USE OF BACTERIALLY-EXPRESSED ANTIGEN FOR DETECTION OF ANTIBODIES TO THE EBV-SPECIFIC DEOXYRIBONUCLEASE IN SERA FROM PATIENTS WITH NASOPHARYNGEAL CARCINOMA
Jy. Chen et al., USE OF BACTERIALLY-EXPRESSED ANTIGEN FOR DETECTION OF ANTIBODIES TO THE EBV-SPECIFIC DEOXYRIBONUCLEASE IN SERA FROM PATIENTS WITH NASOPHARYNGEAL CARCINOMA, Journal of virological methods, 45(1), 1993, pp. 49-66
A cDNA clone, BG9, corresponding to the open reading frame BGLF5 of Ep
stein-Barr virus (EBV) DNase was inserted into an E. coli expression v
ector, pET3a, to generate a recombinant plasmid, pDNase 5. High level
of expression of a DNase activity was detected, in the E. coli transfo
rmed 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 homo
geneous 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 activ
ity 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 exp
ressed EBV DNase may be useful for preparing specific test for large s
cale screening of patients with NPC.