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Cloning, expression, and characterization of the MSP1a and MSP1b recombinant proteins from PR1 Anaplasma marginale
Katia Tamekuni1, Flora S Kano, Alexandre C Ataliba
1Universidade Estadual de Londrina, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Agrárias, Pr 445 Km 380, Cx. Postal 6001, 86051-990 Londrina, PR, Brazil.
Abstract:
The Anaplasma marginale is a bacterium that has obligate intraerythrocytic multiplication in cattle causing important economic loss. The A. marginale major surface protein 1 (MSP1) complex, heterodimer composed of MSP1a and MSP1b, has been identified as adhesins for bovine erythrocytes. The objectives of this study were to sequences the msp1beta gene and produce and characterize recombinant MSP1a and MSP1b from a Brazilian strain of A. marginale, PR1. The msp1alpha and msp1beta genes from the PR1 strain were cloned and expressed in E. coli BL21 Star using the vectors pET102 and pET101/D-TOPO. Antibodies were produced against the recombinant proteins and were shown to react with rMSP1a and rMSP1b demonstrating a molecular mass of 70kDa to 105kDa and 100kDa, respectively for these proteins. Bovine erythrocytes were agglutinated by BL21/rMSP1a and BL21/rMSP1b and, this agglutination was inhibited by the presence of the IgY anti-rMSP1a, confirming the adhesion function of these proteins. Additionally, using the IgY anti-rMSP1a and rMSP1b in a IFI, the presence of rMSP1a and rMSP1b was confirmed on the outer membrane of the recombinant E. coli BL21. Our results show that the msp1beta gene from the PR1 strain has both the conserved region and contain the defined polymorphism regions previously described for other strains of A. marginale. The results from this study confirm adhesive functions for rMSP1a and rMSP1b from PR1 strain in bovine erythrocytes invasion.
Insights
This study characterizes the Anaplasma marginale major surface protein 1 (MSP1) complex from a Brazilian strain, confirming its adhesive role in bovine erythrocyte invasion and economic loss prevention.
Area of Science:
- Veterinary Microbiology
- Molecular Biology
- Bacteriology
Background:
- Anaplasma marginale causes significant economic losses in cattle due to intraerythrocytic multiplication.
- The A. marginale major surface protein 1 (MSP1) complex, comprising MSP1a and MSP1b, acts as an adhesin for bovine erythrocytes.
Purpose of the Study:
- To sequence the msp1beta gene from the Brazilian A. marginale PR1 strain.
- To produce and characterize recombinant MSP1a and MSP1b proteins.
- To confirm the adhesive properties of MSP1a and MSP1b in bovine erythrocyte invasion.
Main Methods:
- Cloning and expression of msp1alpha and msp1beta genes in E. coli.
- Production and characterization of polyclonal antibodies against recombinant MSP1a and MSP1b.
- Analysis of protein molecular mass and agglutination assays with bovine erythrocytes.
- Immunofluorescence inhibition (IFI) assays to confirm protein localization.
Main Results:
- Recombinant MSP1a and MSP1b (rMSP1a, rMSP1b) were successfully produced and characterized.
- Antibodies against rMSP1a and rMSP1b reacted with the recombinant proteins.
- rMSP1a and rMSP1b agglutinated bovine erythrocytes, an effect inhibited by specific antibodies.
- The msp1beta gene from the PR1 strain exhibited conserved and polymorphic regions.
Conclusions:
- The MSP1a and MSP1b proteins from the Brazilian A. marginale PR1 strain possess adhesive functions.
- These findings confirm the role of MSP1 complex in the invasion of bovine erythrocytes.
- Understanding these adhesive mechanisms is crucial for developing strategies against anaplasmosis.
