The cloning and amino acid sequences of the gene encoding p33 of T. sergenti of a Korean isolate has been previously described (Kang et al., 1997). For construction of the transfer vector for baculovirus expression, the cloned p33 in pUC19 plasmid was digested with BamHI (Promega) and EcoRI (Promega, Madison, USA). The digested DNA of p33 was then extracted after gel electrophoresis. The extracted DNA fragments were ligated with Aurographa california nuclear polyhedrosis virus (AcNPV) transfer vector. For site directed ligation, BacPAK8 (Clonteck, Palo Alto, USA) vector was also digested with BamHI and EcoRI within cloning sites before ligation with p33 gene as shown in Fig. 1. Through site-directed ligation, the ATG starting codon of p33 was directly located behind the AcNPV promoter with its own initiation and terminating codon within the transfer vector. The ligated transfer vector was then cotransfected into Spodoptera frugiperda (Sf21) cells (Invitrogen, Carlsbad, USA) with wild-type baculovirus DNA using Lipofectin (GibcoBRL, Gaithersburg, USA). The recombinant baculoviruses were plaque purified twice by the procedures as described previously (Kweon et al., 1997a, 1997b). Plaque purified recombinant baculoviruses were concentrated and subjected to PCR for the identification of an inserted p33 DNA in baculoviruses as described before (Choi et al., 1997; Kweon et al., 1997b). The positive clones were further propagated in Sf21 cells and screened through the indirect immunofluorecent antibody (IFA) assay using positive bovine sera against T. sergenti.

For the identification of expressed p33, Sf21 cells were infected with the recombinant baculovirus at a multiplicity of 0.1-1 per cell. The infected cells were then incubated at 28℃ for 48-72 hr and examined by indirect IFA method. The cells were fixed with cold acetone for 10 min and reacted with a 1:50 dilution of positive bovine sera against T. sergenti for 1 hr at 37℃ followed by washing with PBS. FITC conjugted anti-bovine immunoglobulin (KPL, Gaithersburg, USA) was reacted at the same conditions before examination for fluorescence. The infected cell lysates were also subjected to SDS-PAGE and immunoblotting for the detection of expressed proteins.

To detect immunogenicity of expressed proteins in vivo, the recombinant baculovirus infected Sf21 cells were scraped at 72 hr postinfection. The infected cell lysates were prepared as described previously (Kweon et al., 1997b). Four-week-old guinea pigs were injected using freeze-thawed cell lysates mixed with equal volume of Freund's complete adjuvant (Sigma, St. Louis, USA). The second injection with Freund's incomplete adjuvant (Sigma) followed after three weeks, and the last injections were conducted without any adjuvant. After two weeks, the blood was collected and tested for IFA and immunoblotting. IFA was conducted using the same procedures mentioned above using T. sergenti-infected blood from cattle. SDS-PAGE and immunoblotting were carried out using the procedure of Laemmli (1970) and Towbin et al. (1979), respectively. The subsequent immunodetection procedures were conducted with serum of guinea pigs immunized with p33 expressed Sf21 cell lyates as described previoulsy (Kweon et al., 1997b).

The transfer vector for p33 (860 bp) was constructed with the pBacPAK8 vector using BamHI and EcoRI restriction sites, which contain an ATG initiation codon of p33 of T. sergenti right behind AcNPV polyhedron promoter (Fig. 1).

In a transfection experiment, two recombinant baculoviruses were cloned from inoculation of 10^-5 and 10^-6 dilutions of transfected Sf21 cell-supernatant through plaque assay. When the inserted p33 DNA from recombinant baculoviruses was tested by PCR, the same size of p33 DNA was amplified (Fig. 2), thus confirming that two recombinant baculoviruses contained the gene for p33 of T. sergenti.

Initially, the detection on expression of p33 was screened in recombinant virus infected Sf21 cells by IFA. Positive results were only detected in cells infected with recombinant virus. The immunofluorescence was mainly observed on the cytoplasm and the surface of virus infected cells (Fig. 3). Since the immunofluorescence was observed after the initiation of cytopathic effects in Sf21 cells, positive clones were further selected for the identification of expressed protein by immunolotting.

When the cell lysate was subjected to SDS-PAGE, one extra protein band of 33 kDa was clearly detected in virus infected cells, but not in the control (Fig. 4).

In order to investigate the immunogenicity of expressed p33 protein, guinea pigs were immunized with cell lysates. The sera from immunized guinea pigs were then tested by IFA and immunoblotting. When the sera from guinea pigs were examined by IFA, positive reactions were observed up to 32-64 dilution (Fig. 5). In immunoblotting, although several protein bands were detected, the 33 kDa protein band was also detected with immunized sera (Fig. 6). The detection on several protein bands was rather expected because the immunizing cell lysate was prepared from virus infected cells and resulted in cross-reaction with other proteins of similar immunogenicity. However, sera from immunized guinea pigs also revealed positive titers (1:32 or 1:64) similar to those of bovine sera collected from cattles infected with T. sergenti (data not shown).