Cloning, expression and purification of a chimeric protein based on Rhipicephalus microplus proteins

• Grant number: 16/03736-6
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: May 01, 2016
• End date: April 30, 2017
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Aparecida Sadae Tanaka
• Grantee: Daniel Marconi Mendes
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated research grant: 12/03657-8 - Inhibitor and proteases of ectoparasites: relationship of structure-function and identification of the role of these molecules in the interaction of diseases vector e their etiological agents, AP.TEM

Abstract

The tick R. microplus is consider a parasite of great veterinary importance in tropical and subtropical regions around the world, being responsible for big losses in the production of leather, milk and meat, besides it acts as vector of Babesia sp and Anaplasma sp, which compromise even more the cattle. Until now the tick control has been done by using acaricides, however its improper use may result in the contamination of the environment, bovine derivatives as well as the selection of resistance strains which justify the search for alternative control methods that resulted in the development of two vaccines. However, these vaccines show almost no protection for the national herds, highlighting the need of biochemical studies of the Brazilian R. microplus populations. In the past years our group showed the importance of proteases inhibitors for the physiology of bloodsucking vectors and their potential for vaccine development. Vaccination experiments of bovines using serineproteases Kunitz type inhibitors results in 72% protection efficiency while vaccination with only one inhibitor shows an efficiency of 32%. In attempt to find possible targets for vaccine development, a midgut transcriptome of R. microplus engorged female tick's was performed and two major groups of transcripts were observed, molecules from the serine proteases inhibitor of TIL inhibitor family and molecules similar to microplusin, an antimicrobial protein. Based on this information, the construction of a chimera protein was performed containing the BmSI (TIL inhibitor member) and a second molecule named RmSEI (similar to microplusin) linked by a loop found in the transcripts of Kunitz type inhibitors. The chimera was cloned and express in the bacterial system, using pET14b vector and E. coli BL21 plys S, the results showed only low levels of expression of the recombinant chimera. Therefore, the objectives of this project are the cloning, expression and purification of the chimera in the yeast system using Pichia pastoris, and the use of this recombinant protein in bovine vaccination trials. (AU)