JPH08507681A - Recombinant Epstein-Barr virus capsid antigen useful for diagnostic methods - Google Patents

Abstract

  (57) [Summary] Recombinant EBV-gp125 protein is made in sufficient quantity and purity in a baculovirus expression system. It can be used in assays such as ELISA for the quantitative detection of EBV VCA-IgG and VCA-IgM antibodies. Recombinant gpl25 protein is similar in size to the EBV-gp125 viral capsid antigen isolated from an EBV-infected human cell line and has comparable sensitivity and specificity in immunoassays for VCA-Ig antibodies.

Description

Detailed Description of the Invention Recombinant Epstein-Barr virus capsid antigen useful for diagnostic methods                             Background of the Invention Technical field The present invention relates to the diagnosis and treatment of Epstein-Barr virus (EBV) infection. .Background technology   Epstein-Barr virus is a member of the herpesvirus family. And infect most humans. Most humans are susceptible to the virus in early childhood. Dye and then hold the virus for life. The majority of primary infections in young children are asymptomatic Is. In adolescents and adolescents, most primary EBV infections are contagious. Presents as nucleocytosis (IM). (GH Roberts,Diagnostics and Clinical Te sting  27: 16-21, 1989). EBV also affects rheumatoid arthritis, chronic fatigue syndrome, and bar Kit lymphoma, nasopharyngeal cancer, aplastic anemia, aggressive lymphoproliferative disorder (aggressi ve lymphoproliferative dlsorders), and spleen rupture There is. EBV is known to be an opportunistic pathogen and is often immunosuppressed. Patients with allografts or acquired immunodeficiency syndrome (“AIDS )).   Diagnosis of IM is mainly due to characteristic symptoms including fever, pharyngitis, and cervical lymphadenopathy. It is done based on. However, IM causes splenomegaly, hepatitis, and myalgias, as well as headaches. And even more unclear symptoms can be complicated (S.H. Cheeseman ,.Semin. He matol.  twenty five: 261-268, 1988). In addition, other pathogens, mainly cytomegalovirus As well as adenovirus, rubella virus, human immunodeficiency virus, and Toxoplasma gondii can also cause a similar syndrome. Currently , Frequent tests for heterologous antibodies have been linked to IM with EBV infection. This Such test results are not always definitive for EBV. Because it is like Since antibodies can be induced by other viruses than EBV, and patients with IM Because the incidence of heterologous antibodies during this period is at most 90% (A.S.Evans et al.J. Infect.Dis.  132: 546-553, 1975). Especially young children often have a heterologous antibody response There is no (C.V.Sumaya and Y.Ench,Pediatrics 75: 1011-1019, 1985).   The serological diagnostic method tests for the presence of nonspecific heterologous antibodies. Beginning with EBV-specific serodiagnosis (W. Henle et al.,Hum. Pathol. Five:Five 51-565, 1974). Until now, indirect immunofluorescence (IF) or immunization of EBV infected cells It is mainly performed by the peroxidase (IP) staining method. The standard assay is , A set of EBV-related antigens, especially viral capsid antigen (VCA), early antigens Expresses EA (which includes EA spread and EA restriction), and EBV nuclear antigen (EBNA) Selected EBV infection Fixed cells derived from cultured cells are used. These antigens were used in the late dissolution phase Corresponds to the lytic phase and the latent phase of EBV infection. Then individual antigens or these Of immunoglobulin IgM and / or IgG antibodies against different antigen combinations and Quantitative measurements can identify primary, past, and reactivated EBV infections. However The IF assay is time consuming and not suitable for large scale testing. Because of the subjective interpretation of the variability of antigen-producing cells and the results, these tests It is also difficult to standardize. Furthermore, the IgM-IF test showed that rheumatoid factor It has the drawback that non-specific reactions appear when present (G. Henle et al.Clin. Exp . Immunol.  36: 415-422, 1979).   Various attempts have been made to facilitate specific serodiagnosis of EBV infection. Natural antigens purified from infected cells (G.R.Pearson,J. Virol. Methods twenty one: 91-10 4, 1988) or empirically from the amino acid sequence presumed to be related to EBV-related antigens. Or based on either synthetic peptides selected by computer analysis A microtiter plate assay has been developed (RI Fox et al.,J. Clin. La b. Anal.  1: 140-145, 1987; J.E. Geltofsky et al.J. Clin. Lab. Anal. 1: 153- 162, 1987; J.M. Middledorp and P.M. Herbrink,J. Viro1. Methods twenty one: 133-1 46, 1988).   