CN1194001A - Neutralizing conformational epitopes of chicken anemia virus - Google Patents
Neutralizing conformational epitopes of chicken anemia virus Download PDFInfo
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- CN1194001A CN1194001A CN96195434A CN96195434A CN1194001A CN 1194001 A CN1194001 A CN 1194001A CN 96195434 A CN96195434 A CN 96195434A CN 96195434 A CN96195434 A CN 96195434A CN 1194001 A CN1194001 A CN 1194001A
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Abstract
The production of the conformational neutralizing epitope of chicken anemia virus is described and compositions for preventing or treating CAV infections, in particular vaccines less pathogenic than CAV itself. All these compositions were shown to synthesize the conformational neutralizing epitope, necessary for the protective immune response against CAV infections. The invention provides recombinant DNA molecules derived from the CAV genome, and recombinant DNA fragments of the CAV genome integrated in the genome of other virus vectors. In particular, these compositions comprise a sub-unit vaccine, a recombinant live virus vaccine and an attenuated vaccine against CAV infections. The invention also provides the production of neutralizing antibodies directed against CAV and diagnostic kits for the detection of CAV.
Description
The invention summary
The invention provides cav protein VP1 in and the formation and the evaluation of conformational epitope, it causes protective immune response in immune animal required.
Particularly, the invention describes the generation of anti-CAV neutralizing antibody, in a diagnostic test based on the neutralizing antibody that these produced, detected CAV (particle).
Disclose various anti-CAV vaccine carriers, all these vaccines are pathogenic low than CAV all, all these vaccine carriers all show can produce required CAV in and conformational epitope.
A kind of carrier provides based on the subunit vaccine of expressing the recombinant baculovirus of VP1 and VP2 in same cell simultaneously.Second kind of vaccine is to live in expression vector, and it comprises the stable MarekShi disease carrier that carries VP1 and VP2 encoding sequence.The third CAV vaccine comprises the CAV strain of genetics attenuation, and it has the cytopathic effect of reduction.
Background of invention
Chicken anemia virus (CAV) is a kind of small virus of uniqueness, and its particle diameter is 23-25nm, and genome is formed (Gelder blom et al., 1989, Northern et al., 1991 and Todd et al., 1990) by cyclic single strand (minus strand) DNA.This DNA increases by the double-stranded replicative intermediate of a kind of ring-type in cells infected, and it is recently by clone and all order-checkings.CAV genome length is 2319 Nucleotide (Noteborn and De Boer, 1990), and the DNA analysis from the isolating CAV strain in different continents is shown that fine difference (Meehan et al., 1992 are only arranged between different isolates; Preserving number M81223, Claessens et al., 1991; D10068, Kato et al., 1995; D3 1965 and Pallister etal., 1994, Soine et al., 1993 and 1994; L14767).Recently, CAV has been ranged the new virus family of Orbivirus section based on its genome structure.Yet uncorrelated between other member of CAV and this virus family, this family is made up of animal strand ring-type dna virus, as porcine circovirus and parrot mouth ptilosis (beak-and-feather-disease) virus (Noteborn and Koch, 1995).
The CAV genome contains a tangible promotor/enhancing subarea (Noteborn et al.1994), it is regulated CAV and transcribes, the genomic main transcript of CAV is the not montage polycistronic mRNA of about 2100 Nucleotide, its coding molecule amount is three kinds of protein (Noteborn and Koch of 51.6kDa (VP1), 24.0kDa (VP2) and 13.3kDa (VP3 or cell programme dead albumen (apoptin)), 1995, Noteborn et al.1992, Phenix et al, 1994).The cav protein of all three kinds of expections all has synthetic (Noteborn and Koch, 1995) in the cell that CAV infects.Be used in the same cell simultaneously that synthetic (reorganization) VPl and VP2 immunization can cause protective response, and can be used as anti-chicken infectious anermia subunit vaccine (Koch et al., 1995 and Noteborn and Koch, 1994c).
CAV can cause clinical and non-clinical disease in chicken, and known be worldwide important avian pathogens (McIlroy et al., 1992 and McNulty et al., 1991).The chicken of several ages in days is subject to CAV especially and infects.In these animal lethargys, observe and apocleisis and anaemia are occurring after 10 days with CAV inoculation, infect back mortality ratio maximum and can increase to 50%.Along with the increase at age, resistance also increases (Jeurissen et al., 1992).The hematocrite value of the chicken that is infected by CAV when the 1-3 age in days descends.The chicken of 1-21 age in days infects CAV can cause particularly thymic cortex exhaustion.Yet the age, big slightly chicken can have clinical symptom ground breeding, and the CAV in the age big slightly chicken infects can determining by seroconversion.
The exhaustion of cortical thymocyte is considered to cause immune deficiency, thereby has increased and infect simultaneously and cause immuning failure (Noteborn and Koch, 1995).Thymocyte and the exhaustion that may also have the protoerythrocyte like cell take place by CAV inductive cell programmatic is dead (Jeurissen et al., 1992a).The cell of the CAV coding dead albumen of programming is the main inductor (Noteborn et al, 1994) of this phenomenon.
Found that maternal antibody can give the important protection that anti-CAV infects, the hen of the contacted CAV of people such as Vielitz (1991) report can produce lot of antibodies, and passes to the chicken that each ovum is hatched, the infringement of the not infected property of these antibody protection chickens anaemia relative disease.Detect the CAV neutralizing antibody in the yolk with the egg under the hen of the lysate of following cell inoculation, described cell perhaps mainly produces VP1 and VP2 with CAV recombinant baculovirus coinfection and produce all three kinds of cav proteins.In the filial generation of hatching of attacking, do not show special clinical symptom (Koch et al., 1995) with CAV by these eggs.
Vielitz and Landgrf (1988) have developed a kind of vaccine of anti-infection property anaemia, and it is based on the CAV that breeds in the chicken embryo, and this is unique commercial vaccine at present.The filial generation of the stud bird of immunization is protected, not infected property anaemia infringement.Immunization is possible, because when maternal immunity disappears, the bird susceptible viral infects and do not fall ill.Obviously use based on the living vaccine of attenuated virus not and have danger.To 3 age in week the chicken sexuality that experimentizes dye the obvious decline (McConnell et al., 1993, and 1993a) that can cause function of immune system, but do not fall ill.According to this discovery, McIlroy (1992) provides evidence to show, CAV also can cause considerable financial loss under incidence not, and promptly not having the disease of clinical symptom can the negative impact feed conversion and the medication of mean body weight and increase, and both all cause considerable financial burden.At present, nonpathogenic CAV separates as yet and obtains.
