BABESIA MICROTI GENOMIC CLONES CONTAINING NOVEL ANTIGENS USEFUL IN THE DIAGNOSIS OF BABESIOSIS
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.
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.