Production of Recombinant Protein Pap31 and Its Application for the Diagnosis of Bartonella bacilliformis Infection

Abstract

Tropical bartonellosis is a highly fatal epidemic and endemic infectious disease that occurs throughout the communities of the Andes Mountains in South America. The disease is caused by the facultative intracellular bacteria, Bartonella bacilliformis. The emergence of bartonellosis in new geographic areas and an increase in the number of reported cases suggest the need for a rapid test for epidemiologic study and investigation of the disease burden. The objective of this research is to develop a rapid serologic diagnostic test using recombinant antigens to overcome the limitations of the current standard IFA technique for laboratory diagnosis. Western blot analysis with patient sera of whole cell lysate separated on a 2D gel identified Pap31 as a dominant antigen. PCR primers were designed according to the sequence of ATCC strain 35685 to amplify the gene coding for Pap31 from a local isolate (HOSP 800-09, Peru). The amplicon was subsequently cloned into pET24a, adding the T7 tag, and expressed in E. coli. Patient sera with different IFA titers confirmed the diagnostic band of 31 kDa on a Western blot of SDS-PAGE. The performance of affinity-purified recombinant Pap31 (rPap31) was also evaluated in an ELISA format with 137 patient sera of known IFA titers. The range of ELISA reading from positive sera did not overlap with the range of those from negative sera, suggesting the potential application of rPap31 in both ELISA for high throughput regional hospital settings and in the construction of handheld rapid tests for rural clinical sites.