Expression of pseudorabies virus gE epitopes in Pichia pastoris and its utilization in an indirect PRV gE-ELISA

Journal of Virological Methods 114 (2003) 145–150

Expression of pseudorabies virus gE epitopes in Pichia pastoris and its utilization in an indirect PRV gE-ELISA Jing-qun Ao a , Jing-wen Wang a , Xin-hua Chen b , Xun-zhang Wang a,∗ , Qing-xin Long a b

a State Key Laboratory for Biocontrol, Sun Yat-Sen University, Guangzhou 510275, PR China Key Laboratory of Marine Biotechnology, Third Institute of Oceanography, State Oceanic Administration, 178 Daxue Road, Xiamen 361005, PR China

Received 7 May 2003; received in revised form 18 August 2003; accepted 1 September 2003

Abstract Pseudorabies virus glycoprotein E (PRV gE) has been recognized as a suitable diagnostic antigen for pseudorabies. In order to produce gE antigen in large quantities and at low cost, a gene fragment encoding PRV gE epitopes was expressed in Pichia pastoris expression system. SDS-PAGE and Western blotting revealed that the expression product was two recombinant proteins, approximately 38 and 32 kDa, in the culture supernatant of P. pastoris integrant 72 h after induction. Protein concentration assay showed the expression product amounted to 106.7 mg/l, accounting for 66.67% of total culture supernatant proteins. An indirect PRV gE-ELISA was then established by using the recombinant expression product as a coating antigen. Cross-reactivity assay showed that this antigen was PRV specific. Reproducibility experiment displayed good consistency. Comparison of detection results of 348 field serum samples between PRV gE-ELISA and a commercially available PRV diagnostic kit showed there was no significant difference between these two methods (P > 0.05). © 2003 Elsevier B.V. All rights reserved. Keywords: Pseudorabies virus; Glycoprotein E; Pichia pastoris; Gene expression; ELISA

1. Introduction Pseudorabies virus (PRV), a member of ␣-herpesvirinae, is the causative agent of pseudorabies, which is an economically important swine disease worldwide. Characterized by an acute and often fatal infection in piglets, with a variety of clinical signs in older pigs, including encephalitis, pneumonia, increased susceptibility to other respiratory pathogens, and abortion in pregnant swine, pseudorabies had made a great damage to world stockbreeding (Nauwynck, 1997). Vaccination is used widely in the control of pseudorabies. But since PRV usually establishes latency in the peripheral nervous system (Rziha et al., 1986; Metternleiter, 1991), vaccination can only reduce the virus circulation in the population or reduce the extent of the infection, but cannot lead by itself to the eradication of pseudorabies. van Oirschot et al. (1986) put forward a principle using gene-deleted vaccine combined with accompanying antibody detection in the ∗ Corresponding author. Tel.: +86-20-84113964; fax: +86-20-84113964. E-mail addresses: [email protected] (J.-q. Ao), [email protected] (X.-z. Wang).

0166-0934/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2003.09.012

control of pseudorabies. This made it possible to distinguish infected animals from vaccinated ones and enabled the eradication of pseudorabies. Therefore, it was considered a milestone in the control of psuedorabies (Metternleiter, 2000). PRV gE is an envelope glycoprotein consisting of 588 amnion acids. It is one of PRV virulence determinants and a non-essential protein for in vitro replication as well. When the gE gene is deleted, the propagation and antigenicity of PRV are not affected, but its virulence is reduced greatly (Jacobs et al., 1993; Nauwynck, 1997). On the other hand, gE is present in almost all examined PRV field isolates, and the natural infection always gives rise to detectable antibodies against gE (van Orischot et al., 1990). These characteristics make it ideal for pseudorabies serological diagnosis. A gE-deleted vaccine in combination with gE-specific antibody detection in sera is now used extensively in pseudorabies eradication program and has acquired great success in many countries (van Oirschot et al., 1996; Elbers et al., 2000). Initially, gE antigen was produced mainly by affinity chromatography from virions. It is trivial and costly to produce antigen in this way. In addition, after the eradication of pseudorabies, handling of live virus will be restricted. In this case,

