CN1134175A - Viral vector with bovine viral diarrhea virus (BVDV) antigens - Google Patents
Viral vector with bovine viral diarrhea virus (BVDV) antigens Download PDFInfo
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- CN1134175A CN1134175A CN94193978A CN94193978A CN1134175A CN 1134175 A CN1134175 A CN 1134175A CN 94193978 A CN94193978 A CN 94193978A CN 94193978 A CN94193978 A CN 94193978A CN 1134175 A CN1134175 A CN 1134175A
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Abstract
This invention relates to the field of Bovine Viral Diarrhea Virus (BVDV), and vaccines for the treatment thereof. This invention describes the preparation of live, attenuated Bovine Herpesvirus type 1 (BHV-1) as a virus, vaccine and vector for expression of BVDV antigens. A BVDV cDNA clone containing sequences corresponding to glycoprotein gp53 is inserted into an inactivated BHV-1 virus.
Description
The present invention relates to the field of bovine viral diarrhea virus (BVDV) and treatment vaccine thereof.Documents and materials:
van?Zijl，M.et?al，Live?Attenuated?Pseudorabies?Virus?ExpressingEnvelope?Glycoprotein?E1?of?Hog?Cholera?Virus?Protects?Swine?a-gainst?both?Pseudorabies?and?Hog?Cholera，Journal?of?Virology，Vol.65，No.5，pp.2761-2765(1991).U.S.patent?4,703,011，Kit，M.，and?Kit，S.，Thymidine?Kinase?Deletion?Mutants?of?Bovine?Her-pesvirus-1，issued?27?October?1987.U.S.patent?4,824,667，Kit，M.，and?Kit，S.Thymidine?Kinase?Deletion?Mutants?of?Bovine?Her-pesvirus-1，Vaccines?Against?Infectious?Bovine?Rhinotracheitis?Con-taining?Same?and?Methods?for?the?Production?and?Use?of?Same，issued25?April，1989.Collett，M.S.，et?al.，Proteins?Encoded?by?BcvineViral?Diarrhea?Virus：The?Genomic?Organization?of?a?Pestivirus，Vi-rology，Vol?165?PP·200-208(1988).Collett，M.S.，et?al.，Molec-ular?Cloning?and?NucleotideSequence?of?the?Pestivirus?Bovine?Viral?Di-arrhea?Virus，Virology，Vol.165?PP.191-199(1988).
Bovine viral diarrhea virus (BVDV) is the pestivirus that belongs to yellow fever virus (Flaviviridae) section.It causes many different symptoms in sheep, goat, particularly ox.This symptom depends on the age of animal, physiology and virusology state.In young susceptibility calf and young adult, the disease that it causes be characterized as high incidence and low actual.Symptom can comprise fever, depression, ring nose secreting liquid, and diarrhoea reaches stomatocace once in a while.Except these elementary influences, this virus also causes immunosuppression.Although it is generally quite slight that elementary BVDV infects, the pathogenic of other coinfection microorganism may be strengthened or strengthen to this virus.
In aged or responsive animal, BVDV causes to top in the similar symptom of those symptoms described in the young responsive calf.In addition, in animal pregnancy, virus has the ability of passing placenta and infecting fetus.The consequence of this infection depends on whether gestational age and it are in the stage that its immunity system has enough abilities.Possible the consequence that infects comprises that fetus heavily absorbs, and miscarriage ossifys, birth defect, and birth defects bears the calf of persistent infection BVDV and calf fully normally.The calf of the persistent infection BCDV of birth has embodied most important parts in the pathogenic complex body of this BVDV.The animal of persistent infection is discharged into a large amount of viruses in its environment, the responsive animal of PI.And even the animal of persistent infection has immunotolerance to the virus that infects them in the uterus, but when being infected by the close BVDV biotype of other sibship, they also can form disease.The feature of these infection is sickness rate low (not had many by the animal pregnancy of the normal calf of BVDV persistent infection because just in time produce in the pregnancy duration infection comparatively speaking), but the mortality ratio height.This disease syndromes is called membrane disease, and himself the performance be generally utmost point acute symptom, calf dies from the too much watery diarrhea that contains a large amount of blood.
Cause having formed many vaccines in the trial that this viral importance and its extensively existing in cows attempt to prevent BVDV to infect.These vaccines form according to the traditional concept of deactivation or attenuation, but because the characteristic of BVDV, they have many obvious defects.
The vaccine product of common recognition deactivation is effective not as the living vaccine of attenuation.Inactivation antigen from the inactivated vaccine goods carries out exogenous processing.After being expelled to animal body, antigen becomes the part of animal soluble proteins composition.Enter antigen presenting cell and produce antibody usually by pinocytosis mechanism antigen.Regrettably, because antibody can not enter cell, they generally only can interrupt viral life cycle when ripe virus discharges from cell.On the other hand, come the antigen of the live virus of comfortable time multiplexed cell system to carry out endogenous processing and should mechanism produce preferred cell-mediated immune responses.Cell-mediated immune responses can be discerned by the cell of virus infection and have in the ability that interrupts viral life cycle than stage morning.Therefore, in immune defense, think to many virus infectiones cell-mediated reply of crucial importance.
Because therefore cell-mediated replying should induce effectively cell-mediated replying such as the attenuated live product of vaccine.For BVDV, usually do not stop vaccine virus to cause immunosuppression relevant under the normal circumstances (Roth J.A.and Kaeberle M.L. for producing living vaccine to the attenuation of virus with open-air isolate, Suppression of Neutrophil and Lym-phocyte Function Induced by a Vaccinal Strain of Bovine Viral Diar-rhea Virus With or Without the Adninistration of ACTH, AmericamJournal of Veterinarg Research, Vol.44 PP2366-2372 (1983)).Vaccine stops the failure of immunosuppression reaction to make vaccine produce serious defective.The animal owner may inoculate animal so that its resist the disease, but because this characteristic of vaccine, the owner provides chance on the contrary other eqpidemic disease infection animal.This just logical BVDV vaccine that makes the owner use deactivation, but because the immune mode of action, this vaccine is ineffective especially.
In a word, the vaccine safety of deactivation but ineffective especially, and the living vaccine of attenuation is more effective, but in some cases may be not really safe.
The present invention combines the validity of attenuated live vaccine and the security of inactivated vaccine.Ox herpes virus 1 type (BHV-1) is another kind of main cattle disease substance, and it produces respiratory tract disease.Therefore, be that BHV-1 also duplicates at mucomembranous surface with the BVDV common.We have obtained the gene of coding gp53, gp53 is the main glycoprotein of BVDV virus, and the host produces effective immune response to it, has expressed this albumen in ox herpes virus-1 (BHV-1), this recombinant virus (BHV/BVDV gp53) is as resisting the vaccine of BVDV.(Do-nis, R.O.and Dubovi, E.J., Glycoproteins of BovineViral Diar-rhoea Mucosal Disease Virus in Infected Bovine Cells, Journal of Gen-eral Virology, Vol.68, pp.1607-1616 (1987) and Magar, R., et al., Bovine Viral Diarrhea Virus Proteins:Heterogeneity of Cy-topathogenic and Noncytopathogenic Strains and Evidence of 53KGlycoprotein Neutralization Epitope, the reference that Veterinary Microbiology.Vol.16, pp.303-314. quote is listed this paper in for your guidance).
A kind of not pathogenic virus of rf that is used for the disease that prevention causes by bovine viral diarrhea virus (BVDV), the not pathogenic virus of wherein said rf comprises: take from the gene or the assortment of genes of BVDV virus, the not pathogenic virus of said rf can be expressed the said gene or the assortment of genes on function.The present invention includes the following example: a kind of virus, the not pathogenic virus of wherein said rf is attenuation, is selected from ox herpes virus 1 type (BHV-1) of attenuation, the adenovirus of attenuation, the BMV of attenuation, the bovine papilloma virus of attenuation, or the pseudorabies virus of attenuation.A kind of virus, the not pathogenic virus of wherein said rf be attenuation and contain and express the arbitrary combination of following gene: coding gp48, gp25, p14 capsid protein, p20N-terminal protein enzyme and the proteinic gene of p125/p80.A kind of virus, attenuation wherein is through making thymidine kinase (tk) gene lose function and realize.
A kind of virus, signal peptide wherein are inserted into the gene of coding gp53 or the front of the assortment of genes in said ox herpes virus 1 type (BHV-1).A kind of virus, the gene of wherein said coding gp53 is inserted into thymidine kinase (tk) gene locus of deactivation.A kind of virus, the gene of expressing on function or the assortment of genes that wherein are used to produce this virus comprise the recombinant plasmid with intact virus DNA, said plasmid comprises: a) contain BHV-1 genomic DNA fragment that thymidine kinase (tk) gene and thymidine kinase (tk) gene have disappearance, b) promotor/poly-adenosine signal of insertion thymidine kinase (tk) genetically deficient position, c) be positioned at the gp53 gene or assortment of genes signal peptide gene sequence before, they all are inserted between promotor and the polyadenylation signal.A kind of virus, wherein said plasmid is made of the plasmid with plasmid pHAS4 feature.A kind of virus, wherein said signal peptide gene sequence is taken from the signal peptide sequence of fully identifying arbitrarily, as seen in Perlman, D., etal., the arbitrary sequence in 39 signal peptide sequence examples of the abundant evaluation of J.Mol.Biol.Vol.167pp.391-409 (1983) (quoting) with reference.A kind of virus, wherein said signal peptide gene sequence are taken from pseudorabies virus gIII gene (PRV) and/or middle tethelin (BGH).
A kind of virus, plasmid wherein are selected from following plasmid, a) .pBHVtK ex-1 ∷ BGH/p53; B) pBHVtK ex-1 ∷ gIII/p53; C) pBHVtKex-3 ∷ BGH/p53; Or d) pBHVtKex-3 ∷ gIII/p53.A kind of virus of producing the product of the gene express or the assortment of genes on function, be selected from down a kind of in the influenza virus: T11-3, T11-6 or T11-8.A kind of virus, the gene of expressing on function or the assortment of genes that wherein are used to produce this virus comprise the recombinant plasmid with intact virus DNA, said plasmid comprises: BHV-1 genomic DNA fragment that a) contains thymidine kinase (tk) gene and have thymidine kinase (tk) genetically deficient, b) promotor/poly-adenosine signal that inserts at thymidine kinase (tk) genetically deficient place, c) the gp53 gene or the assortment of genes of between promotor and poly-adenosine signal, inserting.A kind of virus, plasmid wherein are the virus that pBHVtK ex-3 ∷ p53. is selected from down one of influenza virus: T2-3#3 or T2-2#5.A kind of vaccine that is used for the disease that prevention causes by bovine viral diarrhea virus (BDVD) comprises medicinal virus and a kind of carrier of going up significant quantity as herein described.
The above-mentioned vaccine that is used for the disease that prevention causes by bovine viral diarrhea virus (BDVD), comprise above-mentioned medicinal virus and a kind of carrier of going up significant quantity, said carrier comprises any physiological buffer medium, promptly, contain the medium (PH is approximately 7.0 to 7.4) of the serum of 2.5 to 15% nonreactive BHV antibody.
A kind of immune animal is resisted the method for the infectious diseases that is caused by bovine viral diarrhea virus (BDVD), comprises to animal to use medicinal virus or the vaccine of going up significant quantity as herein described.
A kind of method for preparing virus as herein described, comprise: a) separate the gene of on function, expressing or the assortment of genes that causes BVDV, b) the said gene or the assortment of genes are inserted in the not pathogenic virus of rf, c) are chosen in the live virus that to express the product of the said gene or the assortment of genes on the function.
A kind of method for preparing virus as herein described, the gene of expressing on function or the genome compound that wherein are used to produce this virus are produced plasmid and the method that intact virus DNA makes up through comprising, said plasmid comprises: a). BHV-1 genomic DNA fragment that contains thymidine kinase (tk) gene and have thymidine kinase (tk) genetically deficient, b) promotor/polyadenylation signal is inserted thymidine kinase (tk) the genetically deficient place of said plasmid, c) between promotor and polyadenylation signal, insert gp53 gene or genome compound, d) contain the said functioning gene that inserts in the live virus or the recombinant virus of genome compound with said plasmid transfection cell with generation, this live virus does not cause immunosuppression and expresses said functioning gene or the genome compound in common host.
A kind of method for preparing virus as herein described, wherein be used to produce the gene of on function, expressing of this virus or genome compound by comprising that the method with plasmid and intact virus DNA combination produces, said plasmid comprises: a) contain BHV-1 genomic DNA fragment that thymidine kinase (tk) gene and this thymidine kinase (tk) gene have disappearance, b) promotor/polyadenylation signal is inserted thymidine kinase (tk) the genetically deficient place of said plasmid, c) the gp53 gene or the assortment of genes that the front is had the signal peptide gene sequence is inserted between promotor and the polyadenylation signal, d) contain the said functioning gene that inserts in the live virus or the recombinant virus of genome compound with said plasmid transfection cell with generation, this live virus does not cause immunosuppression and expresses said functioning gene or the genome compound in common host.
Fig. 1, be used for foreign gene is inserted the structure of the shuttle vectors of BHV-1.
Fig. 2, on BVDV gp53 gene, add the method for signal peptide sequence.
Fig. 3, be used for gp53 is inserted the collection of illustrative plates of 5 shuttle plasmids of BHV-1:
A. embodiment 1:pBHVtKex-3 ∷ p53
B. embodiment 2:pBHVtKex-1 ∷ BGH/p53
C. embodiment 3:pBHVtKex-1 ∷ gIII/p53
D. embodiment 4:pBHVtKex-3 ∷ BGH/p53
E. embodiment 5:pBHVtKex-3 ∷ gIII/p53
The transcripting spectrum of Fig. 4 .BHV-1/gp53 recombinant virus expection.
Fig. 5 .Northem trace has shown gp53 messenger RNA(mRNA) transcribing in the BHV-1 recombinant chou.
Fig. 6. immunoprecipitation has shown the expression of gp53 protein in the BHV-1 recombinant chou.
Below used whole terms be that those skilled in the art is understandable.At many places, the trade name of device or reagent provides in device of naming or the parenthesis behind the reagent.Normally used term, reagent and damping fluid, as " plasmid ", " Klenow fragment ", " connecting blunt ends again ", " Tris ", and liquid during chelating is slow, as EDTA and EGTA, and normally used chromatographic column is not further explained when mentioning.
In description, use standard molecular biological technique and in this concise and to the point name or describe to the structure that is used for compound of the present invention.The visible standard laboratory handbook of the detailed explanation of these technology is as " Molecular Cloning:a Laboratory Manual " (1989), Sam-brook, et, al., Cold Spring Harbor Press, Cold Spring Harbor, NewYork, or " Current Protocols in Molecwlar Biology " is (l991), Ausubel, F.M., et.al., eds., Wiley Interscience, New York.
The present invention combines the validity of attenuated live vaccine and the security of inactivated vaccine.We have obtained the gene of coding pg53, it is the main glycoprotein of BVDV virus, can produce effective immune response at this albumen host, express this albumen in ox herpes virus-1 (BHV), this recombinant virus (BHV/BVDV pg53) is as the vaccine of anti-BVDV.
Ox herpes virus (BHV) is the another kind of main cattle disease substance that produces respiratory disease.Therefore, identical with BVDV is that BHV also duplicates at mucomembranous surface.For BVDV, duplicate mainly at the gastrointestinal mucosal interface, less duplicating of respiratory tract interface.For BHV, preponderate duplicating of respiratory tract interface.The common mucomembranous immune system has guaranteed still have effectiveness in the immune response that a surface produces on other surface.Therefore, recombinant virus BHV/BVDV gp53 of the present invention (preferably pass through in the nose by way of) when using for milk cow, sheep or goat duplicates and produces immune response at respiratory mucosa.
Before BVDV gp53 gene is expressed in BHV, use known method to make the thymidine kinase gene disappearance of BHV virus with attenuated virus.BHV (a kind of attenuated virus of work) is reproducible and produce cell-mediated reaction.As the part of reproduction process, also can express BVDV pg53 gene, because virus in cell, is used for also can taking place at the correct processing of recombinant virus BVDVgp53 cell-mediated reaction partly.The most important thing is that the possibility of this reaction does not cause immunosuppressant side effect, because only there is a part of BVDV virus.Therefore, the present invention combines the validity of BVDV attenuated live virus vaccines and the security of deactivation goods.
The embodiment that method part is provided only is used to the purpose explained, is not in order to limit the scope of the invention by any way.All media and buffering solution are all prepared in glass distilled water, except as otherwise noted.Composition and medication: the vaccine of medicinal significant quantity of the present invention can be gone up acceptable carrier or thinner use with medicinal, as animal, as the vaccine of anti-BHV-1 and BVDV in ox, sheep or the goat.
The useful in the present invention medicinal example of going up acceptable carrier or thinner comprises any physiological buffer medium, promptly, pH is approximately 7.0 to 7.4, contains about 2.5 to 15% serum, do not contain anti-BHV antibody (being the BHV seronegativity) in the serum.The serum that does not contain gamma Globulin is better than containing the serum of gamma Globulin.The example of employed serum comprises foetal calf serum among the present invention, lamb serum, horse serum, porcine blood serum, and lowlenthal serum.Content can be used for substitute blood serum from about serum protein of 0.5 to 3.0% as pig albumin or bovine serum albumin (hereinafter referred to as " BSA ").Yet, need avoid using the exogenous protein in carrier or the thinner of being present in of when inoculation animal induced hypersensitivity reaction.
Virus can be at any conventional phosphoric acid salt buffer, glutaminate, and casein peptone and sucrose or sorbose, or phosphoric acid salt buffer, lactose dilutes in the stabilizing solution of dextran and glutaminate.
Preferred vaccine virus of the present invention is with at least 10
5To 10
6The titre of PFV/ml is stored in-70 ℃ to-90 ℃ or be stored in 2 ℃ to 7 ℃ with lyophilised state.Freeze-drying available sterile distilled water of virus or use contain the diluent water of preservatives such as gentamicin and amphotericin B or penicillin and Streptomycin sulphate and prepare standby again.
The age of inoculating animal is depended in the variation of the useful dosage of administration, the mode of weight and kind and administration.For example, proper dosage can be about 10
4.5To 10
7The PFV/ animal, preferably approximately 10
4.5To 10
5.5PFV.
But in the vaccine intranasal of the present invention, intravaginal or intramuscular administration, approach is preferred administering mode in the nose.
Purposes of the present invention: the present invention attempts to provide a kind of effective vaccine that is used to prevent the disease that BVDV causes to the user, and vaccine wherein can be safely and effectively through intramuscular, in the nose or intravaginal administration.It in the intranasal preferred route of administration.
The purpose of producing vaccine of the present invention is the treatment disease, preferably reaches therapeutic purpose by prevention.Prevention or prophylactic applications refer to the influence that prevents that the host from forming the symptom of disease or alleviating this disease promptly prevent typical disease symptoms.The meaning of prevention is to stop, and stops or delay the decisive behavior of seizure of disease.Prevention can comprise following notion: hinder, defeat, block, tackle, forbid in addition probably, stop or get rid of.Get rid of show outbreak that morbid state does not take place or in causing morbid state pathogenic agent lost efficacy in a large number.Prevent or prevent to refer to that morbid state is delayed that the meaning is to stop or the anticipatory behavior that wards off disease has taken place but the symptom that produces disease is not eliminated.
