Recombinant VP1 Protein Research Articles

High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

BackgroundChicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention.ResultsSignificantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay.ConclusionsPurified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.

Lactobacillus acidophilus as a live vehicle for oral immunization against chicken anemia virus

The AcmA binding domains of Lactococcus lactis were used to display the VP1 protein of chicken anemia virus (CAV) on Lactobacillus acidophilus. One and two repeats of the cell wall binding domain of acmA gene were amplified from L. lactis MG1363 genome and then inserted into co-expression vector, pBudCE4.1. The VP1 gene of CAV was then fused to the acmA sequences and the VP2 gene was cloned into the second MCS of the same vector before transformation into Escherichia coli. The expressed recombinant proteins were purified using a His-tag affinity column and mixed with a culture of L. acidophilus. Whole cell ELISA and immunofluorescence assay showed the binding of the recombinant VP1 protein on the surface of the bacterial cells. The lactobacilli cells carrying the CAV VP1 protein were used to immunize specific pathogen-free chickens through the oral route. A moderate level of neutralizing antibody to CAV was detected in the serum of the immunized chickens. A VP1-specific proliferative response was observed in splenocytes of the chickens after oral immunization. The vaccinated groups also showed increased levels of Th1 cytokines interleukin (IL)-2, IL-12, and IFN-γ. These observations suggest that L. acidophilus can be used in the delivery of vaccines to chickens.

In vitro and in vivo targeted delivery of IL-10 interfering RNA by JC virus-like particles

BackgroundRNA interference (RNAi) is a powerful tool to silence gene expression post-transcriptionally. Delivering sequences of RNAi in vivo remains a problem. The aim of this study was to use JC virus (JCV) virus-like particles (VLPs) as a vector for delivering RNAi in silencing the cytokine gene of IL-10.MethodsJCV VLPs were generated by recombinant JCV VP1 protein in yeast expression system. DNA fragment containing IL-10 shRNA was packaged into VLPs by osmotic shock.ResultsIn RAW 264.7 cells, IL-10 shRNA was found to reduce IL-10 expression by 85 to 89%, as compared with VLPs alone. IL-10 shRNA did not cross-react with TNF-alpha mRNA or influence the expression of TNF-alpha. In BALB/c mice IL-10 shRNA could reduce 95% of IL-10 secretion. Surprisingly, it also down regulated TNF-alpha expression.ConclusionsWe show for the first time that JCV VLPs empty capsids are competent vectors to deliver RNAi and are nontoxic to cells, suggesting that JCV VLPs is an efficient agent to deliver RNAi in both murine macrophage cells and BALB/c mice. This system provides an efficient means for delivering the RNAi for gene therapy purposes.

Development of immunodot blot assay for the detection of white spot syndrome virus infection in shrimps (Penaeus monodon)

The VP 28 gene encoding a structural envelope protein of the white spot syndrome virus (WSSV) was cloned into a pET32a(+) expression vector for the production of the recombinant VP28 protein. A purified recombinant protein of 39.9 kDa size was used for polyclonal antibody production in rabbit. Specific immunoreactivity of the rabbit anti rVP28 antiserum to the viral antigen was confirmed by a Western blot. The specificity of this polyclonal anti-rVP28 antiserum to detect the presence of the virus in WSSV-infected Penaeus monodon was verified using a immunodot blot assay. Immunodot blot showed a positive reaction in infected shrimp tissues with prominent colour development using 3,3′,5,5′-tetramethylbenzidine (TMB) as a chromogenic substrate when compared with 3–3′ diaminobenzidine tetrahydrochloride (DAB). Highest signal intensities of the immunodots were observed in infected shrimp pleopod extracts and haemolymph. On comparison with polymerase chain reaction (PCR), immunodot blot could detect 76% of PCR-positive WSSV-infected shrimp samples. Immunodot blot was found to be equivalent to first-step PCR sensitivity to detect WSSV particles estimated to contain 1.0 × 105 viral DNA copies.

