Molecular cloning and expression of Campylobacter pylori species-specific antigens in Escherichia coli K-12.
A gene bank of Campylobacter pylori DNA in Escherichia coli was constructed by cloning Sau3A-cleaved DNA fragments into the bacteriophage vector lambda EMBL3. The expression of C. pylori antigens was determined by screening the gene library with adsorbed C. pylori whole-cell rabbit antisera. One recombinant clone which reacted positively (lambda CP2) was studied further. Immunoblot analysis with lambda CP2 showed a polypeptide band of 66 kilodaltons (kDa) reacting antigenically with the adsorbed antiserum. Extraction of DNA from lambda CP2 and digestion with SalI revealed a DNA insert of 17 kilobases (kb). Subcloning with SalI and the E. coli vector pUC18 showed that the DNA also encoded a 31-kDa antigen. The cloned antigens were shown by immunoblotting to have the same molecular weight in E. coli as in C. pylori and to be present in all C. pylori strains. Antiserum was raised against the cloned polypeptides and found to react only with C. pylori when analyzed by dot blotting and indirect immunofluorescence. The cloned antigens were determined to be expressed from the pUC18 lac promoter. The DNA encoding these antigens was radiolabeled with 32P and found to hybridize only to C. pylori strains. Immunoblotting with affinity-purified polyclonal antibody to the urease enzyme of C. pylori revealed that the cloned antigens may be part of the urease enzyme.
Clayton
C L
CL
Department of Medical Microbiology, St. Bartholomew's Hospital Medical College, London, United Kingdom.
Wren
B W
BW
Mullany
P
P
Topping
A
A
Tabaqchali
S
S
D000937:Antigen-Antibody Reactions; D000942:Antigens, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D002167:Campylobacter / Q000201:enzymology / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000235:genetics*; D015151:Immunoblotting; D013045:Species Specificity; D014510:Urease / Q000276:immunology
D000942:Antigens, Bacterial; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D015252:Deoxyribonucleases, Type II Site-Specific; C027046:TCGA-specific type II deoxyribonucleases; D014510:Urease