Can be used as subunit vaccine by baculovirus expression system synthetic recombinant C AV albumen, prove that recombinant C AV albumen VP1 and VP2 can protect chicken (Koch et al., 1995) by maternal immunity.Because baculovirus vector is an insect specificity virus and known to the vertebrates no pathogenicity, thus its can cultivate and supply with chicken and do not have expectation less than danger (Vlak and Keus, 1990).
Usually, live-virus vaccine is induced better immunne response and relatively cheap than subunit vaccine, therefore, can use immunogenicity knowledge architecture attenuation CAV carrier or other recombinant viral vector of various cav proteins, as avian herpetoviruses (Nakamura et al., 1992, Morgan et al., 1993) or fowlpox virus (Nazerian et al., 1992, Boyle andHeine, 1993).These recombinant viruses also should be expressed cav protein VP1 and VP2 except expressing himself albumen, and the vaccine of can be used for laying eggs kind of chicken and fryer kind chicken is resisted infectious anermia to protect its offspring.In addition, this class carrier also can be used as the disease of the no clinical symptom of protection maternal immunity kind chicken opposing.
The detailed description of invention
The present invention relates to chicken anemia virus (CAV) in and the generation and the analysis of conformational epitope structure.
In addition, also disclose the generation at the neutralizing monoclonal antibody of CAV, it is a conformational epitope at cav protein VP1 that these neutralizing antibodies demonstrate.
Also described the diagnostic kit that is used to detect CAV, the invention provides in the neutralizing antibody and the evidence of CAV particulate mechanism based on these neutralizing antibodies.
For form VP1 in and conformational epitope, need synthetic VP2 in same cell.The recombinant baculovirus of in insect cell, only expressing VP1 not with neutralizing antibody reaction at CAV, but when VP1 and VP2 synthesize in a cell, these neutralizing antibodies will react.
But in the CAV of purifying capsid, only there is VP1.The present invention discloses when VP1 is synthetic, and most probably produces in the CAV capsid process when it forms complex body, and VP2 temporarily combines with VP1.CAV capsid sex change meeting is destroyed neutralizing epitope, and the prompting neutralizing epitope is a conformational epitope.
In addition, the present invention relates to prevent or treat vaccine and the composition that the poultry infective virus particularly infects CAV.
Particularly, the present invention relates to than the pathogenic low of CAV itself but still in producing and the vaccine of conformational epitope, can cause producing neutralizing antibody, therefore watch for animals and the offspring resists the CAV infection with the vaccine inoculation chicken of these types.
The present invention relates to contain the baculovirus vector of the sequence that is independent of its genomic coding VP1 or VP2, this recombinant baculovirus can synthesize VP1 and VP2 in same cell, thus formation CAV in and conformational epitope.
The invention still further relates to and make up the synthetic in such a way recombinant C AV albumen of MarekShi syndrome virus (MDV) carrier, particularly this recombinant mdv carrier contain the proteic CAV sequence of coding VP1, VP2, promptly make it to prepare suitably VP1 in and conformational epitope.
In addition, the invention describes the formation of various attenuation CAV strains, these strains are compared with wild-type deutero-CAV strain the cytopathic effect that produces in chicken T cell are reduced.Attenuation CAV strain is to prepare by introduce point mutation in clone's CAV DNA genome, and the sequence that particularly is positioned at promotor/enhancing subarea is suddenlyd change.During the CAV of attenuation sudden change strain can produce and conformational epitope.
Prevention or control method that the CAV in the chicken particularly infects and the method for the reorganization part of the sequence that preparation comprises CAV and the method for preparing vaccine also are themes of the present invention.
Therefore, the invention provides neutralizing antibody or antibody fragment or derivatives thereof with the conformational epitope reaction of the viral protein 1 (VP1) of chicken anemia virus (CAV), described conformational epitope is named as 132-1, the monoclonal antibody identification of 132-2 or 132-3, described monoclonal antibody is by in Pasteur Institut's preservation, preserving number is xxxxxx, yyyyyy, and the hybridoma of zzzzzz produces.Determine a kind of antibody whether be the experiment of antibody of the present invention be observe its whether with antibody cross reaction (or competition) from the hybridoma of preservation.The fragment of antibody and derivative are well known in the art, do not need those skilled in the art are further explained.
The present invention also provides the conformation neutralizing epitope by the viral protein 1 of the chicken anemia virus of above-mentioned antibody recognition, this epi-position can be a more macromolecular part, for example it can combine with a kind of carrier, and perhaps this epi-position can (with certain interval) repeat in a polypeptide chain.Preferred epi-position is the major part of VP1, because this molecule will more easily have correct conformation.
As mentioned above, if conformational epitope preferably produces with VP2 then it exists only on the VP1 from a carrier in a cell.Therefore, the invention provides to produce and comprise the method for the viral protein 1 of conformational epitope as mentioned above, this method comprises to be expressed with the viral protein 2 of described CAV described viral protein 1 in a cell, the genetic information of the VP1 that wherein encodes has an independent existence on a recombinant vectors with the genetic information of coding VP2.
The present invention also comprises the carrier that is used for aforesaid method, described carrier comprise as two independently the coding VP1 of encoding sequence genetic information and the coding VP2 genetic information.Preferably, this carrier is based on MarekShi syndrome virus carrier, thereby can produce the vaccine of the provide protection that can provide anti-two kinds of pathogenic agent.
A kind of very simple and effectively expressing system that is used for viral protein of the present invention is based on the carrier of baculovirus vector, and as previously mentioned, baculovirus is considered to usually and can uses safely in vertebrates, because it does not infect vertebrates.
The present invention also provide produce have very important in and conformational epitope but the vaccine of pathogenic reduction or the approach of vaccine composition (for example attenuated virus).
This point can reach by following method is provided, described method is used for providing and comprises of the present invention and viral protein 1 conformational epitope, described method comprises from the genetic expression of coding VP1 and the VP2 funtion part of VP1 and VP2 at least, wherein at least one gene is under the control of control region of the CAV sequence that is derived from the transcription initiation site upstream, and described control region is modified to reduce its effect.By this way, can produce to duplicate and be not very fast and the therefore recombinant virus of pathogenic reduction (being attenuation).
This virion also is a part of the present invention, as mentioned above, modifies and is preferably located in promotor/enhancing subarea, and most preferably modify the 12bp that is arranged in promotor/enhancing subarea and insert fragment.
The recombinant virus particle that is obtained by any method of the present invention also is a part of the present invention, the nucleic acid that is used for method of the present invention also is a part of the present invention, as comprise the carrier of gene of the funtion part of the gene of the funtion part of VP1 at least of encoding and coding VP2, wherein at least one gene is under the control of regulating and controlling sequence, and described regulating and controlling sequence is modified to reduce its effect.
Antibody of the present invention and epi-position also can be used for whether having CAV or CAV antibody at being used for detection or definite sample in the test kit.
