Identification Of Neutralizing Epitopes And Screening Of Receptor Of Duck Tmebusu Virus Envelope Protein

Duck Tembusu virus(Duck Tembusu virus,DTMUV)is a new flavivirus in China’s Jiangsu and Zhejiang regions in early 2010,causing the egg production of ducks to drop or even stop to cause serious economic losses to our aquaculture industry.In recent years,there have been many reports on the identification,genomic and diagnostic methods,but there are few studies on the neutralize epitopes and receptors in the virus,especially for the study of DTMUV protein receptors.Capsule E protein as the main structural protein of DTMUV,can stimulate the body to produce neutralizing antibodies and combined with the receptor-mediated invasion of the virus,to reveal the virus infection mechanism and immune response is essential.In this study,we cloned and expressed DTMUV E protein,prepared a number of monoclonal antibodies,screening out a neutralizing antibody,and the epitope was located in the Domain I region of the E protein,which was the first neutralizing epitope found in the Domain I region of the flavivirus E protein,which enriched our understanding of the neutralizing epitope of the flavivirus.Through the cloning,sequence analysis and the construction of the recombinant plasmid of Sc Fv,we found that the sequence of the light chain complementarity region was significantly specific,the purified single chain antibody has a certain antiviral effect,laying the foundation for the prevention and treatment of the disease.The receptors of DTMUV were screened by immunoprecipitation and virus-receptor protein binding assay(VOPBA).However,the protein receptors that binding to DTMUV susceptible cells were not detected and hemoglobin hemagglutination test found that E protein has a certain hemagglutination activity,suggesting that the virus receptors may belong to the carbohydrate,for the prevention and control of the disease to provide new ideas.Details are as follows:1.Preparation and identification of monoclonal antibody against DTMUV EIn this study,the recombinant plasmid p ET-28a-E,which was a mature E protein without the signal peptide,was constructed according to the isolated E gene sequence of Gen Bank KY235382.The prokaryotic induction and purification system was used to obtain high purity E protein.The purified E protein was immunized with BALB/c mice to prepare multiple monoclonal antibodies,two hybridoma cell lines stably secreting antibodies were obtained by indirect ELISA,indirect immunofluorescence and western blot,and named as 3B8 G and 3F12 G.The titers of ascites were 1: 51200 and 1: 409600;the results of subclass identification showed that Mc Ab 3B8 G heavy chain was Ig G1,Mc Ab 3F12 G heavy chain was Ig G3,light chain was κ chain;indirect immunofluorescence and western blot results showed that both monoclonal antibodies reacted specifically with the overexpressed E protein and DTMUV.The results of virus neutralization showed that Mc Ab 3B8 G had better neutralization effect on DTMUV.2.Identification of E protein neutralizing epitopesThe spatial conformation of DTMUV protein was analyzed by bioinformatics method.The eukaryotic expression recombinant plasmid of the three domain truncated mutants of the E protein was constructed,indirect ELISA,indirect immunofluorescence and western blot were detected Mc Ab 3B8 G recognizes the Domain I region and Mc Ab 3F12 G recognizes the Domain II region of the E protein.In order to further identify the minimum function area of the neutralizing antibody 3B8 G in the Domain I region,we constructed a series of truncated mutant eukaryotic expression recombinant plasmids covering the full length of Domain I protein,a B-cell linear epitope 1FSCLGMQ7 was precisely positioned by indirect immunofluorescence and western blot.3.Preparation and identification of anti-DTMUV E protein single chain antibodyIn this study,on the basis of successful establishment of stable monoclonal antibody against DTMUV E protein,the VL region of two monoclonal antibodies was amplified,the results showed that the similarity of the two antibodies in the VL region was low,which determined the specificity of the antibody to recognize the E protein domain.Through the genetic engineering method,splicing anti-DTMUV E protein single chain antibody gene,nd constructed recombinant expression vector p ET 28a-DTMUV-E Sc Fv,expression of purified single chain antibodies has good antiviral activity.4.Screening of DTMUV E protein receptorsTo study the DTMUV receptor protein molecules on susceptible cells,purified E protein and secreted Domain III protein and DTMUV susceptibility cells for protein and cell binding indirect immunofluorescence experiments,found that E protein can binding DTMUV susceptible DEF cells,DF-1 cells and Vero cells.The membrane protein was extracted by the membrane protein extraction kit,membrane protein,purified E protein and anti-DTMUV monoclonal antibody were subjected to immunoprecipitation and virus-protein binding screening,none of the specific separable DTMUV protein receptors were screened.Hemagglutination test suggests that DTMUV receptors may be carbohydrate,which provides a thought for further study of viral receptors.In summary,this study identified the DTMUV neutralizing epitope located within the Domain I of the E protein;analyzed the differences of light chain variable region sequences of monoclonal antibodies against different antigen regions,and constructed a single chain antibody fusion expression vector with anti-DTMUV E protein;screened the protein receptor molecules of DTMUV.This study is of great significance to the prevention and treatment of tanbusu virus.