Expression and antibody preparation of nonstructural protein 1 for Japanese encephalitis virus from pigs

Key words: 
• zoology /
• Japanese encephalitis virus /
• nonstructural protein 1 /
• prokaryotic expression /
• purification /
• antibody

Abstract: Japanese encephalitis virus （JEV） in breeding pigs，has caused reproductive disorders，such as orchitis，stillbirths，and mummified fetuses，and has produced encephalitis in piglets. The NS1 （nonstructural protein 1） gene is associated with viral RNA packaging and replication and with viral anti-host immunity.NS1 protein were expressed by prokaryotic expression system and polyclonal antibodies of NS1 were prepared. In this study，the cDNA of JEV was synthesized from a viral genome by reverse transcription-polymerase chain reaction （RT-PCR）. The NS1 gene was cloned from cDNA by PCR and subcloned into pET-28（a） plasmid. The recombinant plasmid pET-28（a）-JEV-NS1 was then transformed into Escherichia coli BL21（DE3）. Next，the recombinant JEV-NS1 protein （whose molecular weight is 46 kDa） was expressed by isopropyl-beta-D-thiogalactopyranoside （IPTG） induction. To improve the expression level of the recombinant JEV-NS1 protein，the 958-1 245 bp of the JEV-NS1 gene was truncated，and sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） was used for the analysis. Also，the protein was immunized into an Institute of Cancer Research（ICR） mouse；then the mouse anti-JEV-NS1 antiserum was prepared；the antiserum specificity were detected with western-blotting. Results showed that the truncated JEV-NS1 expression was greatly increased and the SDS-PAGE analysis confirmed this. In addition，purification production of the recombinant protein was 85% of the total protein content. The antiserum of NS1 can specifically recognized the production of JEV infected cells. This study will assist in JEV-NS1 functional research and exploration of JEV pathogenic mechanism.［Ch，6 fig. 13 ref.］