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Previous issue date: 2017-03-28 === Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES === Rhipicephalus microplus is a tick that infests preferably bovines and cause a great damage in the livestock. The main control method against ticks is the use of acaricides, which lead to the emergence of resistant populations, in addition to increasing costs and risk of contamination of meat, milk and environment. Thus, vaccine antigens have been identified and characterized as an alternative to acaricides. Salivary molecules of hematophagous parasites can inhibit responses associated to homeostasis and modulate the host immune system and, therefore, are options in the search for anti-tick vaccines. This work characterized a glycine rich-protein and the paramyosin of R. microplus. Glycine rich-proteins are secreted in tick saliva and are essential in the attachment and blood feeding. Paramyosin is able to evade the host immune system and proved to be an important allergen of mites and vaccine antigen against helminths.
The cDNA and amino acid sequences of the R. microplus glycine rich-protein (RmGRP) were identified and analyzed, revealing two distinct portions of the protein (glycine residues
presented dispersed in the N-terminal region and in repeated patterns along the C-terminal
region). Cloning, expression and purification of RmGRP were performed and the recombinant protein was tested for recognition by sera from naturally and experimentally infested bovines, indicating their antigenicity, but also displaying a high heterogeneity in protein recognition between individuals and among infestations in the same individual. In addition, recombinant RmGRP was recognized by sera from rabbits immunized with saliva and salivary gland from partially and fully engorged females and eggs, but not from sera from rabbits immunized with gut. Expression of the gene encoding RmGRP (rmgrp) in different female tissues and tick development stages was evaluated by qRT-PCR. Gene transcription was found in eggs, larvae, adult males, salivary glands, fat bodies and ovaries of partially and fully engorged females, with the highest expression levels in 1-day-old larvae and salivary glands of fully engorged females. rmgrp was not expressed in 3- and 6-day-old eggs and in guts of partially and fully engorged females, corroborating to the aforementioned result. Effects of RNAm silencing corresponding to RmGRP and RmPRM were evaluated, which resulted in egg laying reduction in the group in which the RmPRM gene was silenced and in the reduction of larval hatching rate in the two groups. Therefore, it is suggested that both proteins are important during larval development, but only RmPRM is required in egg formation and/or laying. Cloning of the coding DNA regions and expression of N-terminal, internal and Cterminal fragments of R. microplus paramyosin (RmPRM) in Escherichia coli and of the complete RmPRM in Pichia pastoris were confirmed by SDS-PAGE and western-blot. From the data obtained, both RmGRP and RmPRM have characteristics that make them possible candidates to compose a cocktail vaccine against R. microplus. === O Rhipicephalus microplus ? um carrapato que infesta preferencialmente bovinos, causando
um grande preju?zo ? pecu?ria. O principal m?todo de controle ? o uso de acaricidas, os quais
selecionam popula??es resistentes, al?m de aumentar o custo e o risco de contamina??o da
carne, do leite e do ambiente. Desta forma, ant?genos vacinais t?m sido identificados e
caracterizados como alternativa aos acaricidas. Mol?culas salivares de parasitos hemat?fagos
podem inibir respostas associadas ? homeostase e modular o sistema imune do hospedeiro e,
portanto, s?o op??es na busca por vacinas anti-carrapato. Neste contexto, este trabalho
caracterizou uma prote?na rica em glicinas e a paramiosina do R. microplus. Prote?nas ricas
em glicinas s?o secretadas na saliva e s?o essenciais na fixa??o e na alimenta??o do parasito.
J? a paramiosina ? capaz de evadir o sistema imune do hospedeiro e mostrou-se um
importante alergeno em ?caros e ant?geno vacinal contra helmintos. As sequ?ncias de cDNA e
amino?cidos da prote?na rica em glicinas de R. microplus (RmPRG) foram identificadas e
analisadas, revelando duas por??es distintas da prote?na (com glicinas isoladas ao longo da
regi?o N-terminal e padr?es repetidos contendo glicinas ao longo da regi?o C-terminal). A
clonagem, express?o e purifica??o da RmGRP foram realizadas e a prote?na recombinante foi
testada quanto ao reconhecimento por soros de bovinos naturalmente e experimentalmente
infestados, indicando a sua antigenicidade, mas tamb?m uma alta heterogeneidade no
reconhecimento da prote?na entre indiv?duos e entre infesta??es no mesmo indiv?duo. Al?m
disso, a RmGRP recombinante foi reconhecida pelos soros de coelhos imunizados com saliva
e gl?ndula salivar de f?meas parcialmente e totalmente ingurgitadas e ovos, mas n?o por soros
de coelhos imunizados com intestino. A express?o do gene codificante para a RmGRP
(rmgrp) em diferentes tecidos de f?meas e fases do desenvolvimento do carrapato foi avaliada
por qRT-PCR. A transcri??o do gene foi encontrada em ovos, larvas, machos adultos, al?m de
gl?ndulas salivares, corpos gordurosos e ov?rios de f?meas parcialmente e totalmente
ingurgitadas, apresentando os maiores n?veis de express?o nas larvas de 1 dia e nas gl?ndulas
salivares de f?meas totalmente ingurgitadas. rmgrp n?o foi expresso nos ovos de 3 e 6 dias e
no intestino de f?meas parcialmente e totalmente ingurgitadas, corroborando o resultado
anterior. Ainda foram avaliados os efeitos do silenciamento do RNAm correspondente ?
RmGRP e ? RmPRM, o que resultou na redu??o da taxa de postura de ovos no grupo em que
o gene da RmPRM foi silenciado e da eclos?o de larvas em f?meas tratadas nos dois grupos.
Portanto, sugere-se que as duas prote?nas s?o importantes durante o desenvolvimento larval,
mas s? a RmPRM seja necess?ria na forma??o e/ou postura dos ovos. Tamb?m foi realizada a
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clonagem da regi?o codificante e express?o dos fragmentos N-terminal, interno e C-terminal
da paramiosina de R. microplus (RmPRM) em E. coli e da RmPRM completa em P. pastoris,
sendo confirmadas por SDS-PAGE e western-blot. A partir dos dados obtidos, ambas
RmGRP e RmPRM apresentam caracter?sticas que fazem delas poss?veis candidatas a
comporem uma vacina coquetel contra o R. microplus.
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