The present invention also provides the vaccine that is used for the treatment of and prevents the CAV relative disease, and described vaccine comprises the adjuvant of antibody of the present invention or epi-position and suitable administration and/or the excipient of suitable administration; The present invention also provides the vaccine that is used for the treatment of and prevents the CAV relative disease, and described vaccine comprises the adjuvant of recombinant virus particle of the present invention and suitable administration and/or the excipient of suitable administration.
The present invention will illustrate in greater detail based on following experimental section, and this only is for the purpose of description of giving an example, and should not regard the restriction to protection domain as.
Experiment
Purifying CAV particle is to induce the neutralizing monoclonal antibody of anti-CAV
For producing the neutralizing monoclonal antibody of anti-CAV, with the CAV particle injection mouse of purifying.
(Millipore, USA) the MDCC-MSB1 cell culture supernatant that 1 liter of CAV is infected concentrates 40 times, and supernatant is dialysed to 10mMTris (pH8.7)-1mM EDTA (TE) damping fluid with MILLITAN 300-kDa filter membrane.Adding sodium lauryl sulphate (SDS) to final concentration then in this CAV capsid suspension was 0.5%, 37 ℃ of insulations 30 minutes.Deposit C AV capsid on 30% sucrose bed course at last, the throw out that will contain the CAV capsid is resuspended in the 1ml TE damping fluid.Inject mouse twice with 100 μ l CAV capsid suspensions.
External neutralization test
The supernatant of candidate's monoclonal antibody with dilution in 1: 2 and make 2 times of dilution series, is incubated 1 hour (VonBulow et al., 1983, Von Bulow, 1985) with the supernatant and the 104-105 TCID50 CAV-Cux-1 of dilution.About 100,000 these mixtures with the diluted last cleer and peaceful virus of the cell of the T clone MDCC-MSB1 of MarekShi syndrome virus conversion infect (Yuasa, 1983, Yuasa et al, 1983).
Immunofluorescence analysis and immunoperoxidase analysis
Describe with 80% acetone fixed cell and as people such as Noteborn (1990), the goat anti-mouse IgG that cell and CAV monoclonal antibody specific and fluorescein are puted together is carried out immunofluorescence analysis.
As described in people such as Jeurissen (1988), with the recombinate immunoperoxidase staining of the CEF that VP1/VP2-MDV infects of CAV monoclonal antibody specific.
Enzyme Linked Immunoadsorbent Assay (ELISA)
Be used among the 50mM sodium bicarbonate pH9.6 with 1: 10, CAV specificity neutralizing monoclonal antibody 132.1 bags of 000 dilution by the droplet hole (Greiner, FRG), with the tap water hole flushing that contains 0.05%Tween80 three times.With the phosphate-buffered saline that contains 4% horse serum, 51g/l NaCl and 0.05%Tween80 (saturated damping fluid) 100 μ l 30 minutes, with the tap water hole flushing that contains 0.05%Tween80 three times in 37 ℃ of saturated holes.Then, mix the lysate that CAV particulate supernatant or 50 μ l contain reorganization VP1 and the proteic insect cell of VP2 that contains that undiluted chicken serum of 50 μ l and 50 μ l concentrate 30 times, and it is added in every hole, 37 ℃ of insulations 1 hour, with the tap water hole flushing that contains 0.05%Tween80 three times.The anti-mouse immuning ball protein G of the rabbit conjugate that adds the peroxidase labelling of 1: the 2000 times dilution of 100 μ l in saturated damping fluid in every hole is 37 ℃ of insulations 1 hour, once more with the tap water hole flushing that contains 0.05%Tween 80 three times.Xiang Kongzhong added 100 μ l tetramethyl benzidines, sodium acetate and catalatic standardized solution, room temperature insulation 10 minutes.With 10% sulfuric acid blocking reaction.Detect each hole as standard method at 450nm.
Baculovirus and insect cell
Recombinant baculovirus AcRP23-lacZ (Bishop, 1992) derives from doctor R.Possee of England Oxford NERC Ins of Virology, as method purified genomic dna as described in the Summers and Smith (1987).Fall army worm (Sf9) cell derives from U.S. tissue culture preservation center (CRL 1711).As described in Summers and Smith (1987), baculovirus stoste is cultivated on confluent monolayer cells, and cultivates suspension in containing the TC-100 substratum (GIBCO/BRL) of 10% foetal calf serum.
The gram of CAV DNA falls
All CAV dna sequence dnas carry out according to the described method of people such as Maniatis (1982) with all clone's steps that plasmid DNA is carried out in principle at first all from plasmid DNA pIc-20H/CAV-EcoRI (Noteborn and De Boer, 1990).
Carrying out DNA in intestinal bacteria HB101 bacterial strain transforms.All plasmids all increase in the large volume culture that stirs, and pass through then filtering on the Sephacryl-S500 post or filtering and purifying on the QIAGEN chromatography column by the CsCl gradient.
Structure and the screening of reorganization VP1/VP2 baculovirus
With the recombinant transfer vector pAcVP1/VP2 DNA transfection Sf9 cell that has linearizing recombinant baculovirus AcRP23-lacZ DNA, obtain recombinant baculovirus after homologous recombination, its two cav protein VP1 and VP2 that has mixed in the polyhedron unit respectively under the promoter regulation of p10 or polyhedrosis gene replaces lacZ.At first, in the plaque of the insect cell of baculovirus infection, lack betagalactosidase activity by detection and identify recombinant C AV virus, then by in hybrid experiment, determining that with the CAV specificity DNA probing needle CAV dna sequence dna is integrated into the baculovirus genome.
Immunoprecipitation analysis
Infected back two days, with cell and Promix marker (ICN, USA) insulation, after 4 hours at E1A damping fluid (50mM Tris (pH7.5), 0.1%Triton-X-100,250mMNaCl, 50mM NaF and 5mM EDTA) middle lysing cell, and with at the monoclonal antibody 111.1 of VP2 4 ℃ of insulations 2 hours, wash and on the PAA-SDS gel, separate with the E1A damping fluid.
CEF is advanced in MDV DNA transfection
In order to make up reorganization VP1/VP2 MDV, carry out the reorganization VP1/VP2 MDV transfer vector of purifying and the cotransfection of DNA according to the described method of Graham and Van der Eb (1973), described DNA separates the chick embryo fibroblast (CEF) that personal MDV Rispens isolate infects.
CAV genome transfection chicken T cell with sudden change
With the CAV clone that EcoRI digests various sudden changes, recirculation contains the EcoRI fragment of CAV (sudden change) sequence, and by the described DEAE-dextran of people such as Noteborn (1991) method with this fragment transfection MDCC-MSB1 cell.In contrast, with the parallel whole procedure of carrying out of pCAV-EcoRI clone.