One of the best indicators of active EBV infection is a structural protein required for viral replication Quality against the viral capsid antigen The presence of antibodies (G.R. Pearson and J. Luka, characterization of virus-determining antigens). Ke.In Epstein-Barr Virus: Recent Developments, Epstein and Achong , Wiley: New York, pp. 47-73, 1986). Viral capsid antigen is sensitive to EBV Present on all stained cells. VCA-IgG is the first to appear early in primary EBV infection , And it peaks in 4-6 weeks. VCA-IgG is in the recovery phase But it remains detectable for life (G.H. Roberts,Diagnostics and C linicalTesting  27: 16-21, 1989). Almost all subjects in the third week after infection Be positive for VCA-IgG by 4 and become VCA-IgG negative after 4-8 weeks. Can be expected. Detecting VCA-IgG antibody remains the most important factor in monocytosis. Is also a highly sensitive detection method. Because VCA-IgG is detected in the serum The absence of this excludes the diagnosis of primary EBV infection (Id.).   Two major polypeptides associated with the VCA complex have been identified. These are g a glycosylated protein of 125,000 daltons named p125, and p It is a non-glycosylated protein with a size of 160,000 daltons, designated as l60. Studies have shown that gp125 is the most predominant antigen in the VCA protein and hence the most It has been shown to be a strong immunogen (G.R. Pearson et al. (1986), supra). gp125 , Already known to be most useful in diagnosing current or past EBV infections . Tested from confirmed individuals with infectious monocytosis 100% of sera that are positive for IgM antibodies to this glycoprotein. gp12 5 also serves as a reliable substrate for measuring IgA antibodies against VCA. Yes (G.R. Pearson et al. (1986), supra), which is associated with nasopharyngeal cancer. It is known to be a useful marker (GH Roberts (1989), supra).   As is conventional, gp125 has been isolated and purified from EBV infected cells. Cell culture Stems are very expensive and time consuming, however, they produce small amounts of protein. It only gives birth. In addition, there is no suitable culture system for large-scale production of gp125. Therefore, the use of gp125 for routine diagnosis is not supported.   M. Gong et al. (1987),Jour. Viro 1.61: 499-508 is E. EBV open in coli Expression of the reading frame BALF4 is described.   Recently, four diagnostically useful recombinant EBV proteins, typical virus capsi Antigen (p150), diffusion early antigen (p54), major DNA binding protein (p138), and And EBV nuclear antigen (p72) for the detection of immunoglobulin IgM and IgG antibodies Individual enzyme-linked immunosorbent assay (ELISA) has been initiated (M. Gorgievs ki-Hrisoho et al.J. Clin. Microbiol. 28: 2305-2311, 1990). Standard IF or IP In direct comparison with the results obtained by the assay, recombinant EBV ELISA was Provide a method for specific and sensitive serodiagnosis of immunocytosis (IM) Was shown.                             Summary of the invention Disclosure of the invention   In one aspect, in general, the invention provides immunoassays for EBV VCA antibodies. Pure recombinant EBV-gp125 protein with sufficient selectivity and sensitivity to be useful , And EBV VCA-IgG and VCA-IgM antibodies for qualitative and quantitative detection. Sufficient amount available for the assay of the enzyme-linked immunosorbent assay (ELISA) And a method for producing recombinant gp125 protein with purity, which Facilitates diagnosis of past EBV infections. Used for immunoassay of EBV VCA antibody Sometimes, the results were EBV-gpl25 virus that was routinely isolated and purified from EBV transformed cells. Seropositivity and seronegative when immunoassayed with Ruscapsid antigen Recombinant gp125 protein according to the present invention if it shows substantially the same pattern as sex States, "Sufficient selectivity and sensitivity useful in immunoassays for EBV VCA antibodies. Have a degree. "   As used herein, the term "recombinant gp125 protein" and / or " "Recombinant EBV-gp125 protein" is partially or fully glycosylated Full length recombinant gp125 polypeptide, whether partial or Any recombination of gp125 polypeptides, whether fully glycosylated Polypeptide fragment, which is an EBV gp125 virus capsid antigen and immunology Immunologically reactive VCA antibody Reactive is meant to include. The recombinant gp125 protein of the present invention A full-length gp125 virus capsid antigen or a shorter length, A lycosylated or non-glycosylated polypeptide; and, for example, the invention Purified recombinant gp125 protein by A DNA sequence corresponding to the EBV DNA sequence portion between the stop codon of baculovirus system It is prepared by expressing it in E. coli, but it was isolated from EBV-infected cells in VCA antibody ELISA. And can be as effective as with purified EBV gp125 protein. And are indicated.   