Preparation and preliminary application of monoclonal antibody against Vp1 protein of chlamydiaphage ΦCPG1

Objective To express recombinant Vp1 protein of chlamydiaphage φCPG1, prepare monoclonal antibody against Vp1 protein and utilize it to screen clinical isolates of Chlamydia trachomatis. Methods The Vp1 protein was obtained by prokaryotic expression, and monoclonal antibody against this protein was prepared by hybridoma technique. ELISA and Western blot were used to identify monoclonal antibodies. Then,the monoclonal antibody was prepared in quantity by injecting hybridoma cells into the abdominal cavity of BALB/C mice, and purified by using protein G affinity chromatography. Clinical isolates of Chlamydia trachomatis were screened for the chlamydiaphage by immumofluorescence assay using the prepared monoclonal antibody.Results Purified Vp1 protein was obtained. The monoclonal antibody against Vp1 protein was gained after 3times of sub-clone and consistently identified as IgG1. Three hybridoma cell strains that stably secreted monoclonal antibody were generated. Chromosome analysis of hybridoma cells showed that the mean number of chromosome was 96, most of them were telocentric and a few were submetacentric. The titer of purified monoclonal antibody was more than 1: 12 800. Twenty clinical isolates were screened by using the monoclonal antibody and no positive results were obtained. Conclusions The monoclonal antibody against Vp1 protein of chlamydiaphage φCPG1 is successfully prepared, while no chlamydiaphage is detected by immumofluorescence assay using the prepared antibody in 20 clinical isolates of Ct. Key words: Chlamydiaphages; Capsid proteins; Antibodies, monoclonal; Chlamydia trachomatis

Protection of chickens, with or without maternal antibodies, against IBDV infection by a recombinant IBDV-VP2 protein

The use of avian herpesviruses (Marek's disease virus, MDV) as vectors to express the capsid protein of infectious bursal disease virus (IBDV) was well established, and its protection against IBDV challenge has been evaluated previously. However, there is little data about rMDV1 expressing the VP2 protein of IBDV protecting SPF and commercial chickens against virulent IBDV (vIBDV) challenge. In this study, we constructed a stable rMDV1 expressing the VP2 protein of IBDV by inserting the coding sequence within the US10 gene of MDVl by homologous recombination and designated this as rMDVl-US10L, and evaluated effectiveness of the recombinant VP2 protein with SPF chickens and commercial chickens with maternal antibodies in vIBDV challenge. The results can be summarized as follows: (1) We constructed a rMDV1 expressing IBDV-VP2 under the control of the MDV1 glycoprotein B (gB) promoter [rMDV1-US10L]. (2) rMDV-VP2 protein was readily expressed and induced 53% protection against a vIBDV challenge in SPF chickens with 10 3 PFU/chicken, whereas 10 4 PFU induced 73% protection. (3) Vaccination of commercial chickens having maternal antibodies to rMDV1-VP2 induced 87% protection in vIBDV challenge, which was similar to results using the live vaccine, BJ87 IBDV strain, in commercial chickens. These results demonstrate that the VP2 antigen expressed in the MDV vector was an effective and stable vaccine in correlation with the vaccine efficacy against lethal IBDV challenge, and can provide a better protective effect that is likely to persist for the life of the chickens.

A Rapid Assay for Detecting Antibody against Bluetongue Virus with a Latex Agglutination Test Using Recombinant VP7 Antigen

A latex agglutination test (LAT) for detecting antibody against Bluetongue virus (BTV) in ruminants was developed using latex beads coupled with recombinant VP7 protein. Compared with competitive enzyme-linked immunosorbent assay (ELISA), the specificity and sensitivity of the LAT were 99.0% and 93.0%, respectively. There was excellent agreement between the results obtained by competitive ELISA and the LAT (kappa = 0.930). Because it is rapid and easy to use, the LAT could be used for BTV antibody detection, especially for screening many serum samples.