This vaccine provides the ability of effective protection to confirm availability of the present invention in the propagation to the various manifestation of BVDV disease.Because vaccine uses gp53 (a kind of main BVDV glycoprotein), produces effective immune response at gp53 host, therefore for the potential host who is treated, this vaccine will provide a large amount of benefits.Another object of the present invention provides a kind of BVDV vaccine, and it can be safely gives calf and in the cow in calf medication in whole stages of gestation.
Active measurement: this vaccine uses gp53, and it is a kind of main glycoprotein of BVDV, can produce effective immune response at its host.Other authors have disclosed gp53 and have had hyperimmunization originality (Donis, R.O. and Dubori, E, J., Glycoproteins of BovineViral Diarrhoea-Mucosal Disease Virus in Infected Borine Cell, Jour-nal of General Virology, Vol.68, pp.1607-1616 (1987)).The reagent that produces effective immune response can be made into effective vaccine, this is the known (Magar.R. of people, et al., Borine Viral Diarthea Virus Proteins:Heterogeneity of Cy-topathogenil and Noncytopathogenil Strains and EVidence of 53K Gly-coprotein Neutrali2tion Epitope.Veterinary Microbiology, Vol.16, pp.303-314).Vaccine of the present invention contains the gene of expressing a large amount of gp53, as shown in Figure 5.Because the expression of a large amount of gp53, vaccine of the present invention provides a large amount of benefits can for the potential host who is treated.
Preferred mixture: any with tk disappearance attenuation and carry the gp53 gene, have before the gp53 gene signal peptide and to express be that the BHV-1 virus of enough amount gp53 is preferred suitable candidate vaccine.As if signal peptide sequence can be taken from any suitable source.We have selected to check that two kinds of different signal peptides are to guarantee gp53 protein best orientation in vivo.We have selected two kinds of candidate signal peptides, are referred to as " T11-6 " (using) and " T11-3 " (using) in embodiment 2, so that carry out vaccine test in embodiment 3.Preceding a kind of viral nomenclature is preserved in ATCC for VC VR-58.Back a kind of " T11-3 " plasmid has also carried out preservation.The virus that we are labeled as " T11-8 " may contain the clipped form of tk transcripton, and this may show (but may not necessarily be meant), and it has less magnetism as candidate vaccine.When going down to posterity in the substratum of the foetal calf serum that contains the calf of taking from infection, a large amount of existing clones are by the pathogenic BVDV persistent infection of acellular.For the present invention, the BVDV that does not conform to pollution as the virus of the attenuated vaccine that lives is indispensable.
The preparation of mixture
Be used for gene is inserted the structure of the expression shuttle vectors of ox herpes virus 1 type (BHV-1).
We have made up 2 shuttle vectorss foreign gene have been inserted among the BHV-1.Although the present invention has shown the purposes of BHV-1 as the carrier of BVDV gene, many other viruses also can be served as identical role.Other example from ox, sheep and goat can comprise ox, goat and sheep pox virus, adenovirus, BMV, bovine papilloma virus and pseudorabies virus.Non-pathogenic virus refers to anyly have the ability of duplicating but do not produce the virus of any disease symptoms in its a kind of host species in these species.The non-pathogenic virus of this class may produce with pathogenic parental virus through spontaneous mutation, can maybe can cause not pathogenic by using recombinant DNA technology through carrying out mutagenesis to produce non-pathogenic virus such as chemical or light.See 1) viral neutral zone location, E.wimmer, E.A.Emini, and D.C.Dia-mondand.2). the immunogenicity of vaccine product and neutralizing antibody, E Norrby.Two pieces of articles are all edited by Notkins and Oldstone, by Springer-Verleg New YorkInc, publish in 1986.
Because we attempt to come attenuation BHV-1 (M.Kit, et al., United States Patent (USP) 4,703,011 (1983)) by inactivation of viruses thymidine kinase (tk), so we determine to use the BHV-1tk gene as inserting the site.This test has not only guaranteed to make viral tk complete deactivation, also makes us select recombinant chou with the method for setting up, i.e. the negative virus of tk-(M, F.Shih, et al., Proe Natl Acad Sci VSA, 81; 5867-5870 (1948)).The method of other attenuation BHV-1 also can be used for the present invention as the disappearance of other dispensable gene.We begin with plasmid pHAS4, and it contains the BHV-1 Sal I subfragment of 2.7kb, and the clone advances the HindIII-A fragment of plasmid pVC18.E.PetrovKis, data is not published.M.Engels，etal.，Virus?Res，6：57-73(1986)；J.E.Mayfield，et?al.，J.Virol，47：259-264(1983)；A.L.Meyer，et?al.，BiochimBiophys?Acta，1090：267-9(1991)。As shown in Figure 1, this SalI fragment contains complete tk gene, and with the part upstream gene and the part glycoprotein h gene of HSV-1 VL24 dna homolog.L.J.Bello, et al., Virology, 189:407-414 (1992); J.G.Jacobson, et al., J.Virol, 63:1839-1843 (1989); M.Kit, et al., United States Patent (USP) 4,703,011, (1983); A.L.Meyet, et al., Biochim Biophys Acta, 1090:267-9 (1991).
Through the disappearance of 424bP being imported in the tk gene, with Klenow fragment (Klenow) the polishing end of dna polymerase i and reconnect the blunt end fragment of gained with BglII and XhoI digestion pHSA4.This operation recovery the BglII recognition site, but lost XhoI site (Fig. 1).The plasmid called after pHAS4 Δ BX of gained.Select this disappearance to be because it identify before not hindering at tk gene 5 ' end eclipsed VL24 homologue transcription initiation site.L.J.Bello，et?al.，Virology，189：407-414(1992)；J.G.Jacobson，et?al.，J.Virol.63：1839-1843(1989)。In intragenic many other disappearances of BHV-1 tk also is possible.In order to be beneficial to clone operations subsequently, we are through digesting pHAS4ABX with HindIII, thereby with Klenow polishing terminal viscosity and reconnect blunt end and removed the HindIII site among the pVC18.
We have obtained to contain the 1775bp sequence box of main immediate early promoter of people's cytomegalovirus (cmv) and Trobest polyadenylic acid sequence.R.J.Brideau，etal.，J.Gen?Virol，74；471-477(1993)。These genetic expression signals are generally used for the high level expression of foreign gene in many different systems, but other promotor/polyadenylation signal is to also using at this.This sequence box in carrier p3CL-DHFR is the border with single EcoRI and BglII site, and contains single HindIII and SalI restriction site so that clone's foreign gene between promotor and polyadenylation signal.With EcoRI digestion p3CL-DHFR carrier, then polishing and be connected to the BamHI joint (New England Biolabs, Bererly, Massachusetts) on.This operation produces the EcoRI site again.Then with BamHI and BglII digestion construct and the sequence box that discharges is connected on the BglII site of pHAS4 Δ BX (Fig. 1).Connector is transformed into e.colistraindh5.Through asymmetrical restriction site location, we have separated contains the recombinant plasmid that the p3CL that exists with two kinds of orientations with respect to the BHV-1tK gene inserts fragment.So these two kinds constructs that are called pHAS4 Δ BXex-1 and pHAS4 Δ BXex-3 (Fig. 1) contain strong promoter and polyadenylation signal so that can homologous recombination advance the BHV-1 genome on the border of BHV-1 tK gene and flanking region.
Fig. 1 is used for foreign gene is inserted the structure of the shuttle vectors of BHV-1.PHAS4 is the subfragment from the segmental 2.7kb of BHV-1 HindIII-A.Shown the BglII/xhoI subfragment that will lack.The disappearance derivative of pHAS4 is pHAS4 Δ BX.Thymidine kinase (tK) gene of disappearance is with the box indicating of black band point.The sequence box that contains promotor and poly-adenosine signal shows under pHAS4 Δ BX.The CMV immediate early promoter is with the box indicating of white band point, and Trobest (BGH) polyadenylation signal is with scored box indicating.Two insertion fragments of expressing shuttle plasmid pHAS4 Δ BXex1 and pHAS4 Δ BXex3 have been shown at last.
Signal peptide sequence is added on the BVDV gp53 gene
CDNA (the Kennedy that contains from the BVDV gp53 gene of virus strain 2724 (strain of a kind of acellular pathogenic virus) had been described in the past, M.et al, abstracts of theAmerican College of Veterinary Microbiologists, 1992 workshop).Because BVDV rna gene group is generally translated into a long polyprotein, become various virus proteins through posttranslational modification then, so the genomic gp53 of BVDV part does not contain protein translocation required convectional signals peptide, wherein this protein normal expression on film to the cytolemma.However, cDNA can both successfully express in cell free system and baculovirus, as if though lack the normal signal peptide, this protein can both transposition in two kinds of systems, and glycosylation and anchor.Yet we determine to use and do not use various signal peptides to estimate the expression of gp53 in BHV-1.
For the nucleotide sequence with the coded signal peptide is added on the gP53 gene, we import 5 of p53 gene ' end through site-directed mutagenesis with the BamHI site, operate as follows: the p53 gene is connected to plasmid pSP72 (Promega Corp. with blunt end, Madison, Wisconsin) on the BamHI site of polishing, so remove whole BamHI site from the plasmid of gained.Use synthetic oligonucleotide and " Double Take " site-directed mutagenesis test kit (Stratagene, La Jolla CA) to import the change of single base from used initiator codon 11 bases of cDNA, promptly become G from C according to shop instruction.The change of this base is with in the quiding gene of single BamHI site and do not change gp53 aminoacid sequence (Fig. 2 B part).The process nucleotide sequencing has confirmed the change of base, and the plasmid of gained is called pP53mut.We will encode from PRV gIII gene (A.K.Robbins, et al., J.Virol, 58:339-347 (1986)) with from Trobest (R.P.Woychik, et al., the Nucleotide of signal peptide Nucl.Acids Res, 10:7197-7210 (1982)) inserts in the pP53mut sequence (Fig. 2 A part).The complementary oligonucleotide of composite coding two signal peptides is so that the annealed oligonucleotide has SalI5 ' sticky end and BamHI3 ' sticky end (Fig. 2 A part).The pP53mut that these signal peptide sequence boxes are connected to BamHI and SalI digestion goes up and transforms DH5 α.Correct insertion through nucleotide sequencing acknowledge signal peptide sequence box.
The present invention can use the coding complementary oligonucleotide of the abundant signal peptide of identifying arbitrarily.Fully 39 examples of the signal peptide sequence of identifying are seen Perlman, D, et al., J.Mol.Biol.Vol.167 pp.391-409 (1983) (quoting for your guidance).These and any other signal peptide of fully identifying are applicable to implementing the present invention.
Fig. 2. on BVDV gp53 gene, add the method for signal peptide sequence.A part: the corresponding oligonucleotide of signal peptide sequence of synthetic and Trobest (BGH) and pseudorabies virus gIII (PRV gIII).Synthetic complementary oligonucleotide so as annealing to the BamHI site of SalI site and 3 ' end with 5 ' end.Also shown the signal peptide aminoacid sequence of inferring.In each case, the cleavage site of signal peptide expection just in time is positioned at L-Ala (A) (3 terminal amino acid) afterwards.2 amino-acid residues of original natural protein (are F.P among the BGH; Be P.S among the gIII) codon be positioned at the left side of signal peptide sequence to guarantee correct cutting.
B part: the site-directed mutagenesis of the cDNA of coding BVDV gp53 gene.Preceding 60 Nucleotide and the amino acid sequence corresponding that have shown gp53 cDNA.Change the single base pair shown in the arrow so that in sequence, produce the BamHI restriction site, in square frame, show.This variation does not change aminoacid sequence.Use the BamHI digested cdna then as shown in the figure, it is connected with one of signal peptide sequence shown in the A part in reading frame.
Other expressing gene fragment except gp53.
Other BVDV gene or the expression of the assortment of genes in live vector also are embodiment of the present invention.This comprises that the inoculation animal changes any and whole BVDV protein to its induction of immunity reaction.This example includes, but is not limited to other two kinds of BVDV surface glycoproteins, gp48 and gp45 (Collett, M.S., et al., Virology 165:200-208 (1988)), p14 capsid protein matter (Thiel, H., J, et al., J.Virol.65:4705-4712 (1991)) and p20 N-terminal protein enzyme (Wiskerchen, M., et al., J.Vi-rol.65:4508-4514 (1991)).This histone matter can be expressed from single cDNA molecule with the gp53 gene, and the polyprotein of expression correctly oneself is processed into protein separately.Another BVDV candidate protein of expressing in vaccine is non-structure p125/p80 protein (Deregt, D., et al., Can.J.Microbiol.37:815-122 (1991)), and it induces tangible antibody response in the cow that infects.
BVDV gp53 gene is inserted in the BHV-1 expression vector.
Through the polishing carrier and insert fragment sticky end separately then blunt end is connected will be added with or the p53 gene of plus signal peptide sequence be not connected on the HindIII insertion site of pHAS4 Δ BXex-1 and pHAS4 Δ BXex-3.Connector is transformed into e.colistraindh5.We think finally to estimate gp53 in BHV-1 with various orientations and expression with at least 2 unlike signal peptides to guarantee to obtain effectively expressing.Use is made the bacterium colony that probe transforms through the colony hybridization screening with the p53 insertion fragment of digoxigenin-dVTP mark." essence " DNA crossing system (Boeringer Mannheim Bio-chemicals (BMB), Indianapolis, IN) whole other DNA hybridization that can be used for this hybridization and in evaluation of the present invention, describe.Those screen positive recombinant chou with respect to the recombinant chou that CMV promotor and BgH polyadenylation signal carry the gp53 gene with proper orientation through restriction analysis then.Isolate 5 plasmids, they are described in Fig. 3 A-E with the diagrammatic form.It is described below:
Embodiment 1:pBHVtKex-3 ∷ p53: contain the BVDV gp53 gene that between pHAS4 Δ BXex-3 CMV promotor and BGH polyadenylation signal, inserts, not the plus signal peptide.In this construct, original gp53 gene inserts before site-directed mutagenesis, sees Fig. 3 A.This plasmid is used to make up viral T2-3# then.
Embodiment 2:pBHVtKex-1 ∷ BGH/P53: be contained in the gP53 gene that the BGH signal peptide sequence inserts the sudden change of pHAS4 Δ BXex-1 afterwards, see Fig. 3 B.This plasmid is used to make up viral T11-6.The preservation of this virus.
Embodiment 3:pBHVtKex-1 ∷ gIII/p53: contain the gp53 gene that after PRV gIII signal peptide sequence, inserts the mutagenesis of pHAS4 Δ BXex-1.See Fig. 3 C.This plasmid is used to make up viral T11-3.The preservation of this plasmid.
Embodiment 4:pBHVtKex-3 ∷ BGH/P53: the gp53 gene that contains the mutagenesis of after the BGH signal peptide sequence, inserting pHAS4 Δ BXex-3.See Fig. 3 D.
Embodiment 5:pBHVtKex-3 ∷ gIII/p53: contain the gp53 gene that after PRV gIII signal peptide sequence, inserts the mutagenesis of pHAS4 Δ BXex-3.See Fig. 3 E.This plasmid is used to produce viral T11-8.The preservation of this plasmid.
Fig. 3 A-E: the complete collection of illustrative plates that is used for gp53 is inserted five shuttle plasmids of BHV-1.The gp53 gene represents that with solid band the BHV-1 sequence is with the expression of black band point band, and the CMV promoter region is with the expression of white band point band, and BGH polyadenylation signal district represents with the striped band.Plasmid vector pVC18 represents with single line.Shown under every kind of situation transcriptional orientation with respect to the gp53 of BHV-1 tK original transcriptional orientation.Various signal peptide sequences have been shown.
A. embodiment 1.pBHVtKex-3 ∷ p53.
B. embodiment 2.pBHVtKex-1 ∷ BGH/p53
C. embodiment 3.pBHVtKex-1 ∷ gIII/p53
D. embodiment 4.pBHVtKex-3 ∷ BGH/p53
E. embodiment 5.pBHVtKex-3 ∷ gIII/p53
Other possible mode of BVDV gp53 gene being inserted the BHV-1tK gene of these and all is embodiment of the present invention.These plasmids and any plasmid that produces by this way are called " main plasmid vector ", and they are the plasmid vectors that are used to produce virus vaccines of the present invention.
The gp53 gene is imported BHV-1 " Iowa "
Use XbaI will carry 5 kinds of (w.cai that express the shuttle plasmid line styleization and revise of gp53 by Cai, et al., J Virol, 61:714-721 (1987)) standard C aPO4 method (R.I.Graham, et al., Virology, 52:456-467 (1973)) their cotransfections is advanced to have in nose of an ox first (BT) cell from BHV-1 virus strain Iowa (the tK positive) unit length DNA.This cell obtains from ATCC.Then 100Mg/ml5-bromo-2 '-deoxyuridine (BDVR, Sigma Chemical Company, St.Louis, Missouri) under the condition of Cun Zaiing to 143tK-cell (S.K.Mittal, et al., J.Gen Virol, 70:(1989)) or to Rab (RV) cell (S.Kit, et al., Virology, 130:381-389 (1983)) carry out two-wheeled and select to dye son and no longer express the virus of tK to separate.The standard program (M.Kit, et al., United States Patent (USP) 4,703,011 (1983)) that this describes before being.Also can use other tK-clone that allows BHV-1 growth.After process two-wheeled BDVR goes down to posterity, in the perfect medium that contains 1% low melting-point agarose, will still show as the cause a disease transfectant of effect of cell and infect on the BT cell, from each transfectant, choose many single plaques and the p53 gene in dot blotting DNA screening by hybridization viral DNA to obtain single plaque.Although be not whole transfectants in the BDVR middle survival (what particularly those carried out on the 143tK-cell goes down to posterity, because these cells only allow the BHV-1 viral growth on the edge) of going down to posterity, the transfectant of those survivals produces 100% recombinant virus.Separate 4 kinds of different recombinant viruses and done further evaluation:
Embodiment 1.T2-3#3 and T2-2#5 (two kinds identical but virus clone that independent separate goes out): the insertion sequence that in pBHVtKex-3 ∷ p53, comprises recombinate into BHV-1 " Iowa ".Contain not the plus signal peptide sequence, between CMV promotor and BGH polyadenylation signal, the BVDVgp53 gene that transcriptional orientation is identical with BHV-1tK gene direction.
Embodiment 2, T11-6 (this virus is submitted to ATCC with UC VR-58 name): the insertion sequence that pBHVtKex-1 ∷ BGH/p53 comprises recombinate into BHV-1 " Iowa ".Contain the BGH signal peptide sequence that has between CMV promotor and BGH polyadenylation signal and the BVDV gp53 gene opposite with respect to BHV-1 tK genetic transcription direction.
Embodiment 5, the insertion sequence that comprises among the T11-8:pBHVtkex-3 ∷ gIII/p53 recombinate into BHV-1 " Iowa ".Contain the PRV gIII signal peptide sequence and the transcriptional orientation BVDV gp53 gene identical that have between CMV promotor and BGH polyadenylation signal with the BHV-1tK gene.