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it was proposed to use recombinant antigen expressed in heterologous expression system, especially eukaryotic expression system, for gE epitopes are conformational-dependent (Jacobs et al., 1990; Jacobs, 1994). Several researchers have reported the development of diagnostic assays based on baculovirus-expressed PRV glycoproteins (Banks, 1994; Kimman et al., 1996; Gut et al., 1999; Gut-Winiarska et al., 2000). But as far as large-scale production is concerned, baculovirus expression system is not a good choice because of its high culture cost and inconvenience of growing insect cells in fermenter. Pichia pastoris is an alternate. It offers advantages of eukaryotic expression system including post-translational modification together with simple manipulation, low culture cost and facility to fermentation of prokaryotic expression system (Hollenberg and Gellissen, 1997; Cereghino and Cregg, 2000). Recombinant diagnostic antigens such as hepatitis B core antigen (Watelet et al., 2002) and slow catalase of Aspergillus fumigatus (Calera et al., 1997) had been expressed successfully in this system. In this paper, a gene fragment encoding gE epitopes of PRV Fa strain, a Chinese PRV representative strain, was cloned into the P. pastoris expression vector pPICZ␣A, under the control of a methanol inducible alcohol oxidase 1 (AOX1) promoter and ␣-factor secretion signal. The expression product was secreted into medium. After desalination by dialysis, it was prepared as a coating antigen for an indirect PRV gE-ELISA.

2. Materials and methods 2.1. Strains and plasmid P. pastoris strain SMD1168, E. coli strain Top10F and P. pastoris expression vector pPICZ␣A were obtained from Invitrogen.

to the sequence of PRV Fa gE gene (Ao et al., 2002) to amplify the gene fragment encoding gE epitopes located on nucleotides 151–714 (Jacobs et al., 1990; Fuchs et al., 1990). Primer sequences were as following: • forward primer: 5 -CG GAATTC GAG GCC GGC GAC GAT GAC CTC GAC-3 ; • reverse primer: 5 -GC TCTAGA CC GGG CGA GAA GAG CTG CGA GTG-3 . Genomic DNA of PRV Fa strain was extracted from virus-infected Vero cells and used as PCR template. Extraction of viral genomic DNA and PCR condition was described previously (Ao et al., 2002). The amplified PCR product was then cloned into the expression vector pPICZ␣A by EcoRI and XbaI restriction enzyme sites added to the 5 end of PCR primers. The constructed recombinant expression vector pPICZ␣A-FS was sequenced with ␣-factor primer. 2.4. Transformation of P. pastoris strain P. pastoris wild type strain SMD1168 was transformed with SacI (TaKaRa) linearized expression vector pPICZ␣A and pPICZ␣A-FS by electroporation using a GenePulser (Bio-Rad) set at 1500 V, 25 ␮F and 400 . Transformations were spread on YPDS plates containing 100 ␮g/ml ZeocinTM (Invitrogen). After incubation at 30 ◦ C for 2–3 days, the formed colonies were selected for phenotype identification and high-concentration ZeocinTM (500 ␮g/ml) screening according to the manufacture’s instruction. Colonies which can grow in YPDS plates containing 500 ␮g/ml ZeocinTM most probably have multi-copy expression cassettes. 2.5. Expression of the gene fragment encoding PRV gE epitopes in P. pastoris

2.2. Antibodies and sera Porcine anti-PRV polyclonal serum, HRP-conjugated mouse anti-porcine secondary antibody were gifts from College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, PR China. Standard positive and negative sera of PRV gE antibody (gE+ and gE− ), standard positive sera of four epidemic porcine disease: toxoplasmosis, hog cholera, chlamydia disease, porcine reproductive and respiratory syndrome, and field porcine serum samples were provided by the Key Laboratory of Veterinary Biotechnology, GuangDong Province, Guangzhou. 2.3. Construction of recombinant expression vector The recombinant expression vector pPICZ␣A-FS was constructed according to the standard protocols (Sambrook et al., 1989). A pair of primers was designed according

After phenotype identification and high-concentration ZeocinTM screening, about 20 multi-copy P. pastoris integrants (SMD1168 transformed with expression vector pPICZ␣A-FS) with Mut+ phenotype were selected. All the multi-copy integrants and a negative control strain (SMD1168 transformed with expression vector pPICZ␣A) were inoculated in well-aerated flasks containing BMGY, followed by shaking at 30 ◦ C until OD600 of the culture reached 2–6. Cells were harvested by centrifugation and pellets were resuspended with BMMY containing 1% casein (Sigma), making the OD600 = 1. Incubation was then continued at 30 ◦ C with 0.5% methanol added every 24 h to maintain induction. 72 h after induction, culture fluid was transferred to an eppendoff tube and centrifuged at 12,000 rpm for 15 min at room temperature. The supernatant was subjected to immediate assay or stored at −80 ◦ C until used. Culture fluids were withdrawn at 12, 24, 36, 48, 72, 96 and 120 h after induction to determine expression kinetics.

