益生菌Nissle 1917鞭毛蛋白高变区随机缺失及鞭毛展呈外源抗原的构建  

Random deletion of flagellin hypervariable region of probiotic Nissle 1917 and construction of foreign antigen for flagella display

作　　者：杨颖[1,2] 金正雨 邓永军 区炳明 文明 YANG Ying;JIN Zheng-yu;DENG Yong-jun;OU Bing-ming;WEN Ming(College of Animal Science,Guizhou University,Guiyang 550025,China;Research Center of Engineering Technology for Animal Biological Products of Guizhou,Guiyang 550025,China;College of Life Sciences,Zhaoqing University,Zhaoqing,Guangdong 526061,China)

机构地区：[1]贵州大学动物科学学院,贵阳550025 [2]贵州省动物生物制品工程技术研究中心,贵阳550025 [3]肇庆学院生命科学学院,广东肇庆526061

出　　处：《西南农业学报》2022年第3期707-711,共5页Southwest China Journal of Agricultural Sciences

基　　金：贵州省科学技术厅联合基金项目[黔科合LH字(2017)7268号];贵州大学引进人才项目[贵大人基合字(2016)77号];广东省基础与应用基础研究基金联合基金青年基金项目(2019A1515111186)[粤基金字(2020)6号]。

摘　　要：【目的】探索益生菌Nissle 1917无质粒克隆菌株(EcNc)鞭毛蛋白高变区展呈外源抗原的位点,为EcN靶向投递外源抗原提供操作策略,为优化构建遗传稳定、定植力强的功能性益生菌疫苗打下基础。【方法】以重组质粒pBR322-fliC为模板,构建EcNc fliC高变区随机缺失质粒库、筛选有效的fliC高变区缺失区域、构建EcNc FliC高变区突变株展呈外源抗原、通过透射电镜和运动性观察及IPEC-J2细胞黏附试验对重组菌株鞭毛展呈外源抗原的能力进行检测。【结果】EcNc fliC高变区不同部位出现缺失,获得EcNc fliC高变区随机缺失质粒库;经细菌运动性检测和测序分析,筛选到fliC基因缺失910~927 bp处碱基序列后不影响EcNc鞭毛丝的形成和运动性能;将F18大肠杆菌黏附素FedF亚单位与仔猪小肠上皮细胞相互作用的受体结合域(第6~109位氨基酸残基区段)碱基序列插入该位点。【结论】成功将FedF亚单位受体结合域的50个氨基酸展呈于EcNc表面。【Objective】The foreign antigen display sites in flagellin hypervariable region of the probiotic Nissle 1917 plasmid-free clone strain(EcNc)is explored,which provides an operating strategy for targeted delivering foreign antigens using EcN flagella,and lays the foundation for the optimized construction of functional probiotics vaccine with stable genetics and strong colonization ability.【Method】With the recombinant plasmid pBR322-fliC as a template,the plasmid libraries of random deletion in the EcNc fliC hypervariable region was constructed,the effective deletion regions in fliC hypervariable region was screened and the EcNc mutants for displaying exogenous antigens in FliC hypervariable regions was constructed to test the ability to display exogenous antigens in flagella of these recombinant strains by transmission electron microscope,motility observation and in-vitro IPEC-J2 cell adhesion assay.【Result】Different parts in EcNc fliC hypervariable region are deleted,and the plasmid libraries of random deletion of EcNc fliC hypervariable region is obtained.After bacterial motility detection and sequencing analysis,it was found that the absence of the 910;-927;base pairs in fliC gene did not affect the formation of the 910;EcNc flagella and its motility.And the receptor binding domain of FedF adhesin of F18;Escherichia coli(the 6;-109;amino acid residues)and piglet intestinal epithelial cells was inserted into the above-mentioned display site.【Conclusion】These 50 amino acid residues of FedF subunit receptor binding domain is inserted into EcNc fliC hypervariable region and successfully displayed on the surface of EcNc.

关 键 词：EcNc 鞭毛蛋白 鞭毛展呈 

分 类 号：S852.61[农业科学—基础兽医学]