Bioinformatics Analysis of Porcine ETEC K88ac and K99 Adhesin Proteins and Construction of Multi-epitope Vaccine Vector
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(0.906 8),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 an alternative vector protein for multi-epitope vaccines,and future construction of recombinant Lactobacilli expressing ETEC multi-epitope antigens was supported by the design of the multi-epitope peptide and the gain of pET28a-KT plasmid.
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