J.-q. Ao et al. / Journal of Virological Methods 114 (2003) 145–150

2.5.1. SDS-PAGE Culture supernatant was mixed with 2 × SDS-PAGE reducing loading buffer in equal volume, boiled for 5 min and then subjected to SDS-PAGE (12% separating gel with 5% stacking gel). Protein bands were visualized by staining with 0.1% AgNO3 (Frederic et al., 1995). 2.5.2. Western blotting Western blotting was carried out according to the protocols described by Sambrook et al. (1989). Porcine anti-PRV polyclonal serum and HRP-conjugated mouse anti-porcine antibody were used as primary and secondary antibody, respectively. 3,3 -Diaminobenzidine tetrahydrochloride (DAB, Sigma) was used as a substrate to visualize the reaction result. 2.5.3. Analysis of glycosylation For analysis of glycosylation, 50 ␮l culture supernatant, 10 ␮l 10× deglycosylation buffer (1 mol/l NaCl, 2 mol/l Na2 HPO4 ·3H2 O, 0.44 mol/l citric acid, pH 5.4), 38 ␮l deionized water and 2 ␮l deglycosylase Endo-H (0.01U, Sigma) were mixed, incubated at 37 ◦ C for 5 h, then subjected to 12% SDS-PAGE and Western blotting. 2.5.4. Protein concentration assay Protein concentration was determined by thin layer chromatogram scanning and Bradford total protein content assay (Frederic et al., 1995). Bovine serum albumin (BSA, Ameresco) was used as a standard. 2.6. ELISA 2.6.1. Antigen preparation Culture of P. pastoris integrant SMD1168/pPICZ␣A-FS at 72 h after induction was collected and centrifuged. The supernatant was dialyzed into deionized water overnight at 4 ◦ C for desalination. Dialysate was filtrated through 0.2 ␮m-pore-sized filter and the filtered fluid was used as a coating antigen immediately or stored at −80 ◦ C for further use. 2.6.2. Indirect PRV gE-ELISA procedure ELISA plates (Grenier) were coated at 4 ◦ C overnight with the prepared antigen diluted in carbonate buffer (22.4 mmol/l NaHCO3 , 11.9 mmol/l Na2 CO3 , pH 9.6). Each well was then thoroughly washed with phosphate-buffered saline containing 0.05% Tween-20 (PBS-T) and blocked with blocking solution (PBS-T containing 5% newborn bovine serum and 2% culture supernatant of SMD1168/pPICZ␣A) at 37 ◦ C for 1 h. Subsequently, plates were incubated with serum diluted with blocking solution at 37 ◦ C for 2 h and with HRP-conjugated secondary antibody at 37 ◦ C for 1 h. Between each incubation step, the plates were washed with PBS-T thoroughly. After the last incubation, freshly prepared chromogenic substrate (0.1 mg/ml tetramethylbenzidine, 0.04 mol/l citric acid, 0.1 mol/l Na2 HPO4 ·3H2 O, pH

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5.4, 3.15 × 10−3 % H2 O2 ) was added and the plates were incubated at room temperature for 10 min. Then, 50 ␮l 1 mol/l sulfuric acid was added to stop the colorimetric reaction. Optical density of each well was read at 450 nm using a Bio-Rad Microplate Reader Model 550. In addition to the sulfuric acid, all reagents were added at 100 ␮l volume. In each plate, standard gE+ and gE− serum was tested in triplicate as positive and negative controls. 2.6.3. Optimization of ELISA working conditions A panel of checkboard ELISAs was carried out to determine the optimal antigen concentration, sera dilution and blocking buffer composition. HRP-conjugated secondary antibody was fixed at a dilution of 1:8000, the optimum concentration recommended by the provider. 2.6.4. Determination of cut-off value Sixty PRV gE negative porcine sera from pseudorabiesfree herd were used to determine the cut-off threshold. All sera were subjected to gE-ELISA three times to abate the deviation. The S/N (S/N = OD450 of sample/mean OD450 of three negative controls) value was calculated, and the cut-off value was defined as the mean of all S/N values plus three standard deviations between them. 2.6.5. Reproducibility experiments Ten serum samples were tested in four different wells in one ELISA plate or on three different days. For each serum sample, the coefficients of variation of OD450 value between each well and each day were calculated and used for reproducibility assessment of gE-ELISA. 2.6.6. Cross-reactivity assay Standard positive sera of toxoplasmosis, hog cholera, chlamydia disease, porcine reproductive and respiratory syndrome were tested according to gE-ELISA procedure. S/N values were calculated. Each sample was tested three times. 2.6.7. Validation of gE-ELISA and comparison with commercial PRV ELISA Three hundred and forty-eight field serum samples were tested according to the gE-ELISA procedure and manufacturer’s instructions of a commercial PRV diagnostic kit: PRV gPI-Antibody Test Kit (IDEXX). Results were compared for the validation of gE-ELISA.

