摘要
由猪产肠毒素大肠杆菌(ETEC)引起的仔猪腹泻是规模化猪场最常见的疾病之一。目前国内和国际上制备ETEC疫苗的靶基因多为其黏附素基因K88ac、K99。本试验通过分析ETEC K88ac、K99黏附素蛋白的生物学信息,将抗原表位进行组合,设计了一种同时含有K88ac、K99基因的多表位重组疫苗载体。首先应用ProParam、SOPMA和GOR软件分析K88ac、K99蛋白的理化特性及二级结构,应用IEDB与ABCpred软件预测二者的B细胞抗原表位,应用RANKPEP软件预测Th细胞表位,应用IEDB与NetMHC-4.0软件预测CTL细胞表位;然后将预测的B细胞、Th细胞和CTL细胞抗原表位加上接头序列,按照一定顺序进行组合,分别设计多表位连接多肽Ⅰ、Ⅱ和Ⅲ;最后通过分析抗原性、致敏性,筛选抗原性最强的连接多肽,并对其进行表征。结果显示:共筛选出9个抗原表位,按照不同的排列组合获得了3种连接多肽,其中连接多肽Ⅱ抗原性最强,为0.906 8;应用AllerTOP v. 2.0在线工具对连接多肽Ⅱ进行过敏性预测,发现其没有过敏性;应用ZpGJn4在线工具对连接多肽Ⅱ进行三维建模,发现其结构表位暴露性较好,易与抗体结合,在蛋白分子构象上符合表位设计要求。将该多表位肽的氨基酸序列按照乳酸菌密码子嗜好性反向翻译为核苷酸序列,随后插入pET28a载体中获得了p ET28a-KT质粒。结果表明,构建的连接多肽Ⅱ适合作为多表位疫苗的备选载体蛋白。多表位肽的设计及pET28a-KT质粒的获得为下一步构建表达ETEC多表位抗原的重组乳酸菌提供了技术支撑。
Piglet diarrhea caused by porcine enterotoxigenic Escherichia coli(ETEC)is one of the most common diseases in intensive swine farms.The adhesin genes K88ac and K99 are used to prepare ETEC vaccine as the target genes in China and abroad at present.In the study,the biological information of ETEC K88ac and K99 adhesin proteins was analyzed,and the antigenic epitopes were combined to design a recombinant multi-epitope vaccine vector containing both K88ac and K99 genes.Firstly,the physicochemical properties and secondary structures of K88ac and K99 proteins were analyzed by ProParam,SOPMA and GOR software,whose B cell epitopes were predicted by IEDB and ABCpred software,then the Th cell epitopes were by RANKPEP software,and the CTL cell epitopes were by IEDB and NetMHC-4.0 software;then linker sequences were added to the predicted B cell,Th cell and CTL cell antigenic epitopes,after combining them in a certain order,the multi-epitope connecting polypeptides Ⅰ,Ⅱ and Ⅲ were designed,respectively.Finally,the antigenicity and sensitization of the connecting polypeptides were analyzed to select and characterize the one with strongest antigenicity.The results showed that 9 antigenic epitopes were screened,and 3 kinds of connecting polypeptides were obtained according to different arrangement and combination,in which,the connecting polypeptide Ⅱ was with the strongest antigenicity(0.9068),but without hypersensitivity as predicted by AllerTOPv.2.0 online tool;three-dimensional modeling of the polypeptide Ⅱ was carried out by using ZpGJn4 online tool,and good exposure of its structural epitope was found,which was easy to bind to antibodies,reaching the requirements of epitope design from the perspective of protein molecular conformation.The amino acid sequence of the polypeptide Ⅱ was back-translated into nucleotide sequence according to the codon usage bias of Lactobacilli,and subsequently inserted into the pET28a vector to obtain the pET28a-KT plasmid.In conclusion,the polypeptide Ⅱ was suitable to be used as
作者
张铭洋
辛长卫
赵格
曲志娜
赵建梅
宋时萍
彭磊
张喜悦
王君玮
Zhang Mingyang;Xin Changwei;Zhao Ge;Qu Zhina;Zhao Jianmei;Song Shiping;Peng Lei;Zhang Xiyue;Wang Junwei(China Animal Health and Epidemiology Center,Qingdao 266032,Shandong,China;Ningjin Animal Husbandry Development Center,Ningjin 253400,Shandong,China;Bo'ai Vocational Secondary Vocational School,Bo'ai 454450,Henan,China)
出处
《中国动物检疫》
CAS
2024年第7期104-111,共8页
China Animal Health Inspection
基金
国家重点研发计划项目(2022YFC2303900,2022YFD1301003)
山东省重点研发计划项目(2022CXGC010606-01-05)
中国动物卫生与流行病学中心创新基金项目(DW2021001-13)。