A more preferred recombinant gp125 protein is the EBV virus in a baculovirus expression system. Although it can be produced by a DNA sequence encoding the capsid protein, The current product is, for example, E. EBV virus capsid protein coat in coli or yeast It has substantially better specificity and sensitivity than the product obtained with modified DNA. You can Furthermore, the recombinant gp125 protein of the invention is highly expressed in the baculovirus system. It can be expressed in yield.   The recombinant gp125 protein of the present invention can be obtained in large quantities at low cost, and Can be purified to a higher degree of purity than gp125, which is usually obtained from EBV-infected cell culture .   Preferably, a method for producing a recombinant EBV-gp125 protein in large quantities and in high purity Clones and expresses the EBV-gp125 protein in a baculovirus expression system And the resulting protein is subjected to metal affinity chromatography. rough Isolation and purification according to In other general aspects, Ming features recombinant gp125 protein made by such a method .   In another general aspect, the invention provides the gp125 protein in serum from a subject. Using Recombinant gp125 Protein in an Immunoassay to Detect Protein-Binding Antibodies Serological methods to diagnose present or past primary EBV infection in a subject by And an apparatus for carrying out the method. The device is preferably solid Recombinant gp125 tamper supported and blocked immobilized and non-specific antibody And tested blocked immobilized recombinant gp125 protein. Provided with a sample (eg, serum) that has a detectable label A second antibody that binds to IgG and / or IgM is provided; and the label is detected A method is provided.                      Description of preferred embodiments BEST MODE FOR CARRYING OUT THE INVENTION   Purified EBV-gp125 protein   Purified recombinant gp125 protein can be expressed in a suitable host by the method of the invention Insert a sequence encoding gp125 into the vector and transform the host cell with the vector. Can be made in large quantities by substituting and culturing the cells. Then recombination Gpl25 can be purified from lysates of cultured cells.   Expression of EBV-gp125 protein in insect cells The complete (172,282 base pairs) nucleotide sequence of Epstein-Barr virus is definitive. (R. Baer et al.Nature 310: 207-211, 1984). gp125 protein is , Is mapped to the BamHI A DNA fragment. a plasmid encoding gp125 DNA (designated as pTZ19-110) was labeled with Dr. Ferderick Wang (Harvard Medica School, Boston, MA). In this recombinant plasmid, gp125 The 3.5 kb BamHi-EcoRI fragment encoding Cloned (Pharmacia, Piscata Way, NJ). This EBV sequence represents 15 of the EBV genome. It represents 9,469 to 155,969 nucleotides. From this DNA sequence, the protein initiation code ATG (methionine) is at nucleotides 159,322, and the stop codon is at base 15 Turned out to be 6,755. Determine the size of the protein from the DNA sequence by 95,640 daltons. I predicted it. Since gp125 is a glycosylated protein, it is isolated from EBV-infected cells. The size of the released protein was determined to be 125,000 daltons.   Preferably, the gp125 protein is expressed in the baculovirus system.   For expression of the protein in baculovirus, plasmid pTZ19-110, Cleavage by the restriction enzyme NheI at a site located 33 bp downstream from ATG of the gp125 coding sequence did. The coding sequence was then digested with the restriction enzyme EcoRI Separated. The NheI-EcoRI fragment encoding this gp125 was spun SpindBind DNA extraction unit (FMC, Rockla nd, ME) and was isolated from a low melting 0.7% agarose gel. Baculovirus Correct reading in the transfer vector pBlueBacHis C (Invitrogen, San Diego, CA) The DNA fragment was transformed into E. coli vector pTrcHis C (I nvitrogen, CA) at the NheI-EcoRI site. Then NcoI coding for gp125 -HindIII was subcloned at the corresponding sites of pBlueBacHis C. This vector Since it is derived from the E. coli pUC vector, all the procedures were carried out using E. coli. coli Top 10 strain (Invi trogen, CA).   