Revista Peruana de Medicina Experimental y Salud Publica
versión impresa ISSN 1726-4634
GALLEGOS V, Karen; BALDEVIANO V, Christian; MARCELO N, Adolfo y PADILLA R, Carlos. Clonamiento, expresión y seroreactividad del antígeno recombinante flagelina de Bartonella bacilliformis. Rev. perú. med. exp. salud publica [online]. 2005, vol.22, n.1, pp. 39-46. ISSN 1726-4634.
Objectives: To clone the Bartonella bacilliformis flagellin gene (flaA), and to preliminarily express and assess reactivity of the recombinant protein against B. bacilliformis bartonellosis patients' sera. Materials and Methods: A couple of initiating oligonucleotides was designed -BbFlaA1 and BbFlaA2- for complete B. bacilliformis flagelline A gene amplification. The amplification product obtained was cloned in pEGM, and later it was subcloned in pGEX4T-1 expression vector. Fusion protein rBbFlaA-GST expression was induced with isopropyl thio-β -D-galactoside (IPTG). The fusion protein produced was digested with thrombin in order to release its GST contents. Finally, an ELISA test was standardized in order to detect IgG antibodies against fusion protein rBbFlaA-GST and rBbflaA GST-free. Sera from patients with bartonellosis caused by B. bacilliformis (n= 30), sera from healthy individuals (n= 20), and sera from patients with a possible cross-reactivity; i.e., brucellosis (n= 3), leptospirosis (n= 3), and salmonellosis (n= 7) were assessed. Results: It was determined that for optimal expression of fusion protein rBbFlaA in E. coli BL21, it is required that the culture grows in LB/ampicillin broth at a 30º C temperature, supplemented with 2% glucose from a 100 µL pre-inoculum (left to grow overnight), until it reaches a 1 optical density (OD 600), and being induced for two hours at 2,5 mM IPTG. Finally, 57,6% (17 of 30) sera from patients with a confirmed bartonellosis diagnosis reacted with BbFlaA recombinant protein in an ELISA format. Conclusions: B. bacilliformis BbFlaA recombinant protein was successfully expressed in E. coli, and an rBbFlaA expression and purification protocol was determined for producing this protein. Also, rBbFlaA antigen is recognized by antibodies present in sera from bartonellosis patients infected with B. bacilliformis.
Palabras llave : Bartonella bacilliformis; Diagnosis; Flagellin; Enzime-Lynked Immunosorbent Assay; Recombinant Proteins; Carrion's Diseases.