Insect Cell Lysates Research Articles

Identification of Epitope on DNA-binding Protein Expressed in Insect Cell Infected by Baculovirus

DNA-binding protein (DBP) is an early gene product produced during viral replication. Polyclonal anti-DBP was produced using rabbit by intradermal injections of Escherichia coli-expressed purified recombinant DBP. Prepared anti-DBP completely blocked the replication of baculovirus in insect cells. The anti-DBP binding to DBP was confirmed by both Western blotting with Tn-5B1-4 insect cell lysates as well as immunostained baculovirus-infected Tn-5B1-4 insect cells. To determine the anti-DBP epitope 12 peptides were synthesized and their specific-binding activities were measured using ELISA. Based on specific-binding activity against anti-DBP the epitope was predicted to be between amino acid residues 248-265 (QRMSVEDFDRLFEMDKID). Especially from 18 amino acid residues it was further to be narrowed between amino acid residues 260-265 (EMDKID) which showed a critical role in specific-binding activity.

Application of the ferrous oxidation–xylenol orange assay for the screening of 5-lipoxygenase inhibitors

5-Lipoxygenase (5-LO) is the key enzyme involved in leukotriene synthesis and its improper regulation is implicated in several inflammatory diseases. A rapid and sensitive assay for 5-LO activity suitable for high-throughput format is not yet available. In this study, we examined whether the ferrous oxidation–xylenol orange (FOX) assay could be applicable for the high-throughput screening of 5-LO inhibitors. Using insect cell lysates overexpressing rat 5-LO, the effects of cofactors of 5-LO such as ATP, Ca 2+, and l-α-phosphatidylcholine (PC) on the color development of FOX reagents were investigated. ATP quenched substantially color development by hydroperoxide, an intermediate of 5-LO reaction, and an optimum concentration of ATP with little interference was determined as 20 μM. Ethylenediaminetetraacetate (0.4 mM) also affected the complex formation with FOX reagents. On the other hand, neither Ca 2+ nor PC influenced complex formation with FOX reagents. Under optimized assay conditions, zileuton, a 5-LO-specific inhibitor, exhibited inhibitory potency (IC 50 values of 0.1–0.2 μM) similar to that determined by the conventional spectrophotometric assay. Taken together, this study shows that the FOX assay with some modifications can be employed for high-throughput assay format for the measurement of 5-LO activity at the stage of primary screening.

517. Type 1 Rep52 Is Superior to Authentic Rep52 for Producing Recombinant Adeno-Associated Virus Type 5 in Insect Cells

Top of pageAbstract The recently described method for producing recombinant adeno-associated virus (rAAV) type 2 in insect cells facilitates scale-up by using suspension cell cultures and baculovirus expression vectors (Hum Gene Ther 13:1935-1943, 2002). AAV5 is one of the most divergent dependovirus characterized and has been shown to utilize different receptors than AAV2 for example, and has demonstrated different tissue tropism from other serotype rAAVs. In addition to vectors derived from serotype 2, other serotypes constitute a vector set from which an optimal one can be selected for specific applications. Thus, we established a method to generate rAAV5 in insect cells. The current process requires triple infection with recombinant baculoviruses that provide the following: 1. AAV structural proteins that form the virus capsid (VP 1, 2, 3); 2. two of the AAV non-structural proteins for replication and encapsidation (Rep 78 and Rep 52); and 3. the AAV vector DNA which contain the gene of interest flanked by the AAV origins of replication (ITRs). Compared to other rAAV serotypes produced in insect cells, the rAAV5 yield per cell was substantially lower and we tested some modifications of the system. The initial Rep baculovirus drove type 5 Rep72 expression with a truncated promoter for the immediate-early 1 gene of Orgyia pseudotsugata nuclear polyhedrosis virus. The titers of the Rep baculovirus were relatively lower than others. We constructed Rep baculoviruses with a series of truncated p10 promoters for Rep78 and selected one that could produce rAAV5 at a high titer and propagate well. Rep52 or small Rep protein packages AAV genome into preformed empty capsids. We examined Rep52 of other serotypes, 1, 2, 3, 4 for generation of rAAV5 particles. The titer of rAAV5-GFP produced with type 1, 2, 3, or 4 small Rep was 56,000|[plusmn]|3,200, 41,000|[plusmn]|18,900, 42,000|[plusmn]|7,300, or 39,000|[plusmn]|3,500 particles per Sf9 cell while rAAV5-GFP produced by authentic Rep52 was 13,500|[plusmn]|3,200. The rAAV5-GFP produced with either serotype small Rep has an equal ratio of nucleic acid to capsid protein assessed by real-time PCR quantification and silver staining of purified rAAV5 particles. Also, rAAV5-GFP produced with either combination of large and small Rep proteins, transduced the simian Cos cell line with similar efficiency. The analysis by cesium density gradient of insect-cell lysates indicated that approximately 50% of all capsids contained the vector genome. These results indicate that heteroserotypic small Rep polypeptide is able to substitute for AAV5 small Rep and package AAV vector genome with type 5 in Sf9 cells and the new Rep baculovirus expressing type 5 Rep78 under the control of a truncated p10 promoter and type 1 Rep52 will contribute to the development of more efficient production of rAAV5 in insect cells.

