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To obtain chicken CD40L protein, the cDNA was prepared from chicken splenic cells and used as a template to clone and amplify CD40L by PCR. The target gene was cloned into pFastBac vector to construct a pFastBac-chCD40L donor plasmid. Recombinant plasmid was transformed into DH10Bac and recombinant Bacmid-chCD40L was obtained. The Bacmid-chCD40L plasmid was transfected into sf9 insect cells to obtain His-chCD40L protein. In addition, the target gene was cloned into pQM01 vector to construct a pQM01-chCD40L plasmid, recombinant plasmid was transfected into HEK 293T cells to obtain Strep-chCD40L protein. The chCD40L protein was purified by affinity chromatography, and the concentration of purified chCD40L protein was determined to be 0.01 mg/mL. Primary cells were isolated from the bursal tissue of 3-week old SPF chickens, and the chCD40L protein was added to the culture medium to stimulate cells. The chCD40L could bind to CD40 on B cells as examined by Western blotting, indirect immunofluorescence assay and flow cytometry, suggesting that chCD40L protein is biologically active. We successfully obtained chicken CD40L protein of biological activity, which laid the foundation in the in vitro culture of primary B lymphocytes for the isolation and diagnosis of virulent IBDV.
Subject(s)
Animals , Baculoviridae/genetics , CD40 Ligand/genetics , Chickens , Cloning, Molecular , Genetic Vectors/genetics , Recombinant Proteins/geneticsABSTRACT
PLCζ is a new isoenzyme of the PLC family which plays an important role in activating mammalian oocytes. In recent years, large-scale expression and purification of active PLCζ protein in vitro for structural biology research has not been successful. In this study, the recombinant human PLCζ protein was expressed and purified in the baculovirus expression system. First, the full length of human PLCζ gene was cloned into the pFastBac-HTA plasmid to form the recombinant donor plasmid that was further transformed into DH10Bac Escherichia coli cells to construct the recombined bacmid by the site-specific transposition that was screened by resistance and blue-white spots. Then the bacmid was transfected to Sf9 insect cells via cellfectin to package the recombinant baculovirus. After the amplification of the recombinant baculovirous, the recombinant protein was expressed from the cells transduced by the recombinant baculovirus and was purified by Ni-NTA resin. Purified protein was identified by Western blotting and time-of-flight mass spectrometry and the enzyme activity was determined. The results showed that the recombinant PLCζ protein in the Sf9 cells was achieved at 72 hours after baculovirus infection and expressed in secreted form in cell culture medium. The recombinant protein purified by Ni²⁺ affinity column was identified as PLCζ by Western blotting and ionization time-of-flight mass spectrometry and the enzyme activity was up to 326.8 U/mL. The experimental results provide a reference for the large-scale production and biological application of recombinant human PLCζ protein.
Subject(s)
Animals , Humans , Baculoviridae , Genetic Vectors , Recombinant Proteins , Sf9 Cells , SpodopteraABSTRACT
Objective:To construct I-Ad/IgG2b Fc baculovirus expression vector and express I-Ad/IgG2b Fc dimer fusion protein in Sf9 insect cells.Methods:I-Ad α,I-Ad β and IgG2b Fc gene sequences were amplified from BALB/c mouse lymphocytes by RT-PCR.I-Ad α and I-Ad β were connected with the leucine zipper sequence Fos and Jun respectively by overlapping PCR to form I-Ad α-Fos and I-Ad β-Jun.I-Ad α-Fos and IgG2b Fc fragments were ligated by restriction sites Xba I to form I-Ad α-Fos-IgG2b Fc recombination sequence.I-Ad α-Fos-IgG2b Fc and I-Ad β-Jun fragments were inserted to PPH and PP10,which were the downstream of the promoters in the plasmid pFastBacTMDual,to form pFastBacTMDual+[I-Ad/IgG2b Fc] recombinant plasmids.The constructed vector was identified by PCR,restriction endonuclease and sequencing.The recombinant plasmids pFastBacTMDual+[I-Ad/IgG2b Fc] was transferred into the DH10Bac competent cell to form recombinant baculovirus Bacmid+[I-Ad/IgG2b Fc].The recombinant baculovirus was transfected into Sf9 insect cells by liposome transfection reagent.After infected with Sf9 insect cells,the supernatant was collected and concentrated by PEG20000 to obtain I-Ad/IgG2b Fc dimer fusion protein.The fusion protein was detected by double-antibody sandwich ELISA and Western blot.Results:PCR,restriction enzyme digestion and sequencing confirmed that the recombinant vector pFastBacTMDual+[I-Ad/IgG2b Fc] had the correct sequence.The double antibody sandwich ELISA and Western blot showed that recombinant bacmid could successfully infect Sf9 insect cells,and the expressed fusion protein had the correct conformation.Conclusion:The pFastBacTMDual+[I-Ad/IgG2b Fc] baculovirus expression vector was successfully constructed and expressed in Sf9 insect cells,laying a foundation for the study of I-Ad-restricted T cells.
