Malaria plasmodium antigen polypeptide se36, method of purifyng the same and vaccine and diagnostic with the use of the thus obtained antigen

Abstract

The present invention provides a polypeptide SE36 derived from the N-terminal domain (47 kd) of SERA (serine-repeat antigen) produced by malaria parasite, Plasonodium falciparum , at the erythrocyte stage, a process for purifying said polypeptide, and a malaria vaccine and diagnostic agent using as an active component said purified antigen obtained therefrom. SE36 can be produced in Escherichia coli on a large scale by deleting all or part of polymerized serines of the 47 kd serine-repeat region, whereby high purification is permitted. The human IgG3 antibodies specifically binding to SE36 prevents highly effectively growth of the protozoa in the red blood cells to inhibit fever and cerebral malaria, and further prevent the death.

1 . A polypeptide SE36 consisting of the full length of the amino acid sequence of SEQ ID NO: 4.  
     
     
         2 . The polypeptide SE36 of  claim 1 , which has at least one of the following amino acid substitution in the amino acid sequence of SEQ ID NO: 4: 
 the 19th Gly is Val;    the 128th Glu is Lys;    the 157th Gly is Ser;    the 160th Gly is Ser;    the 172nd Pro is Ser;    the 178th Glu is Val;    the 179th Ser is Asn;    the 180th Leu is Pro;    the 185th Pro is Ala;    the 186th Asp is Gly;    the 188th Pro is Thr;    the 189th Thr is Pro;    the 190th Val is Asp;    the 191st Lys is Ala;    the 192nd Pro is Lys;    the 193rd Pro is Lys;    the 194th Arg is Lys;    the 219th Ile is Leu;    the 252nd Ser is Asn;    the 273rd Ala is Ser;    the 274th Leu is Ile; and    the 327th Asn is Lys.    
     
     
         3 . The polypeptide SE36 of  claim 1  or  2 , wherein which an oligopeptide consisting of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6 is added between the 18th Gly and the 19th Gly in the amino acid sequence of SEQ ID NO: 4.  
     
     
         4 . The polypeptide SE36 of  claim 1  or  2 , wherein an oligopeptide consisting of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8 is added between the 42nd Ala and the 43rd Ser in the amino acid sequence of SEQ ID NO: 4.  
     
     
         5 . The polypeptide SE36 of  claim 1  or  2 , wherein an oligopeptide consisting of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6 is added between the 18th Gly and the 19th Gly in the amino acid sequence of SEQ ID NO: 4, and an oligopeptide consisting of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8 is added between the 42nd Ala and the 43rd Ser in the amino acid sequence of SEQ ID NO: 4.  
     
     
         6 . The polypeptide SE36 of any one of  claims 1  to  5 , wherein an oligopeptide consisting of the amino acid sequence of from the 19th Gly to the 26th Gly in the amino acid sequence of SEQ ID NO: 4 is deleted.  
     
     
         7 . The polypeptide SE36 of any one of  claims 1  to  5 , wherein an oligopeptide consisting of the amino acid sequence of from the 163rd Thr to the 175th Asp in the amino acid sequence of SEQ ID NO: 4 is deleted.  
     
     
         8 . The polypeptide SE36 of any one of  claims 1  to  5 , wherein an oligopeptide consisting of the amino acid sequence of the 19th Gly to the 26th Gly in the amino acid sequence of SEQ ID NO: 4 is deleted, and an oligopeptide consisting of the amino acid sequence of the 163rd Thr to the 175th Asp in the amino acid sequence of SEQ ID NO: 4 is deleted.  
     
     
         9 . The polypeptide SE36 of any one of  claims 1  to  8 , wherein the number of serine residues polymerized through a peptide linkage between the 175th Asp and the 178th Glu in the amino acid sequence of SEQ ID NO: 4 is in the range of 0 to 10.  
     
     
         10 . A polypeptide which has an antigenicity crossing with the polypeptide SE36 of  claim 1 , and in which the number of the polymerized serine residue detected by the amino acid homology search is in the range of 0 to 10.  
     
     
         11 . A malaria vaccine, which comprises as an active component at least one of polypeptides selected from the group consisting of the polypeptides SE36 of  claims 1  to  9  and the polypeptide of  claim 10 .  
     
     
         12 . A diagnostic agent for malaria, which comprises as an active component at least one of polypeptides selected from the group consisting of the polypeptides SE36 of  claims 1  to  9  and the polypeptide of  claim 10 .  
     
     
         13 . A synthetic DNA fragment encoding any one of the polypeptides SE36 of  claims 1  to  9 .  
     
     
         14 . A process for purifying a polypeptide SE36, which comprises collecting of the cells from the culture solution of  Escherichia coli  transformed with the synthetic DNA fragment of  claim 13 , and carrying out each step in order of cell destruction, fractionation by salting-out, membrane filtration, column chromatography, hydrophobic chromatography, precipitation by salting-out, membrane filtration, column chromatography, and dialysis.  
     
     
         15 . The process of  claim 14 , wherein the dialysis is conducted after the concentration of the polypeptide SE36 is adjusted at 10 to 100 μg/ml.