酵母双杂交、细胞转染、免疫共沉淀、胶体金免疫共定位等方法从体内和体外探究验证两者互作关系；通过SiRNA干扰、重组同源蛋白模拟粘附蛋白和抗体封闭粘附受体等手段，在对虾体内和Caco-2细胞上探究其在菌体粘附和病原菌抑制的作用。该项目将为阐明乳酸菌在对虾肠道的粘附特性提供重要数据。

乳酸菌是动物肠道中重要的益生菌，其发挥益生作用的前体是粘附定植，而菌体表面的的各种粘附成分是介导定植的物质基础。其中，表面蛋白不仅在细菌细胞壁的合成、细胞形态的维持以及与宿主的信号传递中起着重要作用，还能介导菌体黏附。因此，本项目鉴定了乳酸菌HC-2表面相关蛋白的种类，探究了表面蛋白的功能。解析了HC-2菌株的基因组遗传特征，发现去除表面蛋白导致细菌自聚集能力和表面疏水性，以及对HCT116细胞和对虾肠道上皮细胞的粘附作用显著下降；通过质谱技术共鉴定到201个独特的表面相关蛋白，其中大部分蛋白质参与了菌体的转录和翻译、生物合成或代谢、细胞周期或分裂、细胞壁的合成和应急反应等生命过程；揭示了HC-2在对虾肠道粘附定植规律，发现菌体主要定植于对虾的中肠，定植数是前肠的15倍，而后肠的定植率较低；鉴定到表面相关蛋白延伸因子Tu（EF-Tu）、I型甘油醛-3-磷酸脱氢酶（GAPDH）、小热休克蛋白（sHsp）和30S核糖体蛋白（rpsH）能与对虾肠道上皮细胞发生相互作用，其中延伸因子Tu（EF-Tu）与对虾肠道中的纤维蛋白原相关蛋白2（FREP2）相互作用介导菌体在对虾肠道中粘附定植，还发现重组蛋白rEF-Tu在一定浓度范围内能促进Caco-2细胞的生长，并能显著抑制LPS和病原菌诱导的细胞凋亡。这些结果揭示了表面蛋白在介导乳酸菌在对虾肠道黏附定植及调节肠道黏膜屏障功能过程发挥了重要作用，有助于我们深入理解乳酸菌的黏附特性和益生作用机制，为研发高效益生菌提供了重要理论数据。申请人以独立第一作者发表SCI论文4篇。培养了硕士研究生2名。