Análisis in silico de un candidato a vacuna multi-epítopo contra viruela del mono usando vaculonogía reversa / In silico analysis of a multi-epitope vaccine candidate against monkey pox using reverse vaccinology

Introducción. La viruela del mono es una infección zoonótica con una tasa de transmisión global aumentada durante 2022. Actualmente, la enfermedad no tiene tratamientos específicos disponibles; por lo tanto, se puede lograr un enfoque preventivo a través de la inmunización. Objetivo. Diseño in sílico de una vacuna aplicando técnicas computacionales avanzadas utilizando una construcción de múltiples epítopos del M. virus. Materiales y métodos. Los antígenos se seleccionaron en base a informes sobre proteínas que provocan la activación de linfocitos T y B citotóxicos. Los ensayos inmunoinformáticos fueron antigenicidad, alergenicidad, toxicidad, afinidad de unión al complejo mayor de histocompatibilidad (CMH) y estimulación de IFN-γ. Resultados y discusión. Ocho epítopos de las proteínas M1R, ADN polimerasa, B6R y A35R de M. virus mostraron una respuesta significativa para las células inmunitarias. Se eligieron once epítopos con antigenicidad >0,3, no alergénicos y no tóxicos, de los cuales 4 presentaron alta afinidad por los linfocitos T, 4 generaron alta activación de linfocitos B y 3 se asociaron con resultados de activación de IFN-γ. La construcción in sílico del candidato vacunal de 509 aminoácidos con alta similitud topológica registró principalmente carga negativa, además de ser soluble con índice alifático >80%, estable y particular con activación CMH y alta afinidad molecular con TLR-3, y además presentó multiantigenicidad, similar a las vacunas generadas por esta metodología con M. tuberculosis e Influenza. La simulación de inyección de una dosis de la construcción molecular mostró la activación de las células plasmáticas auxiliares T durante aproximadamente 15 a 25 días y una alta expresión de IFN-γ e IL-2 durante ocho días. Conclusión. Estos resultados indican un excelente proceso de inmunización que podría potenciarse con dosis múltiples.

Introduction. Monkey pox is a zoonotic infection with an increased global transmission rate during 2022, denoted epidemiological trouble in public health. Currently, the disease has no specific treatments available; thus, a preventive approach can be achieved through immunization. Objective. was to design in silico a vaccine applying advanced computational techniques using a multi-epitope construct of the Monkeypox virus. Materials and methods. Antigens were selected based on reports about proteins that cause the activation of cytotoxic T and B lymphocytes. The immunoinformatics assays were antigenicity, allergenicity, toxicity, MHC binding affinity, and IFN-γ stimulation. Results and discussion. Eight epitopes of the M1R, DNA polymerase, B6R, and A35R proteins of the M. virus showed a significant response for immune cells. Eleven epitopes with antigenicity >0.3, non-allergenic and non-toxic were chosen, of which 4 presented high affinity to T lymphocytes, 4 generated high activation of B lymphocytes, and 3 were associated with IFN-γ activation results. The in silico construction of the 509-amino acid vaccine candidate with high topological similarity registered mainly a negative charge, in addition to being soluble with an aliphatic index >80%, stable and particular with MHC activation and high molecular affinity with TLR-3, and also presented multi-antigenicity, similar to vaccines generated by this methodology with M. tuberculosis and Influenza. One-dose injection simulation of the molecular construct showed activation of T helper plasma cells for about 15 to 25 days and high expression of IFN-γ and IL-2 for eight days. Conclusion. These results indicate an excellent immunization process that could be potentiated with multi-dosing.

