Construção de quimeras recombinantes contendo antígenos de Bartonella henselae

Abstract

Bartonella henselae is a facultative gram-negative and intracellular bacterium, where cats are its primary reservoir and humans are accidental hosts. B. henselae is considered one of the most important medical species, causing the zoonosis known as Cat Scratch Disease (CSD). The diagnosis of CSD is still a challenge since conventional techniques for detecting antigens or antibodies have demonstrated limitations. The gold standard diagnosis of CSD is IFI, with good sensitivity, however, with the possibility of cross-reactions with other species. Given this, bioinformatics tools have been used worldwide, optimizing the search for new diagnostic alternatives. Thus, the objective of the present work was to identify, through immuno-bioinformatics analyzes, epitopes of B. henselae, and to build multi-epitope recombinant chimeras for use as biotechnological inputs. The seven most cited antigens in scientific articles (GroEL, 17kDa, P26, BadA, Pap31, OMP89 and OMP43) were chosen for the epitope identification and selection phase. B cell epitopes were selected using two predictors (BepiPred and Iedb). The three-dimensional study of proteins, as well as structural quality analyzes, physical-chemical and antigenicity analyzes were performed using the I-TASSER, QMEAN6 and Protoparam servers, respectively. The analysis of the secondary structures of the mRNA were performed to verify the stability of the translation of proteins using the RNAfold software. Next, the in silico drawing of the chimeras was done and sent to chemical synthesis. Three recombinant chimeras (rC) were formed, being composed of epitopes present in the GroEL, 17kDa and P26 (rC1) proteins; BadA, Pap31 and GroEL (rC2) and P26, OMP89 and OMP43 (rC3). Recombinant chimeras (rC) were cloned into an expression vector in Escherichia coli, pAE. The antigenicity of the proteins was verified through Western Blot (WB) with human serum naturally infected by B. henselae. All chimeric proteins were successfully expressed, insoluble, and with an expected molecular mass of 37 kDa (rC1), 35 kDa (rC2) and 38 kDa (rC3). Quality analyzes showed that rC2 was a slightly better construction than the others. Analyzes of secondary mRNA structures revealed that they had sufficient stability to effectively translate proteins. All chimeras were shown tobe antigenic through the WB. The chimeras produced have potential for use in diagnostic tests.