Construction and immunoscreening of cDNA library of Babesia microti

Abstract

Objective To construct a cDNA library of Babesia microti and immunoscreen candidate antigens for immuno-diagnosis of Babesia infection.Methods The blood samples were collected from the mice infected with B.microti 7 days post infection when the parasitemia density reached 70％.Total RNA of B.microti was extracted and the mRNA obtained by a purifying kit was used to construct the cDNA-library.The library was immunoscreened with pooled sera from B.microti infected mice to obtain positive clones.The inserted fragments of positive clones were identified by PCR amplification,and the obtained genes were sequenced and analyzed for their homology.Results The titer of the library was 5.2 × 101 plaque forming unit (pfu)/ml.The inserted fragment length of the library ranged from 500 to 3 000 bp with a recombination efficiency being 91.0％.Six positive clones,Bm2,Bm4,Bm6,Bm7,Bm9 and Bm15 were found and their inserted fragment length was about 633,614,1 073,890,1 001 and 1 032 bp,respectively.Sequence analysis revealed that all the 6 clones contained open reading frames.The deduced amino acid sequences of the 6 clones contained 159,100,254,217,60 and 244 amino acid residues,with Mr of 17 900,11 400,28 600,24 000,6 800 and 28 900,respectively.Conclusion A cDNA library of B.microti has been constructed and 6 positive clones identified,which can be recognized by the sera of mice infected with B.microti.This study provided preliminary information for further identification of highly reactive antigens for development of immunodiagnostics of B.microti infection. Key words: Babesia microti ; cDNA library ; Immunoscreening