Preparation and characterization of a recombinant poly-epitopic vaccine EgG1Y162-2 (4) against cystic echinococcosis based on the linker GSGGSG / 中国血吸虫病防治杂志

Objective To perform prokaryotic expression and preliminary characterization of the recombinant poly-epitope vaccine EgG1Y162-2 (4) against cystic echinococcosis. Methods The recombinant poly-epitope vaccine EgG1Y162-2 (4) against Echinococcus granulosus based on the linker GSGGSG was subjected to structural three-dimensional (3D) modeling using immunoinformatics to analyze the structural changes and evaluate the antigenicity of the vaccine. The pET30a-EgG1Y162-2 (4) recombinant plasmid was generated using double digestion with EcoR I and Sal I, and then transformed into competent cells. Following protein induction with isopropyl-β-D-thiogalactoside (IPTG), the prokaryotic expression proteins were characterized using Western blotting, and the antigenicity of the recombinant protein was analyzed using sera from cystic echinococcosis patients and health volunteers. Results The four EgG1Y162-2 proteins coupled by the 3D structure of the recombinant vaccine EgG1Y162-2 (4) presented independent and effective expression and good antigenicity. The highest protein expression was detected in the supernatant following induction of the recombinant plasmid pET30a-EgG1Y162-2 (4) by 0.2 mmol/L IPTG at 37 °C for 4 h, and a pure protein component was seen following elution with 60 mmol/L imidazole. Western blotting analysis of the recombinant multiepitope protein HIS-EgG1Y162-2 (4) showed a band at approximately 39 kDa, and this band was recognized by sera from cystic echinococcosis patients. Conclusion A recombinant poly-epitope vaccine EgG1Y162-2 (4) against cystic echinococcosis has been successfully constructed, which provides a preliminary basis for researches on recombinant multi-epitope vaccine against cystic echinococcosis.

Immunogenicity of chimeric peptide gene of Xinjiang hemorrhagic fever virus glycoprotein / 国际生物医学工程杂志

Objective:To investigate the effects of different chimerism strategies and different immune ways on the two antigen-dominant regions of Xinjiang hemorrhagic fever virus (XHFV) glycoprotein.Methods:The 5' end was added or not added with interleukin-2 (IL-2) signal peptide and the general-purpose auxiliary T cell epitopes as different design strategies. GcⅠ and GcⅡ and the epitopes previously identified on GcⅠ (Gc 233-248, Gc 241-256 and Gc 281-296) were fused and constructed into the eukaryotic expression vector pVAX1 and the prokaryotic expression vector pET-28a. The recombinant prokaryotic plasmid transformed into E.coli BL21 was induced and purified, and the recombinant eukaryotes were extracted by indirect immunofluorescent assay. BALB/c mice were immunized by protein immunity, gene immunity, and DNA prime-protein boost immunity. The IgG antibody level was measured by ELISA. The immune effect was evaluated by the proliferation of T-lymphocytes and the content of cytokines in the spleen. Results:The results of double enzyme digestion and sequencing showed that eight recombinant plasmids were successfully constructed, and the recombinant eukaryotes were successfully expressed in vitro by fluorescence microscopy. After three times of immunization, the IgG level and the proliferation of T-lymphocytes in the spleen of mice in the experimental group were significantly higher than those in the control group ( P<0.01). The mass concentration test results of Th2 cytokines IL-4 and Th1 cytokines interferon-gamma (IFN-γ) revealed that the response of the DNA prime-protein boost immunity was biased to Th1. Conclusions:The multi-epitope chimeric vaccine of XHFV glycoprotein was successfully constructed, and the target antigen could be expressed effectively in vivo. The immune groups stimulated stronger humoral and cellular immune responses compared with the control group. Among them, the immune effect of pVAX1-ST(GcⅠe+GcⅡ) combined with recombinant protein r(GcⅠe+GcⅡ) was the best, and it is expected to be a new candidate vaccine for XHFV.

Multi-Epitope Peptide-Based and VacciniaBased Universal Influenza Vaccine Candidates Subjected to Clinical Trials

@#In light of the limited protection conferred by current influenza vaccines, immunisation using universal influenza vaccines has been proposed for protection against all or most influenza sub-types. The fundamental principle of universal influenza vaccines is based on conserved antigens found in most influenza strains, such as matrix 2, nucleocapsid, matrix 1 and stem of hemagglutinin proteins. These antigens trigger cross-protective immunity against different influenza strains. Many researchers have attempted to produce the conserved epitopes of these antigens in the form of peptides in the hope of generating universal influenza vaccine candidates that can broadly induce cross-reactive protection against influenza viral infections. However, peptide vaccines are poorly immunogenic when applied individually owing to their small molecular sizes. Hence, strategies, such as combining peptides as multi-epitope vaccines or presenting peptides on vaccinia virus particles, are employed. This review discusses the clinical and laboratory findings of several multi-epitope peptide vaccine candidates and vaccinia-based peptide vaccines. The majority of these vaccine candidates have reached the clinical trial phase. The findings in this study will indeed shed light on the applicability of universal influenza vaccines to prevent seasonal and pandemic influenza outbreaks in the near future.

