The gene encoding the 65K tegument phosphoprotein (pp65) of human cytomegalovirus (HCMV) was cloned into pAc373 to construct a recombinant baculovirus (Acpp65-3) expressing pp65 in insect Sf9 cells. A baculovirus that carried a fragment of the gene, corresponding to the first 442 amino acids of pp65, was also developed, using vector pVL941 (Acpp65-2). Recombinant proteins migrating in SDS-polyacrylamide gels with an of either 65K (Acpp65-3) or 56K (Acpp65-2) were detected in cytoplasmic and nuclear extracts of infected Sf9 cells. The 56K and 65K proteins were recognized in immunoblots by monoclonal antibodies (MAbs) 28-77 and 28-19, which are specific for pp65. The insect cell-expressed antigens were also analysed on Western blots using MAbs 4D11, 7D2, 8E3, 7B4 and 8E10, which recognize the HCMV antigen GP66 in immunoblots. The truncated pp65 antigen of Acpp65-2 was reactive with MAbs 4D11, 7D2, 8E10 and 7B4. The protein expressed by Acpp65-3 reacted only with MAb 4D11. The data proved that the epitopes recognized by MAbs 4D11, 7D2, 8E3 and 7B4 mapped in the region of pp65, comprising amino acids 1 to 442, and also that GP66 and pp65 represent the same HCMV antigen. Immunoblot analysis of human sera from individuals seropositive for HCMV showed that the recombinant pp65 products were as antigenic as the native 65K phosphoprotein produced in HCMV-infected human embryonic fibroblasts.


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