(embodiment 4) do not isolate virus during with " Iowa " DNA and plasmid pBHVtKex3 ∷ BGH/p53 cotransfection, but the virus of this expection is easy to make up, and this virus all is embodiments of the invention with any other BHV-1 virus that contains the BVDV gp53 gene that is inserted in the thymidine kinase gene.From these viruses respectively purifying its DNA, use gp53 gene and CMV promotor/BgH polyadenylic acid sequence box to make the control pin and check the suitable insertion sequence (data does not show) that exists with suitable orientation through Southern hybridization.These four kinds of viruses are all carried the complete promotor/gene/polyadenylic acid sequence box that is arranged in the BHV-1tK gene, and wherein the tK gene is predicted as according to the restriction fragment size and lacks.As with the contrast of these transfections, the pHAS4 Δ BX plasmid of also having used BHV-1 " Iowa " unit length DNA transfection is also isolated the negative offspring of the tK that carries 424bp disappearance among the tK (also confirming with the southern essay).This viral nomenclature is Iowa Δ BX.These viruses all are through the plaque of twice of limiting dilution purifying on the BT cell.
When going down to posterity in the substratum of the foetal calf serum that contains the calf of taking from infection, a large amount of existing clone is by the pathogenic BVDV persistent infection of acellular.For the present invention, the BVDV virus that does not conform to pollution as the virus of the attenuated vaccine that lives is indispensable.Do not polluted in order to ensure the BHV-1 virus of carrying the BVDV sequence by the pathogenic BVD virus of acellular, we have prepared DNA (comprising parental virus strain Iowa and Iowa Δ BX) and have used clone's RNAse (RNAse ONE from every kind of virus, Promega Corporation, Madison, Wisconsin) carrying out completely to these DNA goods, RNAse handles.Because BVDV only has RNA as its genetic stocks, this operation should be removed any BVDV sequence that may pollute from the viral DNA goods.The viral DNA transfection that these RNAse are handled is advanced to turn out to be in the MDBK cell (ATCC) of no BVD and is selected viral plaque to be used for further operation from transfectant then.
The transcription analysis of gp53 recombinant chou.
Preparation RNA and transcribing from each recombinant virus and parent BHV-1 virus strain Iowa through Northern hybridization evaluation gp53.Possible messenger RNA(mRNA) kind and used probe are as shown in Figure 4.
Fig. 4: the transcripton such as the back of the expection of BHV-1/gp53 recombinant virus are shown in Figure 5.Two kinds of probes are 1) gp53 cDNA and 2) the SalI/BglII part (shown in top collection of illustrative plates) of pHAS4.First collection of illustrative plates has shown the transcripton of viral T11-3 and T11-6 expection, and second collection of illustrative plates shown the transcripton of T11-8 expection.The transcription initiation site that has shown tK and UL24 for your guidance.
All the gp53 recombinant viruses produce the messenger RNA(mRNA) of a 1.6kb. and the gp53 probe hybridization of it and 32p mark, the size of expectation is convenient to transcribe and stop on the BgH polyadenylation site initial on the CMV promotor, as Fig. 5 probe 1.T2-3#3 and T2-2#5 virus do not show.As other main band, T11-3 and T11-6 produce the transcripton of 8.5kb, and T11-8 and T2-3#3 produce the transcripton of 5.6kb.For these transcriptons of recombinant virus is single, and respectively with initial at CMV promotor place, by the reading of BgH polyadenylation signal and in UL24 or tK/gH polyadenylation signal place terminated messenger RNA(mRNA) unanimity.
These homogenies of long messenger RNA(mRNA) have been confirmed with the hybridization of upstream and downstream probe.The p53 probe not with Iowa, Ia Δ BX or infect the stand-in hybridization of RNA.As quantitative contrast, we use probe pHAS6, and it is to be positioned at tK open reading frame downstream and in the SalI fragment (A.L.Meyer, et al., Biochim Biophys Acta, 1090:261-9 (1991)) of a 867bp of gH gene inside.All these viruses produce the gH messenger RNA(mRNA) (data does not show) of the 3.1kb of a great deal of.This probe also with among T11-8 and the T2-3-3 is hybridized than the messenger RNA(mRNA) (with the gH transcripton is that common 3 ' end is arranged, L.J.Bello, et al., Virology, 189:407-414 (1992)) of 4.3Kb tK among long p53 messenger RNA(mRNA) and the Iowa.
In order to check the transcriptional profile of gp53 insertion sequence upstream, use by pHAS4 the probe that fragment is formed, the BglII site (disappearance starting point recombinant virus) (be probe 2) of this fragment from SalI site, upstream to the tK gene.All virus all produces the messenger RNA(mRNA) of an about 4.4Kb, is inferred as UL24 (Fig. 5, probe 2).Yet, this messenger RNA(mRNA) is than (L.J.Bello such as Bello, et al., Virology, 189:407-414 (1992)) 5.2Kb UL24 messenger RNA(mRNA) among the BHV-1 virus strain Cooper that describes wants little and moves jointly with tK messenger RNA(mRNA) among the wild-type virus type strain Iowa.Although not through using single-stranded probe further to estimate the messenger RNA(mRNA) of these common migrations, but only in Iowa DNA, detected the tK transcripton of 4.2Kb and in all viral RNAs, detected the transcripton of identical size, even tK transcripton that other virus can not generation wild-type size with upstream probe with probe pHAS6.In T11-3 and T11-6, upstream probe does not detect the clipped form of any tK messenger RNA(mRNA) and only hybridizes with UL24 messenger RNA(mRNA) and 8.5Kb p53 messenger RNA(mRNA).On the other hand, in T11-8, this probe and about 5.0,3.7,1.8 and (accessory) band hybridization of four kinds of increases of 1.0Kb.
Fig. 5, Northern trace have shown the transcripton of gp53 messenger RNA(mRNA) in the BHV-1 recombinant virus.First group has shown and the transcripton of probe 1 (pg53 cDNA) hybridization that second group has shown the transcripton of hybridizing with probe 2 (the SalI/BglI subfragment of pHAS4).Code name: the cell that the M=simulated virus infects, the cell that I=BHV-1 " Iowa " infects, 3.6,8=T11-3, the cell that T11-6 and T11-8 infect.The RNA size standards provides in every group of left side with kilobase (KB).
The expression of BVDV gp53 protein in BHV-1.
Estimated the expression of gp53 protein in the BHV-1 recombinant chou with immuno-precipitation (IP).The visible canonical reference document of the detailed method of IP (as: Current Protocols inMolecular Biology ", Ausubel, F.M., et.al., eds., Wiley Interscience, New York).With BHV-1 recombinant chou infected B T cell through metabolism with 35S-methionine(Met) (Amersham, Arlington Heights, Illinois) mark.The cell that lytic virus infects and with soluble protein and anti-BVDV hyper-immuneserum (VMRD, Pullman, Washington) reaction from ox or goat.With staphylococcus A (immunoprecipitin, Gibco/BRL, Gaithersburg, Maryland) or albumin A sepharose 4B (Pharmaci-a, Uppsala, Sweden) Precipitation Antigen/antibody complex.Differentiate immunoreactive protein matter through sds polyacrylamide gel electrophoresis (SDS-PAGE) and PF.
Fig. 6 has shown that all 3 kinds of recombinant viruses that carry the gp53 gene behind signal peptide sequence all produce the protein of significant quantity.From carrying the gp53 gene but do not conform to any expression that does not detect gp53 the T2-3#3 of signal peptide or the T2-2#5 virus, even should the synthetic quite a large amount of gp53 messenger RNA(mRNA) of virus.Clone T2-3#3 and T2-2-2#5 are independent isolating clones, and this has got rid of the possibility (data does not show) that a certain specific virus has the defective of repelling the gp53 expression.Remaining possibility is synthetic gp53 from T2-3, but is degraded rapidly, or our used antibody can not detect this proteinic undressed form.
Fig. 6 shows the immunoprecipitation protein that gp53 expresses in the BHV-1 recombinant chou.With the protein of anti-BVDV serum precipitation mark in the polyclone, this serum and BHV-1 antigen also have faint reactivity.Code name: 3.6,8=T11-3, the cell protein that T11-6 and T11-8 infect, the cell protein that IA=BHV-1 " Iowa " infects, the cell protein that the M=simulated virus infects.MW=is similar to the protein molecular weight standard product, and unit is for doing dalton.
T11-3, the gp53 protein belt of T11-6 and T11-8 is roomy, shows that this protein processes, they as if with NADL gp53 protein quite but size (data does not show) inequality.Through using N-dextranase (Genzyme, Cambridge, Massachusetts) digestion is removed the sugar that N-is connected and can not be differentiated this proteinic difference in size from the gp53 protein that BVDV-NADL and BHV-1 express, and processes in an identical manner with recombinant forms but the proportional minimizing of this protein size shows that gp53 is natural.Slight difference in size between reorganization and the natural protein may be since the gp53 gene of BHV-1 virus from the different BVD virus strain of the gp53 with slight difference in size, or the cDNAgp53 clone may not contain the accurate amino acid that is processed into natural gp53 from the BVDV polyprotein.
The present invention is not limited to the clone or the embodiment disclosed herein institute restricted portion of preservation, and they only are the explanations to one aspect of the invention, and suitable clone or embodiment are all within the scope of the invention on any function.In fact, except this paper demonstration and description, be conspicuous to those skilled in the art according to above describing to various modifications of the present invention.This modification will fall within the scope of the appended claims.
What it is also to be understood that is that the Nucleotide that provides and all base pairs and the total number of atnino acid of peptide are approximation and the purpose that is used for description.
Whole documents that this paper quotes are listed in for your guidance.
The preservation of genetic stocks
Through only using given description those skilled in the art can rebuild all various embodiments of the present invention.Yet for the sake of completeness,, some genetic constructs of the present invention is deposited in the depositary institution of approval according to budapest treaty to guarantee exploitativeness and to serve as to make and use other people of the present invention that various chances are provided.
A kind of virus is deposited in American type culture collection (12301 ParklownDrive, Rockville, Maryland, postcode 20852, the U.S.).These preservation product provide following preserving number by the called after UC VR-58 of Vpjohn company and by depositary institution: ATCC No.VR 2436, it with this paper with " T11-6 " (being also referred to as " embodiment 2 ") describe viral corresponding.These preservation product were received by the depositary institution of American type culture collection on October 28th, 1993.
Some plasmids are deposited in farming research and serve society culture collection center (NRRL) (USDA, 1815 North University Street, Peoria, Illinois, postcode 61604, the U.S.).The Upjohn called after pUC1564 of a plasmid, it is culture of Escherichia coli UC15085, is called pBHVtKex-1 ∷ gIII/p53, it is corresponding with the plasmid that is used to make up the virus that this paper describes with " T11-3 " (being also referred to as " embodiment 3 ").This plasmid provides following preserving number by depositary institution, NRRL B-21350.The Upjohn called after pUC1565 of another preservation product, it is culture of Escherichia coli UC15086, is called pBHVtKex-3 ∷ gIII/p53, to be called the plasmid of virus of " T-11-8 " (also claiming " embodiment 5 ") corresponding with being used to make up this paper for it.This plasmid provides following preserving number by depositary institution, NR-RL B-21351.This two plasmids are received by the depositary institution that society culture collection center is served in farming research on October 26th, 1994.
Sequence description (1) physical data:
(i) applicant: The Upjohn Company
Contriver's (being only applicable to the U.S.): Wardley, Richard c.and Haanes,
Elizabeth?J.
(ii) invention exercise question: express bovine viral diarrhea virus (BVDV) by membrane glycoprotein