Preparation of multi-epitope recombinant diagnostic antigen of Mycobacterium tuberculosis / 生物工程学报

Multi-epitope recombinant diagnostic antigen (designated 'B102') of Mycobacterium tuberculosis (Mtb) was prepared and evaluated as a serological diagnostic antigen. With TRX at the N-terminal and His tag at the C-terminal, the multi-epitope Mtb recombinant diagnostic antigen including 11 predicted B-cell epitopes from 6 Mtb antigens (PstS1, ESAT6, CFP10, Ag85B, Ag85A and PPE54) was expressed in Escherichia coli BL21 (DE3) and purified by Ni²⁺-Chelating affinity and DEAE anion exchange chromatography. Based on the antigenicity of B102 confirmed in Western blotting analysis, we constructed and evaluated a double-antigen sandwich ELISA for diagnosis of Mtb infection. The protein B102 exists in the form of inclusion bodies, accounting for 31.25% of the total proteins of the bacteria. After purification and renaturation, protein B102 exists in soluble form with the concentration 3.124 mg/mL and the homogeneity 96.71%. WB analysis demonstrated that protein B102 could react with antibodies in Mtb positive serum. Using the novel antigen in ELISA, we tested 60 Mtb-related positive and negative serum; The results showed the sensitivity, specificity, positive and negative predictive values and coincidence rate of the detection method is 90.00%, 93.33%, 93.10%, 90.32% and 91.67%, respectively. The McNemer analysis suggested there was no statistical difference between the 'Gold standard' and the novel ELISA with kappa 0.833, which suggested the excellent consistency. By prokaryotic expression and chromatography purification, the multi-epitope recombinant antigen B102 was obtained with excellent antigenicity, which could be applied for Mtb-related serological detection.

Multi-Epitope Fusion Protein Eg mefAg-1 as a Serodiagnostic Candidate for Cystic Echinococcosis in Sheep

Cystic echinococcosis (CE) in sheep is a hazardous zoonotic parasitic disease that is caused by Echinococcus granulosus (Eg). At present, serological test is an important diagnostic method for Eg infection in domestic animals. Here, a fusion protein Eg mefAg-1 harboring 8 dominant B-cell epitopes of Eg such as antigen B, tetraspanin 1, tetraspanin 6, reticulon and Eg95 was produced in E. coli and evaluated for CE in sheep by indirect ELISA. Eg mefAg-1 showed in ELISA a high sensitivity (93.41%) and specificity (99.31%), with a coincidence rate of 97.02%. Overall, it is suggested that the Eg mefAg-1 could be a potential antigen candidate for CE serodiagnosis in sheep.

Evaluation of Double Antigen Sandwich Time-resolved Fluoroimmunoassay for Specific Total Antibodies to Treponema Pallidum / 现代检验医学杂志

Objective To evaluation the performance of double antigen sandwich time-resolved fluoroimmunoassay(TRFIA)for specific total antibody to Treponema pallidum (TP).Methods Specific total antibody to TP was detected by a double antigen TRFIA based on recombinant multi epitope chimeric antigen.The methodological precision,low limit of detection,accuracy,linearity,reference standard coincidence rate and other analytical performance indicators were evaluated,and clinical comparison research trials were completed.The x2 test was used for the difference between two methods results,the P ＜0.05 which represents the difference was statistically significant.Results The intra-assay and inter-assay coefficients of var iation (CV) were both less than 10％ respectively.The low limit of detection was 0.05 mIU/ml.The relative deviation of de tecting the national standard was not exceed 10％.The linear range was 1.50～ 155.00 mIU/ml and the linear correlation co efficient could be reached 0.999 9.The performance of detection national reference could meet the national accreditation requirements.The consistent rate was 100 ％ when the TRFIA methodology detected the standardized serum plate.The parallel test of TRFIA and treponema pallidum gelatin agglutination test (TPPA) were completed,the total coincidence rate was 99.56％,and the Kappa index was 0.990 6.Conclusion Their result showed that the TRFIA methodology is high sensitivity,accuracy,wide linear range,and highly clinical coincidence rate,which is valuable for clinical application.