Usefulness ofEscherichia coli-expressed Recombinant VP6 Proteins of Group A Rotavirus in Serodiagosis of Rotavirus Infection

†Department of Microbiology, Chung-Ang University College of Medicine, Seoul, Korea Purpose: The serologic diagnosis of rotaviral infections is not commonly used in clinical practice, but is used in seroepidemiologic studies. In this study, the usefulness of Escherichia coli-expressed recombinant VP6 proteins of group A rotavirus in the serodiagnosis of rotavirus infections by ELISA was evaluated. Methods: The recombinant VP6 proteins of group A rotavirus expressed in E. coli Rosetta II strain were purified and identified. One hundred sera from 22 children (4 healthy neonates, 13 healthy children, and 5 immunocompromised children) who had serial sera samples prior to and after rotavirus infections were provided by the Gyeongsang National University Hospital, a member of the National Biobank of Korea. IgG, IgA, and IgM antibodies against rVP6 were analyzed by ELISA in all of the patients and Western blot analysis in 4 neonates. Results: ELISA tests using rVP6 proteins of group A rotavirus as antigen revealed that IgG, IgA, and IgM antibodies increased after rotaviral infections in most neonates and healthy children. IgG antibodies also increased after rotaviral infections in most immunocompromised children without an adequate increase in IgM or IgA antibodies. Western blot analysis in four neonates revealed very early IgM antibody responses, even in the sera with low optical densities in ELISA tests. Conclusion: Our study showed that ELISA using rVP6 as an antigen is a valid diagnostic tool for seroepidemiologic studies of rotavirus infections and Western blot analysis is a sensitive test in detecting IgG, IgA, and and IgM antibodies in patients with rotavirus infections. (Korean J Pediatr Gastroenterol Nutr 2010; 13: 134∼145)

Expression and immunogenicity of recombinant polypeptide VP1 of human hepatitis A virus in stably transformed fruitfly (Drosophila melanogaster) Schneider 2 cells

We describe the secretory expression and immunogenicity of the recombinant HAV (hepatitis A virus) structural polypeptide VP1 from stably transformed Drosophila melanogaster S2 (Schneider 2) cells. Southern-blot analysis indicated that transformed S2 cells contained multiple copies of the HAV VP1 gene in the genome. Recombinant VP1 was secreted into a culture medium with a molecular mass of 42-49 kDa. A maximum production level of 6.24 mg of recombinant VP1/litre was obtained in a T-flask culture of Drosophila S2 cells 5 days after induction with 0.5 mM CuSO4. The recombinant HAV VP1 protein elicited the production of specific IgA in the small intestine by oral immunization and production of specific IgG in the serum by intraperitoneal immunization. Our findings show that secretory recombinant VP1 from transformed Drosophila S2 cells can be used as an effective experimental immunogen for research in vaccine development.

Application of VP1 protein to develop monoclonal antibody against foot-and-mouth disease virus Asia1 type

In order to develop an anti-FMDV Asia1 type monoclonal antibody (mAb), BABL/c mice were immunized with recombinant FMDV VP1 protein. Three mAbs, 1B8, 5E1 and 5E2, were then further optimized. The result indicated that prepared anti-FMDV Asia1 mAbs had no cross-reactivity with Swine vesicular disease (SVD) and FMDV O, A and C type antigen. Their titers in abdomen liquor were 1:5×106, 1:2×106 and 1:5×106, respectively. 1B8 was found to be of IgG1 subtype, 5E1 and 5E2 belonged to IgG2b subtype. In this study, the prepared mAbs are specific for detecting FMDV type Asia1, and is potentially useful for pen-side diagnosis.

Protection of Pacific White Shrimp, Litopenaeus vannamei against White Spot Virus Following Administration of N-terminus Truncated Recombinant VP28 Protein Expressed in Gram-positive Bacteria, Brevibacillus choshinensis

Protection of Pacific White Shrimp, Litopenaeus vannamei against White Spot Virus Following Administration of N-terminus Truncated Recombinant VP28 Protein Expressed in Gram-positive Bacteria, Brevibacillus choshinensis

Prokaryotic expression of truncated VP7 of bluetongue virus (BTV) and reactivity of the purified recombinant protein with all BTV type-specific sera

