奶牛乳腺炎无乳链球菌Hly基因抗原表位的截短序列表达及抗原性研究  

Expression and study of the antigenicity of a truncated sequence of an antigenic epitope of the Hly gene of Streptococcus agalactiae causing bovine mastitis

作　　者：吴金花[1,2,3] 陈金龙 锡林高娃 王金良[5,4] 布日额 于辰龙[1,2,3] 

机构地区：[1]内蒙古民族大学生命科学学院,内蒙古通辽028043 [2]内蒙古自治区乳源性致病菌防控工程技术研究中心 [3]内蒙古民族大学乳源性致病菌研究所 [4]山东绿都生物科技有限公司 [5]山东省滨州畜牧兽医研究院

出　　处：《中国病原生物学杂志》2018年第1期5-10,共6页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.31560689;31760725);内蒙古自治区乳源性致病菌防控工程技术研究中心开放课题(No.MDK2016037;MDK2016036;MDK2017017;MDK2017016);内蒙古自治区科技计划项目(2017);内蒙古自治区"草原英才"工程创新创业人才团队项目(2017)

摘　　要：目的截短克隆奶牛乳腺炎无乳链球菌溶血素(Hly)基因抗原优势区域,构建重组表达载体,实现原核细胞高效表达,进而对表达产物进行抗原性分析。方法根据GenBank公布的牛源无乳链球菌(CP018623.1)hly基因序列设计引物,以牛乳源性无乳链球菌1886菌株基因组DNA为模板,PCR扩增hly基因片段,构建重组质粒pET-30a-hly并转化至E.coli/BL21(DE3)中,经IPTG诱导高效表达后,并进行SDS-PAGE电泳和Western blot鉴定。表达产物纯化后经水溶性501佐剂乳化,免疫昆明小白鼠,采用间接ELISA法测定血清抗体滴度。结果成功克隆奶牛乳腺炎无乳链球菌1886菌株hly基因,其碱基长度513bp,编码171个氨基酸殘基,与GenBank公布的牛源无乳链球菌(CP018623.1)Hly基因核苷酸序列及其编码氨基酸序列相似性为100%。重组质粒表达的重组蛋白分子质量单位约为30×103,纯化蛋白含量为1.2mg/ml。Western blot检测重组蛋白可被相应抗体识别。ELISA检测重组蛋白免疫小鼠血清抗体滴度为1∶25 600。结论内蒙古分离株无乳链球菌Ia型hly基因抗原优势序列编码多肽具有良好的抗原性,可以作为候选免疫抗原,为研究其致病机制及新型疫苗奠设计奠定了基础。Objeclives To clone a truncated peptide derived from the main antigenic region of the hemolysin （hly） gene of Streptococcus agalactiae causing bovine mastitis, to efficiently express a recombinant protein in a prokaryotic system, and to study the antigenicity of the expressed protein in order to provide an experimental basis and theoretical basis for the pathogenesis of bovine mastitis and to prepare a hemolysin antigen. Methods A pair of primers were designed and syn- thesized based on the sequence of the hly gene of S. agalactiaeECP018623.1] registered in GenBank. A 513-bp DNA frag- ment of the hly gene was amplified with PCR using the genomic DNA of the 1886 strain of S. agalactiae as a template. The target DNA fragment was recovered from agarose gel and cloned into the prokaryotic expression vector pET-30（a） to construct the recombinant plasmid pET 30a-hly. pET 30a-hly was verified as correct with sequencing and then trans- formed into E. coli / BL21 （DE3）. Its expression was induced with IPTG and it was analyzed with SDS-PAGE electro- phoresis. Western blotting was used to verify the antigenieity of the recombinant protein. The recombinant protein was e- mulsified with a water-soluble 501 adjuvant and was used to immunize Kunming mice to investigate its immunogenicity via indirect ELISA. Results The results of sequencing and digestion indicated that the hly gene of the 1886 strain of S. agalactiae was successfully cloned and efficiently expressed in E. coll. The molecular weight of the recombinant protein was about 30）〈 103. The recombinant protein was sueeessfully purified at a level of 1.2 mg/mL after refolding. The re sults of Western blot analysis indicated that the recombinant protein has good antigenicity. The results of indirect ELISA indicated that this recombinant protein has good immunogenicity. The limit of detection in indirect ELISA was 1 .. 25 600. Conclusion The results of Western blot analysis and indirect ELISA indicated that a recombinant protein derived from an epitope of

关 键 词：牛乳腺炎 无乳链球菌 溶血素 抗原性 

分 类 号：R378.12[医药卫生—病原生物学]