科尔沁牛乳腺炎无乳链球菌菌毛岛屿AP2基因部分片段克隆及其抗原性预测  

Cloning and antigenicity prediction of pilus island AP2 gene fragment from Horqin bovine mastitis pathogen(Streptococcus agalactiae)

作　　者：锡林高娃[1] 杜长智[1] 吴金花[1] 王华[1] 白文丽[1] 于辰龙[1] 布日额[1] 

机构地区：[1]内蒙古民族大学生命科学学院/乳源性致病菌研究所,内蒙古通辽028043

出　　处：《南方农业学报》2015年第12期2185-2190,共6页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31260600);内蒙古民族大学市校合作项目(SXYB2012086)

摘　　要：【目的】克隆分析科尔沁牛乳腺炎无乳链球菌菌毛岛屿AP2基因片段,为深入研究奶牛乳腺炎无乳链球菌免疫机制及制备新型免疫抗原提供理论依据。【方法】根据Gen Bank已公布牛源无乳链球菌PI-2a菌毛岛屿的AP2基因序列,采用Primer 5.0软件设计1对特异性引物,以内蒙古通辽地区乳源性无乳链球菌临床菌株总DNA为模板,扩增AP2基因片段,并分析其编码序列及免疫活性。【结果】科尔沁牛乳腺炎无乳链球菌菌毛岛屿AP2基因片段大小为699 bp,共编码233个氨基酸残基,其基因序列与Gen Bank已公布牛源无乳链球菌PI-2a菌毛岛屿AP2基因序列(EU930008)的相似性达99.6%,仅有3个碱基出现变异(402C→A、563C→T和467A→T)。菌毛岛屿辅助蛋白AP2二级结构的α螺旋在C端分布较多;β折叠较丰富,且分布均匀;而无规则卷曲分布在两端。菌毛岛屿辅助蛋白AP2的亲水区分布较广,几乎遍布于整个蛋白区,属于亲水性蛋白;其抗原指数较高的区段较丰富,抗原性良好;蛋白质分子表面可及性也较高,其区域占比达54.0%。【结论】科尔沁牛乳腺炎无乳链球菌菌毛岛屿AP2基因具有高度保守性,其编码蛋白具有良好的抗原性,可作为阻止无乳链球菌感染的候选疫苗靶标抗原。[Objective]The pilus island auxiliary protein gene（AP2） of Horqin bovine mastitis pathogen（Streptococcus agalactiae） were cloned and analysed, in order to provide theoretical basis for further study on immune mechanisms of bovine mastitis pathogen S. agalactiae and preparation of new immunogenic antigen. [ Method ]According to published PI- 2a pilus island AP2 gene sequences of bovine S. agalactiae on GenBank, a pairs of specific primers was designed with bioinformatics software of PrimerS.0. And using total DNA of S. agalactiae clinical strains from Tongliao, Inner Mongolia as template, AP2 gene sequence was amplified. Then its coding sequence and immunological activity were analyzed. [Result] The results showed that, AP2 gene fragment of Horqin bovine mastitis pathogen S. agalactiae was 699 bp in size, encoding total 233 amino acid residues. And it shared similarity of 99.6% with published PI-2a pilus island AP2 gene sequence （EU930008） of bovine S. agalactiae on GenBank, there existed 3 bases to mutate （402C→A, s63C→T and 467A→T）. In the secondary structure of pilus island auxiliary protein AP2, more a-helixes appeared at C-terminus, and a great quantity of β-sheets appeared and were distributed evenly, while the irregular colis appeared at both terminuses. Furthermore, the hydrophilic regions of pilus island auxiliary protein AP2 were distributed widely, and were almost spread across entire pro- tein region, therefore, the auxiliary protein AP2 belonged to hydrophilic protein. Moreover, there was more zones with higher antigen index in its structure, so it had good antigenicity. And its surface accessibility of protein molecule were higher, accounted for 54.0% of all areas. [Conclusion]The PI-2a pilus island AP2 gene of bovine mastitis pathogen S. agalactiae is highly conserved, and its encoded protein has good antigenicity, which can be used as candidate vaccine target antigen to prevent S, agalactiae infection.

关 键 词：科尔沁牛 无乳链球菌 菌毛岛屿 AP2基因 克隆 抗原性 

分 类 号：S858.23[农业科学—临床兽医学]