Purification of bluetongue virus (BTV) group-specific VP7 protein, expressed in prokaryotic system as histidine-tagged fusion protein is described in the present study. The major antigenic portion of VP7 gene of BTV 23 was amplified from the extracted RNA by reverse transcription polymerase chain reaction and cloned. The recombinant expression construct (pET-VP7) was identified by the polymerase chain reaction and sequencing analysis. Expression of histidine-tagged fusion truncated VP7 protein with a molecular mass of 36 kDa was determined by Western blot analysis using anti-His antibody. The expressed VP7 was purified to near homogeneity by chromatography on nickel-agarose column as judged by sodium dodesyl sulfate-polyacrylamide gel electrophoresis analysis. The purified VP7 protein was recognized by antibody to BTV in Western blot analysis. The capability of the recombinant VP7 protein to differentiate hyperimmune serum of rabbit to BTV from normal rabbit serum was evident in the enzyme-linked immunosorbent assay (ELISA). The purified VP7 reacted well with the 24 BTV serotype-specific sera obtained from OIE Reference Laboratory on bluetongue. Our results indicated that the expressed VP7 protein could be used as antigen for development of antibody-capture ELISA for detection BTV group-specific antibodies. This recombinant protein may also be used as antigen in competitive ELISA format.

Production, Characterization and Assay Application of a Purified, Baculovirus-Expressed, Serogroup Specific Bluetongue Virus Antigen

The predominant serodiagnostic assay used in many countries to detect bluetongue virus (BTV) infections is a competitive enzyme-linked immunosorbent assay (c-ELISA) which employs two critical reagents: a cell culture-derived BTV antigen and group-specific monoclonal antibody (Mab). Ongoing difficulties have been reported by laboratories in the production and quality control of the native antigen reagent which relies on the presence of adequate molar quantities and appropriate presentation of the major BTV core protein VP7. To address this important issue, a recombinant baculovirus was constructed containing a cDNA copy of genome segment 7 of BTV serotype 11 and used to infect insect cells which, in turn, expressed high levels of theVP7 protein with an estimated molecular mass of 39 kDa. In its purified form, this recombinant protein could be detected by group-specific Mabs designated 3.17.A3 and 8A3B.6 produced against BTV serotypes 1 and 17, respectively, as well as by polyclonal bovine antibodies raised against North American and South African BTV serotypes. No reactivity was observed by Western blot analysis with these two Mabs suggesting that the common antigenic determinants, on the BTV VP7 protein, were mainly conformational. It was interesting to note that the purified recombinant VP7 protein demonstrated a greater degree of reactivity with Mab 8A3B.6 compared to that exhibited with Mab 3.17.A3 when evaluated in an ELISA. Due to its antigenic similarity to the native antigen, the recombinant protein was found to be a suitable replacement for use in a c-ELISA to detect BTV-specific antibodies with the added advantage that it could be consistently produced and was, therefore, amenable to quality control testing for purity, stability and other standards.

Expression of VP1 protein in the milk of transgenic mice: A potential oral vaccine protects against enterovirus 71 infection

Enterovirus 71 (EV71) is the most common etiological agent detected in cases of hand-foot-and-mouth disease (HFMD) resulting in incidences of neurological complications and fatality in recent years. The clinical data have already shown the significant increase in recent EV71 epidemic activity throughout the Asia-Pacific region. Due to the lack of an effective antiviral agent, primary prevention of the disease, including the development of an effective vaccine, has been the top priority in terms of control strategies. In this study, we first generated a transgenic animal system to produce the EV71 VP1 capsid protein under the control of α-lactalbumin promoter and α-casein leader sequences. A high level of recombinant VP1 protein (2.51 mg/ml) was expressed and secreted into the milk of transgenic mice. Mouse pups that received VP1-transgenic milk orally demonstrated relatively better health conditions after challenge with the respective virus as compared with the non-transgenic milk fed group; moreover, the mice fed with the VP1-milk had body weights similar to those of the PBS placebo control groups. According to the serum-neutralization assay and serum antibody detection, the littermates suckling VP1-milk generated antibodies specific to EV71. Our data suggest that EV71 VP1-containing milk is suitable for development as a potential oral vaccine.