Result and discussion
The generation of the neutralizing monoclonal antibody of anti-CAV
Noteborn and Koch (1994) have described two kinds of CAV monoclonal antibody specifics, a kind of is at VP2, and another kind of at VP3, these monoclonal antibodies are not all showed in the CAV specificity and activity, the more important thing is that these monoclonal antibody nones are at VP1.We think can neutralizing monoclonal antibody at VP1, because capsid mainly contains VP1 (Todd et al., 1990).Below we will describe the generation of anti-CAV neutralizing antibody.
In order to produce the neutralizing monoclonal antibody of anti-CAV, with the CAV particle injection mouse of purifying.
Be the screening first time of (neutralization) monoclonal antibody of carrying out anti-CAV, with insect cell (the stating as follows) bag of the reorganization VP1/VP2 baculovirus infection of synthetic VP1 and VP2 simultaneously by the droplet hole.The dilution CAV specific antisera that has senior middle school and tire reacted (stating as follows) with reorganization VP1 and/or VP2 product specifically in 1: 1000.Obtained specifically several different hybridoma cell line with the reaction of reorganization VP1/VP2 product.
CAV specific serum neutralization test shows three kinds of neutralization activity with anti-CAV in these monoclonal antibodies that obtained, these three kinds of CAV specificity neutralizing monoclonal antibodies are called after 132.1,132.2 and 132.3 (in Pasteur Institut's preservation, preserving number is xxxx, yyyy and zzzz) respectively.
The microscopic examination of the neutralizing monoclonal antibody of anti-CAV
Immunofluorescence analysis shows the special construction in the MDCC-MSB1 cell that three kinds of neutralizing monoclonal antibodies 132.1,132.2 and 132.3 identification CAV infect, these monoclonal antibodies all not with the MDCC-MSB1 cell response of not infecting.
With the neutralizing antibody (132.1) of electron microscope observation and anti-CAV or the purifying CAV particle of monoclonal antibody 111.1 (anti-VP2) or 111.3 (anti-VP3) insulation.People such as Todd (1990) have reported that the CAV capsid of purifying only contains the 50kDa protein that is likely VP1.Detect various monoclonal antibodies by immuno-gold labeling, only find that neutralizing monoclonal antibody 132.1 combines with the CAV particle, monoclonal antibody 132.1 combines with the CAV particulate and causes virolysis.Show that in addition cracked CAV capsid does not combine with the monoclonal antibody of anti-VP2 or VP3 with neutralizing monoclonal antibody 132.1 insulations.
These results have disclosed the mechanism of action of neutralizing monoclonal antibody: they cause the cracking of viral capsid, cause producing non-infectious particle thus.In addition, these data show that the CAV particle (almost) of purifying only contains VP1.
Neutralizing monoclonal antibody is at a conformational epitope on the VP1
Pepscan analyze (Geysen etc., 1984) disclose do not have in 3 kinds of neutralizing monoclonal antibodies a kind of with from VP1 or VP2 or VP3 deutero-12 aggressiveness significant reaction is arranged.The data that only useful for simplicity's sake monoclonal antibody 132.1 obtains are shown in Fig. 1 (at VP1) or Fig. 2 (at VP2).These results show that neutralizing monoclonal antibody is at a conformational epitope.
These data are by following experiment confirm.The CAV particle of the purifying in trace under the natural condition to nylon leaching film still can react with neutralizing monoclonal antibody 132.1, yet after boiling in the presence of SDS, the CAV capsid protein no longer combines with monoclonal antibody 132.1.
Carry out immunoprecipitation experiment as partially purified CAV particle of usefulness as described in (1994b) such as Noteborn and monoclonal antibody 132.1,132.2 or 132.3 under natural condition, the result shows the protein of a kind of about 50kDa of these monoclonal anti physical efficiencys precipitations.
Can sum up neutralizing monoclonal antibody from all results that draw is a conformational epitope at VP1.
Enzyme Linked Immunoadsorbent Assay (ELISA) based on the neutralizing antibody of anti-CAV
We have developed a species complex and have caught blocking-up (CTB)-ELISA, can use enrichment CAV particle that the MDCC-MSB-1 cell that infects from CAV obtains or by above-mentioned rhabdovirus system synthetic reorganization VP1/VP2 albumen.
The antibody that contains all epi-positions on blocking-up CAV capsid or the reorganization VP1/VP2 in the serum of the chicken that infects from CAV, this means that CAV capsid or reorganization VP1/VP2 will not combine with the monoclonal antibody 132.1 of bag quilt, yet negative serum will allow CAV capsid or reorganization VP1/VP2 to combine with 132.1 of bag quilt.To be decided to be less than 0.5 times signal of the signal that detects with negative control sera is positive signal.
The detection level of our CTB-ELISA is the valence value of the 24-25 that measures in serum neutralization test, and this is unusual sensitive.Analyzed more than 400 serum, be positive in CTB-ELISA with the serum that is defined as positive serum in serum neutralization test that shows 96.5% after the serum neutralization test relatively, 98.3% the serum that is defined as negative serum in serum neutralization test is negative in CTB-ELISA.
The structure of reorganization VP1/VP2 transfer vector
The encoding sequence of cav protein VP1 and .VP2 is cloned among the baculovirus transfer vector pAcUW51 (article No. 21205P), and this carrier can be from PharMingen, San Diego, and USA buys.This carrier as shown in Figure 3, it contains the polyhedrin flanking region, is baculovirus polyhedrin body protein promotor and p10 promotor in the middle of this district and is two 3 ' non-encoding transcription sequences that contain poly-adenosine signal that transcription unit is required.This transfer vector contains duplicate required protokaryon sequence in bacterium.
Plasmid pET-16b-VP2 (Noteborn etc., date is unexposed) contain the CAV dna sequence dna of 380-1512 position, this CAV dna fragmentation contains the VP2 encoding sequence, both sides are the non-coding CAV of 484bp3 ' dna sequence dna, at the 106bp place, initiator codon downstream of VP2, found to be arranged in the VP3 initiator codon of another frame.Handle plasmid pET-1 6b-VP2 with restriction enzyme NdeI and NheI, mend flat cohesive end with the Klenow polysaccharase.Be separated to 0.8kb CAV dna fragmentation.With BamHI linearization plasmid pAcUW51, mend flat cohesive end and use alkaline phosphatase (CIP) to handle with the Klenow polysaccharase.0.8kb CAV dna fragmentation is connected with linearizing pAcUW51 DNA, determines VP2 direction among the pAcUW51 by restriction endonuclease analysis.This construction called after pUW-VP2.