The unlikely virus-virus of rf
(iii) sequence number: 2
(iv) contact address
(A) contact person: Thomas A Wootton (1920-32-1), The Upjohn
Company
(B) street: 7000 Portage Road
(C) city: Kalamazoo
(D) state: Michigan
(E) country: the U.S.
(F) postcode: 49001-0199
(V) computer-reader form
(A) media format: Floppy disk
(B) computer: IBM PC compatibility
(C) operating system: PC-DOS/MS-DOS
(D) software: PatentIn Release#1.0, Version#1.25
(vi) nearest application materials:
(A) application number:
(B) applying date:
(C) classification:
(viii) lawyer/proxy's data
(A) name: Wootton, Thomas A.
(B) registration number: 35004
(C) reference/catalog number (Cat.No.): 4748
(ix) telecommunications data:
(A) phone: 616 385-7914
(B) fax: 616 385-6897
(C) fax: 224 401 VPJOHN (2) SEQ ID NO:1 data
(i) sequence signature:
(A) length: 8083 base pairs
(B) type: nucleic acid
(C) chain: two strands
(D) topology: line style
(ii) molecule type: DNA (genome)
(iii) suppose: non-
(iv) antisense: non-
(vi) primary source:
(A) organism: bovine viral diarrhea virus
(B) virus strain: 2724
(C) single isolate: pBHVtkex-3 ∷ p53
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 60
CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT 120
CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180
TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG CCAAGCTAGC 240
TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAGGTTGTCC 300
GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC 360
GTCCGCGTCC CCAACTCCGC GCGAAGACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420
GCGTATAAAA TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480
GCCCAGGCAA GCAAACTCTA AACGCCCGAG CGCCATGGCC CCGATGCCGC CACAAAGAGC 540
GCCGAAATTT CGCCCAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600
CTGTTGCCCG CGTGCCTGCT GGCCGCCCAC CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 660
CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCCACAGC CGCCCTTTTG CCCGTAGCCA 720
GGGGAAGCGG CTGCCCCTTC TGCCGCCGCG GCCGCGGTTG CTCGGCTTTG CGTTTGCCCC 780
GCGGCGATCG CCCCGCTCGC CGCGAACGCG CGCGCGCGAA TGGGGCGTAC TCGGCGAGCC 840
CGGCTATTAT AGCCTCAAGG CGCGCCGCGT TGCTAGCGAT CGTCTGGGCC GGCAGGCGCG 900
TCACTCTGAG CACGCGCATG CCCCGCTGGG AGACGAACAC CTGCACCGGC GCTAGGACCA 960
CCGGGTCTGG GCCCGGGGGG GCGAGATCGC GCACAAGCCG GGCCGAGTCG CGCAGCTGCC 1020
GCAGCCCCCC GAGGCGCTGG TCCATCTTGC TGGGCGTGTT CATGTTCGTT GAAAAACGGC 1080
ACGTCTTCAG CTCCACGATA AGACAGACGG CCCGGGCGTG CCCTGCCTCC GCGACCCGGA 1140
GTAGGCACAC GCAATCGGGC CGCCGGCTTT GCAGGTTTAC CTCAAAGCTC AGAGACACGC 1200
CCACGACCTG CTTAAAAACC TCCGGGGCGC CAAACTTGCC CAAAAGCTGG GCGAGGCGCG 1260
GGCGCAGCTT CTGCGCGCCA ACCGCCGCGC GTGCGTCGCA AGCCAGCGCC TCGTAAAAGC 1320
GGCTGTGGCA CCGGATCCCG GCGCGCAGGC GCGCACGTCG GTCGCGGTCG CGCGCCATGG 1380
CCGAGCCCGC GCGCGCTCTC CGCGTCGTGC GTATCTACCT GGACGGCGCG CACGGGCAGG 1440
GAAAGACAAC AACGGGCCGC GCGCTCGCGG CCGCTTCCAC CGCTGGGGAG GGCGTGCTCT 1500
TTTTCCCGGA GCCGATGGCG TACTGGCGCA CGATGTTTGG TACGGACGCC TTAAGTGGGA 1560
TCCTCGCGGC GTCTGCGCGA TGCGCCGCAG CCTCGCACGG GAGCGCACGC GCGCGGCGGG 1620
CCGGCGCACC GCGCAGACGC GGACGCGGCG GGCCTGGTTG CGTACTACCA GGCCAGGTTC 1680
GCGGCCCCGT ACTTAATTTT GCACGCGCGT GTCCGCGCTG CTGCGCCGCC TGGGCCGGCG 1740
CCGGGCGGCG AGCTGGTGGA CCCTCGTGTT CGACCGCCAC CCCGTGGCGC GCGTGCCTCT 1800
GCTACCCCTT CGCCCGCTAC TGCCTCCGCG AGATCAACGC GGAAGATCCG AATTCCTCGA 1860
CCTGCAGTGA ATAATAAAAT GTGTGTTTGT CCGAAATACG CGTTTGAGAT TTCTGTCCCG 1920
ACTAAATTCA TGTCGCGCGA TAGTGGTGTT TATCGCCGAT AGAGATGGCG ATATTGGAAA 1980
AATCGATATT TGAAAATATG GCATATTGAA AATGTCGCCG ATGTGAGTTT CTGTGTAACT 2040
GATATCGCCA TTTTTCCAAA AGTTGATTTT TGGGCATACG CGATATCTGG CGATACGCTT 2100
ATATCGTTTA CGGGGGATGG CGATAGACGC CTTTGGTGAC TTGGGCGATT CTGTGTGTCG 2160
CAAATATCGC AGTTTCGATA TAGGTGACAG ACGATATGAG GCTATATCGC CGATAGAGGC 2220
GACATCAAGC TGGCACATGG CCAATGCATA TCGATCTATA CATTGAATCA ATATTGGCCA 2280
TTAGCCATAT TATTCATTGG TTATATAGCA TAAATCAATA TTGGCTATTG GCCATTGCAT 2340
ACGTTGTATC CATATCATAA TATGTACATT TATATTGGCT CATGTCCAAC ATTACCGCCA 2400
TGTTGACATT GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT 2460
AGCCCATATA TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG 2520
CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA 2580
GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA 2640
CATCAAGTGT ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC 2700
GCCTGGCATT ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC 2760
GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA 2820
TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG 2880
TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG 2940
CAAATGGGCG GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCG TTTAGTGAAC 3000
CGTCAGATCG CCTGGAGACG CCATCCACGC TGTTTTGACC TCCATAGAAG ACACCGGGAC 3060
CGATCCAGCC TCCGCGGCAA GCTGATCCGT CAGGGGCCAG ATGGTACAGG GCATCCTATG 3120
GCTACTACTG ATAACAGGGG TACAAGGGGA CATTGACTGC AAACCTGAAC ACTCATACGC 3180
CATAGCCAGG AATGATAGAA TTGGCCCATT AGGAGCTGAA GGCCTCACCA CTGTTTGGAA 3240
GGATTACTCA CATGAAATGA AGCTGGAAGA CACAATGGTC ATAGCTTGGT GCAAAGACGG 3300
TAAGTTTACA TACCTCTCAA GGTGCACAAG AGAAACTAGA TATCTTGCAA TTCTGCATTC 3360
AAGAGCCTTG CAGACCAGTG TGGTATTCAA AAAACTTTTC GAGGGGCAAA GGCAAGGGGA 3420
AACATTTGAA ATGGCTGACG ACTTTGAATT TGGACTCTGC CCATGCGATG CCAATCCCGT 3480
AGTAAGAGGG AAGTTCAATA CAACACTGCT AAACGGACCG GCCTTCCAGA TGGTATGCCC 3540
TATAGGATGG ACAGGAACTG TGAGCTGTAT GTTAGCTAAT AGGGACACCC TAGACACAGC 3600
AGTAGTGCGT GTGTATAAGA GGTCCAAACC ATTCCCTTAT AGACAAGGTT GTATCACCCA 3660
AAGAACTCTG GGGGAGGATC TCTATAACTG TGATCTTGGA GGGAATTGGA CTTGTGTGAC 3720
TGGGGACCAG CTACAATACA CAGGAGGCCC TGTCGAATCT TGCAAGTGGT GTGGTTATAA 3780
ATTCCAAAAA AGTGAGGGGT TGCCACACTA CCCCATCGGC AAGTGTAGGT TGAAGAATGA 3840
GACTGGCTAC AGATTTGTAG ACGGCACCAC TTGCAACAGA GAGGGTGTAG CCATAGTACC 3900
ACAAGGATTG GTAAAGTGTA AGATAGGAGA CACAATCGTA CAGGTCATAG CTCTTGACAC 3960
CAAACTTGGG CCTATGCCTT GCAAGCCATA TGAGATCATA CCAAGTGAGG GGCCTGTAGA 4020
AAAGACGGCA TGCACCTTCA ACTACACGAG GACATTAAAA AATAAATATT TTGAGCCCAG 4080
AGACAGTTAC TTCCAGCAAT ACATGCTAAA AGGAGATTAT CAATACTGGT TCGACCTGGA 4140
GGTCACTGAC CATCATCGGG ATTACTTCGC CGAGTCCATA TTGGTGGTGG TGGTAGCTTT 4200
ACTGGGTGGA AGATACGTGC TCTGGTTACT GGTAACATAC ATGGTCCTAT CAGAACAAAA 4260
GGCCTTGGGG ACCCAATATG GGGCAGGGGA AGTGGTGATG ATGGGTAACT TGCTAACACA 4320
TGACAGTATT GAAGTGGTGA CATATTTCTT GTTGTTATAC CTACTGCTAA GAGAGGAGGC 4380
TGTAAAGAAG TGGGTCTTAC TCTTATACCA CCTTGATTGA TTGAGGATCA GCTTATCCAG 4440
GGTCGACCTC AGGCATGCAA GCTCAGATCC GCTGTGCCTT CTAGTTGCCA GCCATCTGTT 4500
GTTTGCCCCT CCCCCGTGCC TTCCTTGACC CTGGAAGGTG CCACTCCCAC TGTCCTTTCC 4560
TAATAAAATG AGGAAATTGC ATCGCATTGT CTGAGTAGGT GTCATTCTAT TCTGGGGGGT 4620
GGGGTGGGGC AGGACAGCAA GGGGGAGGAT TGGGAAGACA ATAGCAGGCA TGCTGGGGAT 4680
GCGGTGGGCT CTATGGGTAC CCAGGTGCTG AAGAATTGAC CCGGTTCCTC CTGGGCCAGA 4740
AAGAAGCAGG CACATCCCCT TCTCTGTGAC ACACCCTGTC CACGCCCCTG GTTCTTAGTT 4800
CCAGCCCCAC TCATAGGACA CTCATAGCTC AGGAGGGCTC CGCTTCAATC CCACCCGCTA 4860
AAGTACTTGG AGCGGTCTCT CCCTCCCTCA TCAGCCCACC AAACCAAACC TAGCCTCCAA 4920
GAGTGGGAAG AAATTAAAGC AAGATAGGCT ATTAAGTGCA GAGGGAGAGA AAATGCCTCC 4980
AACATGTGAG GAAGTAATGA TAGAAATCAT AGAATTGAGA TCTCGAGGTG TTCGTGCTGG 5040
ACGTGTCCGC GGCGCCAGAC GCGTGCGCGG CCGCCGTACT GGACATGCGG CCCGCCATGC 5100
AGGCCGCTTG CGCGGACGGG GCGGCGGGCG CGACGCTGGC GACCCTGGCG CGTCAGTTCG 5160
CGCTAGAGAT GGCGGGGGAG GCCACGGCGG GCCCTAGGGG ACTATAAAGC TGCCCCTGCG 5220
CTCGCTCGCT CGCTGCATTT GCGCCCCGAT CGCCTTACGG GGACTCGGCG CTCGGCGGAT 5280
CCCCTCCCGG CCCCGCCGCG AAGCAGGCCG CCAGACAAAA AAATGCGGCG CCCGCTCTGC 5340
GCGGCGCTAT TGGCAGCGGC TGTCCTCGCG CTCGCCGCGG GCGCCCCCGC CGCCGCCCGC 5400
GGCGGGGGCG CCGAAGCCAG GGCAGCACAG AGACGCCCGA TACGAAATCG AAGAGTGGGA 5460
AATGGTGGTC GGAGCCGGGC CGGCCGTGCA CACGTTCACC ATCCGCTGCC TCGGGCCGCG 5520
GGGCATTGAG CGCGTGGCCC ACATTGCAAA CCTCAGCCGG CTGCTGGACG GGTACATAGC 5580
GGTCCACGTT GACGTTGCGC GCACCTCTGG CCTGCGGGAC GCCATGTTTT TCCTGCCGCG 5640
CGCGGCCGTC GACTCTAGAG GATCCCCGGG TACCGAGCTC GAATTCACTG GCCGTCGTTT 5700
TACAACGTCG TGACTGGGAA AACCCTGGCG TTACCCAACT TAATCGCCTT GCAGCACATC 5760
CCCCTTTCGC CAGCTGGCGT AATAGCGAAG AGGCCCGCAC CGATCGCCCT TCCCAACAGT 5820
TGCGCAGCCT GAATGGCGAA TGGCGCCTGA TGCGGTATTT TCTCCTTACG CATCTGTGCG 5880
GTATTTCACA CCGCATATGG TGCACTCTCA GTACAATCTG CTCTGATGCC GCATAGTTAA 5940
GCCAGCCCCG ACACCCGCCA ACACCCGCTG ACGCGCCCTG ACGGGCTTGT CTGCTCCCGG 6000
CATCCGCTTA CAGACAAGCT GTGACCGTCT CCGGGAGCTG CATGTGTCAG AGGTTTTCAC 6060
CGTCATCACC GAAACGCGCG AGACGAAAGG GCCTCGTGAT ACGCCTATTT TTATAGGTTA 6120
ATGTCATGAT AATAATGGTT TCTTAGACGT CAGGTGGCAC TTTTCGGGGA AATGTGCGCG 6180
GAACCCCTAT TTGTTTATTT TTCTAAATAC ATTCAAATAT GTATCCGCTC ATGAGACAAT 6240
AACCCTGATA AATGCTTCAA TAATATTGAA AAAGGAAGAG TATGAGTATT CAACATTTCC 6300
GTGTCGCCCT TATTCCCTTT TTTGCGGCAT TTTGCCTTCC TGTTTTTGCT CACCCAGAAA 6360
CGCTGGTGAA AGTAAAAGAT GCTGAAGATC AGTTGGGTGC ACGAGTGGGT TACATCGAAC 6420
TGGATCTCAA CAGCGGTAAG ATCCTTGAGA GTTTTCGCCC CGAAGAACGT TTTCCAATGA 6480
TGAGCACTTT TAAAGTTCTG CTATGTGGCG CGGTATTATC CCGTATTGAC GCCGGGCAAG 6540
AGCAACTCGG TCGCCGCATA CACTATTCTC AGAATGACTT GGTTGAGTAC TCACCAGTCA 6600
CAGAAAAGCA TCTTACGGAT GGCATGACAG TAAGAGAATT ATGCAGTGCT GCCATAACCA 6660
TGAGTGATAA CACTGCGGCC AACTTACTTC TGACAACGAT CGGAGGACCG AAGGAGCTAA 6720
CCGCTTTTTT GCACAACATG GGGGATCATG TAACTCGCCT TGATCGTTGG GAACCGGAGC 6780
TGAATGAAGC CATACCAAAC GACGAGCGTG ACACCACGAT GCCTGTAGCA ATGGCAACAA 6840
CGTTGCGCAA ACTATTAACT GGCGAACTAC TTACTCTAGC TTCCCGGCAA CAATTAATAG 6900
ACTGGATGGA GGCGGATAAA GTTGCAGGAC CACTTCTGCG CTCGGCCCTT CCGGCTGGCT 6960
GGTTTATTGC TGATAAATCT GGAGCCGGTG AGCGTGGGTC TCGCGGTATC ATTGCAGCAC 7020
TGGGGCCAGA TGGTAAGCCC TCCCGTATCG TAGTTATCTA CACGACGGGG AGTCAGGCAA 7080
CTATGGATGA ACGAAATAGA CAGATCGCTG AGATAGGTGC CTCACTGATT AAGCATTGGT 7140
AACTGTCAGA CCAAGTTTAC TCATATATAC TTTAGATTGA TTTAAAACTT CATTTTTAAT 7200
TTAAAAGGAT CTAGGTGAAG ATCCTTTTTG ATAATCTCAT GACCAAAATC CCTTAACGTG 7260
AGTTTTCGTT CCACTGAGCG TCAGACCCCG TAGAAAAGAT CAAAGGATCT TCTTGAGATC 7320
CTTTTTTTCT GCGCGTAATC TGCTGCTTGC AAACAAAAAA ACCACCGCTA CCAGCGGTGG 7380
TTTGTTTGCC GGATCAAGAG CTACCAACTC TTTTTCCGAA GGTAACTGGC TTCAGCAGAG 7440
CGCAGATACC AAATACTGTC CTTCTAGTGT AGCCGTAGTT AGGCCACCAC TTCAAGAACT 7500
CTGTAGCACC GCCTACATAC CTCGCTCTGC TAATCCTGTT ACCAGTGGCT GCTGCCAGTG 7560
GCGATAAGTC GTGTCTTACC GGGTTGGACT CAAGACGATA GTTACCGGAT AAGGCGCAGC 7620
GGTCGGGCTG AACGGGGGGT TCGTGCACAC AGCCCAGCTT GGAGCGAACG ACCTACACCG 7680
AACTGAGATA CCTACAGCGT GAGCTATGAG AAAGCGCCAC GCTTCCCGAA GGGAGAAAGG 7740
CGGACAGGTA TCCGGTAAGC GGCAGGGTCG GAACAGGAGA GCGCACGAGG GAGCTTCCAG 7800
GGGGAAACGC CTGGTATCTT TATAGTCCTG TCGGGTTTCG CCACCTCTGA CTTGAGCGTC 7860
GATTTTTGTG ATGCTCGTCA GGGGGGCGGA GCCTATGGAA AAACGCCAGC AACGCGGCCT 7920
TTTTACGGTT CCTGGCCTTT TGCTGGCCTT TTGCTCACAT GTTCTTTCCT GCGTTATCCC 7980
CTGATTCTGT GGATAACCGT ATTACCGCCT TTGAGTGAGC TGATACCGCT CGCCGCAGCC 8040
GAACGACCGA GCGCAGCGAG TCAGTGAGCG AGGAAGCGGA AGA 8083
(2) SEQ ID NO: 2 items
...
(i) sequence signature:
(A) length: 8149 base pairs
(B) type: nucleic acid
(C) chain: two strands
(D) topology: line style
(ii) molecule type: DNA (genome)
(iii) suppose: non-
(iv) antisense: non-
(vi) primary source:
(A) organism: bovine viral diarrhea virus
(B) virus strain: 2724
(C) single isolate: pBHVtkex-l ∷ gBGH/p53
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 60
CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT 120
CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180
TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG CCAAGCTAGC 240
TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAGGTTGTCC 300
GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC 360
GTCCGCGTCC CCAACTCCGC GCGAAGACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420
GCGTATAAAA TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480
GCCCAGGCAA GCAAACTCTA AACGCCCGAG CGCCATGGCC CCGATGCCGC CACAAAGAGC 540
GCCGAAATTT CGCCCAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600
CTGTTGCCCG CGTGCCTGCT GGCCGCCCAC CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 660
CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCCACAGC CGCCCTTTTG CCCGTAGCCA 720
GGGGAAGCGG CTGCCCCTTC TGCCGCCGCG GCCGCGGTTG CTCGGCTTTG CGTTTGCCCC 780
GCGGCGATCG CCCCGCTCGC CGCGAACGCG CGCGCGCGAA TGGGGCGTAC TCGGCGAGCC 840
CGGCTATTAT AGCCTCAAGG CGCGCCGCGT TGCTAGCGAT CGTCTGGGCC GGCAGGCGCG 900
TCACTCTGAG CACGCGCATG CCCCGCTGGG AGACGAACAC CTGCACCGGC GCTAGGACCA 960
CCGGGTCTGG GCCCGGGGGG GCGAGATCGC GCACAAGCCG GGCCGAGTCG CGCAGCTGCC 1020
GCAGCCCCCC GAGGCGCTGG TCCATCTTGC TGGGCGTGTT CATGTTCGTT GAAAAACGGC 1080
ACGTCTTCAG CTCCACGATA AGACAGACGG CCCGGGCGTG CCCTGCCTCC GCGACCCGGA 1140
GTAGGCACAC GCAATCGGGC CGCCGGCTTT GCAGGTTTAC CTCAAAGCTC AGAGACACGC 1200
