J Bacteriol Virol.  2009 Mar;39(1):53-60. 10.4167/jbv.2009.39.1.53.

Expression and Antibody Production of Japanese Encephalitis Virus RNA Polymerase (NS5) Protein

• Kim JM
• Yun SI
• Song BH
• Choi YJ
• Park JS
• Lee YM

• KMID: 1474180
• DOI: http://doi.org/10.4167/jbv.2009.39.1.53

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.

Keyword

Japanese encephalitis virus; Flavivirus; RNA-dependent RNA polymerase; NS5

MeSH Terms

Animals
Antibody Formation
Asia, Southeastern
Asian Continental Ancestry Group
Australia
Clone Cells
DNA
Encephalitis Virus, Japanese
Encephalitis, Japanese
Encephalitis, Viral
Flavivirus
Genome
Humans
Immune Sera
Immunization
Mice
Proteins
RNA
RNA Replicase
Sprains and Strains
Vero Cells
DNA
Immune Sera
Proteins
RNA
RNA Replicase

Figure

• 

  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).

• 

  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.

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  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.

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  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.

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