pBlueBacHis C vector is a recombinant baculovirus clone in insect cells Is designed for efficient protein expression and purification. pBlueBacHis C High level expression of DNA sequences cloned into the vector is achieved by polyhed rin) made possible by the presence of the promoter. Polyhedra proteins are baculo It is the major structural component of the occlusion body of the virus, and SDS-poly All "stainable" of infected Spodoptera frugiperda on acrylamide gels It accounts for over 50% of protein. This vector has a natural polyhedron leader sequence Subsequent (N-terminal to C-terminal 5'to 3'direction) coding sequence and ATG translation Initiation codon, 6 histi that function as metal binding domain in translated protein Gin residue region (tract), transcriptional stabilizing sequence from gene 10 of phage T7, and And an enterokinase cleavage recognition sequence. Multiplexes located downstream of this sequence ( multiple) Cloning region is related to the start codon So that the external gene can be inserted in the correct reading frame. This Is also co-expressing β-galactosidase, When transfected, in the presence of substrate X-gel or blue-gel, Allows rapid identification of replacement plaques.   The sequence flanking the polyhedrin gene in the wild-type baculovirus genome is p It is located 5'to 3'of the expression cassette of the BlueBacHls C transfer vector. Wild type Following co-transformation with Ils DNA, homologous recombination between these sequences occurs. Of the target gene under the control of the viral polyhedron enhancer / promoter element. It produces a recombinant virus in which the gene is expressed. BaculoGold linearized baculovirus DN A (PharmingenN San Diego, CA) cotransfected with pBlueBacHis C. Used for This DNA contains a lethal deletion. Therefore, the transformed Ils DNA is a polyhedron-based pBlueBacHis cotransfected with the deletion.  Not capable of producing infectious viral particles in insect cells unless complemented by C Yes. Insect cell Sf9 transfection and recombinant virus production and Purification was performed by the method described in the Invitrogen manual.   Expression of the gp125-encoding DNA fragment in pBlueBacHls C was analyzed by Western blotting. It was confirmed by a dot analysis. About 1 ml of insect cells infected with recombinant virus 3 days ago Cells and 1 ml of EBV-transformed human B cell line (Raji cell line) were pelleted and Then 10 It was dissolved in 0 μl of Laemmli buffer. Boil sample for 2 minutes, then 7.5% SDS -Loaded on a polyacrylamide gel and electrophoresed at 70 volts overnight. Ta The proteins were electrophoretically transferred to nitrocellulose membranes. 5% degreasing Membrane with TBST ((10mM Tris, pH 8.0), 1mM EDTA, 0.05% Tween-20) containing milk powder. The treatment blocked non-specific binding of proteins. against gp125 Monoclonal antibody from DuPont Company (Cat # NEA 9243) Membrane-bound protein, incubate for 1 hour, and wash 3 times with TBST Then incubate with anti-mouse IgG-alkaline phosphatase conjugate for 30 minutes. Was added. The membrane was washed again 3 times with TBST, and NBT (nitroblue tetrazo ) And BCIP (5-bromo-4-chloro-3-indole phosphate) It was Monochrome in extract of recombinant virus-transfected cell line Sf9 The single protein band that reacted with the primary antibody was the EBV-transformed Raji cell line. And others EBV-gp125 virus capsid protein monoclonal antibody reaction band Move closer to. This result uses the baculovirus system in insect cells to Confirm expression of 125 protein.                  