3. Results 3.1. Expression of the gene fragment encoding PRV gE epitopes in P. pastoris Seventy-two hours after induction, the expression product of the gene fragment encoding PRV gE epitopes could be seen in the culture supernatant of all P. pastoris

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Fig. 1. SDS-PAGE (a) and Western blotting (b) of expression product 72 h after induction. (M) Molecular weight standards; (1) culture supernatant of SMD1168/pPICZ␣A; (2) culture supernatant of SMD1168/ pPICZ␣A-FS-10; (3) deglycosylated culture supernatant of SMD1168/ pPICZ␣A-FS-10.

integrants (result not shown). One integrant designated as SMD1168/pPICZ␣A-FS-10, which exhibited the highest expression level, was selected for the following characterization of expression product. In SDS-PAGE, two differential proteins with molecular weight of approximately 38 and 32 kDa could be visualized in the culture supernatant of SMD1168/pPICZ␣A-FS-10. No similar bands appeared in that of negative control strain SMD1168/pPICZ␣A. After being deglycosylated by Endo-H, the 38 and 32 kDa proteins disappeared, and a new protein of approximately 23 kDa emerged, that was exactly corresponding to the expected molecular weight (Fig. 1a). In Western blotting, 38, 32 and 23 kDa proteins reacted positive with porcine anti-PRV serum (Fig. 1b). In order to determine expression kinetics, culture supernatant of SMD1168/pPICZ␣A-FS-10 was collected at 12, 24, 36, 48, 72, 96 and 120 h after induction and subjected to SDS-PAGE. From the result (Fig. 2), we noted that the molecular weight of the expression product changed greatly at different induction time. At 24 h, the expression product

Fig. 2. SDS-PAGE of expression product at different induction time. (M) Molecular weight standards; (1) culture supernatant of SMD1168/ pPICZ␣A at 72 h; (2–8) culture supernatant of SMD1168/pPICZ␣A-FS-10 at 12, 24, 36, 48, 72, 96 and 120 h.

Fig. 3. SDS-PAGE of culture supernatant before and after deglycosylation at different induction time. (M) Molecular weight standards; (1) culture supernatant of SMD1168/pPICZ␣A at 72 h; (2, 4 and 6) culture supernatant of SMD1168/pPICZ␣A-FS-10 at 24, 48 and 72 h; (3, 5 and 7) deglycosylated culture supernatant of SMD1168/pPICZ␣A-FS-10 at 24, 48 and 72 h.

was composed of three differential proteins with molecular weight of approximately 35, 38 and 40 kDa. At 36 h, a faint 32 kDa protein emerged. 12 h later, the expression product converted to a smear about 32–40 kDa. And at 72 h, it existed as two proteins of approximately 38, 32 kDa and changed little later. The result of thin layer chromatogram scanning combined with that of Bradford protein assay indicated that the expression product reached the maximum level at 72 h, amounting to 106.7 mg/ml and accounting for 66.67% of total culture supernatant proteins. When the culture supernatants at 24, 48 and 72 h were deglycosylated by Endo-H, all high molecular weight proteins converted to lower molecular weight ones (Fig. 3). The deglycosylated expression product at 24 h was composed of two proteins of approximately 32 and 23 kDa, and the former is about twice the latter in quantity. At 48 h, it consisted of a predominant 23 kDa protein with a spot of proteins of approximately 28 and 32 kDa. While at 72 h, the deglycosylated expression product was almost composed of solely 23 kDa protein. 3.2. gE-ELISA In checkboard ELISAs, antigen was diluted in two-fold dilutions from 1:20 to 1:2560, and sera were diluted from 1:5 to 1:40. In each dilution, the OD450 ratio between positive and negative serum reached or exceeded a value 2.1. The dilutions of antigen and serum were then set at 1:320 and 1:10, respectively, in which the OD450 value of the standard gE+ serum is 1.009, the smallest one greater than 1.0. On this test condition, each well was coated with 35.2 ng antigen, and PBS-T containing 5% NBS and 2% culture supernatant of SMD1168/pPICZ␣A provided the best blocking effect. On the optimized test conditions described above, the mean S/N value and standard deviation of sixty PRV gE negative sera were 0.9187 and 0.1478, respectively. The cut-off value was 1.3620. For a specific serum sample, when its reactive S/N value was greater than or equal to 1.3620, the

