Recombinant vaccine against japanese encephalitis virus [jev] infection and a method thereof

Abstract

The present invention relates to a method for preparing recombinant adenovirus (RadEs) vaccine to protect against JEV infection. The vaccine produces secretory envelop protein (ES) of JEV.

1 - 20 . (canceled) 
     
     
         21 . A method of preparing a recombinant adenovirus (RAdR5) vaccine (ECACC Accession Number 04121701) to protect against Japanese encephalitis virus (JEV) infection, wherein said vaccine produces secretory envelop protein (Es) of JEV, said method comprising the steps of:
 a) digesting plasmid pMEs from Japanese encephalitis virus with restriction enzymes Kpn I and Bam HI to obtain cDNA encoding JEV proteins prM and Es,   b) ligating the cDNA to adenovirus shuttle plasmid pShuttle digested with restriction enzymesKpn I and Hind III at the Kpn I end,   c) adding nucleotides at the free Bam HI and Hind III ends with T4 DNA polymerase to create blunt ends,   d) ligating the blunt ends together to yield shuttle plasmid pSEs with JEV cDNA encoding the proteins prM and Es,   e) digesting the shuttle plasmid pSEs with restriction enzymes I-Ceu I and Pl-Sce Ito obtain expression cassette containing the JEV cDNA together with the CMV promoter/enhancer and BGH polyadenylation signal,   f) ligating the digested shuttle plasmid with I-Ceu I and Pl-Sce I digested adenovirus plasmid pAdeno-X to generate plasmid pAdEs containing Es expression cassette,   g) digesting the plasmid pAdEs with Pac I,   h) transfecting the monolayers HEK 293 cells with digested plasmid pAdEs for about one week, and   i) obtaining the recombinant virus RAdEs vaccine.   
     
     
         22 . A method as claimed in  claim 21 , wherein the transfection is at about 37° C. 
     
     
         23 . A method as claimed in  claim 21 , wherein the JEV proteins are under the control of human CMV IE promoter/enhancer.