China ; epidemiology ; Hong Kong ; epidemiology ; Humans ; Severe Acute Respiratory Syndrome ; epidemiology

OBJECTIVETo express the cloned gene glycoprotein I (gpI) of varicella-zoster virus (VZV), Beijing VZV 84-7 strain in insect cells and to purify its expression product.

METHODSThe gene coding for gpI of VZV was amplified from viral DNA by PCR and cloned into baculovirus transfer vector (pBacPAK9), and recombinant transfer vector plasmid pBacVZVgpI was obtained. The inserted gpI gene in the pBacVZVgpI was sequenced. Insect cells Sf 9 were co-transfected with the recombinant transfer vector plasmid pBacVZVgpI and wild type linear baculovirus BacPAK6 (digested with Bsu36I) DNA. The recombinant baculoviruses containing the VZV 84-7 gpI gene was isolated through several rounds of limited dilution. Recombinant protein gpI was expressed in insect cells Sf 9, postinfected with recombinant baculoviruses. The expressed recombinant gpI was purified by lectin affinity chromatography and its antigenicity and immunogenicity were investigated.

RESULTSThe gene coding for gpI of VZV was obtained by PCR and the gpI gene of pBacPAK9 was confirmed by DNA sequencing. The recombinant gpI was expressed in insect cells Sf 9, post-infected with recombinant baculovirus and identified by SDS-PAGE and western blotting, with its product in cell culture reaching the peak in 72 hours and with a molecular mass of 58 kd and 70 kd, the same as theoretical values. Results of immunoassay with cell lysates infected by recombinant baculoviruses indicated that recombinant protein expressed in insect cells had ability of eliciting specific antibodies against native VZV in mice and complement-dependent neutralizing antibodies. The purified recombinant gpI gave a product with a purity of more than 80%. ELISA and Western-blot analysis demonstrated that purified protein had specific VZV antibody-binding activity. This suggested that the recombinant gpI expressed in insect cells had the same biological characteristics as its native counterpart.

CONCLUSIONBaculovirus-insect cells could be used to express the gene of VZV gpI, which could provide a basis for quantitative analysis of VZV antigen, and preparation of its subunit vaccine.

Animals ; Cell Line ; DNA, Viral ; genetics ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; genetics ; Genetic Vectors ; genetics ; Polymerase Chain Reaction ; Recombinant Proteins ; genetics ; isolation & purification ; Spodoptera ; cytology ; genetics ; Viral Envelope Proteins ; genetics ; isolation & purification