Cloning, expression, and characterization of the MSP1a and MSP1b recombinant proteins from PR1 Anaplasma marginale strain, Brazil.
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.
Tamekuni
Katia
K
Universidade Estadual de Londrina, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Agrárias, Pr 445 Km 380, Cx. Postal 6001, 86051-990 Londrina, PR, Brazil.
Kano
Flora S
FS
Ataliba
Alexandre C
AC
Marana
Elizabete R M
ER
Venâncio
Emerson J
EJ
Vidotto
Marilda C
MC
Garcia
João Luis
JL
Headley
Selwyn A
SA
Vidotto
Odilon
O
D000595:Amino Acid Sequence; D042322:Anaplasma marginale / Q000235:genetics*; D000818:Animals; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D002418:Cattle Diseases / Q000097:blood / Q000382:microbiology*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004912:Erythrocytes / Q000382:microbiology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect / Q000662:veterinary; D007136:Immunoglobulins / Q000276:immunology; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D016415:Sequence Alignment
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; C016512:IgY; D007136:Immunoglobulins; D011994:Recombinant Proteins; C449089:major surface protein 1a, Anaplasma; C450434:major surface protein 1b, Anaplasma