CCACGACCTG CTTAAAAACC TCCGGGGCGC CAAACTTGCC CAAAAGCTGG GCGAGGCGCG 1260
GGCGCAGCTT CTGCGCGCCA ACCGCCGCGC GTGCGTCGCA AGCCAGCGCC TCGTAAAAGC 1320
GGCTGTGGCA CCGGATCCCG GCGCGCAGGC GCGCACGTCG GTCGCGGTCG CGCGCCATGG 1380
CCGAGCCCGC GCGCGCTCTC CGCGTCGTGC GTATCTACCT GGACGGCGCG CACGGGCAGG 1440
GAAAGACAAC AACGGGCCGC GCGCTCGCGG CCGCTTCCAC CGCTGGGGAG GGCGTGCTCT 1500
TTTTCCCGGA GCCGATGGCG TACTGGCGCA CGATGTTTGG TACGGACGCC TTAAGTGGGA 1560
TCCTCGCGGC GTCTGCGCGA TGCGCCGCAG CCTCGCACGG GAGCGCACGC GCGCGGCGGG 1620
CCGGCGCACC GCGCAGACGC GGACGCGGCG GGCCTGGTTG CGTACTACCA GGCCAGGTTC 1680
GCGGCCCCGT ACTTAATTTT GCACGCGCGT GTCCGCGCTG CTGCGCCGCC TGGGCCGGCG 1740
CCGGGCGGCG AGCTGGTGGA CCCTCGTGTT CGACCGCCAC CCCGTGGCGC GCGTGCCTCT 1800
GCTACCCCTT CGCCCGCTAC TGCCTCCGCG AGATCAACGC GGAAGATCTC AATTCTATGA 1860
TTTCTATCAT TACTTCCTCA CATGTTGGAG GCATTTTCTC TCCCTCTGCA CTTAATAGCC 1920
TATCTTGCTT TAATTTCTTC CCACTCTTGG AGGCTAGGTT TGGTTTGGTG GGCTGATGAG 1980
GGAGGGAGAG ACCGCTCCAA GTACTTTAGC GGGTGGGATT GAAGCGGAGC CCTCCTGAGC 2040
TATGAGTGTC CTATGAGTGG GGCTGGAACT AAGAACCAGG GGCGTGGACA GGGTGTGTCA 2100
CAGAGAAGGG GATGTGCCTG CTTCTTTCTG GCCCAGGAGG AACCGGGTCA ATTCTTCAGC 2160
ACCTGGGTAC CCATAGAGCC CACCGCATCC CCAGCATGCC TGCTATTGTC TTCCCAATCC 2220
TCCCCCTTGC TGTCCTGCCC CACCCCACCC CCCAGAATAG AATGACACCT ACTCAGACAA 2280
TGCGATGCAA TTTCCTCATT TTATTAGGAA AGGACAGTGG GAGTGGCACC TTCCAGGGTC 2340
AAGGAAGGCA CGGGGGAGGG GCAAACAACA GATGGCTGGC AACTAGAAGG CACAGCGGAT 2400
CTGAGCTTGC ATGCCTGAGG TCGACCCTGG ATAAGCTGAT CCTCAATCAA TCAAGGTGGT 2460
ATAAGAGTAA GACCCACTTC TTTACAGCCT CCTCTCTTAG CAGTAGGTAT AACAACAAGA 2520
AATATGTCAC CACTTCAATA CTGTCATGTG TTAGCAAGTT ACCCATCATC ACCACTTCCC 2580
CTGCCCCATA TTGGGTCCCC AAGGCCTTTT GTTCTGATAG GACCATGTAT GTTACCAGTA 2640
ACCAGAGCAC GTATCTTCCA CCCAGTAAAG CTACCACCAC CACCAATATG GACTCGGCGA 2700
AGTAATCCCG ATGATGGTCA GTGACCTCCA GGTCGAACCA GTATTGATAA TCTCCTTTTA 2760
GCATGTATTG CTGGAAGTAA CTGTCTCTGG GCTCAAATA TTTATTTTTT AATGTCCTCG 2820
TGTAGTTGAA GGTGCATGCC GTCTTTTCTA CAGGCCCCTC ACTTGGTATG ATCTCATATG 2880
GCTTGCAAGG CATAGGCCCA AGTTTGGTGT CAAGAGCTAT GACCTGTACG ATTGTGTCTC 2940
CTATCTTACA CTTTACCAAT CCTTGTGGTA CTATGGCTAC ACCCTCTCTG TTGCAAGTGG 3000
TGCCGTCTAC AAATCTGTAG CCAGTCTCAT TCTTCAACCT ACACTTGCCG ATGGGGTAGT 3060
GTGGCAACCC CTCACTTTTT TGGAATTTAT AACCACACCA CTTGCAAGAT TCGACAGGGC 3120
CTCCTGTGTA TTGTAGCTGG TCCCCAGTCA CACAAGTCCA ATTCCCTCCA AGATCACAGT 3180
TATAGAGATC CTCCCCCAGA GTTCTTTGGG TGATACAACC TTGTCTATAA GGGAATGGTT 3240
TGGACCTCTT ATACACACGC ACTACTGCTG TGTCTAGGGT GTCCCTATTA GCTAACATAC 3300
AGCTCACAGT TCCTGTCCAT CCTATAGGGC ATACCATCTG GAAGGCCGGT CCGTTTAGCA 3360
GTGTTGTATT GAACTTCCCT CTTACTACGG GATTGGCATC GCATGGGCAG AGTCCAAATT 3420
CAAAGTCGTC AGCCATTTCA AATGTTTCCC CTTGCCTTTG CCCCTCGAAA AGTTTTTTGA 3480
ATACCACACT GGTCTGCAAG GCTCTTGAAT GCAGAATTGC AAGATATCTA GTTTCTCTTG 3540
TGCACCTTGA GAGGTATGTA AACTTACCGT CTTTGCACCA AGCTATGACC ATTGTGTCTT 3600
CCAGCTTCAT TTCATGTGAG TAATCCTTCC AAACAGTGGT GAGGCCTTCA GCTCCTAATG 3660
GGCCAATTCT ATCATTCCTG GCTATGGCGT ATGAGTGTTC AGGTTTGCAG TCAATGTCCC 3720
CTTGTACCCC TGTTATCAGT AGTAGCCATA GGATCCCTGG GAAGGCGCCC ACCACCTGAG 3780
TCCAGGGCAG GCAGAGCAGG GCGAAAGCCA GGAGCAGGGA GGTCCGGGGG CCTGCAGCCA 3840
TCATGTCGAA GCTTGCCGCG GAGGCTGGAT CGGTCCCGGT GTCTTCTATG GAGGTCAAAA 3900
CAGCGTGGAT GGCGTCTCCA GGCGATCTGA CGGTTCACTA AACGAGCTCT GCTTATATAG 3960
ACCTCCCACC GTACACGCCT ACCGCCCATT TGCGTCAATG GGGCGGAGTT GTTACGACAT 4020
TTTGGAAAGT CCCGTTGATT TTGGTGCCAA AACAAACTCC CATTGACGTC AATGGGGTGG 4080
AGACTTGGAA ATCCCCGTGA GTCAAACCGC TATCCACGCC CATTGATGTA CTGCCAAAAC 4140
CGCATCACCA TGGTAATAGC GATGACTAAT ACGTAGATGT ACTGCCAAGT AGGAAAGTCC 4200
CATAAGGTCA TGTACTGGGC ATAATGCCAG GCGGGCCATT TACCGTCATT GACGTCAATA 4260
GGGGGCGTAC TTGGCATATG ATACACTTGA TGTACTGCCA AGTGGGCAGT TTACCGTAAA 4320
TACTCCACCC ATTGACGTCA ATGGAAAGTC CCTATTGGCG TTACTATGGG AACATACGTC 4380
ATTATTGACG TCAATGGGCG GGGGTCGTTG GGCGGTCAGC CAGGCGGGCC ATTTACCGTA 4440
AGTTATGTAA CGCGGAACTC CATATATGGG CTATGAACTA ATGACCCCGT AATTGATTAC 4500
TATTAATAAC TAGTCAATAA TCAATGTCAA CATGGCGGTA ATGTTGGACA TGAGCCAATA 4560
TAAATGTACA TATTATGATA TGGATACAAC GTATGCAATG GCCAATAGCC AATATTGATT 4620
TATGCTATAT AACCAATGAA TAATATGGCT AATGGCCAAT ATTGATTCAA TGTATAGATC 4680
GATATGCATT GGCCATGTGC CAGCTTGATG TCGCCTCTAT CGGCGATATA GCCTCATATC 4740
GTCTGTCACC TATATCGAAA CTGCGATATT TGCGACACAC AGAATCGCCC AAGTCACCAA 4800
AGGCGTCTAT CGCCATCCCC CGTAAACGAT ATAAGCGTAT CGCCAGATAT CGCGTATGCC 4860
CAAAAATCAA CTTTTGGAAA AATGGCGATA TCAGTTACAC AGAAACTCAC ATCGGCGACA 4920
TTTTCAATAT GCCATATTTT CAAATATCGA TTTTTCCAAT ATCGCCATCT CTATCGGCGA 4980
TAAACACCAC TATCGCGCGA CATGAATTTA GTCGGGACAG AAATCTCAAA CGCGTATTTC 5040
GGACAAACAC ACATTTTATT ATTCACTGCA GGTCGAGGAA TTCGGATCTC GAGGTGTTCG 5100
TGCTGGACGT GTCCGCGGCG CCAGACGCGT GCGCGGCCGC CGTACTGGAC ATGCGGCCCG 5160
CCATGCAGGC CGCTTGCGCG GACGGGGCGG CGGGCGCGAC GCTGGCGACC CTGGCGCGTC 5220
AGTTCGCGCT AGAGATGGCG GGGGAGGCCA CGGCGGGCCC TAGGGGACTA TAAAGCTGCC 5280
CCTGCGCTCG CTCGCTCGCT GCATTTGCGC CCCGATCGCC TTACGGGGAC TCGGCGCTCG 5340
GCGGATCCCC TCCCGGCCCC GCCGCGAAGC AGGCCGCCAG ACAAAAAAAT GCGGCGCCCG 5400
CTCTGCGCGG CGCTATTGGC AGCGGCTGTC CTCGCGCTCG CCGCGGGCGC CCCCGCCGCC 5460
GCCCGCGGCG GGGGCGCCGA AGCCAGGGCA GCACAGAGAC GCCCGATACG AAATCGAAGA 5520
GTGGGAAATG GTGGTCGGAG CCGGGCCGGC CGTGCACACG TTCACCATCC GCTGCCTCGG 5580
GCCGCGGGGC ATTGAGCGCG TGGCCCACAT TGCAAACCTC AGCCGGCTGC TGGACGGGTA 5640
CATAGCGGTC CACGTTGACG TTGCGCGCAC CTCTGGCCTG CGGGACGCCA TGTTTTTCCT 5700
GCCGCGCGCG GCCGTCGACT CTAGAGGATC CCCGGGTACC GAGCTCGAAT TCACTGGCCG 5760
TCGTTTTACA ACGTCGTGAC TGGGAAAACC CTGGCGTTAC CCAACTTAAT CGCCTTGCAG 5820
CACATCCCCC TTTCGCCAGC TGGCGTAATA GCGAAGAGGC CCGCACCGAT CGCCCTTCCC 5880
AACAGTTGCG CAGCCTGAAT GGCGAATGGC GCCTGATGCG GTATTTTCTC CTTACGCATC 5940
TGTGCGGTAT TTCACACCGC ATATGGTGCA CTCTCAGTAC AATCTGCTCT GATGCCGCAT 6000
AGTTAAGCCA GCCCCGACAC CCGCCAACAC CCGCTGACGC GCCCTGACGG GCTTGTCTGC 6060
TCCCGGCATC CGCTTACAGA CAAGCTGTGA CCGTCTCCGG GAGCTGCATG TGTCAGAGGT 6120
TTTCACCGTC ATCACCGAAA CGCGCGAGAC GAAAGGGCCT CGTGATACGC CTATTTTTAT 6180
AGGTTAATGT CATGATAATA ATGGTTTCTT AGACGTCAGG TGGCACTTTT CGGGGAAATG 6240
TGCGCGGAAC CCCTATTTGT TTATTTTTCT AAATACATTC AAATATGTAT CCGCTCATGA 6300
GACAATAACC CTGATAAATG CTTCAATAAT ATTGAAAAAG GAAGAGTATG AGTATTCAAC 6360
ATTTCCGTGT CGCCCTTATT CCCTTTTTTG CGGCATTTTG CCTTCCTGTT TTTGCTCACC 6420
CAGAAACGCT GGTGAAAGTA AAAGATGCTG AAGATCAGTT GGGTGCACGA GTGGGTTACA 6480
TCGAACTGGA TCTCAACAGC GGTAAGATCC TTGAGAGTTT TCGCCCCGAA GAACGTTTTC 6540
CAATGATGAG CACTTTTAAA GTTCTGCTAT GTGGCGCGGT ATTATCCCGT ATTGACGCCG 6600
GGCAAGAGCA ACTCGGTCGC CGCATACACT ATTCTCAGAA TGACTTGGTT GAGTACTCAC 6660
CAGTCACAGA AAAGCATCTT ACGGATGGCA TGACAGTAAG AGAATTATGC AGTGCTGCCA 6720
TAACCATGAG TGATAACACT GCGGCCAACT TACTTCTGAC AACGATCGGA GGACCGAAGG 6780
AGCTAACCGC TTTTTTGCAC AACATGGGGG ATCATGTAAC TCGCCTTGAT CGTTGGGAAC 6840
CGGAGCTGAA TGAAGCCATA CCAAACGACG AGCGTGACAC CACGATGCCT GTAGCAATGG 6900
CAACAACGTT GCGCAAACTA TTAACTGGCG AACTACTTAC TCTAGCTTCC CGGCAACAAT 6960
TAATAGACTG GATGGAGGCG GATAAAGTTG CAGGACCACT TCTGCGCTCG GCCCTTCCGG 7020
CTGGCTGGTT TATTGCTGAT AAATCTGGAG CCGGTGAGCG TGGGTCTCGC GGTATCATTG 7080
CAGCACTGGG GCCAGATGGT AAGCCCTCCC GTATCGTAGT TATCTACACG ACGGGGAGTC 7140
AGGCAACTAT GGATGAACGA AATAGACAGA TCGCTGAGAT AGGTGCCTCA CTGATTAAGC 7200
ATTGGTAACT GTCAGACCAA GTTTACTCAT ATATACTTTA GATTGATTTA AAACTTCATT 7260
TTTAATTTAA AAGGATCTAG GTGAAGATCC TTTTTGATAA TCTCATGACC AAAATCCCTT 7320
AACGTGAGTT TTCGTTCCAC TGAGCGTCAG ACCCCGTAGA AAAGATCAAA GGATCTTCTT 7380
GAGATCCTTT TTTTCTGCGC GTAATCTGCT GCTTGCAAAC AAAAAAACCA CCGCTACCAG 7440
CGGTGGTTTG TTTGCCGGAT CAAGAGCTAC CAACTCTTTT TCCGAAGGTA ACTGGCTTCA 7500
GCAGAGCGCA GATACCAAAT ACTGTCCTTC TAGTGTAGCC GTAGTTAGGC CACCACTTCA 7560
AGAACTCTGT AGCACCGCCT ACATACCTCG CTCTGCTAAT CCTGTTACCA GTGGCTGCTG 7620
CCAGTGGCGA TAAGTCGTGT CTTACCGGGT TGGACTCAAG ACGATAGTTA CCGGATAAGG 7680
CGCAGCGGTC GGGCTGAACG GGGGGTTCGT GCACACAGCC CAGCTTGGAG CGAACGACCT 7740
ACACCGAACT GAGATACCTA CAGCGTGAGC TATGAGAAAG CGCCACGCTT CCCGAAGGGA 7800
GAAAGGCGGA CAGGTATCCG GTAAGCGGCA GGGTCGGAAC AGGAGAGCGC ACGAGGGAGC 7860
TTCCAGGGGG AAACGCCTGG TATCTTTATA GTCCTGTCGG GTTTCGCCAC CTCTGACTTG 7920
AGCGTCGATT TTTGTGATGC TCGTCAGGGG GGCGGAGCCT ATGGAAAAAC GCCAGCAACG 7980
CGGCCTTTTT ACGGTTCCTG GCCTTTTGCT GGCCTTTTGC TCACATGTTC TTTCCTGCGT 8040
TATCCCCTGA TTCTGTGGAT AACCGTATTA CCGCCTTTGA GTGAGCTGAT ACCGCTCGCC 8100
GCAGCCGAAC GACCGAGCGC AGCGAGTCAG TGAGCGAGGA AGCGGAAGA 8149
(2) SEQ ID NO: 3 Data
...
(i) sequence signature:
(A) length: 8135 base pairs
(B) type: nucleic acid
(C) chain: two strands
(D) topology: line style
(ii) molecule type: DNA (genome)
(iii) suppose: non-
(iv) antisense: non-
(vi) primary source:
(A) organism: bovine viral diarrhea virus
(B) virus strain: 2724
(C) single isolate: pBHVtkex-1 ∷ gIII/p53
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 60
CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT 120
CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180
TGTGAGCGGA TAACAATTTC ACACAGGAAAC AGCTATGAC CATGATTACG CCAAGCTAGC 240
TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAGGTTGTCC 300
GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC 360
GTCCGCGTCC CCAACTCCGC GCGAAGACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420
GCGTATAAAA TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480
GCCCAGGCAA GCAAACTCTA AACGCCCGAG CGCCATGGCC CCGATGCCGC CACAAAGAGC 540
GCCGAAATTT CGCCCAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600
CTGTTGCCCG CGTGCCTGCT GGCCGCCCAC CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 660
CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCCACAGC CGCCCTTTTG CCCGTAGCCA 720
GGGGAAGCGG CTGCCCCTTC TGCCGCCGCG GCCGCGGTTG CTCGGCTTTG CGTTTGCCCC 780
GCGGCGATCG CCCCGCTCGC CGCGAACGCG CGCGCGCGAA TGGGGCGTAC TCGGCGAGCC 840
CGGCTATTAT AGCCTCAAGG CGCGCCGCGT TGCTAGCGAT CGTCTGGGCC GGCAGGCGCG 900
TCACTCTGAG CACGCGCATG CCCCGCTGGG AGACGAACAC CTGCACCGGC GCTAGGACCA 960
CCGGGTCTGG GCCCGGGGGG GCGAGATCGC GCACAAGCCG GGCCGAGTCG CGCAGCTGCC 1020
GCAGCCCCCC GAGGCGCTGG TCCATCTTGC TGGGCGTGTT CATGTTCGTT GAAAAACGGC 1080
ACGTCTTCAG CTCCACGATA AGACAGACGG CCCGGGCGTG CCCTGCCTCC GCGACCCGGA 1140
GTAGGCACAC GCAATCGGGC CGCCGGCTTT GCAGGTTTAC CTCAAAGCTC AGAGACACGC 1200
CCACGACCTG CTTAAAAACC TCCGGGGCGC CAAACTTGCC CAAAAGCTGG GCGAGGCGCG 1260
GGCGCAGCTT CTGCGCGCCA ACCGCCGCGC GTGCGTCGCA AGCCAGCGCC TCGTAAAAGC 1320
GGCTGTGGCA CCGGATCCCG GCGCGCAGGC GCGCACGTCG GTCGCGGTCG CGCGCCATGG 1380
CCGAGCCCGC GCGCGCTCTC CGCGTCGTGC GTATCTACCT GGACGGCGCG CACGGGCAGG 1440
GAAAGACAAC AACGGGCCGC GCGCTCGCGG CCGCTTCCAC CGCTGGGGAG GGCGTGCTCT 1500
TTTTCCCGGA GCCGATGGCG TACTGGCGCA CGATGTTTGG TACGGACGCC TTAAGTGGGA 1560
TCCTCGCGGC GTCTGCGCGA TGCGCCGCAG CCTCGCACGG GAGCGCACGC GCGCGGCGGG 1620
CCGGCGCACC GCGCAGACGC GGACGCGGCG GGCCTGGTTG CGTACTACCA GGCCAGGTTC 1680
GCGGCCCCGT ACTTAATTTT GCACGCGCGT GTCCGCGCTG CTGCGCCGCC TGGGCCGGCG 1740
CCGGGCGGCG AGCTGGTGGA CCCTCGTGTT CGACCGCCAC CCCGTGGCGC GCGTGCCTCT 1800
GCTACCCCTT CGCCCGCTAC TGCCTCCGCG AGATCAACGC GGAAGATCTC AATTCTATGA 1860
TTTCTATCAT TACTTCCTCA CATGTTGGAG GCATTTTCTC TCCCTCTGCA CTTAATAGCC 1920
TATCTTGCTT TAATTTCTTC CCACTCTTGG AGGCTAGGTT TGGTTTGGTG GGCTGATGAG 1980
GGAGGGAGAG ACCGCTCCAA GTACTTTAGC GGGTGGGATT GAAGCGGAGC CCTCCTGAGC 2040
TATGAGTGTC CTATGAGTGG GGCTGGAACT AAGAACCAGG GGCGTGGACA GGGTGTGTCA 2100
CAGAGAAGGG GATGTGCCTG CTTCTTTCTG GCCCAGGAGG AACCGGGTCA ATTCTTCAGC 2160
ACCTGGGTAC CCATAGAGCC CACCGCATCC CCAGCATGCC TGCTATTGTC TTCCCAATCC 2220
TCCCCCTTGC TGTCCTGCCC CACCCCACCC CCCAGAATAG AATGACACCT ACTCAGACAA 2280
TGCGATGCAA TTTCCTCATT TTATTAGGAA AGGACAGTGG GAGTGGCACC TTCCAGGGTC 2340
AAGGAAGGCA CGGGGGAGGG GCAAACAACA GATGGCTGGC AACTAGAAGG CACAGCGGAT 2400
CTGAGCTTGC ATGCCTGAGG TCGACCCTGG ATAAGCTGAT CCTCAATCAA TCAAGGTGGT 2460
ATAAGAGTAA GACCCACTTC TTTACAGCCT CCTCTCTTAG CAGTAGGTAT AACAACAAGA 2520
AATATGTCAC CACTTCAATA CTGTCATGTG TTAGCAAGTT ACCCATCATC ACCACTTCCC 2580
CTGCCCCATA TTGGGTCCCC AAGGCCTTTT GTTCTGATAG GACCATGTAT GTTACCAGTA 2640
ACCAGAGCAC GTATCTTCCA CCCAGTAAAG CTACCACCAC CACCAATATG GACTCGGCGA 2700
AGTAATCCCG ATGATGGTCA GTGACCTCCA GGTCGAACCA GTATTGATAA TCTCCTTTTA 2760
GCATGTATTG CTGGAAGTAA CTGTCTCTGG GCTCAAAATA TTTATTTTTT AATGTCCTCG 2820
TGTAGTTGAA GGTGCATGCC GTCTTTTCTA CAGGCCCCTC ACTTGGTATG ATCTCATATG 2880
GCTTGCAAGG CATAGGCCCA AGTTTGGTGT CAAGAGCTAT GACCTGTACG ATTGTGTCTC 2940
CTATCTTACA CTTTACCAAT CCTTGTGGTA CTATGGCTAC ACCCTCTCTG TTGCAAGTGG 3000
TGCCGTCTAC AAATCTGTAG CCAGTCTCAT TCTTCAACCT ACACTTGCCG ATGGGGTAGT 3060
GTGGCAACCC CTCACTTTTT TGGAATTTAT AACCACACCA CTTGCAAGAT TCGACAGGGC 3120
CTCCTGTGTA TTGTAGCTGG TCCCCAGTCA CACAAGTCCA ATTCCCTCCA AGATCACAGT 3180
TATAGAGATC CTCCCCCAGA GTTCTTTGGG TGATACAACC TTGTCTATAA GGGAATGGTT 3240
TGGACCTCTT ATACACACGC ACTACTGCTG TGTCTAGGGT GTCCCTATTA GCTAACATAC 3300
AGCTCACAGT TCCTGTCCAT CCTATAGGGC ATACCATCTG GAAGGCCGGT CCGTTTAGCA 3360
