Use of bacterially-expressed antigen for detection of antibodies to the EBV-specific deoxyribonuclease in sera from patients with nasopharyngeal carcinoma.
A cDNA clone, BG9, corresponding to the open reading frame BGLF5 of Epstein-Barr virus (EBV) DNase was inserted into an E. coli expression vector, pET3a, to generate a recombinant plasmid, pDNase 5. High level of expression of a DNase activity was detected in the E. coli transformed with pDNase 5 following induction with IPTG. The enzyme activity was purified using DEAE-cellulose, phosphocellulose and DNA-cellulose column chromatography. The purified protein appeared to be nearly homogeneous in SDS-PAGE using Coomassie blue staining. The requirement for divalent cations and optimum pH as well as inhibitory concentrations of ionic strength and polyamines for the purified enzyme activity were determined and seemed to be very similar to those of the enzyme activity purified from an EBV producing lymphoblastoid cell line. Using the purified enzyme as an antigen and anti-IgA as the secondary antibody, 82% (64/78) and 91% (71/78) of sera from patients with nasopharyngeal carcinoma (NPC) were shown to be positive by dot immunobinding assay and ELISA, respectively. The results suggest that purified E. coli expressed EBV DNase may be useful for preparing specific test for large scale screening of patients with NPC.
Chen
J Y
JY
Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, People's Republic of China.
Liu
M Y
MY
Hsu
T Y
TY
Cho
S M
SM
Yang
C S
CS
D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral / Q000096:biosynthesis / Q000276:immunology; D003851:Deoxyribonucleases / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D006566:Herpesviridae Infections / Q000276:immunology; D004854:Herpesvirus 4, Human / Q000201:enzymology / Q000276:immunology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009303:Nasopharyngeal Neoplasms / Q000276:immunology* / Q000382:microbiology; D011073:Polyamines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D012492:Salts / Q000494:pharmacology; D014412:Tumor Virus Infections / Q000276:immunology
D000914:Antibodies, Viral; D000956:Antigens, Viral; D011073:Polyamines; D011994:Recombinant Proteins; D012492:Salts; D003851:Deoxyribonucleases