Generation Of Monoclonal Antibodies And Epitope Mapping For ORF2-V1 Recombinant Proteins Of Swine Hepatitis E Virus

Hepatitis E is a self-limited acute illness which caused by Hepatitis E virus (HEV). The illness may be particularly severe among pregnant women, with mortality rates reaching as high as 20%. Human healthy will be severe harmed. The antibody against HEV is widely detected in the body of primates, rodents, domestic animals and poultry, so it is considered as a possible diseases contracted commonly by both human beings and livestockHepatitis E virus (HEV) was previously considered an enterically transmitted non-A, non-B hepatitis virus. Using an immunoelectronmicroscopic technique. Balagan et al observed a type of particles 27 to 30 min diameter in the stool sample of a volunteer subject. They further proved this virus was a new non-A, non-B type that represents a serious threat to human health. Reyes et al cloned the genome of this virus. The virus and the hepatitis caused by the virus were named as HEV and hepatitis E, respectively, at an international conference held in Tokyo Japan in 1989. The HEV genome is a single-stranded, positive-sense RNA molecule of approximately 7.5 kb. The viral genome contains three open reading frames (ORFs) flanked by two non-coding regions (NCRs) at the 5′- and 3′-ends. The order of the sequence is 5′-NCR, ORF1, ORF2, ORF3, 3′-NCR and a polyadenylated tail.HEV is found all around the world and causes great damage. Although methods of HEV diagnosis were always improved, the primary measure of control and prevention is to develop special vaccine. The basis of HEV diagnosis reagent and vaccine is making clear the HEV antigenic epitopes. The comprehensive studies have been conducted to further elaborate the antigenic composition of the ORF2-encoded protein with recombinant proteins and synthetic peptides, confirming the existence of a strong antigenic region located at the C terminus. The ORF2 protein existed many antigenic epitopes, complex structure, and was riched in B-cell antigenic epitopes in approximately two thirth c-terminal region of the ORF2 protein.In this research, the immunogenic antigen was ORF2-V1pritein ,which was approximately 220aa c-terminal region of the ORF2 protein. Six hybridoma cell lines designated steadily secreting monoclonal antibody (McAb) against ORF2-V1 protein of swine Hepatitis E virus were obtained by hybridomatechnology. Antigenic epitope information of the virus is not only useful for investigating the relationship between antigenic structure and function of the HEV but also useful for diagnosis of HEV infection and developing safe and effective multi-epitope vaccine against the disease.In this study, recombinant plasmids harboring structural protein of HEV swDQ strain designated as pET32a-V1, were constructed and expressed in E.coli. The expressed fusion proteins were detected with sera of HEV infected pigs by Western-blotting. BALB/c mice were immunized with expressed fusion proteins and monoclonal antibodies (MAbs) were developed. 6 anti-ORF2-V1 MAbs were identified. This study identified the antigenic epitopes of 6 McAbs to HEV ORF2-V1(αC11,αC12,γH1,γF8,BC4,CH8) with pep scan.The ORF2-V1 gene were divided into two overlapping fragments and expressed in E. coli respectively. The reactivity of different fragments expressed in E. coli were probed with 6 anti-ORF2-V1 MAbs by Western-blot. Three out of 6 MAbs could react with ORF2-M1 and ORF2-M2 fusion Proteins. The results showed that both fragments could react with MAbsαC11,αC12andγF8 , suggesting that the epitope recognized by MAbsαC11,αC12andγF8 located in the overlaping region (ORF2 492-500aa) of the two fragments. The ORF2-M1 protein(386-492aa)and ORF2-M2 protein(500-606aa)was dissected into 25 overlapping fragments, expressed as fusion products in E. coli, and used for epitope mapping by pepscan analysis. Three antigenic epitopes were identified by ELISA and Western blot assays. MAb CH8 could react with E4 and E5 short synthetic peptides. The results showed that the epitope recognized by MAb CH8 located in the overlapingregion(ORF2 418~425aa)of the two fragments. MAbγH1 could only react with E1 short synthetic peptides,and could not react with other short synthetic peptides. The results showed that the epitope recognized by MAbγH1 located in the HEV ORF2-encoded protein(ORF2 386~394aa). MAb BC4 could react with E24 and E25 short synthetic peptides. The results showed that the epitope recognized by MAb BC4located in the overlapingregion(ORF2 588~595aa)of the two fragments. The epitope 418~425aa and the epitope492-500aa were identified first.Immunization of mice with each of the four antigenic epitope-fused proteins revealed that all four proteins could elicit short peptide specific antisera.The identified epitopes were synthesized as polypeptides. SDS-PAGE analysis showed that the four antigenic epitope-fused proteins was expressed in E.coli B121(DE3)and identified positive antisera of the swine by the Western-blot.Identification of HEV envelope protein antigenic epitopes may provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic clinical techniques for HEV, and further structural and function analysis of envelope protein.