J.-q. Ao et al. / Journal of Virological Methods 114 (2003) 145–150 Table 1 Detection results of 348 samples by gE-ELISA and PRV gPI-Antibody Test Kit gPI-Antibody Test Kit

gE-ELISA Positive

Negative

Total

Positive Negative

133 19

23 173

156 192

Total

152

196

348

sample was scored as PRV gE antibody positive. Otherwise it was determined to be PRV gE antibody negative. In reproducibility experiments, the coefficients of variation of ten serum samples tested in four different wells in an ELISA plate or on three different days were 1.52–7.47% and 4.98–14.55%. This indicated the indirect gE-ELISA yielded a low and acceptable variation. S/N values of standard positive sera of four other porcine disease were 0.5392 ± 0.0180, 0.5636 ± 0.0122, 0.4991 ± 0.0202 and 0.5030 ± 0.0130, respectively. All were significantly less than the cut-off value, proving that the recombinant antigen was PRV specific. There was no observed cross-reactivity between the coating antigen and other porcine disease antibodies. Among 348 samples, 152 were detected as gE antibody positive and 196 as negative by gE-ELISA. For the PRV gPI-Antibody Test Kit, 156 were positive and 192 were negative. Among them 133 samples were detected as positive by both methods, and 173 samples were judged as negative by both methods (summarized in Table 1). Total coincidence rate between these two methods was 87.93%. χ2 tests found no significant difference existed between them (P > 0.05).

4. Discussion PRV gE is a typical membrane glycoprotein which spanned 577 amino acids. At its N-terminus there is an immunodominant region consisting of five distinct, conformation-dependent epitopes (Jacobs et al., 1990; Fuchs et al., 1990). Computer analysis showed there were two putative N-glycosylation sites in PRV Fa gE epitopes (deduced by PIR Pattern/Peptide Match at http://www-nbrf. georgetown.edu/pirwww/search/patmatch.html). The calculated molecular weight of gE epitopes expressed in P. pastoris is about 23 kDa, while SDS-PAGE and Western blotting revealed that the expression product was two recombinant proteins of approximately 38 and 32 kDa at 72 h after induction. Generally, P. pastoris adds N-linked oligosaccharide side chains composed of mannose residues to the expressed glycoproteins (Cereghino and Cregg, 2000) and Endo-H removes mannose glycans specifically. Based on that and the result of deglycosylation analysis, it could be inferred that both 38 and 32 kDa proteins were expression product of the gene fragment encoding gE epitopes

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in different glycosylated states. Interestingly, the molecular weight of the expression product changed over time. At 24 h after induction, it was composed of three high molecular weight proteins, which changed into two low molecular weight ones at 72 h. Deglyscosylation analysis proved that it was also because they were in different glycosylated state, for molecular weight of all differential proteins decreased after Endo-H treatment. No reports in the literature could be found documenting the molecular weight of glycoproteins expressed in P. pastoris changing over time. We postulated that the newly added oligosaccharide side chain might be unstable. With time, they experience a cleavage process and reach a steady state eventually. But how this process occur is not clear yet. Many of the gE-ELISAs developed are blocking ELISAs based on MAbs against one or two of the five epitopes (van Oirschot et al., 1988; Morenkovs et al., 1997; Gut-Winiarska et al., 2000). But as gE displays an antigenic drift (Ben-Porat et al., 1986; Mettenleiter et al., 1987), and antibody response against different epitopes of gE are considerably variable among individual pigs (Jacobs, 1994; Jacobs and Kimman, 1994), blocking ELISAs may give some false results. At this point, binding ELISAs based on the detection of antibody against whole protein or whole immunodominant region, such as indirect ELISA or indirect double-antibody sandwich ELISA, are superior because they are less dependent on gE antigenic modifications or the variation of epitope-specific antibody response. Since it was difficult to express full-length gE in heterologous expression systems (Kimman et al., 1996), and gE epitopes had been identified clearly (Jacobs et al., 1990; Fuchs et al., 1990), we expressed a gene fragment encoding gE epitopes in P. pastoris and developed an indirect PRV gE-ELISA with the recombinant expressed product as coating antigen. Results of reactions between this antigen and PRV gE standard positive or negative serum proved that the antigen could differentiate them quite effectively. Cross-reactivity assay revealed that this antigen was highly PRV specific. In a validation experiment, χ2 tests found that no significant difference existed between the detection results of gE-ELISA and a commercial PRV diagnostic kit (P > 0.05), despite some detection results are not in concordance. According to gene sequences of PRV glycoproteins in GenBank (data not shown), Fa strain is somewhat genetically distant from PRV isolate from other area of the world, this may account for some of the difference between detection results of current gE-ELISA and those of IDEXX kit. On the other hand, all sera used in this study were from China, in order to further warrant the effectiveness of gE-ELISA, antisera produced with PRV strains from other areas of the world should be included in the next test. Compared with former method of producing PRV gE antigen, recombinant expression in P. pastoris is superior due to its low cost, simple manipulation and high yields. The expressed gE epitopes accounted for approximately two thirds of total supernatant proteins. Just after centrifugation and desalination by dialysis, the culture supernatant could be