Evaluation of the immune response to human metapneumovirus multi-epitope antigen in an mouse model / 中华微生物学和免疫学杂志

Objective To evaluate the immune response triggered by an in-house constructed hu-man metapneumovirus multi-epitope antigen ( MEA) in a mouse model .Methods Female SPF BALB/c mice at age 4-6 weeks were used in the study and divided into 7 groups.Mice in the five groups including MEA+oligodeoxynucleotides containing CpG motifs ( CpG ODN) intraperitoneal injection ( i.p.) treatment group, MEA+Alum i.p.treatment group, MEA+Alum+CpG ODN i.p.treatment group, MEA+CpG ODN intranasal (i.n.) treatment group and MEA+Alum+CpG ODN i.n.treatment group were immunized three times on days 0, 14 and 21, and those in the other experimental group were immunized intramuscularly with MEA+Quickantibody5W on days 0 and 21.A control group without treatment was set up accordingly .All mice were sacrificed two weeks after the last immunization .Antibodies including IgG , IgG1, IgG2a and IgA in serum samples were detected by ELISA .MTS assay was performed to analyze the proliferation of lympho-cytes.The cytotoxicity of cytotoxic T lymphocytes (CTL) was measured by LDH assay.Flow cytometry was used to detect T lymphocyte subsets .The cytokines secreted by T helper cells ( Th1 and Th2) were analyzed with Bio-Rad Liquid Chips.Results High titers of IgG, IgG1 and IgG2a antibodies were produced in MEA treated mice except for those in intranasal treatment groups .Serum samples from three groups including the MEA+Alum i.p., MEA+Alum+CpG ODN i.p.and MEA+Quickantibody5W i.m.treatment groups were positive for IgA antibody .The highest titer of IgA antibody was detected in mice from the MEA+Alum+CpG ODN i.p.treatment group, which was 2.15×103.Compared with the control group, significantly enhanced proliferation of lymphocytes was observed in the MEA+Alum i.p., MEA+Alum+CpG ODN i.p.and MEA+Quickantibody5W i.m.treatment groups (P<0.05).Enhanced cytotoxic activities of CTL were observed in mice with ip.and i.m.treatments as compared with those in control group (P<0.05).The levels of CD4+/CD8+T cells were slightly increased in mice from the MEA+CpG ODN i.p., MEA+Alum+CpG ODN i.p. and MEA+Quickantibody5W i.m.treatment groups as compared with those in control group (P<0.05).In-creased secretion of IL-2, IFN-γand Th2-type cytokines including IL-4, IL-5 and IL-10 were detected in mice from the MEA+CpG ODN i.p.treatment group.The MEA+Alum i.p.treated mice showed a slightly increased secretion of IFN-γand significantly increased secretions of IL-4, IL-5 and IL-10.Significantly in-creased secretions of IFN-γ, IL-4, IL-5 and IL-10 were detected in mice from the MEA+Alum+CpG ODN i.p.treatment group.Significantly increased secretions of IFN-γ, IL-5, IL-10 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were detected in mice from the MEA+Quickantibody5W i.m.treatment group.Conclusion MEA together with different adjuvants could stimulate high titers of specific antibodies , increase the proliferation of lymphocytes and enhance the cytotoxic activities of CTL .CpG ODN could bal-ance the Th1/Th2-mediated immune responses , and the balance could be enhanced when using CpG ODN in combination with Alum .A similar effect could be achieved by using the commercial adjuvant Quickanti -body5w.This study has paved the way for further investigation on the development of hMPV epitope vaccines and diagnostic reagents for hMPV as well as the epidemiological study of hMPV .

Induction of protective T-helper 1 immune responses against Echinococcus granulosus in mice by a multi-T-cell epitope antigen based on five proteins

In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3) were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST). The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund's adjuvant. Mouse splenocytes were then cultured in Dulbecco's Modified Eagle's Medium in the presence of the antigen. The production of interferon-γ was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL), but interleukin (IL)-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6%) was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj) ]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model.