Plasmid pET-16b-VP1 (Noteborn etc., the date is unexposed) contains the CAV dna sequence dna of 853-2319 position, and this CAV DNA inserts fragment and contains the proteic complete encoding sequence of VP1, and both sides are 117bp 3 ' the non-coding CAV dna sequence dna.Handle plasmid pET-16b-VP1 with restriction enzyme NdeI and EcoRI, mend flat cohesive end with the Klenow polysaccharase.Be separated to 1.45kb CAV dna fragmentation.With EcoRI linearization plasmid pUW-VP2, mend flat cohesive end and use alkaline phosphatase (CIP) to handle with the Klenow polysaccharase.The 1.45kbCAV dna fragmentation is connected with linearizing pUW-VP2, determines and the reciprocal VP1 direction of p10 promotor unit that by restriction endonuclease analysis this final construction pAcVP1/VP2 is shown in Fig. 3.
The structure of reorganization VP1/VP2 baculovirus
The open reading frame of coding VP1 and VP2 is cloned into single baculovirus transfer vector pAcUW51 by oneself, and the CAV sequence of coding VP1 is under the regulation and control of baculovirus p10 promotor, and VP2 is under the regulation and control of baculovirus polyhedrin body protein promotor.With " exposing " baculovirus DNA and transfer vector DNA transfection Sf 9 insect cell, mixed the baculovirus of VP1/VP2 ceneme after the homologous recombination, the polyhedrin district that this VP1/VP2 ceneme has been integrated into recombinant baculovirus has replaced lacZ unit.The evaluation recombinant baculovirus has been lost betagalactosidase activity and has been integrated the CAV dna sequence dna.
Cav protein VP1 and the VP2 expression in the Sf9 cell
By coomassie brilliant blue staining and with Promix (ICN, USA) or
3The H-leucine (Amersham, UK) express in cav protein VP1 and the VP2 in the Sf9 cell of reorganization VP1/VP2 baculovirus infection by labelled protein and PAA-SDS gel electrophoresis analysis.
In PCT/NL94/00168, show in the lysate with the insect cell of reorganization VP1 baculovirus infection and contain the CAV specific proteins of 52kD, and in the insect cell that infects, produce the main specificity product of 30kDa by recombinant VP 2 baculovirus expression VP2.Two kinds of CAV specific proteinses that cause synthetic 52kD and 30kD with reorganization VP1/VP2 baculovirus infection insect cell.CAV specificity product can be used as the protein band of radiolabeled protein band or coomassie brilliant blue staining and detects.Back one result shows that two kinds of products all produce with higher relatively level in the insect cell of reorganization VP1/VP2 baculovirus infection.Sf9 cell with reorganization lacZ baculovirus infection does not contain these CAV specific proteinses.
Therefore we obtain following evidence, promptly inoculate the neutralizing antibody that hen can be induced anti-CAV with the thick lysate of the Sf9 cell of reorganization VP1/VP2 baculovirus infection.
The effect of VP2 in the conformation neutralizing epitope that forms VP1
As among the PCT/NL94/00168 and people (1995) such as Koch report; obtain to synthesize when neutralization and protective immune response need recombinant C AV albumen VP1 and VP2 but not simple the mixing; these data promptings VP2 is a Nonstructural Protein; its a certain stage in infection is that the correct conformation of virus assembling and/or VP1 is required, causes the formation of neutralizing epitope thus.An explanation that needs VP2 may be that it is a kind of scaffolding protein, needs it still not have (Leibowitz and Horwitz, 1975) in the assembling of virion in final product.The example of scaffolding protein have the IVa2 of adenovirus and 39kDa albumen (D ' Halluinet al., 1978; Persson et al., 1979).These albumen are still removed in next step as the support that forms so-called light capsid.VP2 may play similar effect in forming CAV virion process, but we can not get rid of following possibility fully in this stage, promptly at the CAV of purifying preparation (Todd et al., 1990) in the electroblotting, or in the cracked CAV particulate electron micrograph of the VP2 monoclonal antibody specific insulation of above-mentioned and immuno-gold labeling, have detection less than (very) a spot of VP2 combine formation conformation neutralizing epitope with VP1.Recently, reported the not strong evidence (Buchholz, 1994) of cogency that in the CAV of gradient purifying, has VP2.
In following experiment, provide when VP2 is synthetic simultaneously only (advantage) to have the evidence of the neutralizing epitope of VP1.Add the VP2 infected insect cell with the recombinant C AV baculovirus (seeing PCT/NL94/00168) or the VP1 that express VP1, VP2, after infection, collected the Sf9 cell that infects in 3 or 4 days, and it is carried out the immunofluorescence test with CAV specificity neutralizing monoclonal antibody 132.1.The cell that only contains CAV specific proteins VP2 does not react with monoclonal antibody 132.1, the cell that only contains VP1 only demonstrates very faint immunofluorescence signal after being incubated with monoclonal antibody 132.1, yet very strong with combining of neutralizing monoclonal antibody 132.1 with the insect cell of the reorganization VP1/VP2 baculovirus infection of expressing VP1 and VP2 simultaneously.Express the parallel PAA-SDS gel electrophoresis of radio-labeling lysate that VP1, VP2 or VP1 add the insect cell of VP2 and show that its expression level is identical when expressing when the VP1 single expression or with VP2.
The interaction of VP1 and VP2 is temporary transient
The neutralizing epitope of VP1 only could form when VP2 exists, so it is the epi-position that conformation is distinguished, this prompting VP1 and VP2 interosculated in a short period of time.By the immunoprecipitation under condition as mild as a dove, we have detected whether VP1 can combine with VP2, add the recombinate shape virus infection Sf9 insect cell of VP2 with synthetic VP1, VP2 or VP1.
The result clearlys show when VP2 is synthetic separately or in the presence of VP1, monoclonal antibody 111.1 can precipitate VP2.Also expressing except VP2 under the situation of VP1, VP1 seldom with the VP2 co-precipitation.When at VP2 in the presence of not during synthetic VP1, monoclonal antibody 111.1 can not precipitate VP1.These data show that VP1 interosculates with VP2 (only with relative less amount), and during this combination, VP1 may obtain to produce the conformation of neutralizing epitope.
Develop the basis of the vaccine of anti-CAV infection
The above-mentioned result who provides is shown as the neutralizing antibody of inducing anti-CAV, needs VP1 that one special conformation is arranged.In baculovirus expression system, this correct VP1 conformation only is only possible when synthesizing VP1+VP2 or VP1+VP2+VP3 simultaneously.Also can destroy the neutralizing epitope of VP1 with for example sodium lauryl sulphate sex change VP1, prompting VP1 neutralizing epitope is a kind of conformational epitope.
