Expression And Purification Of Nonstructural Protein 2B (NS2B) Of Dengue Type 2 Virus And Preparation Of The Polyclonal Antibodies Against NS2B

Dengue virus (DV), a member of the family Flaviviridae, is transmitted by mosquitos Aedes aegypti and Aedes albopictus. There are four serotypes (types 1 to 4) that cause widespread human diseases such as dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). About 40% of the world population living in tropical and subtropical regions is at risk of infection. Of the 100 million cases of DHF cases per year, about 5% are fatal. There is currently no effective vaccine or antiviral drug to protect against dengue diseases. Therefore, DV infection has become the severe problem of the public health.DV2 is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 11 kb. The genome has only one open reading frame and encodes a polyprotein which is post- and co-translationally cleaved by both host and viral proteases to three structural proteins (capsid, C; membrane protein, M; and envelope, E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The presence of a small activating protein or cofactor is a prerequisite for optimal catalytic activity of the flaviviral proteases with natural polyprotein substrates. DV2 NS3 contains a serine protease domain and necessary for the processing of its polyprotein. As a cofactor, the NS2B is essential for the catalytic activity of NS3 protease. The presence of the NS2B cofactor was shown that enzymatic cleavage of dibasic peptides, NS2B–NS3 protease complex, is markedly enhanced. Therefore, the NS2B–NS3 complex is an attractive target for therapeutic intervention in flavivirus infections.NS2B is an endoplasmic reticulum resident integral membrane protein. On the basis of their relative hydrophobicity, the NS2B protein contains seven domains (domains I–VII). The hydrophobic core residues, belonging to domain IV (G69–E80), were proposed to interact with NS3pro. This domain is flanked by two hydrophilic stretches (domains III and V). Studies using mutant plasmids transfected into cells have shown that the fragment of 40 residues of the NS2B, encompassing domains III to V, is the minimal region necessary for inducing the protease activity of NS3pro. Moreover, the NS2B–NS3 association, demonstrated by co-immunoprecipitation experiments, is also mediated by this hydrophilic region. Currently, the molecular details of the mechanism by which the NS2B cofactor stimulates the activity of the protease have not yet known. To examine the role of NS2B cofactors, other experiments indicate that in addition to activating proteolytic activity, NS2B is necessary for promoting membrane association of the NS3 complex, and the importance of NS2B as a cofactor to NS3 protease-induced apoptosis.It has remained unanswered as to whether NS2B cofactor can be supplied in DV2 replication because of absence of specific visualization assays. To further investigate the role of NS2B protein in pathogenesis of DV2 infections and develop a new diagnostic assay with NS2B antigen detection, in this study, DV2 NS2B gene was amplified by molecular biology technique and subcloned in into prokaryotic, eukaryotic expression vectors. DV2 NS2B protein was expressed, and purified. BALB/c mouses were immunized and then polyclonal antibodies (PAbs) were generated.RESULTS1. Production and characterization of the polyclonal antibodies against DV2 NS2B protein.(1) Production of antibodies against DV2 NS2B①The NS2B gene was successfully amplified from genomic cDNA of DV2 by PCR.②The PCR product of NS2B gene was inserted into pQE31 vector and transformed into E.coli JM109. The recombinant pQE-NS2B/JM109 expressed the fusion protein, nearly all of which was in the inclusion-body.○3 The inclusion body of fusion protein was extracted from cultures of stably transformed E.coli JM109, and then dissolved in 8mol/L urea. The fusion NS2B protein was captured by Ni+-NTA column.④The purified fusion protein was renatured through dialysis.⑤Five BALB/c mouses were immunized subcutaneously with NS2B protein. After three-time immunization, the antiserum was taken. Antiserum preparations were titrated by ELISA assay. The titration of the mouse antisera was more than is 1:12800. The characteristic of the NS2B antibody was analysed by Western blot and immunofluorescence. The productions of antisera were specific to NS2B protein of DV2 and could recognize native NS2B protein in ECV304 cells infected DV2, suggesting that these antibodies might be a useful tool to study the role of DV2 NS2B in DV2 infections. (2) Characterization of DV2 specific antisera against NS2B①It was not obvious that DV2 NS2B antisera neutralize DV2 by PRNT, indicating that the antisera don't have the ability of neutralization.②To unravel the role of DV2 NS2B in the viral life cycle, we detect DV2 NS2B protein using the antisera in DV2-infected ECV304 cells. DV2 NS2B protein was detected post infection12h. Cells show increased fluorescence after 24 h and 48h by immunofluorescence. By using confocal laser scanning microscopy, we found the colocalization pattern between NS2B and NS3 in peri-nuclear and cytoplasm of infected ECV 304 post infection 12h, 24h and 48h2. Construction of eukaryotic expression plasmid NS2B vector and DNA immunization(1) Construction of eukaryotic expression plasmid NS2B vector①The NS2B gene was successfully amplified from pQE-NS2B vector by PCR. The PCR product of NS2B gene was inserted into pCAGGS-P7 vector and transformed into E.coli XL-Blue. The recombinant pCAGGS-NS2B/ XL-Blue was obtained.②The expression vector pCAGGS-NS2B containing DV2 NS2B cDNA was transfected into Vero cell line by lipid-mediated gene transfection method. DV2 NS2B protein was detected.(2) DNA immunizationFive BALB/c mouses were immunized pCAGGS-NS2B. After three-time immunization, the antiserum was taken. Antiserum preparations were titrated by ELISA assay. The titration of the mouse antisera was more than is 1:2000. The characteristic of the NS2B antibody was analysed by Western blot and immunofluorescence. The productions of antisera were specific to NS2B protein of DV2 and could recognize native NS2B protein in ECV304 cells infected DV2.In this study, we constructed prokaryotic and eukaryotic expression plasmid NS2B vector. DV2 NS2B protein was expressed, and purified under denaturing condition. The polyclonal antibodies (PAbs) were generated by DV2 NS2B protein or DNA immunization. Our results showed that the production of PAbs were specific to NS2B protein of DV2 and could recognize native NS2B protein, suggesting that these antibodies might be a useful tool to study the role of DV2 NS2B in DV2 infections.