In silico experiment with an-antigen-toll like receptor-5 agonist fusion construct for immunogenic application to Helicobacter pylori.

BACKGROUNDS: Helicobacter pylori colonize the gastric mucosa of half of the world's population. Although it is classified as a definitive type I carcinogen by World Health Organization, there is no effective vaccine against this bacterium. H. pylori evade the host immune response by avoiding toll-like detection, such as detection via toll-like receptor-5 (TLR-5). Thus, a chimeric construct consisting of selected epitopes from virulence factors that is incorporated into a TLR-5 ligand (Pseudomonas flagellin) could result in more potent innate and adaptive immune responses. MATERIALS AND METHODS: Based on the histocompatibility antigens of BALB/c mice, in silico techniques were used to select several fragments from H. pylori virulence factors with a high density of B- and T-cell epitopes. RESULTS: These segments consist of cytotoxin-associated geneA (residue 162-283), neutrophil activating protein (residue 30-135) and outer inflammatory protein A (residue 155-268). The secondary and tertiary structure of the chimeric constructs and other bioinformatics analyses such as stability, solubility, and antigenicity were performed. The chimeric construct containing antigenic segments of H. pylori proteins was fused with the D3 domain of Pseudomonas flagellin. This recombinant chimeric gene was optimized for expression in Escherichia coli. The in silico results showed that the conserved C- and N-terminal domains of flagellin and the antigenicity of selected fragments were retained. DISCUSSION: In silico analysis showed that Pseudomonas flagellin is a suitable platform for incorporation of an antigenic construct from H. pylori. This strategy may be an effective tool for the control of H. pylori and other persistent infections.

Immunogenicity and protective effect induced by co-immunization with HCV recombinant protein antigens / 中华微生物学和免疫学杂志

Objective To investigate the cellular and humoral immune responses and protective effect induced by co-immunization with two multi-epitope combinant antigens. Methods Mice were co-im-munized with the muhi-epitope HCV-T and HCV-E1 antigens three times. Sera antibodies IgG, IgG1 and IgG2a were tested by ELISA. Spleens from BALB/c mice immunized were removed 10 days after the last im-munization. CTL activity was assessed using LDH cytotoxicity assay kit. IFN-γ- and IL-4-secreting cells were quantified using ELISPOT kit. Two weeks after the final immunization, the mice were challenged sub-cutaneously(s, c. ) at the back with 106 SP2/0-NS3 cells, and protective effect was observed. For therapy, 106 SP2/0-NS3 cells were implanted into the back of BALB/c mice. Seven days later, mice were immuniza-tion three times. Therapy effect was observed. Results Co-immunization with HCV-T and HCV-E1 induced high tiers of HCV-El-specific IgG, IgG1 and IgG2a antibodies, and high level of CTL activity. Synergistic effect in frequencies of both specific IFN-γ-secreting cells and IL-4-secreting cells was observed in mice co-immunized. Prophylactic as well as therapeutic administration of mT + mE1 in mice led to protecting mice against SP2/0-NS3 cells. These results suggested that mT + mE1 was potential as a prophylactic as well as therapeutic HCV vaccine. Conclusion Co-immunization with HCV-T + HCV-EI induced protective humor-al and cellular immune response. HCV-T + HCV-E1 was potential as a recombinant HCV vaccine.

Study on a Novel Hepatitis B Virus Multi-epitope DNA Vaccine with siRNA,hIL-12 Coexpressing / 中国生物工程杂志