Purification of recombinant gp125 protein   For example, recombinant g expressed in transfected insect cell culture Sf9 as described above. Is p125 in culture, for example, by affinity ram chromatography methods? Can be purified from   Vector pBlueBacHis C is expressed immediately after the translation initiation codon DNA sequence producing 6 histidine peptides in the N-terminal region of the prepared protein Contains rows. This area is²Charged ProBond resin (Invitrogen, CA) It has an affinity to bind to and thus allows a one-step purification. About 50 ml insect cells 2x10 Sf9 in a 100 ml spinner flask⁶Cultivated to a density of cells / ml. Fine Vesicles were infected at a multiplicity of infection of 6 and incubated at 27 ° C for 3 days. Centrifuge cells Pellet, and suspend in 20 mM sodium phosphate and 500 mM NaCl, and Cells were lysed by sonication and centrifuged to remove cell debris. In Recombinant protein from supernatant through ProBond resin by the method described in vitrogen Was purified. The N-terminal fusion peptide is cleaved off by the enterokinase gp125 tamper It was removed from the matrix and then purified through ProBond resin. With this method, 50 ml About 1 mg of protein was obtained from a culture of It was confirmed by polyacrylamide gel.Industrial applicability             ELISA using recombinant gp125 protein   The following ELISA is included in sera from subjects known to have EBV infection The recombinant gp125 protein of the invention was used to assay EBV serological parameters. It will be verified by the embodiment.   Approximately 100 μl of recombinant gp125 protein prepared as above The solution (1 μg / ml purified recombinant gp125 protein in PBS buffer pH 7.5) was added to Maxia Add to bsorb Nunc microwell strip and incubate overnight at 4 ° C. 200μl of TBST buffer (10mM Tris pH8.0, 1mM EDTA) 50mM for each plate  Washing with NaCl and 0.05% Tween-20) for 5 minutes at room temperature was performed twice. Respectively Plate with 300 μl 0.1% nonfat dry milk in TBST buffer at 4 ° C overnight. I'm sorry. A 100 μl aliquot of diluted test serum (1:21 dilution in TBST buffer) Add and incubate at 37 ° C for 1 hour, then wells at 37 ° C for 5 minutes TB Washed 3 times with ST. Then 100 μl of anti-human Ig bound to alkaline phosphatase Aliquots of M and IgG were added and incubated at 37 ° C for 30 minutes. 3 wells Wash 3 times with TBST for 5 minutes at 7 ° C, then 100 μl of alkaline phosphatase substrate (5 mg p-nitrophenyl phosphate and 1 ml 5x diethanolamine buffer) Add fur (Kierkegaard and Perry, Gaithersburg, MD) to 4 mg of distilled water Was prepared) and incubated at 37 ° C for 30 minutes. Absorbance 40 It was measured at 5 nm.   The results are shown in the table. The table shows the ELISA method using the recombinant gp125 protein of the present invention [ "TTI"] and commercially available using gp125 isolated and purified from EBV infected cells. A comparison with available Gull immunofluorescence and ELISA kits is shown. Gull's key And the results for the method of the invention are similar. Single cells from EBV infected cells Since the protein obtained by separation is small, And the cell culture and isolation methods used to obtain this material were expensive For that reason, Gull's kit is very expensive. According to the method of the present invention, a large amount of tamper Quality can be produced relatively cheaply, in large quantities and at very efficient costs. Kit is provided. Traditional methods, for example using immunofluorescence, are more time consuming. Less costly, more labor intensive, and less reliable because of the substantial cross-reactivity. It is only a bad measurement.                             Other embodiments   Other embodiments are within the following claims. For example, recombinant gp125 tamper The cytoplasm can be made using expression systems other than the baculovirus system described above. Departure Expression in the light baculovirus system is described, for example, in E. coli or yeast or mammal More preferable than expression in cells. Because the baculovirus system has a high yield In addition, a high-purity recombinant gp125 protein can be produced. In addition, baculo Configuration of recombinant gp125 protein produced by the viral system (folding, green (Cosylation) can provide a high degree of specificity and selectivity in immunoassay systems. Sufficiently similar to that of the EBV gp125 virus capsid antigen from transformed human cells. ing. E. Recombinant gp125 proteins expressed in E. coli or yeast are highly specific Apparently lacking some of the conformational properties required for sex and selectivity There is. Moreover, the yield of recombinant gp125 protein is low in yeast expression systems. mammalian Expression systems are difficult and expensive to maintain, and recombinant expressed in mammalian cells E Purification of gp125 is expensive and time consuming.   For example, E. Recombinant gp125 expressed in E. coli has sufficient selectivity and sensitivity Therefore, it was found to be insufficient for immunoassay of EBV VCA antigen.   