Immune identification and mass spectrometric analysis of the soluble antigens from Babesia microti

Abstract

Objective To analyze the fractional proteins and immunoreactivity of the soluble antigens from Babesia microti (B. microti), and find the candidate antigens for diagnosis with high sensitivity and specificity. Methods BALB/c mice were inoculated with B. microti-infected red blood cells by intraperitoneal injection. The B. microti were collected from the infected red blood cells when the infection rate reached its peak (infection rate > 70%), then the soluble antigens were extracted by repeated freezing-thawing and ultrasonic method. The mice sera before and after the infection with B. microti for 7, 14, 21, 28, 35, 42, 49 and 56 days were also collected. The polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze protein components of the soluble antigens of B. microti and the Western blot was used to analyze the immunoreactivity of the soluble antigens with the pooled mice sera before and after the infection. The specific positive protein bands were identified by Liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS), and the amino acid sequences of the proteins were analyzed by bioinformatics tools. Results The results from SDS-PAGE analysis indicated that the soluble antigens of B. microti showed distinct protein bands with the range between 12 and 185 × 103 (kDa, relative molecular mass, Mr), among which 9 main bands and 12 minor bands were obtained. In the Western blot analysis, the protein bands with Mr at 40 and 45 kDa could be recognized by pooled mice sera 7 days after infection; the protein bands with Mr at 40, 45, 54 and 95 kDa could be recognized by pooled mice sera 14 days after infection; the protein bands with Mr at 27, 40, 45, 54, 95 and 110 kDa could be recognized by pooled mice sera 21 days after infection. While, the protein bands with Mr at 27, 40, 45, 54, 95, 110 kDa and other weak-reactive bands were recognized by pooled mice sera 28-56 days after infection, and the reaction became stronger with the infection continued. There were 336 proteins, including surface antigen, heat shock protein 70, seroreactive antigen, Eta subunit of chaperonin containing t-complex polypeptide 1 and unnamed protein products, were identified as the components of soluble antigens after mass spectrometry and sequence analysis. Conclusion The 40, 45 and 54 kDa protein components from the soluble antigens of B. microti may be ideal candidate antigens for diagnosis, and their potential applications in diagnosing of human babesiosis deserve further study. Key words: Babesia microti; Soluble antigens; Western blot; Mass spectrometry