Effect Of Nuclear Localization Signal On Expression Of Viral Capsid Protein In E.coli And Immunological Activity Of Recombinant Capsid Protein

Nuclear localization signal(NLS)is a fragment of peptide,which can lead protein into the nucleus of the cells.NLS is usually rich in alkaline amino acids,such as arginine.NLS also present in capsid proteins(CP)of many viruses,such as porcine circovirus 2b capsid protein(PCV2b-CP)and porcine circovirus 3 capsid protein(PCV3-CP).In this study,we found that NLS could result in low expression of recombinant PCV3-CP expressed in E.coli,and the protein expression could be improved by restructuring of NLS sequence.Then,we evaluated the immunological activity of vaccines and adjuvants in mouse model and zebrafish model.At first,we analyzed the sequence and predicted the epitope of PCV3-CP,and designed a recombinant PCV3-CP with enhanced epitope.We constructed and expressed the recombinant PCV3-CP in E.coli,but the protein expression was low.In order to improve the expression,we optimized the codons of recombinant PCV3-CP gene,and found optimized codons weren't able to improve the expression.Next,we analyzed the NLS sequences of recombinant PCV3-CP and recombinant PCV2b-CP,and designed two recombinant NLS sequences.The recombinant PCV3-CP with reconstructed NLS was constructed and expressed.The results showed that the level of protein expression was greatly improved.The recombinant PCV3-CP was dissolved in different solutions and identified by SDS-PAGE.It was found that the recombinant PCV3-CP showed weak band in Solution A,and it could be solved by increasing the concentration of SDS in loading buffer.Then,we evaluated the immunological activities of recombinant PCV3-CP and adjuvants in mouse model and zebrafish model.First,the mice were immunized with recombinant PCV3-CP mixed with adjuvant,and its specific antibodies in serum were detected by ELISA.The spleen cells of mice were isolated and stimulated by recombinant PCV3-CP,and the activated B cells were detected by flow cytometry.The results showed that recombinant PCV3-CP could induce specific antibodies in mice and significantly activate B cells.We use the zebrafish whose neutrophils were marked green fluorescent protein for evaluating the immunological activities of adjuvants.The zebrafish were injected with ODN,or injected with ODN plus recombinant PCV3-CP.Its neutrophils were observed and recorded by fluorescence microscopy.The results showed that different ODN had different effects on the recruitment of neutrophils,and combined using of recombinant PCV3-CP and ODN can also induce neutrophil aggregation,which has potential value as a vaccine.In summary,this study indicates that the NLS sequence has important influence on the protein expression in E.coli,and provide a new strategy for achieving high soluble expression.This study also successfully constructed a recombinant PCV3-CP with satisfactory immunological activity,and preliminarily verified the feasibility of applying zebrafish model for evaluation of vaccines and adjuvants.