ANAIS 2014

SEQUENCE ANALYSIS, ANTIGENICITY AND PRODUCTION OF IXODES PERSULCATUS, IXODES OVATUS, RHIPICEPHALUS APPENDICULATUS CYSTATINS Autor(es): Gabriela Alves Sabadin, Luis Fernando Parizi, Naftaly Githaka, Adriana Seixas, Carlos Jorge Logullo de Oliveira , Satoru Konnai, Kazuhiko Ohashi, Italajara da Silva Vaz Júnior

SEQUENCE ANALYSIS, ANTIGENICITY AND PRODUCTION OF IXODES PERSULCATUS, IXODES OVATUS, RHIPICEPHALUS APPENDICULATUS CYSTATINS

» Á�rea de pesquisa: ACAROLOGIA

» Instituição: Universidade Federal do Rio Grande do Sul

» Agência de fomento e patrocinadores: INCT-Entomologia Molecular, FINEP, CAPES, CNPq, FAPERJ and FAPERGS (Brazil) and MEXT (Japan).

Sequence analysis, antigenicity and production of Ixodes persulcatus, Ixodes ovatus, Rhipicephalus appendiculatus cystatins Sabadin, G.A. 1; Parizi, L.F. 1; Githaka N.W. 2; Seixas, A. 3; Logullo, C. 4,6; Konnai, S. 2; Ohashi, K. 2; da Silva Vaz Jr I. 1,5,6 1 Centro de Biotecnologia, UFRGS, RS, Brazil; 2 Laboratory of Infectious Diseases, Hokkaido University, Japan; 3 Departamento de Farmacociências, UFCSPA, RS, Brazil; 4 Laboratório de Química e Função de Proteínas e Peptídeos, UENF, RJ, Brazil; 5 Faculdade de Veterinária, UFRGS, RS, Brazil; 6 Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Brazil. Cystatins are a family of cysteine peptidase inhibitors that regulates a number of physiological processes. The role of cystatins from different tick species was reported to be important for tick blood-feeding and development. Furthermore, there are evidences of their potential use as a tick vaccine. In this work, the cystatins coding sequences from Ixodes persulcatus (JpIpcys2a, JpIpcys2b, JpIpcys2c), Ixodes ovatus (JpIocys2a), and Rhipicephalus appendiculatus (QnRacys2a) ticks were analyzed concerning cystatin motifs and antigenic regions. The deduced amino acid sequences were aligned with twenty three amino acid sequences of tick cystatins from Genbank. The N-terminal glycine and the motif QxVxG showed conserved in JpIpcys2a, JpIpcys2b, JpIocys2a and QnRacys2a, and the PW motif in JpIpcys2b, JpIocys2a and QnRacys2a. The antigenic regions of cystatins were predicted using Jameson–Wolf algorithm to select sequences that could be useful in a multi specie vaccine. Five similar regions between JpIocys2a and JpIpcys2b amino acid sequences, and three similar regions among QnRacys2a, Rhipicephalus pulchellus and Rhipicephalus microplus amino acid sequences showed higher antigenicity. JPIpcys2a, JPIpcys2b and QnRacys2a cystatin ORFs were cloned in pET43a expression vector. The presence of regions with high similarity and antigenicity suggests that these proteins could be useful as antigens in a cross protection tick vaccine. In addition, the JpIpcys2a was expressed in Escherichia coli C43 and C41 strains at 37 ºC during 2, 5 and 24 hours at 0.4 mM of IPTG. Our perspectives are to characterize JPIpcys2a inhibition profile by enzymatic assay and use this protein as antigen in vaccination trials. Keywords: Cystatin, tick, vaccine.