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Objective S gene of hantavirus(HV) was expressed in insect cells by genetic engineering technology.The expression product of S gene was used as antigen to detect anti-HV specific antibody IgG in serum.Methods Gene encoding NP of the strain HV-Z10 was amplified by PCR and then its eukaryotic expression system rBAC-Z 10S-TN was constructed by using the routine genetic engineering method.SDS-PAGE was applied to measure the expression of rNP.Ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product.Indirect immuno-fluorescence assay (IFA) was used to determine the specific immune-reactivity of rNP.WB assay was established to detect the serum samples from 95 confirmed HFRS patients.Parameters related to the outcomes of detection were compared with the routine HV-IgG IFA method.Results rBAC-Z10S-TN was able to express rNP with high efficiency.The purified rNP only showed a single protein fragment in the gel after SDS-PAGE.HV IgG could efficiently recognize rNP and hybridize with the recombinant protein.97.67％ of the serum samples from the HFRS patients were positive confirmed by WB.Conclusions We successfully constructed a high efficient prokaryotic expression system of NP encoding gene from hantavirus strain HV-Z10.WB assay which was established in this study could be used as a new serological test for HFRS diagnosis,thanks to the simplicity,safety,sensitivity and specificity of this method.
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Objective S gene of hantavirus(HV) was expressed in insect cells by genetic engineering technology.The expression product of S gene was used as antigen to detect anti-HV specific antibody IgG in serum.Methods Gene encoding NP of the strain HV-Z10 was amplified by PCR and then its eukaryotic expression system rBAC-Z 10S-TN was constructed by using the routine genetic engineering method.SDS-PAGE was applied to measure the expression of rNP.Ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product.Indirect immuno-fluorescence assay (IFA) was used to determine the specific immune-reactivity of rNP.WB assay was established to detect the serum samples from 95 confirmed HFRS patients.Parameters related to the outcomes of detection were compared with the routine HV-IgG IFA method.Results rBAC-Z10S-TN was able to express rNP with high efficiency.The purified rNP only showed a single protein fragment in the gel after SDS-PAGE.HV IgG could efficiently recognize rNP and hybridize with the recombinant protein.97.67％ of the serum samples from the HFRS patients were positive confirmed by WB.Conclusions We successfully constructed a high efficient prokaryotic expression system of NP encoding gene from hantavirus strain HV-Z10.WB assay which was established in this study could be used as a new serological test for HFRS diagnosis,thanks to the simplicity,safety,sensitivity and specificity of this method.
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Objective:To express the glycoprotein D of herpes simplex virus type 2 (gD2) in the insect cells,and to determine its immunogenicity.Methods:HSV-2 genome was used as the template for amplification of gD2 extracellular domain fragment gene by PCR.The PCR product was inserted into the vector Bacmind,and the constructed recombinant plasmid gD2-Bacmind was transfected into the sf9 cells to package the recombinant baculovirus.The Sf9 cells were infected by recombinant baculovirus seed derived from the forth passage(P4),the titer of P4 recombinant baculovirus was detected by a plaque assay and the expression of recombinant protein gD2 was determined by Western blotting method.The supernatant of infected cells was collected and purified by Ni-NTA affinity chromatography to obtain the target protein gD2,the purified gD2 protein was used to immunize the BALB/c mice in 0, 2, 4 weeks (gD2 group),and PBS was used as negative control(PBS group);the titers of gD2 specific IgG in serum were detected by ELISA assay.Results: The PCR analysis and sequencing results proved that gD2-Bacmind was constructed correctly.The titer of recombinant baculovirus was 2.0×109 pfu·mL-1,the purified gD2 was about 37 000 with expectation,the percentage of gD2 in total protein was 90%.The average value of Log10 of titer of gD2 specific IgG in serum detected by ELISA assay in gD2 group at the sixth week was 4.34,and there was significant difference compared with PBS group(P<0.01).Conclusion: The gD2 expressed by insect-baculovirus expression vector system has the immunogenicity and can be selected as candidate protein for HSV-2 vaccine.
