鸭源鸡杆菌tolC基因序列特征分析、原核表达及免疫原性分析  

Sequence Characterization,Prokaryotic Expression and Immunogenicity Analysis of Gallibacterium anatis tolC Gene

作　　者：史姿聪 刘盼盼 王新卫[1] 翟雪滢 陈桦源 刘红英[1] 马立静 杨霞[1] 陈陆[1] SHI Zi-Cong;LIU Pan-Pan;WANG Xin-Wei;ZHAI Xue-Ying;CHEN Hua-Yuan;LIU Hong-Ying;MA Li-Jing;YANG Xia;CHEN Lu(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China;College of Agricultural,Ningxia University,Yinchuan 750021,China)

机构地区：[1]河南农业大学动物医学院,郑州450046 [2]宁夏大学农学院,银川750021

出　　处：《农业生物技术学报》2022年第9期1823-1833,共11页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31302090);河南省自然科学基金(162300410153);河南省高等学校重点科研项目(22A230004)。

摘　　要：tolC基因广泛存在于革兰氏阴性菌中,其表达的TolC蛋白(TolC family protein,TolC)是一种外膜通道蛋白,介导多种不同类型化合物的胞外转运过程,在细菌的生物学功能中发挥着重要作用。解析鸭源鸡杆菌(Gallibacterium anatis)tolC基因特征及TolC蛋白的特性对于该病防控具有重要意义。为研究鸭源鸡杆菌tolC基因及其编码蛋白的结构特征和其作为抗原的潜能,本研究对28株鸭源鸡杆菌分离株tolC基因序列进行测定及系统发育分析;以PDS-RZ-1-SLG菌株为研究对象,利用生物信息学方法对TolC蛋白的理化性质及结构进行预测,并进行克隆、表达及抗原特性分析。结果表明,tolC基因在鸭源鸡杆菌中国分离株中普遍存在,全长1371 bp,核苷酸序列高度保守,同源性为92.1%~100%,与鸡杆菌复合群(Gallibacterium genomosp)1和2亲缘关系最近;TolC蛋白由456个氨基酸编码,理论分子质量为51.1 kD,是一个稳定的、亲水的α型分泌性膜蛋白,无跨膜结构域,但有1个信号肽序列和10个主要B细胞表位。SDSPAGE和Western blot结果表明获得了约45 kD的重组蛋白,且该重组蛋白与鸭源鸡杆菌阳性血清可发生特异性反应,且能与TolC多克隆抗体兔血清发生特异性结合,证实了TolC的反应原性与免疫原性。本研究为进一步研究TolC蛋白生物功能提供基础信息,同时为鸭源鸡杆菌新型疫苗的开发提供理论依据。The tolC gene is widespread in gram-negative bacteria,and TolC family protein(TolC)expressed by tolC is an outer membrane channel protein,which mediates the extracellular transport of various compounds and plays an important role in the biological functions of bacteria.The identification of the characteristic of tolC gene of Gallibacterium anatis and its protein would play crucial roles in efforts to prevent and control this disease.In order to analyze the structural characteristics of gene and amino acid and potential as an antigen of tolC of G.anatis,in this study,the sequences and phylogenetic analysis of tolC gene of 28G.anatis isolates were carried out.Taking G.anatis PDS-RZ-1-SLG as the research object,the physicochemical properties and structures of TolC were predicted by bioinformatics methods.In addition,the tolC gene sequences were cloned and expressed,and its antigenic properties were analyzed.The results showed that the tolC gene,with 1371 bp in length,was ubiquitous in Chinese isolates of G.anatis and its nucleotide sequences were highly conserved(similarity:92.1%~100%),which had a close relationship with G.genomosp 1 and 2.Moreover,the tolC gene of G.anatis encoded a protein of 456 amino acids,with a calculated molecular mass of 51.1 kD,and the TolC protein was a stable,hydrophilic,α-type and secretory membrane protein without transmembrane domains,but with a signal peptide sequence and 10 main B cell epitopes.SDS-PAGE and Western blot analysis showed the recombinant protein was about 45 k D,and the recombinant protein could react specifically with positive serum of G.anatis and the polyclonal anti-TolC antiserum raised in rabbits had a specific binding with G.anatis,which verified the reactogenicity and immunogenicity of the TolC protein.This study provides basic information for further research on the biological functions of the TolC,and establishes a theoretical basis for development of new vaccines against G.anatis.

关 键 词：鸭源鸡杆菌 TolC蛋白 生物信息学分析 原核表达 免疫原性 

分 类 号：S855.1[农业科学—临床兽医学]