Objective:To construct a DNA vaccine co-expressing the HBV compound multi-epitope antigen gene, the hIL-12 and the anti-HBV siRNA genes, and to express this DNA vaccine in HepG2 cells. Methods:The HBV multi-epitope antigen gene was designed and synthesized before it was fused with enhanced green fluorescent protein(EGFP) gene, and cloned into the multi-clone site(MCS) of the eukaryotic expression vector pVAX1. The expressinig units of hIL-12 and siRNA were cloned into the BspH I and Mlu I site of pVAX1 respectively. Then the recombinant plasmid pVAX1-siHBV-HB-EGFP-hIL12 was transiently transfected HepG2 cells. The expression of HBV compound multi-epitope gene was observed through EGFP report gene. The expression of hIL-12 was analyzed by ELISA and the effects of anti-HBV siRNA was confirmed with rtPCR . Results: The analysis of enzyme digestion and sequencing both demonstrated that the trible-expressing HBV DNA vaccine has been constructed successfully. The green fluorescent image was detected in the transfected cells which could confirm the expression of the multi-epitope antigen gene. The amount of hIL-12 secretion was 1289pg/ml in supernatant at 48h after transfection and 1712pg/ml at 72h after transfection. The mRNA amount of HBV S gene, which was the siRNA target, had been obviously knockdown. Conclusion: The DNA vaccine co-expressing the HBV compound multi-epitope antigen gene, the hIL-12 and the siRNA genes was constructed and transiently expressed in HepG2 cells, and siRNA had shown us a good anti-HBV effect. It laid a foundation of further study on anti-HBV effect of the new DNA vaccine.

Expression and Purification of a Novel Influenza Virus Subunit Vaccine / 中国生物工程杂志

The influenza A virus matrix protein2 gene(M2)which deleted transmembrane region was amplified by overlap extending PCR,and the multiepitope gene of hemagglutinin(HA)was PCR amplified with seven continuous synthesized segments by designing primer.The two gene segments were separately cloned into pMD18T vector to sequence analysis and prokarytic expression vector pET28a+ to construct the recombinant plasmid pET28a+M2dHA.The recombinant plasmid was transformed into E.coli BL21(DE3),and the high expression strain was obtained by screening monoclones.The recombinant protein existed as inclusion bodies,which accounted about 45% of the total cellular protein.The inclusion bodies were washed with 1% Triton X100 solution twice,and dissolved in 8 mol/L urea solution.The solution protein was purified by Ni+2 affinity chromatography,and refolded by dilution renaturation,then purified by Q Sepharose FF cation exchange column.The purity of the protein was over 90% by HPLC analysis.The result of Western blot showed it has good antigenicity and specificity.These results strongly supported for the further study of the broadspectrum influenza virus subunit vaccine.

Construction and Expression of Multi-epitopes Gene of Treponema pallidum and Immunoresponse of Recombinant Protein / 中华医院感染学杂志

OBJECTIVE To develop the strong and the specific multi-epitope antigen for the exploiting diagnosis of Treponema pallidum.METHODS The immuno-dominant epitopes of Tp0453 and Tp17 were amplified by PCR respectively,and subcloned into the expression vector pQE32 to generate multi-epitopes recombinant plasmid pQE32/Tp0453-17.The recombinant protein was expressed in Escherichia coli M15.The immunoresponse of recombinant fusion protein was analyzed by Western blot.RESULTS The multi-epitopes recombinant plasmid was successfully constructed,enzyme digestion analysis and sequencing showed that the inserted target genes were Tp0453 and Tp17 gene,compared with the gene reported by GenBank,it had 100% similarity;SDS-PAGE analysis showed the recombinant plasmid could be expressed in M15,its relatively molecular mass(Mr) of expressed product was about 52.0?103.The Western blot result showed the recombinant protein could be recognized by anti-T.pallidum positive serum.CONCLUSIONS The expressed multi-epitopes recombinant antigen showed excellent immunoresponse.The results lay the foundation for research on development of quick diagnostic kit applying to detection of T.pallidum infection.

Design and Construction of G2 Glycoprotein Multi-epitope Antigen Gene of Hantavirus / 环境与健康杂志

Objective To construct the multi-epitope antigen(mea) gene of G2 glycoprotein of Hantavirus SEO type L99 strain.Methods The B cell epitopes was chosen and connected by three-peptide GPG after the amino acid sequence of G2 protein was analyzed and predicted by bioinformatical soft wares.The corresponding gene mea was constructed by overlap PCR,and cloned into the prokaryotic expression plasmid pET32a(+).Results The mea gene was successfully constructed by the five epitopes being chosen.The recombinant plasmid pET32a-mea was acquired by directional cloning.Conclusion The mea and its expression system E.coli BL21/pET32a-mea were constructed for the first time.The foundation was laid for the expression of the mea and its immunological applications.