GTGTTGTATT GAACTTCCCT CTTACTACGG GATTGGCATC GCATGGGCAG AGTCCAAATT 3420
CAAAGTCGTC AGCCATTTCA AATGTTTCCC CTTGCCTTTG CCCCTCGAAA AGTTTTTTGA 3480
ATACCACACT GGTCTGCAAG GCTCTTGAAT GCAGAATTGC AAGATATCTA GTTTCTCTTG 3540
TGCACCTTGA GAGGTATGTA AACTTACCGT CTTTGCACCA AGCTATGACC ATTGTGTCTT 3600
CCAGCTTCAT TTCATGTGAG TAATCCTTCC AAACAGTGGT GAGGCCTTCA GCTCCTAATG 3660
GGCCAATTCT ATCATTCCTG GCTATGGCGT ATGAGTGTTC AGGTTTGCAG TCAATGTCCC 3720
CTTGTACCCC TGTTATCAGT AGTAGCCATA GGATCCCCGA CGGCGCCGCG GCGATGGCCG 3780
CCGCGTAGAG CGCCAGCAGA GCGAGCATCG CACGCGCGAG CGAGGCCATG GTCGAAGCTT 3840
GCCGCGGAGG CTGGATCGGT CCCGGTGTCT TCTATGGAGG TCAAAACAGC GTGGATGGCG 3900
TCTCCAGGCG ATCTGACGGT TCACTAAACG AGCTCTGCTT ATATAGACCT CCCACCGTAC 3960
ACGCCTACCG CCCATTTGCG TCAATGGGGC GGAGTTGTTA CGACATTTTG GAAAGTCCCG 4020
TTGATTTTGG TGCCAAAACA AACTCCCATT GACGTCAATG GGGTGGAGAC TTGGAAATCC 4080
CCGTGAGTCA AACCGCTATC CACGCCCATT GATGTACTGC CAAAACCGCA TCACCATGGT 4140
AATAGCGATG ACTAATACGT AGATGTACTG CCAAGTAGGA AAGTCCCATA AGGTCATGTA 4200
CTGGGCATAA TGCCAGGCGG GCCATTTACC GTCATTGACG TCAATAGGGG GCGTACTTGG 4260
CATATGATAC ACTTGATGTA CTGCCAAGTG GGCAGTTTAC CGTAAATACT CCACCCATTG 4320
ACGTCAATGG AAAGTCCCTA TTGGCGTTAC TATGGGAACA TACGTCATTA TTGACGTCAA 4380
TGGGCGGGGG TCGTTGGGCG GTCAGCCAGG CGGGCCATTT ACCGTAAGTT ATGTAACGCG 4440
GAACTCCATA TATGGGCTAT GAACTAATGA CCCCGTAATT GATTACTATT AATAACTAGT 4500
CAATAATCAA TGTCAACATG GCGGTAATGT TGGACATGAG CCAATATAAA TGTACATATT 4560
ATGATATGGA TACAACGTAT GCAATGGCCA ATAGCCAATA TTGATTTATG CTATATAACC 4620
AATGAATAAT ATGGCTAATG GCCAATATTG ATTCAATGTA TAGATCGATA TGCATTGGCC 4680
ATGTGCCAGC TTGATGTCGC CTCTATCGGC GATATAGCCT CATATCGTCT GTCACCTATA 4740
TCGAAACTGC GATATTTGCG ACACACAGAA TCGCCCAAGT CACCAAAGGC GTCTATCGCC 4800
ATCCCCCGTA AACGATATAA GCGTATCGCC AGATATCGCG TATGCCCAAA AATCAACTTT 4860
TGGAAAAATG GCGATATCAG TTACACAGAA ACTCACATCG GCGACATTTT CAATATGCCA 4920
TATTTTCAAA TATCGATTTT TCCAATATCG CCATCTCTAT CGGCGATAAA CACCACTATC 4980
GCGCGACATG AATTTAGTCG GGACAGAAAT CTCAAACGCG TATTTCGGAC AAACACACAT 5040
TTTATTATTC ACTGCAGGTC GAGGAATTCG GATCTCGAGG TGTTCGTGCT GGACGTGTCC 5100
GCGGCGCCAG ACGCGTGCGC GGCCGCCGTA CTGGACATGC GGCCCGCCAT GCAGGCCGCT 5160
TGCGCGGACG GGGCGGCGGG CGCGACGCTG GCGACCCTGG CGCGTCAGTT CGCGCTAGAG 5220
ATGGCGGGGG AGGCCACGGC GGGCCCTAGG GGACTATAAA GCTGCCCCTG CGCTCGCTCG 5280
CTCGCTGCAT TTGCGCCCCG ATCGCCTTAC GGGGACTCGG CGCTCGGCGG ATCCCCTCCC 5340
GGCCCCGCCG CGAAGCAGGC CGCCAGACAA AAAAATGCGG CGCCCGCTCT GCGCGGCGCT 5400
ATTGGCAGCG GCTGTCCTCG CGCTCGCCGC GGGCGCCCCC GCCGCCGCCC GCGGCGGGGG 5460
CGCCGAAGCC AGGGCAGCAC AGAGACGCCC GATACGAAAT CGAAGAGTGG GAAATGGTGG 5520
TCGGAGCCGG GCCGGCCGTG CACACGTTCA CCATCCGCTG CCTCGGGCCG CGGGGCATTG 5580
AGCGCGTGGC CCACATTGCA AACCTCAGCC GGCTGCTGGA CGGGTACATA GCGGTCCACG 5640
TTGACGTTGC GCGCACCTCT GGCCTGCGGG ACGCCATGTT TTTCCTGCCG CGCGCGGCCG 5700
TCGACTCTAG AGGATCCCCG GGTACCGAGC TCGAATTCAC TGGCCGTCGT TTTACAACGT 5760
CGTGACTGGG AAAACCCTGG CGTTACCCAA CTTAATCGCC TTGCAGCACA TCCCCCTTTC 5820
GCCAGCTGGC GTAATAGCGA AGAGGCCCGC ACCGATCGCC CTTCCCAACA GTTGCGCAGC 5880
CTGAATGGCG AATGGCGCCT GATGCGGTAT TTTCTCCTTA CGCATCTGTG CGGTATTTCA 5940
CACCGCATAT GGTGCACTCT CAGTACAATC TGCTCTGATG CCGCATAGTT AAGCCAGCCC 6000
CGACACCCGC CAACACCCGC TGACGCGCCC TGACGGGCTT GTCTGCTCCC GGCATCCGCT 6060
TACAGACAAG CTGTGACCGT CTCCGGGAGC TGCATGTGTC AGAGGTTTTC ACCGTCATCA 6120
CCGAAACGCG CGAGACGAAA GGGCCTCGTG ATACGCCTAT TTTTATAGGT TAATGTCATG 6180
ATAATAATGG TTTCTTAGAC GTCAGGTGGC ACTTTTCGGG GAAATGTGCG CGGAACCCCT 6240
ATTTGTTTAT TTTTCTAAAT ACATTCAAAT ATGTATCCGC TCATGAGACA ATAACCCTGA 6300
TAAATGCTTC AATAATATTG AAAAAGGAAG AGTATGAGTA TTCAACATTT CCGTGTCGCC 6360
CTTATTCCCT TTTTTGCGGC ATTTTGCCTT CCTGTTTTTG CTCACCCAGA AACGCTGGTG 6420
AAAGTAAAAG ATGCTGAAGA TCAGTTGGGT GCACGAGTGG GTTACATCGA ACTGGATCTC 6480
AACAGCGGTA AGATCCTTGA GAGTTTTCGC CCCGAAGAAC GTTTTCCAAT GATGAGCACT 6540
TTTAAAGTTC TGCTATGTGG CGCGGTATTA TCCCGTATTG ACGCCGGGCA AGAGCAACTC 6600
GGTCGCCGCA TACACTATTC TCAGAATGAC TTGGTTGAGT ACTCACCAGT CACAGAAAAG 6660
CATCTTACGG ATGGCATGAC AGTAAGAGAA TTATGCAGTG CTGCCATAAC CATGAGTGAT 6720
AACACTGCGG CCAACTTACT TCTGACAACG ATCGGAGGAC CGAAGGAGCT AACCGCTTTT 6780
TTGCACAACA TGGGGGATCA TGTAACTCGC CTTGATCGTT GGGAACCGGA GCTGAATGAA 6840
GCCATACCAA ACGACGAGCG TGACACCACG ATGCCTGTAG CAATGGCAAC AACGTTGCGC 6900
AAACTATTAA CTGGCGAACT ACTTACTCTA GCTTCCCGGC AACAATTAAT AGACTGGATG 6960
GAGGCGGATA AAGTTGCAGG ACCACTTCTG CGCTCGGCCC TTCCGGCTGG CTGGTTTATT 7020
GCTGATAAAT CTGGAGCCGG TGAGCGTGGG TCTCGCGGTA TCATTGCAGC ACTGGGGCCA 7080
GATGGTAAGC CCTCCCGTAT CGTAGTTATC TACACGACGG GGAGTCAGGC AACTATGGAT 7140
GAACGAAATA GACAGATCGC TGAGATAGGT GCCTCACTGA TTAAGCATTG GTAACTGTCA 7200
GACCAAGTTT ACTCATATAT ACTTTAGATT GATTTAAAAC TTCATTTTTA ATTTAAAAGG 7260
ATCTAGGTGA AGATCCTTTT TGATAATCTC ATGACCAAAA TCCCTTAACG TGAGTTTTCG 7320
TTCCACTGAG CGTCAGACCC CGTAGAAAAG ATCAAAGGAT CTTCTTGAGA TCCTTTTTTT 7380
CTGCGCGTAA TCTGCTGCTT GCAAACAAAA AAACCACCGC TACCAGCGGT GGTTTGTTTG 7440
CCGGATCAAG AGCTACCAAC TCTTTTTCCG AAGGTAACTG GCTTCAGCAG AGCGCAGATA 7500
CCAAATACTG TCCTTCTAGT GTAGCCGTAG TTAGGCCACC ACTTCAAGAA CTCTGTAGCA 7560
CCGCCTACAT ACCTCGCTCT GCTAATCCTG TTACCAGTGG CTGCTGCCAG TGGCGATAAG 7620
TCGTGTCTTA CCGGGTTGGA CTCAAGACGA TAGTTACCGG ATAAGGCGCA GCGGTCGGGC 7680
TGAACGGGGG GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA CGACCTACAC CGAACTGAGA 7740
TACCTACAGC GTGAGCTATG AGAAAGCGCC ACGCTTCCCG AAGGGAGAAA GGCGGACAGG 7800
TATCCGGTAA GCGGCAGGGT CGGAACAGGA GAGCGCACGA GGGAGCTTCC AGGGGGAAAC 7860
GCCTGGTATC TTTATAGTCC TGTCGGGTTT CGCCACCTCT GACTTGAGCG TCGATTTTTG 7920
TGATGCTCGT CAGGGGGGCG GAGCCTATGG AAAAACGCCA GCAACGCGGC CTTTTTACGG 7980
TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC ATGTTCTTTC CTGCGTTATC CCCTGATTCT 8040
GTGGATAACC GTATTACCGC CTTTGAGTGA GCTGATACCG CTCGCCGCAG CCGAACGACC 8100
GAGCGCAGCG AGTCAGTGAG CGAGGAAGCG GAAGA 8135
(2) SEQ ID NO: 4 INFORMATION
...
(i) sequence signature:
(A) length: 8149 base pairs
(B) type: nucleic acid
(C) chain: two strands
(D) topology: line style
(ii) molecule type: DNA (genome)
(iii) suppose: non-
(iv) antisense: non-
(vi) primary source:
(A) organism: bovine viral diarrhea virus
(B) virus strain: 2724
(C) single isolate: pBHVtkex-3 ∷ BGH/p53
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 60
CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT 120
CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180
TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG CCAAGCTAGC 240
TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAGGTTGTCC 300
GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC 360
GTCCGCGTCC CCAACTCCGC GCGAAGACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420
GCGTATAAAA TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480
GCCCAGGCAA GCAAACTCTA AACGCCCGAG CGCCATGGCC CCGATGCCGC CACAAAGAGC 540
GCCGAAATTT CGCCCAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600
CTGTTGCCCG CGTGCCTGCT GGCCGCCCAC CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 660
CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCCACAGC CGCCCTTTTG CCCGTAGCCA 720
GGGGAAGCGG CTGCCCCTTC TGCCGCCGCG GCCGCGGTTG CTCGGCTTTG CGTTTGCCCC 780
GCGGCGATCG CCCCGCTCGC CGCGAACGCG CGCGCGCGAA TGGGGCGTAC TCGGCGAGCC 840
CGGCTATTAT AGCCTCAAGG CGCGCCGCGT TGCTAGCGAT CGTCTGGGCC GGCAGGCGCG 900
TCACTCTGAG CACGCGCATG CCCCGCTGGG AGACGAACAC CTGCACCGGC GCTAGGACCA 960
CCGGGTCTGG GCCCGGGGGG GCGAGATCGC GCACAAGCCG GGCCGAGTCG CGCAGCTGCC 1020
GCAGCCCCCC GAGGCGCTGG TCCATCTTGC TGGGCGTGTT CATGTTCGTT GAAAAACGGC 1080
ACGTCTTCAG CTCCACGATA AGACAGACGG CCCGGGCGTG CCCTGCCTCC GCGACCCGGA 1140
GTAGGCACAC GCAATCGGGC CGCCGGCTTT GCAGGTTTAC CTCAAAGCTC AGAGACACGC 1200
CCACGACCTG CTTAAAAACC TCCGGGGCGC CAAACTTGCC CAAAAGCTGG GCGAGGCGCG 1260
GGCGCAGCTT CTGCGCGCCA ACCGCCGCGC GTGCGTCGCA AGCCAGCGCC TCGTAAAAGC 1320
GGCTGTGGCA CCGGATCCCG GCGCGCAGGC GCGCACGTCG GTCGCGGTCG CGCGCCATGG 1380
CCGAGCCCGC GCGCGCTCTC CGCGTCGTGC GTATCTACCT GGACGGCGCG CACGGGCAGG 1440
GAAAGACAAC AACGGGCCGC GCGCTCGCGG CCGCTTCCAC CGCTGGGGAG GGCGTGCTCT 1500
TTTTCCCGGA GCCGATGGCG TACTGGCGCA CGATGTTTGG TACGGACGCC TTAAGTGGGA 1560
TCCTCGCGGC GTCTGCGCGA TGCGCCGCAG CCTCGCACGG GAGCGCACGC GCGCGGCGGG 1620
CCGGCGCACC GCGCAGACGC GGACGCGGCG GGCCTGGTTG CGTACTACCA GGCCAGGTTC 1680
GCGGCCCCGT ACTTAATTTT GCACGCGCGT GTCCGCGCTG CTGCGCCGCC TGGGCCGGCG 1740
CCGGGCGGCG AGCTGGTGGA CCCTCGTGTT CGACCGCCAC CCCGTGGCGC GCGTGCCTCT 1800
GCTACCCCTT CGCCCGCTAC TGCCTCCGCG AGATCAACGC GGAAGATCCG AATTCCTCGA 1860
CCTGCAGTGA ATAATAAAAT GTGTGTTTGT CCGAAATACG CGTTTGAGAT TTCTGTCCCG 1920
ACTAAATTCA TGTCGCGCGA TAGTGGTGTT TATCGCCGAT AGAGATGGCG ATATTGGAAA 1980
AATCGATATT TGAAAATATG GCATATTGAA AATGTCGCCG ATGTGAGTTT CTGTGTAACT 2040
GATATCGCCA TTTTTCCAAA AGTTGATTTT TGGGCATACG CGATATCTGG CGATACGCTT 2100
ATATCGTTTA CGGGGGATGG CGATAGACGC CTTTGGTGAC TTGGGCGATT CTGTGTGTCG 2160
CAAATATCGC AGTTTCGATA TAGGTGACAG ACGATATGAG GCTATATCGC CGATAGAGGC 2220
GACATCAAGC TGGCACATGG CCAATGCATA TCGATCTATA CATTGAATCA ATATTGGCCA 2280
TTAGCCATAT TATTCATTGG TTATATAGCA TAAATCAATA TTGGCTATTG GCCATTGCAT 2340
ACGTTGTATC CATATCATAA TATGTACATT TATATTGGCT CATGTCCAAC ATTACCGCCA 2400
TGTTGACATT GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT 2460
AGCCCATATA TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG 2520
CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA 2580
GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA 2640
CATCAAGTGT ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC 2700
GCCTGGCATT ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC 2760
GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA 2820
TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG 2880
TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG 2940
CAAATGGGCG GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCG TTTAGTGAAC 3000
CGTCAGATCG CCTGGAGACG CCATCCACGC TGTTTTGACC TCCATAGAAG ACACCGGGAC 3060
CGATCCAGCC TCCGCGGCAA GCTTCGACAT GATGGCTGCA GGCCCCCGGA CCTCCCTGCT 3120
CCTGGCTTTC GCCCTGCTCT GCCTGCCCTG GACTCAGGTG GTGGGCGCCT TCCCAGGGAT 3180
CCTATGGCTA CTACTGATAA CAGGGGTACA AGGGGACATT GACTGCAAAC CTGAACACTC 3240
ATACGCCATA GCCAGGAATG ATAGAATTGG CCCATTAGGA GCTGAAGGCC TCACCACTGT 3300
TTGGAAGGAT TACTCACATG AAATGAAGCT GGAAGACACA ATGGTCATAG CTTGGTGCAA 3360
AGACGGTAAG TTTACATACC TCTCAAGGTG CACAAGAGAA ACTAGATATC TTGCAATTCT 3420
GCATTCAAGA GCCTTGCAGA CCAGTGTGGT ATTCAAAAAA CTTTTCGAGG GGCAAAGGCA 3480
AGGGGAAACA TTTGAAATGG CTGACGACTT TGAATTTGGA CTCTGCCCAT GCGATGCCAA 3540
TCCCGTAGTA AGAGGGAAGT TCAATACAAC ACTGCTAAAC GGACCGGCCT TCCAGATGGT 3600
ATGCCCTATA GGATGGACAG GAACTGTGAG CTGTATGTTA GCTAATAGGG ACACCCTAGA 3660
CACAGCAGTA GTGCGTGTGT ATAAGAGGTC CAAACCATTC CCTTATAGAC AAGGTTGTAT 3720
CACCCAAAGA ACTCTGGGGG AGGATCTCTA TAACTGTGAT CTTGGAGGGA ATTGGACTTG 3780
TGTGACTGGG GACCAGCTAC AATACACAGG AGGCCCTGTC GAATCTTGCA AGTGGTGTGG 3840
TTATAAATTC CAAAAAAGTG AGGGGTTGCC ACACTACCCC ATCGGCAAGT GTAGGTTGAA 3900
GAATGAGACT GGCTACAGAT TTGTAGACGG CACCACTTGC AACAGAGAGG GTGTAGCCAT 3960
AGTACCACAA GGATTGGTAA AGTGTAAGAT AGGAGACACA ATCGTACAGG TCATAGCTCT 4020
TGACACCAAA CTTGGGCCTA TGCCTTGCAA GCCATATGAG ATCATACCAA GTGAGGGGCC 4080
TGTAGAAAAG ACGGCATGCA CCTTCAACTA CACGAGGACA TTAAAAAATA AATATTTTGA 4140
GCCCAGAGAC AGTTACTTCC AGCAATACAT GCTAAAAGGA GATTATCAAT ACTGGTTCGA 4200
CCTGGAGGTC ACTGACCATC ATCGGGATTA CTTCGCCGAG TCCATATTGG TGGTGGTGGT 4260
AGCTTTACTG GGTGGAAGAT ACGTGCTCTG GTTACTGGTA ACATACATGG TCCTATCAGA 4320
ACAAAAGGCC TTGGGGACCC AATATGGGGC AGGGGAAGTG GTGATGATGG GTAACTTGCT 4380
AACACATGAC AGTATTGAAG TGGTGACATA TTTCTTGTTG TTATACCTAC TGCTAAGAGA 4440
GGAGGCTGTA AAGAAGTGGG TCTTACTCTT ATACCACCTT GATTGATTGA GGATCAGCTT 4500
ATCCAGGGTC GACCTCAGGC ATGCAAGCTC AGATCCGCTG TGCCTTCTAG TTGCCAGCCA 4560
TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC TTGACCCTGG AAGGTGCCAC TCCCACTGTC 4620
CTTTCCTAAT AAAATGAGGA AATTGCATCG CATTGTCTGA GTAGGTGTCA TTCTATTCTG 4680
GGGGGTGGGG TGGGGCAGGA CAGCAAGGGG GAGGATTGGG AAGACAATAG CAGGCATGCT 4740
GGGGATGCGG TGGGCTCTAT GGGTACCCAG GTGCTGAAGA ATTGACCCGG TTCCTCCTGG 4800
GCCAGAAAGA AGCAGGCACA TCCCCTTCTC TGTGACACAC CCTGTCCACG CCCCTGGTTC 4860
TTAGTTCCAG CCCCACTCAT AGGACACTCA TAGCTCAGGA GGGCTCCGCT TCAATCCCAC 4920
CCGCTAAAGT ACTTGGAGCG GTCTCTCCCT CCCTCATCAG CCCACCAAAC CAAACCTAGC 4980
CTCCAAGAGT GGGAAGAAAT TAAAGCAAGA TAGGCTATTA AGTGCAGAGG GAGAGAAAAT 5040
GCCTCCAACA TGTGAGGAAG TAATGATAGA AATCATAGAA TTGAGATCTC GAGGTGTTCG 5100
TGCTGGACGT GTCCGCGGCG CCAGACGCGT GCGCGGCCGC CGTACTGGAC ATGCGGCCCG 5160
CCATGCAGGC CGCTTGCGCG GACGGGGCGG CGGGCGCGAC GCTGGCGACC CTGGCGCGTC 5220
AGTTCGCGCT AGAGATGGCG GGGGAGGCCA CGGCGGGCCC TAGGGGACTA TAAAGCTGCC 5280
CCTGCGCTCG CTCGCTCGCT GCATTTGCGC CCCGATCGCC TTACGGGGAC TCGGCGCTCG 5340
GCGGATCCCC TCCCGGCCCC GCCGCGAAGC AGGCCGCCAG ACAAAAAAAT GCGGCGCCCG 5400