Be used to inoculate lay eggs the recombinant C AV product VP1+VP2 of hen or VP1+VP2+VP3 can be synthetic by rhabdovirus system, these cav proteins also can be synthetic by other expression system, yeast cell for example is by (reverse transcription) virus infection or the gene amplification (CHO-dhfr system) in the mammal cell line system.
In theory, the expression of VP1 fragment (with VP2 or VP2 and VP3 associating) is enough to induce protective immune response.12 aggressiveness of VP1 can not react this true prompting with the neutralizing antibody of anti-CAV needs bigger VP1 fragment just can obtain correct VP1 conformation to form neutralizing epitope.Yet be understood that the disappearance of small amino acid mutation or several amino acid can not influence the formation of VP1 neutralizing epitope.
2 or 3 albumen by CAV open reading frame coding can induce this fact of protective immune response also to can be used for developing live vector.The encoding sequence of VP1+VP2 or VP1+VP2+VP3 can be cloned in the live vector.
In live vector, a kind of in the cav protein, VP1, VP2 or VP3 also may be suitable for inducing the protective immune response of anti-CAV separately, and the fragment of expressing one of above-mentioned cav protein by live vector also may be enough to induce protective immune response.
VP3 causes this fact of the monocytic apoptosis of chicken (PCT/NL94/00168; Noteborn etc. 1994a) help making up the live vector of not expressing VP3.Duplicating of MarekShi syndrome virus may be by the negative impact of VP3 inductive apoptosis.
Perhaps, can produce cause that anti-CAV infects in and the attenuation CAV of the required expression VP1 of protective immune response with required conformation.
The structure of recombinant mdv-VP1/VP2 transfer vector
With the sequence clone of coding cav protein VP1 and VP2 to MarekShi syndrome virus (MDV) transfer vector pMD-US10-SV (Koch, published material not) in, this transfer vector contains the protokaryon sequence that there is the SV40 early promoter in MDV US10 district both sides and is used for increasing on bacterium.The synoptic diagram of this transfer vector is seen Fig. 4.
The CAV encoding sequence of 368-2319 position Nucleotide is inserted in the pMD-US10-SV transfer vector, makes it under the regulation and control of SV40 early promoter.This recombinant transfer vector detects with digestion with restriction enzyme, and called after pMD-US10-SV-VP1/VP2.
By introducing another terminator codon (Noteborn, not published material) 549 introducing one point mutation (T becomes A) in the gene of coding VP3, therefore the CAV sequence of inserting is only expressed VP1 and VP2.Indirect immunofluorescence analysis with the chick embryo fibroblast (CEF) of carrier pMD-US10-SV-VP1/VP2 transfection confirms that the expression of VP1 and VP2 is arranged really, and does not express VP3.
The structure of reorganization VP1/VP2 MDV
Use calcium phosphate transfection method with recombinant transfer vector pMD-US10-SV-VP1/VP2DNA and MDV (Rispens isolate) transfection CEF cell.After the homologous recombination, obtain recombinant mdv, it has mixed CAV encoding sequence (Nakamura et al., 1992) in the US10 district.Verify that this reorganization VP1/VP2 MDV expresses VP2.By parallel plaque purification and the immunoperoxidase analysis of using at the monoclonal antibody of VP2, obtained the reorganization VP1/VP2 MDV independence strain of several 100% purifying.
The evaluation of reorganization VP1/VP2 MDV
Determine that by PCR and restriction endonuclease analysis the CAV-DNA sequence correctly is integrated into the MDV genome.Follow-up the going down to posterity of reorganization VP1/VP2 MDV by various purifying shows that they are stable.Do not obtain (part) and lost the recon of VP1/VP2 ceneme.
Express cav protein VP1 and VP2 the time in the CEF cell that has confirmed to infect by indirect immunofluorescence, used the monoclonal antibody CVI-CAV-111.1 (111.1) of anti-VP2 and the monoclonal antibody CVI-CAV-132.1 (132.1) of anti-VP1 with reorganization VP1/VP2 MDV.
Monoclonal antibody 132.1 is neutralizing epitopes at VP1, and the required neutralizing epitope of this true prompting of VP1 that neutralizing antibody 132.1 can be discerned and VP2 while synthetic recombinant mdv is expressed is present on the VP1 of MDV expression.Therefore, will cause inducing neutralization/protection antibody to reply with reorganization VP1/VP2 MDV immunization chicken.Except synthetic CAV VP1 and VP2 albumen, reorganization VP1/VP2 MDV will synthesize the required MDV albumen of protective immune response of inducing anti-MDV to infect.
CAV genome pCAV/EcoRI based on the clone produces attenuation CAV
Produce the infectious CAV particle of living with CAV-EcoRI clone, for this reason, it is that 2319bp EcoRI fragment is separated from the bacterium sequence and handled recirculation with archaeal dna polymerase that complete CAV is inserted fragment.The EcoRI fragment transfection of using recirculation then is the MDCC-MSB1 cell for example, will produce wild-type CAV after several days.
Can in CAV-EcoRI clone's CAV sequence, import sudden change, the MDCC-MSB1 cell is advanced in separation then, recirculation and transfection, CAV that can obtain suddenling change rather than wild-type CAV in theory, and if the pathogenic reduction of mutant, attenuation CAV then obtained.The complete strategy that produces attenuation CAV is seen shown in Figure 5.
The genomic structure of CAV that contains the promotor/enhancing subarea of sudden change
A notable feature in the promotor of CAV/enhancing subarea is by 12bp insertion sequence 4 or 5 sequence (Noteborn et al. that intimate 21bp tumor-necrosis factor glycoproteins is completely formed at interval, 1991), the regulation and control that this zone participation CAV transcribes (Noteborn et al., 1994b).
For producing attenuation CAV, we have imported sudden change (Noteborn et al., 1991) in 12bp insertion sequence/direct iteron of CAV-EcoRI clone, and the promotor of the sudden change of wild-type CAV and various CAV mutant/enhancing subarea is shown in Fig. 6.The Nae mutant does not contain the direct iteron of 12bp/ fully, the substitute is and has introduced a NaeI site (5 '-GCCGGC-3 '), and it is shown " N " in Fig. 6.
CAV-EcoRI contains 5 direct repeat sequences, and all other mutant contain 4 primary direct repeat sequences.The various CAV mutant that are called " 6bp ", " 12bp ", " 24bp " contain the 12bp insertion sequence that changes.Mutant " wt in Nae " and the original CAV-EcoRI clone who is called " wild-type " contain original 12bp insertion sequence 5 '-AAGAGGCGTTCC-3 '.
CAV mutant " 6bp ", " 12bp ", " 18bp ", " 24bp " and " wtin Nae " all contain the additional sequences that is positioned at 12bp insertion sequence/direct both sides, iteron, joint 5 '-GCCCATGT-3 ' has been introduced in 5 ' site in this zone, has introduced joint 5 '-GATCCGCC-3 ' in 3 ' site.