The VP1 protein of avian encephalomyelitis virus is a major host-protective immunogen that serves as diagnostic potential

Avian encephalomyelitis virus (AEV) is an important pathogen of poultry and is classified as a member of Picornaviridae. To investigate the protective immunity induced by AEV structural proteins, recombinant VP1, VP0, and VP3 proteins were expressed in a baculovirus system. The result of in vivo protection assays shows that the VP1 protein is a major host-protective immunogen against AEV challenge and demonstrates further that the antibody raised against VP1 protein could neutralize more effectively AEV infection than antibody against VP3 or VP0 protein in a virus neutralization test. These purified recombinant proteins were subsequently evaluated as enzyme-linked immunosorbent assay (ELISA) antigens for detection of AEV infection. A total number of 50 positive sera and 30 negative sera were tested for ELISA validation. Results obtained by testing 193 sera from chickens suspected of being infected AEV further showed that the diagnostic sensitivities of the VP1, VP3, and VP0 protein-based ELISAs were 98.1, 80.6, and 51.9%, and their specificities were 100, 87.9, and 81.8%, respectively. Both sensitivity and specificity of the VP1 protein-based ELISA were comparable with a commercially available test, indicating that the VP1 protein has a highly promising and reliable diagnostic potential, and thus is a suitable antigen for ELISA detection of AEV antibodies in chickens.

Characterization of virus-like particle assembly for DNA delivery using asymmetrical flow field-flow fractionation and light scattering

This study illustrates the application of asymmetrical flow field–flow fractionation (AF4) and light scattering analysis during the development of a gene delivery vehicle based on virus-like particles (VLPs) derived from the human polyoma JC virus. The analytical system was created by connecting an AF4 apparatus to the following detectors: diode array, fluorescence, multiangle light scattering, dynamic light scattering, and refractometer. From a single analysis, the molar mass, root mean square and hydrodynamic radii, composition, and purity of the sample could be determined. The VLPs were purified from baculovirus-infected Sf158 insect cells overexpressing the recombinant VP1 protein using weak anion exchange chromatography. The VLPs were dissociated to VP1 pentamers, and the contaminating DNA and proteins were removed using strong anion exchange chromatography. The gene delivery vehicle was created by reassembling the VP1 pentamers in the presence of the desired DNA. The newly formed VLPs encapsulated the DNA and were shown to be capable of delivering the gene of interest to target cells where it was translated into protein. This paper describes the scalable process that was derived to produce the VLPs and demonstrates how the AF4-based analytical characterization was indispensable during the development process.

Seroprevalence of antibody against human bocavirus in Beijing, China

To find out the importance of human bocavirus (HBoV) as an infectious agent for population in Beijing, China, seroprevalence study was conducted by using expressed recombinant major capsid VP2 protein as an antigen. Serum specimens collected from infants and children who visited the Children's Hospital Affiliated to the Capital Institute of Pediatrics for health check up and adults visited the Xuanwu Hospital, Beijing for diseases other than respiratory infections from April 1996 to March 1997 were used for investigation. The major capsid protein VP2 from HBoV was expressed in E. coli strain BL21 (DE3) with the transformed PET30b vector inserted with full-length VP2 gene of HBoV and the specific antigenicity of this expressed protein was validated by previous study. Western blot was used to detect specific IgG antibody against HBoV in collected serum specimens diluted to 1:200. Mock expressed protein was E. coli cells strain BL21 (DE3) with the transformed PET30b vector without insert. Anti-His monoclonal antibody and rabbit anti-HBoV VP2 polypeptides hyper-immune serum were used as positive control for antibody detection. Out of 677 serum specimens tested, 400 (59.1%) were positive by Western blot. About 45.3% (34/75) of the newborns under 1 month of age had anti-HBoV antibodies, and antibody positive rates were lower in the age groups of 1 and 2 months (41.4% and 31.3%, respectively) and were higher in the following ages from 6 months to 7 years (from 45.6% to 69.7%). The antibody positive rates were at a relatively constant level (about 70%) in the age groups from 7 years to 40 years and became lower (61.8% - 62.8%) in groups of age over 50 years. The high seroprevalence against recombinant HBoV VP2 protein and early age antibody acquisition indicate that HBoV has been circulating in Beijing, China as early as in 1996 and most of children had been exposed to HBoV by the age of 7 years. Infants under the age of 6 months were susceptible to infection with this virus.