Design of a Novel Class of Peptide Inhibitors of Cyclin-dependent Kinase/Cyclin Activation

Cyclin dependent kinases (CDKs) are key regulators of the cell cycle progression and therefore constitute excellent targets for the design of anticancer agents. Most of the inhibitors identified to date inhibit kinase activity by interfering with the ATP-binding site of CDKs. We recently proposed that the protein/protein interface and conformational changes required in the molecular mechanism of CDK2-cyclin A activation were potential targets for the design of specific inhibitors of cell cycle progression. To this aim, we have designed and characterized a small peptide, termed C4, derived from amino acids 285-306 in the alpha5 helix of cyclin A. We demonstrate that this peptide does not interfere with complex formation but forms stable complexes with CDK2-cyclin A. The C4 peptide significantly inhibits kinase activity of complexes harboring CDK2 in a competitive fashion with respect to substrates but does not behave as an ATP antagonist. Moreover, when coupled with the protein transduction domain of Tat, the C4 peptide blocks the proliferation of tumor cell lines, thereby constituting a potent lead for the development of specific CDK-cyclin inhibitors.

Expression, purification, and inhibition of human RET tyrosine kinase

Tyrosine kinases are emerging as frequent targets of primary oncogenic events and therefore represent an optimal focus of therapeutical intervention. Genetic alterations that cause dysregulated activation of the RET tyrosine kinase are responsible for a significant fraction of thyroid carcinomas. In an effort towards therapeutic RET inactivation, we have developed a method for expression and purification of recombinant RET catalytic domain for structural purposes and for use in the screening of potential inhibitors of RET kinase activity. His-tagged RET kinase domain was purified from Sf9 insect cell lysate using a two-step chromatographic protocol and characterised. Purified recombinant RET phosphorylated itself and exogenous substrates at physiological pH. A specific peptide substrate, derived from RET activation loop, was identified and experimentally validated. These reagents were used to develop a rapid ELISA-based kinase assay for screening potential inhibitors. Novel RET inhibitors were identified using this assay.

Evidence for a direct interaction between HSV-1 ICP27 and ICP8 proteins

Herpes simplex virus 1 (HSV-1) ICP27 and ICP8 proteins have both been implicated in the transcription of late genes and regulation of viral gene expression. We showed previously that ICP27 and ICP8 associate with the RNAP II holoenzyme (Zhou and Knipe, J. Virol. 76, 5893–5904). Here, we demonstrate that ICP27 and ICP8 coprecipitate from lysates of HSV-1-infected HEp2 cells and from lysates of insect cells expressing ICP27 and ICP8, the latter being in the absence of other HSV-1 proteins. By expressing and purifying hexahistidine-tagged ICP8 (His-ICP8) and maltose binding protein (MBP)-tagged ICP27 (MBP-27) proteins and performing in vitro immunoprecipitation and pull-down assays, we also demonstrate that ICP27 and ICP8 coprecipitate in the absence of other viral or cellular proteins. Taken together, these data provide evidence that ICP27 and ICP8 interact directly in vitro and in infected cells. We hypothesize that the ICP27–ICP8 interaction plays a role in the stimulation of late gene transcription.