CTCTGCGCGG CGCTATTGGC AGCGGCTGTC CTCGCGCTCG CCGCGGGCGC CCCCGCCGCC 5460
GCCCGCGGCG GGGGCGCCGA AGCCAGGGCA GCACAGAGAC GCCCGATACG AAATCGAAGA 5520
GTGGGAAATG GTGGTCGGAG CCGGGCCGGC CGTGCACACG TTCACCATCC GCTGCCTCGG 5580
GCCGCGGGGC ATTGAGCGCG TGGCCCACAT TGCAAACCTC AGCCGGCTGC TGGACGGGTA 5640
CATAGCGGTC CACGTTGACG TTGCGCGCAC CTCTGGCCTG CGGGACGCCA TGTTTTTCCT 5700
GCCGCGCGCG GCCGTCGACT CTAGAGGATC CCCGGGTACC GAGCTCGAAT TCACTGGCCG 5760
TCGTTTTACA ACGTCGTGAC TGGGAAAACC CTGGCGTTAC CCAACTTAAT CGCCTTGCAG 5820
CACATCCCCC TTTCGCCAGC TGGCGTAATA GCGAAGAGGC CCGCACCGAT CGCCCTTCCC 5880
AACAGTTGCG CAGCCTGAAT GGCGAATGGC GCCTGATGCG GTATTTTCTC CTTACGCATC 5940
TGTGCGGTAT TTCACACCGC ATATGGTGCA CTCTCAGTAC AATCTGCTCT GATGCCGCAT 6000
AGTTAAGCCA GCCCCGACAC CCGCCAACAC CCGCTGACGC GCCCTGACGG GCTTGTCTGC 6060
TCCCGGCATC CGCTTACAGA CAAGCTGTGA CCGTCTCCGG GAGCTGCATG TGTCAGAGGT 6120
TTTCACCGTC ATCACCGAAA CGCGCGAGAC GAAAGGGCCT CGTGATACGC CTATTTTTAT 6180
AGGTTAATGT CATGATAATA ATGGTTTCTT AGACGTCAGG TGGCACTTTT CGGGGAAATG 6240
TGCGCGGAAC CCCTATTTGT TTATTTTTCT AAATACATTC AAATATGTAT CCGCTCATGA 6300
GACAATAACC CTGATAAATG CTTCAATAAT ATTGAAAAAG GAAGAGTATG AGTATTCAAC 6360
ATTTCCGTGT CGCCCTTATT CCCTTTTTTG CGGCATTTTG CCTTCCTGTT TTTGCTCACC 6420
CAGAAACGCT GGTGAAAGTA AAAGATGCTG AAGATCAGTT GGGTGCACGA GTGGGTTACA 6480
TCGAACTGGA TCTCAACAGC GGTAAGATCC TTGAGAGTTT TCGCCCCGAA GAACGTTTTC 6540
CAATGATGAG CACTTTTAAA GTTCTGCTAT GTGGCGCGGT ATTATCCCGT ATTGACGCCG 6600
GGCAAGAGCA ACTCGGTCGC CGCATACACT ATTCTCAGAA TGACTTGGTT GAGTACTCAC 6660
CAGTCACAGA AAAGCATCTT ACGGATGGCA TGACAGTAAG AGAATTATGC AGTGCTGCCA 6720
TAACCATGAG TGATAACACT GCGGCCAACT TACTTCTGAC AACGATCGGA GGACCGAAGG 6780
AGCTAACCGC TTTTTTGCAC AACATGGGGG ATCATGTAAC TCGCCTTGAT CGTTGGGAAC 6840
CGGAGCTGAA TGAAGCCATA CCAAACGACG AGCGTGACAC CACGATGCCT GTAGCAATGG 6900
CAACAACGTT GCGCAAACTA TTAACTGGCG AACTACTTAC TCTAGCTTCC CGGCAACAAT 6960
TAATAGACTG GATGGAGGCG GATAAAGTTG CAGGACCACT TCTGCGCTCG GCCCTTCCGG 7020
CTGGCTGGTT TATTGCTGAT AAATCTGGAG CCGGTGAGCG TGGGTCTCGC GGTATCATTG 7080
CAGCACTGGG GCCAGATGGT AAGCCCTCCC GTATCGTAGT TATCTACACG ACGGGGAGTC 7140
AGGCAACTAT GGATGAACGA AATAGACAGA TCGCTGAGAT AGGTGCCTCA CTGATTAAGC 7200
ATTGGTAACT GTCAGACCAA GTTTACTCAT ATATACTTTA GATTGATTTA AAACTTCATT 7260
TTTAATTTAA AAGGATCTAG GTGAAGATCC TTTTTGATAA TCTCATGACC AAAATCCCTT 7320
AACGTGAGTT TTCGTTCCAC TGAGCGTCAG ACCCCGTAGA AAAGATCAAA GGATCTTCTT 7380
GAGATCCTTT TTTTCTGCGC GTAATCTGCT GCTTGCAAAC AAAAAAACCA CCGCTACCAG 7440
CGGTGGTTTG TTTGCCGGAT CAAGAGCTAC CAACTCTTTT TCCGAAGGTA ACTGGCTTCA 7500
GCAGAGCGCA GATACCAAAT ACTGTCCTTC TAGTGTAGCC GTAGTTAGGC CACCACTTCA 7560
AGAACTCTGT AGCACCGCCT ACATACCTCG CTCTGCTAAT CCTGTTACCA GTGGCTGCTG 7620
CCAGTGGCGA TAAGTCGTGT CTTACCGGGT TGGACTCAAG ACGATAGTTA CCGGATAAGG 7680
CGCAGCGGTC GGGCTGAACG GGGGGTTCGT GCACACAGCC CAGCTTGGAG CGAACGACCT 7740
ACACCGAACT GAGATACCTA CAGCGTGAGC TATGAGAAAG CGCCACGCTT CCCGAAGGGA 7800
GAAAGGCGGA CAGGTATCCG GTAAGCGGCA GGGTCGGAAC AGGAGAGCGC ACGAGGGAGC 7860
TTCCAGGGGG AAACGCCTGG TATCTTTATA GTCCTGTCGG GTTTCGCCAC CTCTGACTTG 7920
AGCGTCGATT TTTGTGATGC TCGTCAGGGG GGCGGAGCCT ATGGAAAAAC GCCAGCAACG 7980
CGGCCTTTTT ACGGTTCCTG GCCTTTTGCT GGCCTTTTGC TCACATGTTC TTTCCTGCGT 8040
TATCCCCTGA TTCTGTGGAT AACCGTATTA CCGCCTTTGA GTGAGCTGAT ACCGCTCGCC 8100
GCAGCCGAAC GACCGAGCGC AGCGAGTCAG TGAGCGAGGA AGCGGAAGA 8149
(2) SEQ ID NO: 5 Data
...
(i) sequence signature:
(A) length: 8135 base pairs
(B) type: nucleic acid
(C) chain: two strands
(D) topology: line style
(ii) molecule type: DNA (genome)
(iii) suppose: non-
(iv) antisense: non-
(vi) primary source:
(A) organism: bovine viral diarrhea virus
(B) virus strain: 2724
(C) single isolate: pBHVtkex-3 ∷ gIII/p53
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA 60
CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT 120
CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT 180
TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG CCAAGCTAGC 240
TTGCATGCCT GCAGGTCGAC TTCCGCGCCC GCGGCGTCTG CCTTCGCCAG CAGGTTGTCC 300
GCGGCCGCTG CCGGCCTGGT TCCGCGCCCG CCGCCTCGCG GCCAGCTCCC GCGCGGGCGC 360
GTCCGCGTCC CCAACTCCGC GCGAAGACGG GCTCGTCCCA GAAGCGCAGC GGAAAGGCCG 420
GCGTATAAAA TTTCGCTCGT CCGGTACAAA GACGCGGTCC GCGACTGCGT GGATGTCCAC 480
GCCCAGGCAA GCAAACTCTA AACGCCCGAG CGCCATGGCC CCGATGCCGC CACAAAGAGC 540
GCCGAAATTT CGCCCAGGCA CGCCGCGCCG CCCGACGCGT CTTTAGCGCA CCCGCCGGCG 600
CTGTTGCCCG CGTGCCTGCT GGCCGCCCAC CGGCGGCCGC TGTCCCCGGC CTCAGCAGGG 660
CCGGGGTCGC CGGCGGGCGG CCGCGGGGTG CGGCCACAGC CGCCCTTTTG CCCGTAGCCA 720
GGGGAAGCGG CTGCCCCTTC TGCCGCCGCG GCCGCGGTTG CTCGGCTTTG CGTTTGCCCC 780
GCGGCGATCG CCCCGCTCGC CGCGAACGCG CGCGCGCGAA TGGGGCGTAC TCGGCGAGCC 840
CGGCTATTAT AGCCTCAAGG CGCGCCGCGT TGCTAGCGAT CGTCTGGGCC GGCAGGCGCG 900
TCACTCTGAG CACGCGCATG CCCCGCTGGG AGACGAACAC CTGCACCGGC GCTAGGACCA 960
CCGGGTCTGG GCCCGGGGGG GCGAGATCGC GCACAAGCCG GGCCGAGTCG CGCAGCTGCC 1020
GCAGCCCCCC GAGGCGCTGG TCCATCTTGC TGGGCGTGTT CATGTTCGTT GAAAAACGGC 1080
ACGTCTTCAG CTCCACGATA AGACAGACGG CCCGGGCGTG CCCTGCCTCC GCGACCCGGA 1140
GTAGGCACAC GCAATCGGGC CGCCGGCTTT GCAGGTTTAC CTCAAAGCTC AGAGACACGC 1200
CGACGACCTG CTTAAAAACC TCCGGGGCGC CAAACTTGCC CAAAAGCTGG GCGAGGCGCG 1260
GGCGCAGCTT CTGCGCGCCA ACCGCCGCGC GTGCGTCGCA AGCCAGCGCC TCGTAAAAGC 1320
GGCTGTGGCA CCGGATCCCG GCGCGCAGGC GCGCACGTCG GTCGCGGTCG CGCGCCATGG 1380
CCGAGCCCGC GCGCGCTCTC CGCGTCGTGC GTATCTACCT GGACGGCGCG CACGGGCAGG 1440
GAAAGACAAC AACGGGCCGC GCGCTCGCGG CCGCTTCCAC CGCTGGGGAG GGCGTGCTCT 1500
TTTTCCCGGA GCCGATGGCG TACTGGCGCA CGATGTTTGG TACGGACGCC TTAAGTGGGA 1560
TCCTCGCGGC GTCTGCGCGA TGCGCCGCAG CCTCGCACGG GAGCGCACGC GCGCGGCGGG 1620
CCGGCGCACC GCGCAGACGC GGACGCGGCG GGCCTGGTTG CGTACTACCA GGCCAGGTTC 1680
GCGGCCCCGT ACTTAATTTT GCACGCGCGT GTCCGCGCTG CTGCGCCGCC TGGGCCGGCG 1740
CCGGGCGGCG AGCTGGTGGA CCCTCGTGTT CGACCGCCAC CCCGTGGCGC GCGTGCCTCT 1800
GCTACCCCTT CGCCCGCTAC TGCCTCCGCG AGATCAACGC GGAAGATCCG AATTCCTCGA 1860
CCTGCAGTGA ATAATAAAAT GTGTGTTTGT CCGAAATACG CGTTTGAGAT TTCTGTCCCG 1920
ACTAAATTCA TGTCGCGCGA TAGTGGTGTT TATCGCCGAT AGAGATGGCG ATATTGGAAA 1980
AATCGATATT TGAAAATATG GCATATTGAA AATGTCGCCG ATGTGAGTTT CTGTGTAACT 2040
GATATCGCCA TTTTTCCAAA AGTTGATTTT TGGGCATACG CGATATCTGG CGATACGCTT 2100
ATATCGTTTA CGGGGGATGG CGATAGACGC CTTTGGTGAC TTGGGCGATT CTGTGTGTCG 2160
CAAATATCGC AGTTTCGATA TAGGTGACAG ACGATATGAG GCTATATCGC CGATAGAGGC 2220
GACATCAAGC TGGCACATGG CCAATGCATA TCGATCTATA CATTGAATCA ATATTGGCCA 2280
TTAGCCATAT TATTCATTGG TTATATAGCA TAAATCAATA TTGGCTATTG GCCATTGCAT 2340
ACGTTGTATC CATATCATAA TATGTACATT TATATTGGCT CATGTCCAAC ATTACCGCCA 2400
TGTTGACATT GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT 2460
AGCCCATATA TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG 2520
CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA 2580
GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA 2640
CATCAAGTGT ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC 2700
GCCTGGCATT ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC 2760
GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA 2820
TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG 2880
TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG 2940
CAAATGGGCG GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCG TTTAGTGAAC 3000
CGTCAGATCG CCTGGAGACG CCATCCACGC TGTTTTGACC TCCATAGAAG ACACCGGGAC 3060
CGATCCAGCC TCCGCGGCAA GCTTCGACCA TGGCCTCGCT CGCGCGTGCG ATGCTCGCTC 3120
TGCTGGCGCT CTACGCGGCG GCCATCGCCG CGGCGCCGTC GGGGATCCTA TGGCTACTAC 3180
TGATAACAGG GGTACAAGGG GACATTGACT GCAAACCTGA ACACTCATAC GCCATAGCCA 3240
GGAATGATAG AATTGGCCCA TTAGGAGCTG AAGGCCTCAC CACTGTTTGG AAGGATTACT 3300
CACATGAAAT GAAGCTGGAA GACACAATGG TCATAGCTTG GTGCAAAGAC GGTAAGTTTA 3360
CATACCTCTC AAGGTGCACA AGAGAAACTA GATATCTTGC AATTCTGCAT TCAAGAGCCT 3420
TGCAGACCAG TGTGGTATTC AAAAAACTTT TCGAGGGGCA AAGGCAAGGG GAAACATTTG 3480
AAATGGCTGA CGACTTTGAA TTTGGACTCT GCCCATGCGA TGCCAATCCC GTAGTAAGAG 3540
GGAAGTTCAA TACAACACTG CTAAACGGAC CGGCCTTCCA GATGGTATGC CCTATAGGAT 3600
GGACAGGAAC TGTGAGCTGT ATGTTAGCTA ATAGGGACAC CCTAGACACA GCAGTAGTGC 3660
GTGTGTATAA GAGGTCCAAA CCATTCCCTT ATAGACAAGG TTGTATCACC CAAAGAACTC 3720
TGGGGGAGGA TCTCTATAAC TGTGATCTTG GAGGGAATTG GACTTGTGTG ACTGGGGACC 3780
AGCTACAATA CACAGGAGGC CCTGTCGAAT CTTGCAAGTG GTGTGGTTAT AAATTCCAAA 3840
AAAGTGAGGG GTTGCCACAC TACCCCATCG GCAAGTGTAG GTTGAAGAAT GAGACTGGCT 3900
ACAGATTTGT AGACGGCACC ACTTGCAACA GAGAGGGTGT AGCCATAGTA CCACAAGGAT 3960
TGGTAAAGTG TAAGATAGGA GACACAATCG TACAGGTCAT AGCTCTTGAC ACCAAACTTG 4020
GGCCTATGCC TTGCAAGCCA TATGAGATCA TACCAAGTGA GGGGCCTGTA GAAAAGACGG 4080
CATGCACCTT CAACTACACG AGGACATTAA AAAATAAATA TTTTGAGCCC AGAGACAGTT 4140
ACTTCCAGCA ATACATGCTA AAAGGAGATT ATCAATACTG GTTCGACCTG GAGGTCACTG 4200
ACCATCATCG GGATTACTTC GCCGAGTCCA TATTGGTGGT GGTGGTAGCT TTACTGGGTG 4260
GAAGATACGT GCTCTGGTTA CTGGTAACAT ACATGGTCCT ATCAGAACAA AAGGCCTTGG 4320
GGACCCAATA TGGGGCAGGG GAAGTGGTGA TGATGGGTAA CTTGCTAACA CATGACAGTA 4380
TTGAAGTGGT GACATATTTC TTGTTGTTAT ACCTACTGCT AAGAGAGGAG GCTGTAAAGA 4440
AGTGGGTCTT ACTCTTATAC CACCTTGATT GATTGAGGAT CAGCTTATCC AGGGTCGACC 4500
TCAGGCATGC AAGCTCAGAT CCGCTGTGCC TTCTAGTTGC CAGCCATCTG TTGTTTGCCC 4560
CTCCCCCGTG CCTTCCTTGA CCCTGGAAGG TGCCACTCCC ACTGTCCTTT CCTAATAAAA 4620
TGAGGAAATT GCATCGCATT GTCTGAGTAG GTGTCATTCT ATTCTGGGGG GTGGGGTGGG 4680
GCAGGACAGC AAGGGGGAGG ATTGGGAAGA CAATAGCAGG CATGCTGGGG ATGCGGTGGG 4740
CTCTATGGGT ACCCAGGTGC TGAAGAATTG ACCCGGTTCC TCCTGGGCCA GAAAGAAGCA 4800
GGCACATCCC CTTCTCTGTG ACACACCCTG TCCACGCCCC TGGTTCTTAG TTCCAGCCCC 4860
ACTCATAGGA CACTCATAGC TCAGGAGGGC TCCGCTTCAA TCCCACCCGC TAAAGTACTT 4920
GGAGCGGTCT CTCCCTCCCT CATCAGCCCA CCAAACCAAA CCTAGCCTCC AAGAGTGGGA 4980
AGAAATTAAA GCAAGATAGG CTATTAAGTG CAGAGGGAGA GAAAATGCCT CCAACATGTG 5040
AGGAAGTAAT GATAGAAATC ATAGAATTGA GATCTCGAGG TGTTCGTGCT GGACGTGTCC 5100
GCGGCGCCAG ACGCGTGCGC GGCCGCCGTA CTGGACATGC GGCCCGCCAT GCAGGCCGCT 5160
TGCGCGGACG GGGCGGCGGG CGCGACGCTG GCGACCCTGG CGCGTCAGTT CGCGCTAGAG 5220
ATGGCGGGGG AGGCCACGGC GGGCCCTAGG GGACTATAAA GCTGCCCCTG CGCTCGCTCG 5280
CTCGCTGCAT TTGCGCCCCG ATCGCCTTAC GGGGACTCGG CGCTCGGCGG ATCCCCTCCC 5340
GGCCCCGCCG CGAAGCAGGC CGCCAGACAA AAAAATGCGG CGCCCGCTCT GCGCGGCGCT 5400
ATTGGCAGCG GCTGTCCTCG CGCTCGCCGC GGGCGCCCCC GCCGCCGCCC GCGGCGGGGG 5460
CGCCGAAGCC AGGGCAGCAC AGAGACGCCC GATACGAAAT CGAAGAGTGG GAAATGGTGG 5520
TCGGAGCCGG GCCGGCCGTG CACACGTTCA CCATCCGCTG CCTCGGGCCG CGGGGCATTG 5580
AGCGCGTGGC CCACATTGCA AACCTCAGCC GGCTGCTGGA CGGGTACATA GCGGTCCACG 5640
TTGACGTTGC GCGCACCTCT GGCCTGCGGG ACGCCATGTT TTTCCTGCCG CGCGCGGCCG 5700
TCGACTCTAG AGGATCCCCG GGTACCGAGC TCGAATTCAC TGGCCGTCGT TTTACAACGT 5760
CGTGACTGGG AAAACCCTGG CGTTACCCAA CTTAATCGCC TTGCAGCACA TCCCCCTTTC 5820
GCCAGCTGGC GTAATAGCGA AGAGGCCCGC ACCGATCGCC CTTCCCAACA GTTGCGCAGC 5880
CTGAATGGCG AATGGCGCCT GATGCGGTAT TTTCTCCTTA CGCATCTGTG CGGTATTTCA 5940
CACCGCATAT GGTGCACTCT CAGTACAATC TGCTCTGATG CCGCATAGTT AAGCCAGCCC 6000
CGACACCCGC CAACACCCGC TGACGCGCCC TGACGGGCTT GTCTGCTCCC GGCATCCGCT 6060
TACAGACAAG CTGTGACCGT CTCCGGGAGC TGCATGTGTC AGAGGTTTTC ACCGTCATCA 6120
CCGAAACGCG CGAGACGAAA GGGCCTCGTG ATACGCCTAT TTTTATAGGT TAATGTCATG 6180
ATAATAATGG TTTCTTAGAC GTCAGGTGGC ACTTTTCGGG GAAATGTGCG CGGAACCCCT 6240
ATTTGTTTAT TTTTCTAAAT ACATTCAAAT ATGTATCCGC TCATGAGACA ATAACCCTGA 6300
TAAATGCTTC AATAATATTG AAAAAGGAAG AGTATGAGTA TTCAACATTT CCGTGTCGCC 6360
CTTATTCCCT TTTTTGCGGC ATTTTGCCTT CCTGTTTTTG CTCACCCAGA AACGCTGGTG 6420
AAAGTAAAAG ATGCTGAAGA TCAGTTGGGT GCACGAGTGG GTTACATCGA ACTGGATCTC 6480
AACAGCGGTA AGATCCTTGA GAGTTTTCGC CCCGAAGAAC GTTTTCCAAT GATGAGCACT 6540
TTTAAAGTTC TGCTATGTGG CGCGGTATTA TCCCGTATTG ACGCCGGGCA AGAGCAACTC 6600
GGTCGCCGCA TACACTATTC TCAGAATGAC TTGGTTGAGT ACTCACCAGT CACAGAAAAG 6660
CATCTTACGG ATGGCATGAC AGTAAGAGAA TTATGCAGTG CTGCCATAAC CATGAGTGAT 6720
AACACTGCGG CCAACTTACT TCTGACAACG ATCGGAGGAC CGAAGGAGCT AACCGCTTTT 6780
TTGCACAACA TGGGGGATCA TGTAACTCGC CTTGATCGTT GGGAACCGGA GCTGAATGAA 6840
GCCATACCAA ACGACGAGCG TGACACCACG ATGCCTGTAG CAATGGCAAC AACGTTGCGC 6900
AAACTATTAA CTGGCGAACT ACTTACTCTA GCTTCCCGGC AACAATTAAT AGACTGGATG 6960
GAGGCGGATA AAGTTGCAGG ACCACTTCTG CGCTCGGCCC TTCCGGCTGG CTGGTTTATT 7020
GCTGATAAAT CTGGAGCCGG TGAGCGTGGG TCTCGCGGTA TCATTGCAGC ACTGGGGCCA 7080
GATGGTAAGC CCTCCCGTAT CGTAGTTATC TACACGACGG GGAGTCAGGC AACTATGGAT 7140
GAACGAAATA GACAGATCGC TGAGATAGGT GCCTCACTGA TTAAGCATTG GTAACTGTCA 7200
GACCAAGTTT ACTCATATAT ACTTTAGATT GATTTAAAAC TTCATTTTTA ATTTAAAAGG 7260
ATCTAGGTGA AGATCCTTTT TGATAATCTC ATGACCAAAA TCCCTTAACG TGAGTTTTCG 7320
TTCCACTGAG CGTCAGACCC CGTAGAAAAG ATCAAAGGAT CTTCTTGAGA TCCTTTTTTT 7380
CTGCGCGTAA TCTGCTGCTT GCAAACAAAA AAACCACCGC TACCAGCGGT GGTTTGTTTG 7440
CCGGATCAAG AGCTACCAAC TCTTTTTCCG AAGGTAACTG GCTTCAGCAG AGCGCAGATA 7500
CCAAATACTG TCCTTCTAGT GTAGCCGTAG TTAGGCCACC ACTTCAAGAA CTCTGTAGCA 7560