E. For expression of the protein in E. coli, plasmid pTZ19-110 was Modified. Approximately 183 bp sequence was obtained by using plasmid pTZ19-110 with restriction enzymes NheI and BamHI. By cutting with This site was deleted, but the Nhel site was 33 bp downstream from ATG and the BamHI site was AT. 150 bp upstream from G. Both sites are blunted with TDNA polymerase and bound And E. coli with the resulting circular plasmid. coli NM 522 was transformed. This operation Resulted in the formation of a new BamHI site. The plasmid was then BamHI and EcoRI Was cleaved double-stranded with the expression vector and the fragment encoding gp125 was inserted into the expression vector pTrHisC. (Invitrogen) from San Diego, CA) in the same translation reading frame Cloned. This vector regulates the inducible tac (trp-lac) promoter Below E. Designed to express recombinant proteins in E. coli at high levels . This vector also contains a translation initiation codon, a polyhistidine metal binding domain, and And an enterokinase cleavage site.   Western blot expression of a DNA fragment encoding gp125 in pTrHisC Confirmed by analysis. In this procedure, transformed cells were OD in LB at 30 ° C._6oo0 Incubate to 0.5 and then add 1 mM final concentration of IPTG to induce expression of gp125. Led. After the incubation, the cells were incubated at 30 ° C for 3 hours. About 200 μl The culture was centrifuged and suspended in SDS buffer. Boil the sample for 2 minutes , Then loaded on 7.5% SDS-polyacrylamide gel, and at 70 volts overnight Electrophoresed. Proteins were electrophoretically transferred to nitrocellulose membranes. For non-specific binding of proteins, TBST (10 mM Tr1s pH8.0, 1 mM EDTA, 0.05% Twee n-20) with 5% skim milk powder in I clicked. Serum that had been previously confirmed to contain VCA-IgM by IF test was applied to the membrane. Add to bound protein and incubate for 1 hour and wash 3 times with TBST Then incubate for 30 minutes with anti-human IgM-alkaline phosphatase complex It was Serum was washed again with TBST three times, and NBT (nitroblue tetrazolium) And developed with 5-bromo-4-chloro-3-indole phosphate. VCA- A single protein band with a molecular weight of approximately 95 kd was obtained which reacted with IgM serum. However, this is because gp125 is E. Suggested to be produced as a 95kd protein in E. coli It However, the size of the gp125 molecule isolated from EBV infected cells was 125 kd. This size difference is probably due to E. Protein is not glycosylated in coli It is probably because   For example, the E. Recombinant 95kd gp125 protein expressed in E. coli culture The quality is determined from the culture using methods such as affinity column chromatography. Can be purified by using. Recombinant 95kd gp125 was created by the above method E. Purified from E. coli transformed cells according to the following protocol based on the examples did.   Add approximately 1.2 liters of LB broth + glucose and ampicillin to the medium, Cells containing fusion plasmid containing 2 ml of overnight culture were inoculated. Cells at 30 ° C for 3 After culturing for a time, IPTG was added at a final concentration of 1 mM to induce expression. Cells at 30 ° C Incubate for a further 3 hours, then harvest and lyse buffer (10 mM phosphate buffer). Sodium acid pH 7.0, 30 mM NaCl, 0.25% Tw een-20, 10 mM β-mercaptoethanol and 1 mM PMSF (phenylmethylsulfone) Nilfluoride)). Lyse cells by sonication and centrifuge did.   E. The recombinant gp125 protein expressed in E. coli was found to be insoluble and And was present in the pellet obtained from the crude fraction obtained as described above. Bae Ret containing 8M urea, 20mM sodium phosphate pH 7.8, and 0.5M NaCl It was dissolved in denaturing buffer. This is then used for ProBond metal affinity chromatography. A chromatography resin column (Invitrogen) San Diego, CA). Wash the column Clean and lyse the protein using the procedure described in the Invitrogen manual. I put it out.   As mentioned above, E. Recombinant gp125 protein expressed in E. coli is EBV VCA antibody Can not be used in the immunoassay. Because it ’s consistent and reliable This is because there is not sufficient selectivity and sensitivity to give good results. Im like this The inability to perform a non-assay is partly denatured during processing. , To the fact that they no longer have a configuration that can provide the appropriate antigen binding site I think it depends.   