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OBJECTIVE To establish an in vitro screening system for activin receptor-like kinase 4,5 and 7 (ALK4,ALK5 and ALK7) inhibitors.METHODS The insect expression systems for kinase domain of ALK4,5,7 and Smad2/3 proteins were established using the Bac to Bac baculovirus expression system.The desired proteins were expressed in Sf9 insect cells and purified by GST affinity.The screening system was composed of the kinase,Smad3 protein,ATP as well as the compound.The impact of the compound on the activities of ALK kinase domains was examined by measuring the amount of remnant ATP in the system as ALKs catalyzed the phosphorylation of Smad3 protein and consumed ATP during the process.The screening conditions were optimized,and validation of the screening system was conducted using known ALKs inhibitors.RESULTS All the reconstructed Bacmids were identified to be correct by PCR and restriction enzyme digestion.All the proteins were expressed in Sf9 insect cells after transfection,and purified proteins were achieved by GST affinity purification.For the screening system,the optimized kinase concentration and Smad3 concentration were 10 mg· L-1 and the optimized ATP concentration was 10 nmol·L-1.The Z'factor for ALK4,ALK5,and ALK7 kinase inhibitors screening system was 0.71,0.51 and 0.74,respectively.The well-known ALK inhibitor SB431542 inhibited the catalytic activities of ALK4,ALK5,and ALK7 with IC50 values of 22,188 and 91 nmol· L-1,respectively.CONCLUSION The in vitro screening system for ALK4,ALK5 and ALK7 inhibitors is successfully established.
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Objective:In the present study,Bac-to-Bac baculovirus expression system was used to obtain recombinant human Cosmc extracellular domain protein,which can lay the foundation for the research about the structure and function of Cosmc protein in vitro,and simultaneously provide ideas for the research of O-glycosylation and related diseases. Methods: The Cosmc extracellular domain ( Cosmc-ED) gene was cloned into a transfer vector pFastBac1 to form the recombinant donor plasmid pFastBac1-Cosmc ED, which was transformed into competent cells DH10Bac. By using blue-white selection and PCR analysis,we could obtain recombinant shuttle vector rBacmid-Cosmc ED. Then, the recombinant gene DNA of rBacmid-Cosmc ED was used to transfect Sf-9 mediated by cationic lipid formulation,and the recombinant baculovirus bacmid was obtained,which was further used to infect the serum-free cell Sf-9 to express Cosmc-ED in the supernatant. Then the protein of interest was detected by SDS-PAGE and Western blot and purified with Ni-NTA affinity column. Results:SDS-PAGE and Western blot analysis showed a specific band about 33 kD,consistent with the interest protein. Mass spectrometry results further prove that the protein was Cosmc extracellular domain protein. Conclusion: Human Cosmc-ED protein can be successfully expressed in Sf-9 insect cells and laid basis for subsequent studies.
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OBJECTIVE@#To express human vascular endothelial growth factor121 (VEGF121) in insect cells.@*METHODS@#A gene construct containing VEGF was cloned in the pFastBac-HTA vector, followed by transformation in DH10BAC. The recombinant bacmid was then extracted, and transfected into Sf9 insect cells. The transfected cells were harvested, and then VEGF expression was confirmed by western blotting using specific antibodies. The tube formation assay was used for functional assessment of VEGF.@*RESULTS@#Our results showed that VEGF could be successfully expressed in the baculovirus system. Purified VEGF was able to stimulate in vitro tube formation of human endothelial cells.@*CONCLUSIONS@#Results from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes.