No requirement of HCV 5'NCR for HCV-like particles assembly in insect cells.

To express all three HCV structural proteins in the presence or absence of HCV 5'NCR to investigate the requirement of 5'NCR for the assembly of HCV-like particles in insect cells. HCV structural protein encoding sequences CE1E2 and 5'NCR-CE1E2 were amplified with PCR. Recombinant baculovirus were constructed with recombinant DNA techniques. HCV structural proteins expressed in insect cells were analyzed by immunofluorescence and SDS-PAGE. Immunoprecipitation experiment of insect cell lysates with anti-E2 monoclonal antibody (MAb) was carried out and the immunoprecipitated proteins were subjected to SDS-PAGE and immunoblotting with anti-C, anti-E2 MAbs and HCV positive serum. The virus-like particles in insect cells were visualized by electron microscopy (EM). The HCV-like particles were purified by sucrose gradient centrifugation and identified by EM and immune aggregation EM. The recombinant baculovirus reBV/CE1E2 containing HCV C, E1, E2 genes and reBV/CS containing the same structural protein genes plus 5'NCR were constructed. The insect cells infected with either reBV/CE1E2 or reBV/CS expressed HCV C, E1 and E2 proteins with a molecular weight of 20 kD, 35 kD and 66 kD respectively. The results of immunoprecipitation and the immunoblotting revealed the coimmunoprecipitation of C, E1, and E2 proteins, indicating the interaction of HCV structural proteins expressed in insect cells. Electron microscopy of insect cells infected with reBV/CE1E2 or reBV/CS demonstrated spherical particles (40 to 60 nm in diameter) similar to the HCV virions from sera or hepatic tissues of HCV infected humans. The HCV-like particles were partially purified by sucrose gradient centrifugation, and the purified VLPs showed immuno-reactivity with anti-HCV antibodies. HCV 5'NCR is not required for the assembly of HCV-like particles in insect cells, HCV core and envelope proteins are sufficient for viral particle formation.

Antipeptide antibodies for detecting crab ( Callinectes sapidus) molt-inhibiting hormone

In crustaceans, the synthesis of ecdysteroid molting hormones is regulated by molt-inhibiting hormone (MIH), a neuropeptide produced by an eyestalk neuroendocrine system, the X-organ/sinus gland complex. Using sequence analysis software, two regions of the blue crab ( Callinectes sapidus) MIH peptide were selected for antibody production. Two 14-mer peptides were commercially synthesized and used to generate polyclonal antisera. Western blot analysis revealed that each antiserum bound to proteins of the predicted size in extracts of C. sapidus sinus glands, and lysates of insect cells containing recombinant MIH. Thin section immunocytochemistry using either antiserum showed specific immunoreactivity in X-organ neurosecretory cell bodies, their associated axons and collaterals, and their axon terminals in the sinus gland.

Avian polyomavirus agnoprotein 1a is incorporated into the virus particle as a fourth structural protein, VP4.

Agnoproteins, encoded by the 5'-region of the late bicistronic mRNA of some polyomaviruses, are small proteins with largely unknown functions. In avian polyomavirus (APV)-infected cells, mRNAs of seven putative agnoproteins have been observed. Recently, it has been shown that agnoprotein 1a and its truncated variant agnoprotein 1b, encoded by the predominant mRNA species, are essential for APV replication. Here, the presence of agnoprotein 1a is demonstrated in the nucleus of APV-infected cells and in purified APV particles. Interaction between agnoprotein 1a and the major structural protein, VP1, was demonstrated by co-immunoprecipitation experiments using lysates of recombinant baculovirus-infected insect cells. With proteins expressed in E. coli, binding to double-stranded DNA in a sequence-unspecific manner was shown for agnoprotein 1a, whereas agnoprotein 1b failed to bind. A leucine zipper-like motif present in agnoprotein 1a is considered to be involved in DNA binding. Due to the absence of any structural or functional homologies between APV agnoprotein 1a and the agnoproteins of mammalian polyomaviruses, it is suggested that this protein should be renamed VP4, indicating its function as a fourth structural protein of APV.

