Demonstration of a heat-stable 120-kilodalton protein of Rickettsia japonica as a spotted fever group-common antigen.
Genomic libraries of Rickettsia japonica were cloned into an expression vector lambda gt11. A clone expressing a protein reactive with antiserum against 120-kilodalton (kDa) proteins, a mixture of heat-modifiable and heat-stable polypeptides, was selected and designated as lambda Rj120-1. The expressed protein has a molecular mass of 180 kDa. Western immunoblotting demonstrated that the expressed protein was a fusion protein with beta-galactosidase. The antiserum against 120-kDa proteins was absorbed by the induced lysogen, resulting in the removal of reactivity to the heat-stable 120-kDa polypeptide. The antiserum against the expressed protein reacted with heat-stable 120- to 130-kDa polypeptides of spotted fever group (SFG) rickettsiae in addition to R. japonica. The findings indicated that the protein expressed from the cloned gene of R. japonica possessed the antigenicity group-common to SFG rickettsiae. Primers designed from the gene coding for R. conorii heat-stable 120-kDa protein (Schuenke, K.W., and Walker, D.H., Infect. Immun. 62: 904-909, 1994) and lambda gt11 lacZ gene amplified the lambda Rj120-1 DNA by the polymerase chain reaction (PCR). Analysis of restriction fragment length polymorphism (RFLP) of the PCR-amplified products revealed that the cloned DNA corresponds to a portion of the gene coding for the heat-stable 120-kDa protein of R. conorii with 2,519 nucleotides beginning at nucleotide 190 of the open reading frame. RFLP demonstrated that the cloned gene was highly homologous to the corresponding gene of R. conorii.
Uchiyama
T
T
Department of Virology, University of Tokushima, Japan.
Zhao
L
L
Uchida
T
T
D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D015153:Blotting, Western; D002522:Chlorocebus aethiops; D017931:DNA Primers; D004269:DNA, Bacterial; D005798:Genes, Bacterial; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010455:Peptides; D011995:Recombination, Genetic; D012281:Rickettsia / Q000235:genetics / Q000276:immunology*; D012373:Rocky Mountain Spotted Fever / Q000382:microbiology; D014709:Vero Cells
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D010455:Peptides