Babesia microti genomic clones containing novel antigens useful in the diagnosis of babesiosis

Abstract

Disclosed are the cloning and expression of novel antigens in Babesia microti . The recombinant polypeptides are highly immunogenic. The polypeptides of the present invention provide the basis of a diagnostic assay that is sensitive, rapid and accurate using patient's sera. Also disclosed is an IgG and IgM ELISA using two novel recombinant antigens in the diagnosis of Babesia infection.

What is claimed is: 
     
         1 . An isolated polynucleotide, said polynucleotide encodes an isolated polypeptide having an amino acid selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4. 
     
     
         2 . The isolated polynucleotide of  claim 1 , wherein said polynucleotide encodes an isolated polypeptide having an amino acid of SEQ ID NO: 2. 
     
     
         3 . The isolated polynucleotide of  claim 2 , wherein said polynucleotide having a nucleotide sequence set forth in SEQ ID NO: 1. 
     
     
         4 . The isolated polynucleotide of  claim 1 , wherein said polynucleotide encodes an isolated polypeptide having an amino acid of SEQ ID NO: 4. 
     
     
         5 . The isolated polynucleotide of  claim 4 , wherein said polynucleotide having a nucleotide sequence set forth in SEQ ID NO: 3. 
     
     
         6 . A vector comprising the isolated polynucleotide of  claim 3 . 
     
     
         7 . A vector comprising the isolated polynucleotide of  claim 5 . 
     
     
         8 . The vector of  claim 6 , further comprising a promoter of DNA transcription operably linked to said isolated polynucleotide. 
     
     
         9 . The vector of  claim 8 , further comprising a promoter of DNA transcription operably linked to said isolated polynucleotide. 
     
     
         10 . The vector of  claim 6 , wherein said vector is selected from the group consisting of pET, pENTR, and pCR®8/GW/TOPO® and said promoter is selected from the group consisting of lac promoter, tip promoter and tac promoter. 
     
     
         11 . The vector of  claim 10 , wherein vector is pET and said promoter is lac promoter. 
     
     
         12 . The vector of  claim 7 , wherein said vector is selected from the group consisting of pET, pENTR, and pCR®8/GW/TOPO® and said promoter is selected from the group consisting of lac promoter, trp promoter and tac promoter. 
     
     
         13 . The vector of  claim 12 , wherein vector is pET and said promoter is lac promoter. 
     
     
         14 . A host cell comprising the vector of  claim 11 . 
     
     
         15 . The host cell of  claim 14 , wherein said host cell is  E. coli.    
     
     
         16 . The host cell of  claim 15 , wherein said  E. coli  is NovaBlue K12 strain, BL21 (DE3) or BL21 pLyss (DE3). 
     
     
         17 . A host cell comprising the vector of  claim 13 . 
     
     
         18 . The host cell of  claim 17 , wherein said host cell is  E. coli.    
     
     
         19 . The host cell of  claim 18 , wherein said  E. coli  is NovaBlue K12 strain, BL21 (DE3) or BL21 pLyss (DE3). 
     
     
         20 . A method of producing an isolated polypeptide having an amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4, comprising:
 (i) introducing an isolated polynucleotide into a host cell, said isolated polynucleotide having nucleotide sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; and   (ii) growing said host cell in a culture under suitable conditions to permit production of said isolated polypeptide;   (iii) isolating said isolated polypeptide.   
     
     
         21 . The method of  claim 20 , wherein said growing step further comprising the step of adding glucose prior to IPTG to said culture.