Cloning of three novel neuronal Cdk5 activator binding proteins

Neuronal Cdc2-like kinase (Nclk) is involved in the regulation of neuronal differentiation and neuro-cytoskeleton dynamics. The active kinase consists of a catalytic subunit, Cdk5, and a 25 kDa activator protein (p25 nck5a) derived from a 35 kDa neuronal-specific protein (p35 nck5a). As an extension of our previous study (Qi, Z., Tang, D., Zhu, X., Fujita, D.J., Wang, J.H., 1998. Association of neurofilament proteins with neuronal Cdk5 activator. J. Biol. Chem. 270, 2329–2335), which showed that neurofilament is one of the p35 nck5a-associated proteins, we now report the isolation of three other novel p35 nck5a-associated proteins using the yeast two-hybrid screen. The full-length forms of these three novel proteins, designated C42, C48 and C53, have a molecular mass of 66, 24, and 57 kDa, respectively. Northern analysis indicates that these novel proteins are widely expressed in human tissues, including the heart, brain, skeletal muscle, placenta, lung, liver, kidney and pancreas. The bacterially expressed glutathione S-transferase (GST)-fusion forms of these three proteins were able to co-precipitate p35 nck5a complexed with Cdk5 from insect cell lysate. Among these three proteins, only C48 and C53 can be phosphorylated by Nclk, suggesting that they may be the substrates of Nclk. Sequence homology searches have suggested that the C48 protein is marginally related to restin protein, whereas the C42 protein has homologues of unknown function in Caenorhabditis elegans and Arabidopsis thaliana.

Expression of YAV proteins and vaccination against viral ascites among cultured juvenile yellowtail.

Yellowtail ascites virus (YAV) is a member of the family Birnaviridae and causes viral ascites among juvenile yellowtail (Seriola quinqueradiata). We have reported the cloning and expression of two viral cDNAs, the first being segment A encoding a polyprotein of viral capsid proteins (VP2 and VP3) and a protease (NS), and the second being VP2-epitope encoding serotype-specific epitope region on VP2, using a baculovirus expression system. Another viral cDNA encoding a polyprotein of NS and VP3 was cloned and expressed in this study. For the expression of NS/VP3 (YAV nt 1626 to 3066) in insect cells a 31-kDa protein, corresponding to VP3 was detected, indicating an appropriate posttranslational processing of NS/VP3 polypeptide by NS protease itself. The analysis of the N-terminal amino acid sequence of this protein showed that NS protease may cleave an Ala-Ser bond. A study of the potential for vaccination of yellowtail fry by injection of insect cell lysates infected with baculovirus, containing either cDNA of segment A, VP2-epitope, or NS/VP3 was undertaken. Only a vaccination with cell lysates infected with a recombinant virus carrying the full length of YAV segment A gene demonstrated approximately the same effect as that of inactivated YAV. This result suggested that all proteins VP2, VP3, and NS are required for an effective vaccination.

Cingulin contains globular and coiled-coil domains and interacts with ZO-1, ZO-2, ZO-3, and myosin.