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used as coating antigen. This is the first report on the expression of a PRV gE gene fragment in P. pastoris. To our knowledge, this expression level is the highest among all reported recombinant expression of PRV gE in eukaryotic system. ELISA tests showed that the recombinant antigen was PRV specific and sensitive. In conclusion, we provide a new method to produce PRV diagnostic antigen in large quantities and at low cost. It is believed that this gE antigen will play a significant role in the eradication program of PRV after a further improvement of the ELISA system. Acknowledgements The authors appreciate Dr. Lou Gao-Ming for providing standard and field sera samples, and Mr. Du Wei-Xian and Yang Ao-Bing for their help in the ELISA. We are also grateful to Dr. Karen Duca, Sarah Penich and Simon Stevens for English language editing. References Ao, J.Q., Lou, G.M., Yang, L., Du, W.X., Long, Q.X., Wang, X.Z., Xie, M.Q., 2002. Cloning and sequencing of gE gene fragment encoding epitopes of pseudorabies virus Fa strain. Chin. J. Anim. Vet. Sci. 33, 92–495. Banks, M., 1994. Aujeszky’s disease ELISA using baculovirus expressed glycoproteins. Acta Vet. Hung. 42, 359–367. Ben-Porat, T., DeMarchi, J.M., Lomniczi, B., Kaolan, A.S., 1986. Role of glycoproteins of pseudorabies virus in eliciting neutralizing antibodies. Virology 154, 325–334. Calera, J.A., Paris, S., Monod, M., Hamilton, A.J., 1997. Cloning and disruption of the antigenic catalase gene of Aspergillus fumigatus. Infect. Immunol. 65, 4718–4724. Cereghino, J.L., Cregg, J.M., 2000. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 24, 45–66. Elbers, A.R., Braamskamp, J., Dekkers, L.J., Voets, R., Duinhof, T., Hunneman, W.A., Stegeman, J.A., 2000. Aujeszky’s disease virus eradication campaign successfully heading for last stage in the Netherlands. Vet. Q. 22, 103–107. Frederic, M.A., Robert, E.K., Seidman, J.G., Kevin, S., Roger, B., David, D.M., John, A.S., 1995. Short Protocols in Molecular Biology, third ed. Wiley, New York. Fuchs, W., Rziha, H.J., Lukàcs, N., Braunschweiger, I., Visser, N., Lütticken, D., Schreurs, C.S., Thiel, H.J., Mettenleiter, T.C., 1990. Pseudorabies virus glycoprotein gI: in vitro and in vivo analysis of immunorelevant epitopes. J. Gen. Virol. 71, 1140–1151. Gut, M., Jacobs, L., Tyborowska, J., Szewczyk, B., Bienkowska-Szewczyk, K., 1999. A highly specific and sensitive competitive enzyme-linked immunosorbent assay (ELISA) based on baculovirus expressed pseudorabies virus glycoprotein gE and gI complex. Vet. Microbiol. 69, 239–249. Gut-Winiarska, M., Jacobs, L., Kerstens, H., Bienkowska-Szewczyk, K., 2000. A highly specific and sensitive sandwich blocking ELISA based

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