Detection of Antibodies to Infectious Bursal Disease Virus (IBDV) by Agar Gel Immunodiffusion using Recombinant VP2 Protein

Infectious bursal disease virus (IBDV) causes a highly contagious and immunosuppressive disease of chicken. Agar gel immunodiffusion using IBDV antigen extracted from bursa of Fabricius of infected chicken has been used officially for diagnosis of IBDV in Korea. In this study, in order to replace the IBDV whole virus antigen with non-infectious antigen, recombinant VP2 protein (rVP2) of IBDV was produced using recombinant baculovirus expression system. Purified baculovirus-expressed rVP2 was used as an antigen in an agar gel immunodiffusion (AGID). rVP2 antigen precipitated specifically IBDV antibodies. AGID using rVP2 antigen detected anti-IBDV antibodies from 6 dpi to 28 dpi (termination of the experiment) when specific pathogen free chickens were experimentally infected with IBDV 52/70 strain. This was consistent with result by AGID using IBDV antigen, virus neutralization test (VNT) and a commercial ELISA kit (except for one serum). The sensitivity of rVP2 was the same with that of IBDV antigen when field sera (n=324) were tested by AGID. However, AGID using rVP2 antigen detected maternal antibodies from broiler chickens (n=20) on a broiler farm up to 15 days old, although the detection rate of the AGID was relatively low compared to a commercial ELISA kit. Our results indicate that IBDV whole virus antigen from IBDV infected chickens would be replaced with recombinant VP2 protein as an antigen for AGID.

Mapping of epitopes of VP2 protein of chicken anemia virus using monoclonal antibodies

To map the epitopes of VP2 protein of chicken anemia virus (CAV), VP2 was expressed as a fusion protein in Escherichia coli BL21 (DE3). The Western blot demonstrated that recombinant VP2 protein could be recognized by sera of chickens infected with CAV. Female BALB/c mice were immunized with purified recombinant VP2 produced in E. coli BL21 (DE3) and seven VP2-specific monoclonal antibodies (MAbs) were developed. The results of Western blot showed that all the seven MAbs recognized the recombinant VP2 protein expressed in the baculovirus and reacted with MDCC-MSB1 cells infected with CAV by indirect immunofluorescence assay. The VP2 protein was dissected into 21 overlapping fragments, expressed as fusion peptides in E. coli and used for epitope mapping by pepscan analysis. ELISA and Western blot assays indicated that most of MAbs reacted with the 12th and 13th fragments (amino acids 111–136) and one of them reacted with the 3rd fragment (amino acids 21–36). The linear immunodominant epitope of VP2 was located mainly in amino acid residues 111–126 and 121–136.

Expression of the VP2 gene of classical D78 infectious bursal disease virus in the methylotrophic yeast Pichia pastoris as a secretory protein

Infectious bursal disease virus (IBDV) is the causative agent of Gumboro disease, an infectious disease of global economic importance in poultry. The expression of heterologous proteins in P.pastoris is fast, simple and inexpensive. In this study, VP2 encoding gene of classical D78 IBDV was amplified using reverse transcription (RT) polymerase chain reaction (PCR) and cloned into pPICZαA vector. Recombinant plasmid DNA was integrated into the chromosome of the transformed Pichia pastoris by electroporation and expressed protein identified by SDS- PAGE and western blotting. A recombinant protein was secreted into the supernatant from the yeast when induced with methanol. The expressed target protein in supernatant was bound with chicken anti IBDV Polyclonal antibodies. Western blotting with antibodies against D78 IBDV indicated that the recombinant VP2 protein retained its antigenicity. The concentration of secreted VP2 protein was 0.67mg/l.The production of recombinant VP2 protein indicated that P. pastoris was an efficient secreted expression system for D78 IBDV.