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Objective To express human vascular endothelial growth factor121 (VEGF121) in insect cells. Methods A gene construct containing VEGF was cloned in the pFastBac-HTA vector, followed by transformation in DH10BAC. The recombinant bacmid was then extracted, and transfected into Sf9 insect cells. The transfected cells were harvested, and then VEGF expression was confirmed by western blotting using specific antibodies. The tube formation assay was used for functional assessment of VEGF. Results Our results showed that VEGF could be successfully expressed in the baculovirus system. Purified VEGF was able to stimulate in vitro tube formation of human endothelial cells. Conclusions Results from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes.
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This study employed a Bac-to-Bac/Bombyx mori bioreactor to mass-produce immunogenic urease subunit B (UreB) from Helicobacter pylori. The signal peptide bombyxin from B. mori was used to promote secretory expression to improve expression levels and was designed and integrated into the UreB gene to generate the Bacmid/BmNPV/(signal peptide)-UreB baculovirus expression system. To determine whether the bombyxin signal peptide resulted in secretory expression of recombinant UreB (rUreB) and to determine the secretory efficiency, we tested the secretory expression level of rUreB in Bm5 cells using ELISA. To further investigate whether secretory expression affected cell viability, cells were evaluated using 0.4% trypan blue staining, and Bacmid/BmNPV/UreB without the signal peptide served as a control. The above recombinant bacmid constructs were injected to silkworm larvae, and the secretory expression level of rUreB was detected using SDS-PAGE and semi-quantitative western blot analysis. The results indicated that the bombyxin signal peptide directed the secretory expression of rUreB and that this expression improved the viability of Bm5 cells. Moreover, the results showed that the expression level of rUreB was 1.5 times higher with the Bacmid/BmNPV constructs containing the bombyxin signal sequence than those without the signal sequence. These results demonstrate that secretory expression can enhance rUreB expression levels and is likely to aid in the large-scale expression and yield of rUreB in silkworm larvae.
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Hemagglutination inhibition (HI) test employing whole virus antigen is a prescribed serological test for serotyping, diagnosis and surveillance for avian paramyxoviruses (APMVs). For use as alternative to the virus antigen, hemagglutinin-neuraminidase (HN) protein gene of the wild duck isolate APMV-6/WB12-163FS of APMV serotype 6 (APMV-6) was amplified, cloned and expressed in Spodoptera frugiperda insect cells. The HN gene of 1,842 bps in length showed nucleotide and amino acid homology of 93.4% and 97.1%, respectively with that of APMV-6 prototype strain. Putative sialic acid binding motif and potential N-linked glycosylation sites were conserved. In Western blot analysis, the expressed protein had a molecular mass of 66 kDa and reacted specifically with antiserum to APMV-6. In addition, the recombinant HN protein showed biological properties such as hemagglutination (HA) and elution. The recombinant HN protein produced from infected cells showed high HA titers (approximately 2(13) HA unit/ml). The HA activity of the recombinant HN protein was inhibited by antisera to APMV-6. In cross HA inhibition test, the recombinant HN protein had the highest titers with antisera to homologous APMV serotype, although there was weak cross reaction with some of antisera to other APMV serotypes. Our results indicated that recombinant APMV-6 HN protein would have the potential as alternative to the APMV-6 antigen in HI assays.
Subject(s)
Avulavirus , Baculoviridae , Blotting, Western , Clone Cells , Cross Reactions , Diagnosis , Ducks , Glycosylation , Hemagglutination , HN Protein , Immune Sera , Insecta , N-Acetylneuraminic Acid , Serologic Tests , Serotyping , SpodopteraABSTRACT
Objective To express and characterize the virus-like particles( VLPs) of H5 subtype containing of hemagglutinin ( HA ) and matrix 1 ( M1 ) protein by using Baculovirus-insect cells .Methods Full length genes encoding HA protein from the A/Indonesia/05/2005(H5N1) strain and the M1 protein from the A/Anhui/01/2005 ( H5N1 ) strain were cloned into a baculovirus expression vector to construct pFBD-M1-HA.The expression of HA and M1 proteins were detected by Western blot and indirect immunoflu-orescence after the transfection of Spodoptra frugiperda (Sf9) insect cells with recombinant baculovirus.Pu-rified VLPs were analyzed by SDS-PAGE and visualized with transmission electron microscope.The biologi-cal activity of purified VLPs was detected by hemagglutination test.Results The HA and M1 proteins of H5 subtype expressed by baculovirus-insect cells could be self-assembled into the functional mature VLPs.The hemagglutination titer of VLPs was as high as 1024 HAU/50μl.Conclusion The H5 subtype VLPs as pre-pared in this study would pave a way for the development of a candidate recombinant A ( H5) vaccine.
