Design, construction and purification of a chimeric slayer Trypanosoma cruzi protein for immunoprophilactic applications against Chagas disease

ZABALA B; VAZQUEZ ME; MESIAS AC; PEREZ BRANDAN C; ACUÑA LA

Evento virtual

Congreso; LVI SAIB Meeting - XV SAMIGE Meeting; 2021

Sociedad argentina de investigación bioquímica y biología molecular (SAIB)

Chagas disease (CD) is an endemic malady in Argentina and there are no vaccines for human application. Though heterologous expression of specific antigens in generally recognized as safe bacteria (GRAS) represents a tempting alternative for vaccine formulations, the engineering of Gram positive strains represents a real challenge. In this work, we present a similar approach that combines the immunogenicity of a specific antigen of Trypanosoma cruzi with the beneficial adjuvant properties of a probiotic bacterium. In order to obtain a system for antigen- self-assembly that enables spontaneous adhesion on multiple surfaces, we developed a genetic construction. For that, we engineered a structure-based chimeric antigen between the SpyTag peptide, a bond-forming subunit of Streptococcus pyogenes followed by the N-terminus fragment of Tc52 (N-Tc52), an immunogenic protein of T. cruzi, and SlpA, a surface layer protein of Lactobacillus acidophilus. The final transcriptional fusion was carried out by successive asymmetric PCRs. In the first step, the sequence encoding to N-Tc52 was amplified by PCR from T. cruzi CL Brener strain using specific primers to incorporate cloning sites, the sequence encoding to SpyTag and a fragment of SlpA gene. In the second step, the gene encoding to SlpA was amplified by PCR from L. acidophillus ATCC 4356 using specific primers to incorporate cloning sites and a fragment of N-Tc52. Finally, we fused the obtained genes by using different combinations and concentrations of primers in an asymmetric PCR. Once obtained, the final fragment was cloned in pRSET-A and inserted into Escherichia coli DH5α. The recombinant plasmid containing the hybrid gene 6HisSpyTag-NTc52-SlpA was purified and inserted into E. coli BL21 [DE3]. Expression of the 6His-SpyTag-NTc52-SlpA protein was carried out by the addition of IPTG 1 mM at 28 °C. After 4 h of induction, cells were collected by centrifugation in phosphate buffered saline and lysed by repetitive cycles of sonication, freezing and thawing. Subsequently, the lysate was centrifuged and the pellet, containing the protein in inclusion bodies, was resuspended in Urea 8M. The protein was purified through a Ni-NTA agarose cartridge