Immunological characterization of the epstein‐barr virus phosphoprotein pp58 and deoxyribonuclease expressed in the baculovirus expression system

Abstract

The open reading frames of the phosphoprotein pp58 (BMRFI) and the deoxyribonuclease (BGLF5) of the Epstein-Barr-virus (EBV) strain M-ABA were cloned in the baculovirus expression vectors pAc373 and pAc360 and expressed in the Spodoptera frugiperda (SF158) insect cells. The recombinant phosphoprotein pp58 expressed in SF158 cells was recognized by the anti-pp58 rabbit anti-sera which were generated by immunizing rabbits with a TrpE-BMRFI fusion protein expressed in E. coli. DNA-cellulose chromatography showed that the recombinant pp58 exhibited DNA-binding activities. Immunofluorescence, immunoblot and ELISA analysis indicated that sera from patients with nasopharyngeal carcinoma (NPC) contained antibodies against pp58. The recombinant EBV DNase expressed in SF158 cells was recognized by the anti-EBV DNase rabbit anti-sera which were generated by immunizing rabbits with a TrpE-C-terminal part of BGLF5 fusion protein expressed in E. coli. The anti-EBV DNase rabbit anti-sera recognized also a protein of about 52 kDa in the EBV-harboring human B-cell lines Raji, Jijoye, B95-8, M-ABA and BL74 induced by TPA and n-butyrate. The recombinant EBV DNase exhibited exonuclease and endonuclease activities, a requirement for magnesium, and a high pH optimum (8.0). Its enzyme activities could be inhibited by sera from NPC patients and anti-EBV DNase rabbit anti-sera. Comparable studies of Raji EBV-DNase and recombinant EBV-DNase implied that recombinant EBV-DNase could also be used in the enzyme activity assay for the detection of NPC. In contrast to the enzyme inhibition test, immunofluorescence and immunoblot analysis demonstrated that the recombinant EBV DNase exhibited only a weak immunological reaction with NPC sera.

TheBFRF1Gene of Epstein-Barr Virus Encodes a Novel Protein

“…The humoral response to BFRF1 may be a consequence of an active and prolonged viral replication going on in NPC and in BL that could be necessary to stimulate the production of BFRF1 antibodies to a detectable level. Moreover, previous studies have revealed antibodies to other EBV lytic proteins, such as ZEBRA, DNase, ribonucleotide reductase, and thymidine kinase, in NPC patients (5,10,14,17). On the other hand, the same antibodies are less frequently detected in healthy individuals.…”

Distribution of Epstein-Barr virus antigenic sites on the carboxyl terminal end of ribonucleotide reductase against nasopharyngeal carcinoma serum antibodies using an immunoabsorption method

“…Employing gene cloning and expression, several groups had used various recombinant EBV polypeptides to evaluate their reactivity with the antibodies from NPC patients with varying success. Such studies included polypeptides of the VCA complex [Uen et al, 1988], the membrane antigen (MA) complex [Uen et al, 1988;Littler et al, 1991;Yao et al, 1991;Durda et al, 1993], the EA complex [Liu et al, 1989;Ginsburg, 1990;Baylis et al, 1991;Chen et al, 1991;Joab et al, 1991;Littler et al, 1991;Chen et al, 1993;Gan et al, 1994;Matthew et al, 1994;Liu et al, 1997], the EBV nuclear antigen (EBNA) complex [Foong et al, 1990], and others [reviewed by Gan et al, 1996].…”

Epstein-Barr Viral Antigens Used in the Diagnosis of Nasopharyngeal Carcinoma

“…One polypeptide belonging to this complex, pp58, was expressed by the baculovirus vector in insect cells [2], Upon testing it by IFA, immunoblot and ELISA, the immunoblot and ELISA IgG tests appeared to give the most comparable results with the IgA-EA-D IFA, with a sensitivity and specificity of 88 and 96%, respectively, for the immunoblot assay and a sensitivity and specificity of 75 and 100%, respectively, for the ELISA (table 3). l also had antibodies against the EA-R complex.…”