We characterized the sequence and protein interactions of cingulin, an M(r) 140-160-kD phosphoprotein localized on the cytoplasmic surface of epithelial tight junctions (TJ). The derived amino acid sequence of a full-length Xenopus laevis cingulin cDNA shows globular head (residues 1-439) and tail (1,326-1,368) domains and a central alpha-helical rod domain (440-1,325). Sequence analysis, electron microscopy, and pull-down assays indicate that the cingulin rod is responsible for the formation of coiled-coil parallel dimers, which can further aggregate through intermolecular interactions. Pull-down assays from epithelial, insect cell, and reticulocyte lysates show that an NH(2)-terminal fragment of cingulin (1-378) interacts in vitro with ZO-1 (K(d) approximately 5 nM), ZO-2, ZO-3, myosin, and AF-6, but not with symplekin, and a COOH-terminal fragment (377-1,368) interacts with myosin and ZO-3. ZO-1 and ZO-2 immunoprecipitates contain cingulin, suggesting in vivo interactions. Full-length cingulin, but not NH(2)-terminal and COOH-terminal fragments, colocalizes with endogenous cingulin in transfected MDCK cells, indicating that sequences within both head and rod domains are required for TJ localization. We propose that cingulin is a functionally important component of TJ, linking the submembrane plaque domain of TJ to the actomyosin cytoskeleton.

Substitutions in the C-terminal portion of the catalytic domain partially reverse assembly defects introduced by mutations in the N-terminal linker sequence of cytochrome P450 2C2.

Mutations in a 7-amino acid linker segment, immediately following the N-terminal signal anchor sequence of cytochrome P450 2C2, have been shown to affect proper assembly of hemoprotein and decrease activity of the mutants expressed in COS cells. In contrast, C2pmBalC1, in which cytochrome P450 2C1 residues were substituted for those of cytochrome P450 2C2 in the C-terminal region, exhibited increased activity when expressed in COS-1 cells. To examine further the basis for the increased activity of C2pmBalC1 in COS-1 cells, the protein was expressed in insect cells and Escherichia coli. The amounts of the functional P450 species of C2pmBalC1 expressed in these systems and the ratios of P450 to P420 were greater than those of cytochrome P450 2C2, indicating that more efficient assembly underlies the increased activity of C2pmBalC1. To determine whether the C-terminal substitutions could compensate for the decreased assembly mediated by the N-terminal linker mutations, the linker mutations were introduced into C2pmBalC1. If all 7 amino acids in the linker were deleted, no enzymatically active cytochrome P450 2C2 or C2pmBalC1 was detected in COS-1, insect, or bacterial cells expressing the mutants. The mutant C2A2, in which two alanines were substituted for the linker, had no detectable laurate hydroxylase activity in COS-1 cells, and minor amounts of hemoprotein for this mutant were expressed in E. coli and insect cells. In contrast, the same mutation in C2pmBalC1 reduced activity only 50% in COS-1 cells and markedly elevated levels of P450 expression in bacteria and insect cells. The A2 mutation did not affect the enzymatic activity of either cytochrome P450 2C2 or C2pmBalC1 assayed in whole cell lysates of insect cells but reduced the activity of partially purified enzymes assayed in a reconstituted assay system. These findings indicate that mutations introduced into the C-terminal region of P450 2C2 can facilitate assembly of the proteins and partially reverse the decreased assembly resulting from the N-terminal mutations.

The p16(INK4a) tumour suppressor protein inhibits alphavbeta3 integrin-mediated cell spreading on vitronectin by blocking PKC-dependent localization of alphavbeta3 to focal contacts.

Expression of full-length p16(INK4a) blocks alphavbeta3 integrin-dependent cell spreading on vitronectin but not collagen IV. Similarly, G1-associated cell cycle kinases (CDK) inhibitory (CKI) synthetic peptides derived from p16(INK4a), p18(INK4c) and p21(Cip1/Waf1), which can be delivered directly into cells from the tissue culture medium, do not affect non-alphavbeta3-dependent spreading on collagen IV, laminin and fibronectin at concentrations that inhibit cell cycle progression in late G1. The alphavbeta3 heterodimer remains intact after CKI peptide treatment but is immediately dissociated from the focal adhesion contacts. Treatment with phorbol 12-myristate 13-acetate (PMA) allows alphavbeta3 to locate to the focal adhesion contacts and the cells to spread on vitronectin in the presence of CKI peptides. The cdk6 protein is found to suppress p16(INK4a)-mediated inhibition of spreading and is also shown to localize to the ruffling edge of spreading cells, indicating a function for cdk6 in controlling matrix-dependent cell spreading. These results demonstrate a novel G1 CDK-associated integrin regulatory pathway that acts upstream of alphavbeta3-dependent activation of PKC as well as a novel function for the p16(INK4a) tumour suppressor protein in regulating matrix-dependent cell migration.