The CAV genome of sudden change causes producing the infectious CAV of attenuation
For whether the CAV genome of determining sudden change can produce CAV particle alive, at first detect the synthetic of VP3.On each time point after the transfection, part with the MDCC-MSB1 cell culture of the various transfections of acetone fixed, and with the monoclonal antibody of anti-VP3 through indirect immunofluorescence analysis, simultaneously every kind of culture of 1ml is joined 9ml and adds in the fresh RPMI substratum of 10% foetal calf serum.
After 6 days and 11 days, there is the cultivation MDCC-MSB1 cell of the CAV genomic dna transfection of 15% and 90% usefulness " wild-type " recirculation to contain VP3 (cell transfecting is after once go down to posterity) respectively approximately.
The CAV mutator gene group " Nae " that discovery lacks complete 12bp insertion sequence/direct iteron does not produce infectious viral particle.After the transfection 6 days, there is 2 ℃ cell to produce VP3 at most, this is because due to the transient expression of " Nae " DNA.(Noteborn etal., 1994b) experiment of carrying out has obtained similar per-cent for VP3 and the known expression plasmid pRSV-VP3 that can not duplicate in eukaryotic cell with only encoding.After 3 weeks and after going down to posterity several times, in the MDCC-MSB1 of Nae-DNA transfection cell culture, no longer there is VP3.
In the various timed intervals after transfection, significantly be lower than the per-cent of the VP3 positive cell in the culture of usefulness wild-type DNA genome transfection with the per-cent of VP3 positive cell in the MDCC-MSB1 culture of DNA genome " 6bp ", " 12bp ", " 18bp " of sudden change, " 24bp " and " wt in Nae " transfection.The various CAV mutant of these Notes of Key Datas are slower than wild-type CAV reproduction speed, and particularly mutant " 6bp " and " 18bp " have reduced their multiple-copy rate.The sudden change of 12bp insertion sequence cause the suddenling change virus disseminating of CAV reduces probably.These result of experiment are shown in Fig. 7.
Afterwards, we are used for the supernatant of sudden change CAV-DNA genome cells transfected of personal all detections and infect fresh MDCC-MSB1 culture, except the supernatant from " Nae "-DNA cells transfected, all supernatants all prove and contain infectious viral particle.For these cultures that infects with the supernatant that contains " mutated viruses ", can show by indirect immunofluorescence to have the cell that contains VP3.
The CAV mutant has the cytopathic effect of reduction
With implement to detect VP3 synthetic experiment parallel analysis transfectional cell whether can apoptosis.Known CAV is 2-3 days meeting inducing apoptosis after infection.For this reason, with the iodate third ingot staining cell, iodate third ingot " normally " DNA that can dye by force, and dye more weak and/or irregular to the DNA of apoptosis.Apoptosis all takes place in the MDCC-MSB1 cell of 11 days nearly all usefulness " wild-type " DNA transfection after the transfection, and with the major part of various sudden change CAV-DNA genome cells transfected apoptosis does not take place.The ability of finding the inducing apoptosis of CAV mutant " 6bp " and " 18bp " reduces greatly.
The DNA analysis of attenuation CAV
Show by polymerase chain reaction with to the sequential analysis of iteron of 12bp insertion sequence/directly and flanking sequence, for various CAV mutant " 6bp ", " 12bp ", " 18bp ", " 24bp " and " wt in Nae ", change of primary sudden change.The various CAV mutant of these results suggest are stable, and cultivating in MDCC-MSB1 at least after about 1 month is so, and these CAV mutant be really live and the cytopathic effect in the chicken T cell of cultivating reduce.
Show that from the Southern engram analysis of the MDCC-MSB1 culture separated DNA that infects with various CAV mutant all CAV mutant of living produce all CAV DNA that wild-type CAV are produced, and all detect double-stranded replicative intermediate and single stranded DNA in wild-type and sudden change CAV.
Attenuation CAV synthesizes CAV specificity neutralizing epitope
The indirect immunofluorescence analysis that carries out with the monoclonal antibody CVI-CAV85.1 of the monoclonal antibody CVI-CAV111.2 of neutralizing monoclonal antibody CVI-CAV132.1 and anti-VP2 and anti-VP3 shows that the CAV genome of all sudden changes all can synthesize cav protein VP1, VP2 and VP3.The more important thing is neutralizing monoclonal antibody 132.1 and the cell response of infecting with various CAV mutant; the CAV of this discovery prompting sudden change will produce required in the CAV specificity epitope; this epi-position will be by the immune system recognition at the chicken of CAV immunization, thereby causes protective immune response.
Can draw as drawing a conclusion, can prepare the CAV genome of sudden change based on CAV clone pCAV-EcoRI, cause producing sudden change CAV particle alive, the reproduction ratio wild-type CAV in the chicken T cell of cultivating is slow for these CAV mutant, the cytopathic effect that is caused by these CAV mutant is also low than wild-type CAV, all CAV mutant all can produce all cav proteins, thereby produce the neutralizing epitope on the required VP1.
Therefore, these CAV mutant attenuation version that is CAV.
Accompanying drawing is described
Fig. 1 illustrates the pepscan analysis to 132.1 type neutralizing monoclonal antibodies of the peptide (12 aggressiveness) used from VP1.
Fig. 2 illustrates the pepscan analysis to 132.1 type neutralizing monoclonal antibodies of the peptide (12 aggressiveness) used from VP2.
Fig. 3 illustrates the synoptic diagram of recombinant transfer vector pUW-VP1/VP2.
Fig. 4 illustrates the synoptic diagram of recombinant transfer vector pMD-US10-SV-VP1/VP2.
Fig. 5 illustrates the strategy that produces attenuation CAV mutant based on the plasmid pCAV/EcoRI of the 2319bp that contains wild-type CAV.
Fig. 6 illustrates schematic structure various sudden changes and CAV promotor/enhancing subarea wild-type CAV strain.
The VP3 of various sudden change CAV and wild-type CAV expresses speed behind that Fig. 7 is illustrated in transfection sudden change or the wild-type cyclized DNA, and VP3 expresses speed and provides with the per-cent of the positive MDCC-MSB1 cell of VP3.
Fig. 8 is the sequence of CAV.
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Claims (15)
1, a kind of neutralizing antibody or antibody fragment or derivatives thereof, a conformational epitope reaction of the viral protein 1 (VP1) of itself and chicken anemia virus (CAV), described conformational epitope is named as 132-1, the monoclonal antibody identification of 132-2 or 132-3, described monoclonal antibody is by in Pasteur Institut's preservation, preserving number is xxxxxx, yyyyyy, and the hybridoma of zzzzzz produces.