E. Recombinant gp125 protein expressed in coll and expressed in baculovirus system Differences with other recombinant gp125 proteins are apparently due to post-translational modifications . As described above, E. The recombinant gp125 protein expressed in E. coli is a molecule of approximately 94 kd. Size, but is the product of expression of the DNA sequence. Corresponds to the predicted molecular size of the polypeptide. Main issue with baculovirus system Recombinant gp125 protein produced by M. a. Created with coli It has much better specificity and sensitivity than Rus-expressed protein, which is an EBV gp125 viral vector isolated from EBV-infected cells. Has a molecular size comparable to the pseudoid antigen) and is substantially glycosylated It can be E. coli expressed gp125 protein and baculovirus expressed gp125 protein There may be differences in folding with the protein, which results in conformation of the antibody binding site. Formation, and thus the specificity or sensitivity of antibody binding at that site. Can affect.   As has been described, it encodes the EBV gp125 viral capsid antigen of the invention. Recombinant gp125 protein produced by expression of a portion of the DNA shorter than full length is Antibody binding specificity comparable to EBV gp125 virus capsid antigen isolated from stained cells. Sex and sensitivity. Fragments smaller than those described in the examples also Within the scope of the claims, and as a convention, for example by the following protocol Can be understood. First, the amino acid sequence is enriched on the surface of immunologically active molecules. Can be investigated for hydrophilic (and hydrophobic) regions that appear less (less), Therefore, more (less) antibody binding domain moieties can be made. Then, the result Sequences that are less likely to create part of a synthetic domain use standard techniques. Can be deleted, and the resulting candidate fragment or A number of fragments can be expressed, purified and isolated, then as described above. These VCA-Ig binding specificities and sensitivity were determined using the general assay described above. The degree can be measured.   Alternatively, candidate polypeptide fragments may be generated using well-known and established techniques and techniques. Can be constructed synthetically by mobilization techniques and then described in general as described above. Assays can be used to measure these VCA-Ig-binding specificities and sensitivities. Shi However, as is well known, recombinant gp125 protein (or any of its Post-translational modification of (fragment) affects the specificity and selectivity of the resulting molecule. To the extent possible, synthetically produced polypep tides, as suggested above, Peptide fragments are, for example, in the baculovirus system as described in detail above. E as expressed, recombinant gp125 protein (or recombinant fragment thereof) It cannot be effective in immunoassays for BV VCA antibodies.

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Claims (1)

[Claims] 1. Has sufficient selectivity and sensitivity to be useful in EBV VCA antibody immunoassays A purified recombinant EBV-gp125 protein produced in a baculovirus expression system. 2. Purified recombinant EBV-gp125 protein made using the baculovirus expression system Pak quality. 3. A method for producing a purified EBV-gp125 protein, the method comprising: Process for producing recombinant EBV-gp125 protein in Rus expression system and resulting product A method comprising the step of purifying A. by affinity chromatography. 4. A method of diagnosing a subject's current or past EBV infection, comprising the steps of: a) Obtaining a suitable sample from the subject; b) Baculovirus system under conditions suitable for antibody-EBV-gp125 protein complex formation Contacting the sample with the EBV-gp125 protein produced in c) measuring the presence of any complex in the sample, and Indicates past or current EBV infection; The method of including. 5. The method according to claim 4, wherein the measuring step includes an enzyme-linked assay. 6. 6. The method of claim 5, wherein the enzyme linked assay comprises an ELISA. 7. The method of claim 4, wherein the measuring step comprises an immunofluorescence assay. 8. A device for making a serological diagnosis of a present or past EBV infection in a subject. , Recombinant EBV-gp12 produced in a baculovirus expression system and immobilized on a support A device containing 5 proteins.