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<p><b>INTRODUCTION</b>Herpes simplex virus type 2 (HSV-2) is the most common cause of genital herpes. Glycoprotein G (gG) is a prototype antigen for type-specific serodiagnosis distinguishing between HSV type 1 (HSV-1) and HSV-2 infections. As immunological diagnosis kits for accurate differentiation between HSV-1 and HSV-2 antibodies can be expensive, there is a need to develop a convenient, sensitive, specific and cost-effective serodiagnostic kit.</p><p><b>METHODS</b>We successfully expressed a fragment of gG comprising residues 321-580 of HSV-2 with histidine tag (gG(321-580His)) in a Bac-to-Bac baculovirus expression system, which had an antigenicity similar to its native counterpart. An indirect enzyme-linked immunosorbent assay (ELISA) was developed using gG(321-580His) as the diagnostic antigen and evaluated by comparison with a commercial HerpeSelect 2 ELISA immunoglobulin G kit as reference.</p><p><b>RESULTS</b>In testing 318 field serum samples, the diagnostic relative sensitivity and specificity of the developed gG(321-580His)-ELISA test in qualitative comparison with the commercial kit were 93.81% and 96.74%, respectively, and the accuracy was 94.65%.</p><p><b>CONCLUSION</b>The study indicates that gG(321-580His) has a high diagnostic potential for HSV-2 virus serodiagnosis in humans.</p>
Subject(s)
Adult , Female , Humans , Male , Antibodies, Viral , Blood , Enzyme-Linked Immunosorbent Assay , Herpes Genitalis , Diagnosis , Virology , Herpes Simplex , Diagnosis , Virology , Herpesvirus 1, Human , Herpesvirus 2, Human , Immunoglobulin G , Chemistry , Polymerase Chain Reaction , Reagent Kits, Diagnostic , Reference Values , Reproducibility of Results , Sensitivity and Specificity , Serologic Tests , MethodsABSTRACT
Objective:To express mycobacterium tuberculosis protein MPB 64 in baculovirus insect cell expression system ,and identify its immunogenicity.Methods:The target gene MPB64 was connected to pFastBac vector ,then the pFastBac-MPB64 plasmid which was harvested would transformed to DH10Bac competent,and the target gene was transposition into Bacmid by Tn7 transposase fragment,therefore Bacmid-MPB64 Shuttle vector was obtained.The shuttle vector was packaged by liposomes and transfected Sf 9 cells to harvest P1-generation virus ,then high titers of P4 generation virus was harvested by repeat transfected Sf 9 cells three times.The target protein MPB64 was purified from the supernatant by Q Sepharose FF and Ni affine chromatography ,which were used to immunize BALB/c mice.Antibody changes in serum would be detected ,and the proliferation of immunized mice spleen cells would be detected by MTT,detected the IFN-γsecretion by MPB64 stimulated spleen cells by ELISA method.Results: MPB64 successfully expressed in insect cells.The purity of target protein was over 90% and yield up to 35 mg/L after purification.Purified protein can effectively stimulate BALB/c mice to produce antibodies , increase the content of IFN-γmedium in mice spleen cells ,and significantly promoting proliferation in spleen cells between 0.2-100 μg/ml.Conclusion: MPB64 which has immunogenicity was successfully expressed in baculovirus insect cell expression system ,that open a new avenue for tuberculosis vaccine production.
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Human norovirus (HuNoV) is the major etiological agent of nonbacterial gastroenteritis worldwide. However, due to the absence of a rapid and sensitive diagnostic system, detection and monitoring have been limited. The HuNoV genome is composed of three open reading frames (ORFs). And major capsid protein, ORF2, is designated as a viral protein 1 (VP1). In this study, the baculovirus expression system was used for expression of the HuNoV capsid protein, VP1. Recombinant baculoviruses can be used as potent tools in HuNoV studies.
