Journal List > J Bacteriol Virol > v.39(1) > 1033957

Kim, Yun, Song, Choi, Park, and Lee: Expression and Antibody Production of Japanese Encephalitis Virus RNA Polymerase (NS5) Protein

Abstract

Japanese encephalitis virus (JEV), a member of mosquito-borne flaviviruses, is the leading cause of viral encephalitis in a large geographic area of Southeast Asia and Australia. JEV contains a single-stranded positive-sense RNA genome, which encodes its own RNA-dependent RNA polymerase (NS5) that is required for genomic RNA replication. In this study, we have described a pair of mouse antisera specific to the N- or C-terminal region of the NS5. Initially, two hydrophilic regions corresponding to the N-terminus and C-terminus of the NS5 protein were individually amplified by reverse transcription-PCR from the genomic RNA of JEV K87P39 strain. The amplified DNA fragments were cloned into a prokaryotic expression vector, pGEX-4T-1; the resulting constructs were used for the expression of GST fusion proteins, designated GST/NS5N and GST/NS5C, in E. coli BL-21 strain. Following immunization of three BALB/c mice with each of the purified GST/NS5N and GST/NS5C, we obtained two pools of the antisera, specifically recognizing the ~103-kDa NS5 and several smaller NS5-related proteins in BHK-21 and Vero cells infected with JEV K87P39 strain. Overall, we have successfully expressed the N- and C-terminal regions of JEV NS5 fused to the C-terminus of GST and generated the mouse antisera capable of recognizing the NS5 and its related proteins in JEV-infected cells. This would provide a valuable reagent for the study of JEV NS5 in the viral life cycle.

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Figure 1.
PCR amplicons of JevNS5N and JevNS5C (A) and schematic presentation of pGEX/JevNS5N and pGEX/JevNS5C constructs (B). The JEV NS5N and NS5C regions were PCR-amplified with an appropriate pair of primers as indicated in the text. The amplicons were separated by 1% agarose gel electrophoresis and stained with ethidium bromide (A). pGEX/JevNS5N and pGEX/JevNS5C constructs were designed to express the N-terminal region (275 amino acids) and C-terminal region (461 amino acids) of JEV NS5 protein, respectively (B).
jbv-39-53f1.tif
Figure 2.
Expression of GST/JevNS5N and GST/JevNS5C proteins (coommasie staining). The E. coli. BL-21 cells were transformed with the parental pGEX-4T-1 or one of two recombinant plasmids designated pGEX/JevNS5N and pGEX/JevNS5C. The expression of the recombinant proteins (GST/JevNS5N and GST/JevNS5C) was analyzed by 12% SDS-PAGE and coommasie staining. The molecular weight markers in kDa were indicated on the left and the sizes of GST, GST/JevNS5N, and GST/JevNS5C were shown on the right.
jbv-39-53f2.tif
Figure 3.
Identification of GST/JevNS5N and GST/JevNS5C proteins (immunoblotting). The parental pGEX-4T-1 or one of two recombinants (pGEX/JevNS5N and pGEX/JevNS5C) was transformed into E. coli BL-21 cells. Each culture was incubated in the presence of IPTG to induce the GST fusion proteins designated GST/JevNS5N and GST/JevNS5C. Following purification of the recombinant proteins with a glutathione sepharose column, the GST fusion proteins were separated by 12% SDS-PAGE and immuno-staining with a GST-specific mouse monoclonal antibody.
jbv-39-53f3.tif
Figure 4.
Immunoblotting with two mouse antisera raised against GST/JevNS5N and GST/JevNS5C. BHK-21 or Vero cells were mock-infected or infected with JEV K87P39 strain at an MOI of 1. At 20 h post-infection, cells were directly lysed with 1 × sample loading buffer and the cell lysates were separated by 12% SDS-PAGE. The NS5 and NS5-related proteins (asterisks) were recognized by two mouse antisera (α-NS5N and α-NS5C) raised against GST/JevNS5N and GST/JevNS5C, respectively. Molecular weight markers (in kDa) were shown on the left and the sizes of NS5 and its related proteins were presented on the right.
jbv-39-53f4.tif
Table 1.
Primers used for PCR amplification in this study
Primer Sequencea Polarity Size (mer)
B-ns5-F 5′-GATGGATCCGGAAGGCCTGGGGGCAGG-3′ Sense 27
B-Nns5-R 5′-ATCCTCGAGCTAGTGGTATGTCCAAGTGCG-3′ Antisense 30
B-Cns5-F 5′-GATGGATCCGAGTGTCACACATGTATC-3′ Sense 27
B-ns5-R 5′-ATCCTCGAGTTACTAGATGACCCTGTC-3′ Antisense 27

JEV-specific sequences are indicated in boldface types.

Restriction recognition sites used for cloning are underlined

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