2, a kind of conformation neutralizing epitope of viral protein 1 of chicken anemia virus of the antibody recognition by claim 1.
3, a kind of method that is used to produce the viral protein 1 of the conformational epitope that comprises claim 2, described method is included in the cell described viral protein 1 is expressed with the viral protein 2 of described CAV, and the genetic information of the genetic information of the VP1 that wherein encodes and coding VP2 has an independent existence in a recombinant vectors.
4, a kind of carrier that is used for the method for claim 3 comprises as the genetic information of the coding VP1 of two absolute coding sequences and the genetic information of coding VP2.
5, the carrier of claim 4, it is based on the carrier of MarekShi syndrome virus carrier.
6, the carrier of claim 4, it is based on the carrier of baculovirus vector.
7, a kind of provide comprise claim 1 in and the method for the viral protein 1 of conformational epitope, described method comprises from the genetic expression of coding VP1 and the VP2 funtion part of VP1 and VP2 at least, wherein said gene is under the control of regulating and controlling sequence of the CAV sequence that is derived from the transcription initiation site upstream, and described regulating and controlling sequence is modified to reduce its effect.
8, claim 3 or 7 method, wherein VP1 is that form with virion provides.
9, the method for claim 7, wherein said modification are positioned at promotor/enhancing subarea.
10, the method for claim 9, wherein said modification are arranged in the 12bp insertion sequence that promotor strengthens the subarea.
11, the recombinant virus particle that obtains by each described method of claim 7-10.
12, a kind of nucleic acid that is used for each described method of claim 7-10, described nucleic acid comprises the gene of the funtion part of the gene of the funtion part of VP1 at least of encoding and coding VP2, at least a described gene is under the control of a regulating and controlling sequence, and described regulating and controlling sequence is through modifying with the reduction effect.
13, a kind ofly be used for detecting or whether definite sample exists the diagnostic kit of CAV or anti-CAV antibody, it uses the antibody of claim 1 and/or the epi-position of claim 2.
14, a kind of vaccine that is used for the treatment of or prevents the CAV relative disease comprises antibody or the epi-position of claim 2 and the adjuvant and/or the excipient of suitable administration of claim 1.
15, a kind of vaccine that is used for the treatment of or prevents the CAV relative disease comprises the recombinant virus particle of claim 11 and the adjuvant and/or the excipient of suitable administration.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/480,020 | 1995-06-07 | ||
| US08/480,020 US5932476A (en) | 1990-09-12 | 1995-06-07 | Cloning of chicken anemia virus DNA |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1194001A true CN1194001A (en) | 1998-09-23 |
Family
ID=23906352
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN96195434A Pending CN1194001A (en) | 1995-06-07 | 1996-06-07 | Neutralizing conformational epitopes of chicken anemia virus |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0832242A1 (en) |
| JP (1) | JPH11510367A (en) |
| CN (1) | CN1194001A (en) |
| AU (1) | AU720640B2 (en) |
| CA (1) | CA2221570A1 (en) |
| NZ (1) | NZ309172A (en) |
| WO (1) | WO1996040931A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113897356A (en) * | 2021-10-20 | 2022-01-07 | 佛山科学技术学院 | Fluorescent quantitative PCR kit and primers for detecting chicken infectious anemia virus |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7192594B2 (en) | 1997-10-03 | 2007-03-20 | Merial Limited | Postweaning multisystemic wasting syndrome and porcine circovirus from pigs |
| FR2772047B1 (en) | 1997-12-05 | 2004-04-09 | Ct Nat D Etudes Veterinaires E | GENOMIC SEQUENCE AND POLYPEPTIDES OF CIRCOVIRUS ASSOCIATED WITH PIGLET LOSS DISEASE (MAP), APPLICATIONS TO DIAGNOSIS AND TO PREVENTION AND / OR TREATMENT OF INFECTION |
| DE69837934T2 (en) * | 1997-12-11 | 2008-02-21 | University Of Saskatchewan, Saskatoon | MULTISYSTEMIC TUMBLER SYNDROME THROUGH VIRUSES IN PIGS |
| JP2001275664A (en) * | 2000-02-29 | 2001-10-09 | Akzo Nobel Nv | Chicken anaemia virus having low pathogenicity |
| AUPR567401A0 (en) * | 2001-06-14 | 2001-07-12 | University Of Melbourne, The | Circovirus vaccines |
| JP2007524609A (en) * | 2003-04-10 | 2007-08-30 | トランスフォーム・ファーマシューティカルズ・インコーポレイテッド | Conformational variant profiling, antibody composition |
| BR112016006192A2 (en) | 2013-09-25 | 2017-09-26 | Zoetis Services Llc | pcv2b divergent vaccine composition and methods of use |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0473210B1 (en) * | 1990-07-30 | 1994-04-13 | Akzo Nobel N.V. | Recombinant Marek's disease virus |
| NL9301272A (en) * | 1993-07-20 | 1995-02-16 | Aesculaap Bv | Chicken Anemia Virus mutants and vaccines and uses based on the viral proteins VP1, VP2 and VP3 or coding sequences of that virus. |
| NL9002008A (en) * | 1990-09-12 | 1992-04-01 | Stichting Centr Diergeneeskund | RECOMBINANT DNA AND RNA AND PRODUCTS DERIVED THEREFROM. |
| ZA918426B (en) * | 1990-10-31 | 1992-12-30 | Akzo Nv | Chicken anemia virus vaccine and diagnostic |
-
1996
- 1996-06-07 JP JP9500328A patent/JPH11510367A/en active Pending
- 1996-06-07 EP EP96916379A patent/EP0832242A1/en not_active Withdrawn
- 1996-06-07 AU AU59137/96A patent/AU720640B2/en not_active Ceased
- 1996-06-07 WO PCT/NL1996/000230 patent/WO1996040931A1/en not_active Application Discontinuation
- 1996-06-07 CN CN96195434A patent/CN1194001A/en active Pending
- 1996-06-07 NZ NZ309172A patent/NZ309172A/en unknown
- 1996-06-07 CA CA002221570A patent/CA2221570A1/en not_active Abandoned
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113897356A (en) * | 2021-10-20 | 2022-01-07 | 佛山科学技术学院 | Fluorescent quantitative PCR kit and primers for detecting chicken infectious anemia virus |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2221570A1 (en) | 1996-12-19 |
| EP0832242A1 (en) | 1998-04-01 |
| AU5913796A (en) | 1996-12-30 |
| WO1996040931A1 (en) | 1996-12-19 |
| JPH11510367A (en) | 1999-09-14 |
| NZ309172A (en) | 1999-11-29 |
| AU720640B2 (en) | 2000-06-08 |
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