Method of producing virus-like particles of picornavirus using a small-ubiquitin-related modifier fusion protein expression system

Abstract

A method for producing picornaviral capsid protein complexes (e.g., picornavirus like particles) in E. coli using a small-ubiquitin-related fusion protein expression system and an E. coli strain used in practicing this method. Also disclosed is use of the picornaviral capsid protein complexes like thus prepared for eliciting immune responses.

What is claimed is: 
     
         1 . A method of eliciting an immune response against a foot-and-mouth disease virus in a subject, comprising administering to a subject in need thereof an effective amount of a virus-like particle of a foot-and-mouth disease virus. 
     
     
         2 . The method of  claim 1 , wherein the subject is a cloven-hoofed animal. 
     
     
         3 . The method of  claim 1 , wherein the virus-like particle of a foot-and-mouth disease virus (FMDV) is produced by:
 providing an  E. coli  strain containing a plurality of nucleotide sequences, each of which is operably linked to a promoter and contains a first fragment encoding a Smt3 protein and a second fragment encoding a capsid protein of a FMDV, the first fragment being upstream to the second fragment, wherein the second fragments in all of the nucleotide sequences, taken together, encode all of the capsid proteins for a virus-like particle of the FMDV, wherein, in at least one of the nucleotide sequences, the first and second fragments are linked via an SfoI restriction site,   culturing the  E. coli  strain to allow expression of fusion proteins, each containing Smt3 and one of the capsid proteins, and subsequent formation of capsid protein complexes, wherein each complex is composed of the fusion proteins;   collecting the cells of the  E. coli  strain for isolation of the capsid protein complexes; and   isolating the capsid protein complexes and treating the complexes with an U1p1 protease, thereby producing a virus-like particle composed of the capsid proteins,   
     
     
         4 . The method of  claim 3 , wherein the step of U1P1 protease treatment is performed in the presence of a therapeutic agent and the virus-like particle thus produced encapsulates the therapeutic agent. 
     
     
         5 . The method of  claim 3 , wherein at least one of the nucleotide sequences contains a third fragment encoding a protein tag. 
     
     
         6 . The method of  claim 5 , wherein the third fragment is upstream to the first fragment. 
     
     
         7 . The method of  claim 5 , wherein the protein tag is selected from the group consisting of hexa-His, maltose binding protein, N-utilizing substance A, thioredoxin, calmodulin-binding protein, glutathione S-transferase, and α-factor. 
     
     
         8 . The method of  claim 3 , wherein the virus-like particle is composed of VP0, VP1, and VP3 capsid proteins. 
     
     
         9 . The method of  claim 8 , wherein the step of U1P1 protease treatment is performed in the presence of a therapeutic agent and the virus-like particle thus produced encapsulates the therapeutic agent.