Subject(s)
Humans , Baculoviridae , Capsid , Capsid Proteins , Gastroenteritis , Genome , Norovirus , Open Reading FramesABSTRACT
To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity, its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system. The S8 gene was subcloned into the pFastBacTM1 vector, to produce the recombinant baculovirus transfer vector pFB-S8. After transformation, pFB-S8 was introduced into the competent cells (E. coli DH10Bac) containing a shuttle vector, Bacmid, generating the recombinant bacmid rbpFB-S8. After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection, Sf9 cells were collected at different times and analyzed by SDS-PAGE, Western blotting and immunofluorescence microscopy. The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells. Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.
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A baculovirus expression vector system (BEVS) is used routinely to produce recombinant proteins in the milligram scale. Dual Ig domain containing cell adhesion molecule (DICAM) belongs to the type I class of transmembrane proteins. It consists of a signal peptide, two V-type Ig domains in the extracellular region, and a short cytoplasmic tail of 442 amino acids. To purify the recombinant DICAM protein from cells overexpressing the mouse full-length DICAM gene, recombinant baculovirus is infected and recovered in the Sf9 cells. As a result, mouse DICAM protein was efficiently expressed and extracted from the insect cells using the BEVS. This recombinant protein can be used in further studies for functional test of DICAM protein in the cells.
Subject(s)
Animals , Mice , Amino Acids , Baculoviridae , Cell Adhesion , Cytoplasm , Insecta , Protein Sorting Signals , Proteins , Recombinant Proteins , Sf9 CellsABSTRACT
Background & objectives: An inability or decreased ability of spermatozoa to bind to the zona pellucida (ZP), an extracellular glycoproteinaceous matrix surrounding egg, is one of the plausible causes of idiopathic infertility. It will be clinically useful to distinguish this condition from other causes of infertility. An assay system, investigating binding of human sperm with ZP glycoprotein may prove useful in this regard. We attempted to develop a simple assay system to analyse the binding of capacitated human spermatozoa to human zona pellucida glycoprotein-3 (ZP3) using baculovirus-expressed recombinant human ZP3 coated beads. Methods: Recombinant baculovirus-expressed ZP3 was purified, labelled with biotin and coated on streptavidin sepharose beads. An in vitro assay system was optimized to study binding of capacitated human sperm to ZP3 coated beads. Results: A higher percentage of baculovirus-expressed recombinant human ZP3 coated beads showed significant (P<0.05) binding of capacitated human sperm as compared to beads coated with fetuin. An inhibition in the binding of sperm to ZP3 coated beads was observed in presence of cold recombinant human ZP3. Further, prior incubation of ZP3 coated beads with monoclonal antibodies (MAbs) against ZP3 but not against ZP2 resulted in the decrease in number of sperm bound to bead. Interpretation & conclusion: An in vitro assay system to study the binding of human sperm to ZP3- primary sperm receptor was established, which may be useful to determine the functional competence of spermatozoa.
Subject(s)
Egg Proteins/genetics , Egg Proteins/metabolism , Humans , Male , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Protein Binding , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sperm Capacitation/physiology , Spermatozoa/cytology , Spermatozoa/metabolism , Zona Pellucida/metabolism , alpha-Fetoproteins/metabolismABSTRACT
Bovine leukemia virus (BLV) envelope glycoprotein (gp51/gp30T-), consisting of BLV gp51 and BLV gp30 that lacked its C-terminal transmembrane domain, was expressed in insect cells under the control of the baculovirus polyhedron promoter. Recombinant BLV gp51/gp30T- secreted from insect cells was determined by immunofluorescence, enzyme-linked immunosorbent and western blot assays using a BLV-specific monoclonal antibody and BLV-positive bovine antibodies. An agar gel immunodiffusion (AGID) test using gp51/gp30T- as the antigen for the detection of BLV antibodies in serum was developed and compared to traditional AGID, which uses wild type BLV antigen derived from fetal lamb kidney cells. AGID with the recombinant BLV gp51/gp30T- was relatively more sensitive than traditional AGID. When the two methods were tested with bovine sera from the field, the recombinant BLV gp51/gp30T- and traditional antigen had a relative sensitivity of 69.8% and 67.4%, respectively, and a relative specificity of 93.3% and 92.3%. These results indicated that the recombinant BLV gp51/gp30T- is an effective alternative antigen for the diagnosis of BLV infection in cattle.