A Bacterial Signal Peptidase Enhances Processing of a Recombinant Single Chain Antibody Fragment in Insect Cells

The production of an antibody single chain fragment (scFv) in insect cells was accompanied by the formation of an insoluble intracellular precursor even with the inclusion of the bee melittin signal peptide. The presence of the precursor polypeptide suggests a limitation in the processing of the signal peptide so a baculovirus containing a signal peptidase fromBacillus subtilis(SipS) was constructed for expression studies. When the wild type SipS was coexpressed with scFv, preprocessed scFv fragments were no longer detected in insect cell lysates. Conversely, coexpression of scFv alone or with an inactive mutant SipS resulted in at least 30% of the intracellular polypeptide in an unprocessed form at 3 days post infection. Production of scFv in the medium was also enhanced in the presence of SipS; however, low secretion levels indicate the presence of a post-processing bottleneck.

Fast isolation of recombinant baculovirus by antibody screening.

We describe a rapid and efficient scheme for the isolation and purification of recombinant baculoviruses. The method is based on the detection of foreign proteins in cellular lysates of baculovirus-infected insect cells by antibody screening. The recombinant virus is purified by repeated serial dilutions. The method allows the identification and purification of recombinant viruses within 2 to 3 wk. This procedure selects for recombinant baculoviruses that highly overproduce the desired protein product.

The G11 Gene Located in the Major Histocompatibility Complex Encodes a Novel Nuclear Serine/Threonine Protein Kinase

Protein kinases are involved in signal transduction pathways and play fundamental roles in the regulation of cell functions. Here we report that the gene G11 located in the human major histocompatibility complex encodes a novel Ser/Thr protein kinase. The G11 gene products of 41.5 and 30 kDa were expressed in insect cells using the baculovirus system and transiently in the mammalian cell line COS-7. It was found that after immunoprecipitation of the G11 polypeptides from recombinant baculovirus-infected insect cell lysates or transfected COS-7 cell lysates the immunoprecipitates contained a Mn2+-dependent protein kinase activity that phosphorylated alpha-casein at Ser/Thr residues and histone at Ser residues. Furthermore, mutation of the ATP-binding site by converting the invariant lysine in the catalytic domain (amino acid 317) to a proline resulted in the complete ablation of the enzyme activity. This was consistent with the observation that the G11 polypeptide can be covalently modified by the reactive ATP analogue 5'-p-fluorosulfonylbenzoyladenosine in the absence of ATP, and that this modification is prevented in the presence of 1 mM ATP, indicating that the kinase domain of the G11 polypeptide is capable of binding ATP. Immunofluorescence staining of transfected COS-7 cells transiently expressing G11 revealed that this novel Ser/Thr protein kinase is localized predominantly in the nucleus.

The Late Expression Factors 8 and 9 and Possibly the Phosphoprotein p78/83 of Autographa californica Multicapsid Nucleopolyhedrovirus Are Components of the Virus-Induced RNA Polymerase

Attempts were made to identify some of the subunits of the baculovirus-induced RNA polymerase following purification of its enzymatic activity by conventional chromatography. Polymerase activity was extracted from lysates of insect cells infected with Autographa californica multicapsid nucleopolyhedrovirus by polyethylenimine precipitation and subsequently purified by phosphocellulose, anion exchange, poly(A) Sepharose affinity, and gel filtration chromatography. The presence of the polymerase was monitored by its α-amanitin-resistant activity in in vitro transcription assays. A number of polypeptides associated with the enzymatic activity were identified. Peptide-specific antibodies were generated against a variety of late-expression factors (LEFs) and these antibodies, along with antisera directed against several other baculovirus proteins, were used in an immunoblot analysis of the purified polymerase. The results revealed that both the viral helicase (p143) and the virogenic stroma protein, pp31, copurify with the baculovirus-induced RNA polymerase activity through several chromatographic steps and may be loosely associated with the RNA polymerase. LEF8, LEF9 and p78/83, a nucleocapsid-associated phosphoprotein, were found to associate with the viral-induced polymerase activity. LEF8 and LEF9 contain regions of sequence homology with components of other DNA-directed RNA polymerases, while a portion of p78/83 exhibits some homology to the sigma factor of bacterial RNA polymerase, the RAP30 protein found in the mammalian transcription complex TFIIF, and the RAP94 polypeptide associated with vaccinia virus RNA polymerase. The p78/83 protein has previously been shown by our laboratory to be a capsid protein, but it may also play some role with the RNA polymerase. These results represent a first attempt to identify specific components of the RNA polymerase associated with infections of insect cells by A. californica nucleopolyhedrovirus.

Antigenic and Immunogenic Properties of Baculovirus-Expressed Infectious Bursal Disease Viral Proteins

Genomic segment A of the variant Md infectious bursal disease virus (IBDV) was amplified by reverse transcriptase/polymerase chain reaction, cloned, and expressed in the baculovirus expression system. Three different baculovirus recombinants expressing the genes encoding VP2, VP2/VP4, and the complete polyprotein (VP2/VP4/VP3) were prepared. The three antigens were used in separate enzyme-linked immunosorbent assays, and each detected antibodies against the variant IBDV strains Md, Del-A, Del-E, and GLS and the classic IBDV strain D78 throughout a 14-wk period following vaccination. Eight-week-old specific-pathogen-free (SPF) chickens were inoculated subcutaneously using the VP2/VP4 or VP2/VP4/VP3 baculovirus recombinant or wild-type baculovirus-infected insect cell lysates. Virus-neutralizing antibodies were detected in the VP2/VP4 and VP2/VP4/VP3 baculovirus-inoculated birds at 13 days postinoculation (PI) and at 43 days PI when titrated against Md IBDV. No virus-neutralizing antibody titer was observed in the negative controls or wild-type baculovirus-inoculated birds. One-week-old SPF chickens were inoculated with the VP2, VP2/VP4/VP3 baculovirus recombinants or wild-type baculovirus-infected insect cell lysates. The birds were boosted 2 wk later and challenged at 4 wk of age with 0.5 ml/bird classic STC IBDV (10(2) median embryo infective dose [EID50]/ml) or variant Md IBDV (10(5) EID50/ml) via the oral/nasal route. The VP2 and VP2/VP4/VP3 baculovirus-inoculated birds were partially protected against challenge with the classic STC IBDV. The birds were protected against clinical disease and death but not bursal damage, whereas the wild-type baculovirus-inoculated birds exhibited clinical signs of disease, 13% mortality, and bursal damage. When challenged with the variant Md IBDV, neither the VP2, VP2/VP4/VP3 baculovirus recombinants nor the wild-type baculovirus elicited protection against bursal damage and atrophy.

Development of an enzyme-linked immunosorbent assay using recombinant chicken anemia virus proteins expressed in a baculovirus vector system.

Recombinant baculoviruses were constructed to express the putative proteins VP1, VP2 or VP3 of the chicken anemia virus (CAV). The recombinant VP1, VP2 or VP3 were detected by SDS-PAGE, and their molecular weights were 50, 30/27 and 16 kDa, respectively. The VP2 and VP3 reacted with sera from CAV-infected chickens in Western blot analysis and when used as an enzyme-linked immunosorbent assay (ELISA) antigen, but VP1 did not. Antibodies to CAV were detected, by ELISA using crude insect cell lysates containing VP2 or VP3, from 2 to 20 weeks or 2 to 7 weeks after CAV infection, respectively. These findings indicate that recombinant VP2 and VP3 expressed in the baculovirus vector system can be used as antigens to detect anti-CAV antibodies in ELISA.