CCGCCTACAT ACCTCGCTCT GCTAATCCTG TTACCAGTGG CTGCTGCCAG TGGCGATAAG 7620
TCGTGTCTTA CCGGGTTGGA CTCAAGACGA TAGTTACCGG ATAAGGCGCA GCGGTCGGGC 7680
TGAACGGGGG GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA CGACCTACAC CGAACTGAGA 7740
TACCTACAGC GTGAGCTATG AGAAAGCGCC ACGCTTCCCG AAGGGAGAAA GGCGGACAGG 7800
TATCCGGTAA GCGGCAGGGT CGGAACAGGA GAGCGCACGA GGGAGCTTCC AGGGGGAAAC 7860
GCCTGGTATC TTTATAGTCC TGTCGGGTTT CGCCACCTCT GACTTGAGCG TCGATTTTTG 7920
TGATGCTCGT CAGGGGGGCG GAGCCTATGG AAAAACGCCA GCAACGCGGC CTTTTTACGG 7980
TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC ATGTTCTTTC CTGCGTTATC CCCTGATTCT 8040
GTGGATAACC GTATTACCGC CTTTGAGTGA GCTGATACCG CTCGCCGCAG CCGAACGACC 8100
GAGCGCAGCG AGTCAGTGAG CGAGGAAGCG GAAGA 8135
...
Claims (30)
1. the not pathogenic virus of rf is used for the disease that prevention is caused by bovine viral diarrhea virus (BVDV), and the not pathogenic virus of wherein said rf comprises:
Take from the gene or the assortment of genes of BVDV virus, the not pathogenic virus of said rf is expressed the said gene or the assortment of genes on function.
2. the virus of claim 1, the not pathogenic virus of wherein said rf is attenuation.
3. the virus of claim 2, the not pathogenic virus of wherein said rf is selected from ox herpes virus 1 type (BHV-1), the adenovirus of attenuation, the BMV of attenuation, the bovine papilloma virus of attenuation or the pseudorabies virus of attenuation of attenuation.
4. the virus of claim 2, the not pathogenic virus of wherein said rf be attenuation and contain and express the arbitrary combination of following gene: coding gp48, gp25, p14 capsid protein matter, the proteinic gene of p20N-terminal protein enzyme and p125/p80.
5. the virus of claim 3, the non-Causative virus of wherein said rf be attenuation and contain and express the arbitrary combination of following gene: coding gp48, gp25, p14 capsid protein matter, p20N-terminal protein enzyme and p125/p80 protein.
6. the virus of claim 2, wherein said attenuation loses function and causes through making thymidine kinase (tK) gene.
7. the virus of claim 3, wherein said attenuation loses function and causes through making thymidine kinase (tK) gene.
8. the virus of claim 4, wherein said attenuation loses function and causes through making thymidine kinase (tK) gene.
9. the virus of claim 5, wherein said attenuation loses function and causes through making thymidine kinase (tK) gene.
10. the virus of claim 9, the not pathogenic virus of wherein said rf is ox herpes virus 1 type (BHV-1) of attenuation
11. the virus of claim 10, the not pathogenic virus of wherein said rf contain and express the gene of coding bovine viral diarrhea virus (BVDV) glycoprotein gp53.
12. the virus of claim 11 is wherein inserted signal peptide before the gene or the assortment of genes of coding gp53 in said ox herpes virus 1 type (BHV-1).
13. the virus of claim 12, the gene of wherein said coding gp53 is inserted into intestines glycosides kinases (tK) gene locus of deactivation.
14. the virus of claim 13 wherein is used to produce this virus, the gene of expressing on function or the assortment of genes comprise the recombinant plasmid with intact virus DNA, and said plasmid comprises:
A) the BHV-1 genomic DNA fragment that contains thymidine kinase (tK) gene and on thymidine kinase (tK) gene, have disappearance,
B) be inserted into the promotor/polyadenylation signal at thymidine kinase (tK) genetically deficient place,
C) be positioned at the gp53 gene or assortment of genes signal peptide gene sequence before, it all is inserted between promotor and the polyadenylation signal.
15. the virus of claim 14, wherein said signal peptide gene sequence is taken from the signal peptide sequence of any abundant evaluation, as seen in Perlman, and D., et al., any one in 39 signal peptide sequences of fully identifying of J.Mol.Biol.Vol.167pp.391-409 (1983).
16. the virus of claim 15, wherein said signal peptide gene sequence are taken from pseudorabies virus gIII gene (PRV) and/or Trobest (BGH).
17. the virus of claim 16, plasmid wherein is selected from following plasmid,
a)pBHVtKex-1∷BGH/P53；
b)pBHVtKex-1∷gIII/P53；
C) pBHVtKex-3 ∷ BGH/P53 or
d)pBHVtKex-3∷gIII/p53。
18. the virus of claim 17, the wherein said virus that is created in the product of the gene of expressing on the function or the assortment of genes are to be selected from one of following virus: T11-3, T11-6 or T11-8.
19. the virus of claim 18, the gene of wherein said coding gp53 is inserted into thymidine kinase (tK) gene locus of deactivation.
20. claim 11 virus, the gene of wherein said coding gp53 is inserted into thymidine kinase (tK) gene locus of deactivation.
21. the virus of claim 20 wherein is used to produce the gene or the assortment of genes expressed on the function of virus and comprises the recombinant plasmid with intact virus DNA, said plasmid comprises:
A) the BHV-1 genomic DNA fragment that contains thymidine kinase (tK) gene and on thymidine kinase (tK) gene, have disappearance,
B) promotor/polyadenylation signal that inserts at thymidine kinase (tK) genetically deficient place.
The gp53 gene or the assortment of genes of c) between promotor and polyadenylation signal, inserting.
22. the virus of claim 21, wherein said plasmid is made of the plasmid with plasmid pHAS4 feature.
23. the virus of claim 22, wherein said plasmid are pBHVtKex-3 ∷ P53.
24. the virus of claim 23, wherein said virus is selected from down one of influenza virus: T2-3#3 or T2-2#5.
25. a vaccine that is used for the disease that prevention causes by bovine viral diarrhea virus (BDVD) comprises medicinal virus and a kind of carrier of going up significant quantity of claim 1.
26. the vaccine that is used for the disease that prevention causes by bovine viral diarrhea virus (BD-VD) that requires in the claim 25, medicinal virus and a kind of carrier of going up significant quantity that comprises claim 1, said carrier comprises any physiological buffer medium, and the pH that promptly contains the serum of about 2.5 to 15% nonreactive BHV antibody is approximately 7.0 to 7.4 buffer medium.
27. an immune animal resists the method for the infectious diseases that is caused by bovine viral diarrhea virus (BDVD), comprises to animal to use the medicinal virus that goes up the claim 1 of significant quantity.
28. a method for preparing the virus of claim 1 comprises:
A) separate the gene or the assortment of genes of expressing on the function that produces BVDV,
B) the said gene or the assortment of genes are inserted in the not pathogenic virus of rf,
C) live virus of the product of the said gene of expression or the assortment of genes on the selection function.
29. the method for virus of preparation claim 11 wherein is used to produce the gene of expressing on this viral function or the assortment of genes and produces to comprise the method that reorganization has the plasmid of intact virus DNA, said plasmid comprises:
A) contain the BHV-1 genomic DNA fragment that thymidine kinase (tK) gene and thymidine kinase (tK) gene have disappearance,
B) promotor/polyadenylation signal is inserted into the disappearance place of thymidine kinase (tK) gene of said plasmid,
C) between promotor and polyadenylation signal, insert the gp53 gene or the assortment of genes,
D) with said plasmid transfection cell to produce recombinant virus, this virus contains the functioning gene or the assortment of genes that is inserted in the live virus, this live virus does not cause immunosuppression and expresses the said functioning gene or the assortment of genes in common host.
30. the method for virus of preparation claim 12 wherein is used to produce the gene of expressing on the function of virus or the assortment of genes by comprising that the method that reorganization has the plasmid of intact virus DNA produces, said plasmid comprises:
A) the BHV-1 genomic DNA fragment that contains thymidine kinase (tK) gene and on thymidine kinase gene, have disappearance,
B) promotor/polyadenylation signal is inserted into thymidine kinase (tK) the genetically deficient place of said plasmid,
C) the gp53 gene or the assortment of genes that the front is had a signal peptide gene sequence is inserted between promotor and the polyadenylation signal,
D) contain the said functioning gene that is inserted in the live virus or the recombinant virus of the assortment of genes with said plasmid transfection cell with generation, this live virus does not cause immunosuppression and expresses the said functioning gene or the assortment of genes in common host.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US14781093A | 1993-11-05 | 1993-11-05 | |
| US147,810 | 1993-11-05 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1134175A true CN1134175A (en) | 1996-10-23 |
Family
ID=22522990
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN94193978A Pending CN1134175A (en) | 1993-11-05 | 1994-10-31 | Viral vector with bovine viral diarrhea virus (BVDV) antigens |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0725831A1 (en) |
| JP (1) | JPH09504435A (en) |
| KR (1) | KR960705944A (en) |
| CN (1) | CN1134175A (en) |
| AU (1) | AU688819B2 (en) |
| CA (1) | CA2172815A1 (en) |
| MX (1) | MXPA94008605A (en) |
| NZ (1) | NZ276234A (en) |
| WO (1) | WO1995012682A2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113913461A (en) * | 2021-11-15 | 2022-01-11 | 贵州大学 | Construction method of bovine viral diarrhea E0-E2 gene recombinant adenovirus vaccine |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6060457A (en) * | 1996-06-20 | 2000-05-09 | Universite De Montreal | DNA plasmid vaccine for immunization of animals against BVDV |
| EP1026252A1 (en) * | 1999-02-02 | 2000-08-09 | Akzo Nobel N.V. | Synthetic gene of bovine viral diarrhoea virus |
| EP1104676A1 (en) * | 1999-11-30 | 2001-06-06 | Boehringer Ingelheim Vetmedica Gmbh | Safe attenuated bovine viral diarrhea viruses for use in pregnant cows |
| KR100331176B1 (en) * | 1999-12-15 | 2002-04-06 | 대한민국(관리청:특허청장, 승계청:국립수의과학검역원장) | A diagnostic method of bovine viral diarrhea using recombination protein as an antigen |
| EP1170367A1 (en) | 2000-06-27 | 2002-01-09 | Bayer Ag | BVDV virus-like particles |
| PL216541B1 (en) | 2002-08-26 | 2014-04-30 | Pfizer Prod Inc | Vaccine for respiratory and reproductive system infections in cattle |
| US20050281832A1 (en) * | 2003-12-05 | 2005-12-22 | Campbell Robert L | Methods of enhancing immune response in the intradermal compartment and compounds useful thereof |
| KR101876535B1 (en) * | 2012-06-14 | 2018-07-09 | 베트올 (주) | Antibody for detecting of bovine viral diarrhea virus(bvdv), bvdv antigen detecting method and test kit using thereof |
| AU2014234982A1 (en) | 2013-03-15 | 2015-09-24 | Zoetis Services Llc | Cross-protection of bovines against B. trehalosi infection by a multi-valent vaccine |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1237668A (en) * | 1983-02-25 | 1988-06-07 | Novagene Inc. | Thymidine kinase-negative temperature resistant bovine herpes-virus-1 mutant as vaccine |
| GB8818415D0 (en) * | 1988-08-03 | 1988-09-07 | Animal Health Inst | Vaccine |
| FR2693472B1 (en) * | 1992-06-26 | 1994-12-23 | Rhone Merieux | Mutants of the infectious bovine rhinotracheitis virus, deleted in one of the genes of minor glycoproteins, vaccines prepared from these strains, production methods and methods of use. |
| SE9002060D0 (en) * | 1990-06-08 | 1990-06-08 | Statens Veterinaermedicinska A | A METHOD OF DETECTING AN INFECTION CAUSED BY A SPECIFIC TYPE OF VIRUSES, PRIMERS, TESTS AND A TEST KIT |
-
1994
- 1994-10-31 NZ NZ276234A patent/NZ276234A/en unknown
- 1994-10-31 AU AU10423/95A patent/AU688819B2/en not_active Ceased
- 1994-10-31 CA CA002172815A patent/CA2172815A1/en not_active Abandoned
- 1994-10-31 CN CN94193978A patent/CN1134175A/en active Pending
- 1994-10-31 EP EP95901037A patent/EP0725831A1/en not_active Withdrawn
- 1994-10-31 KR KR1019960702343A patent/KR960705944A/en not_active Application Discontinuation
- 1994-10-31 WO PCT/US1994/012198 patent/WO1995012682A2/en not_active Application Discontinuation
- 1994-10-31 JP JP7513263A patent/JPH09504435A/en active Pending
- 1994-11-07 MX MXPA94008605A patent/MXPA94008605A/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113913461A (en) * | 2021-11-15 | 2022-01-11 | 贵州大学 | Construction method of bovine viral diarrhea E0-E2 gene recombinant adenovirus vaccine |
Also Published As
| Publication number | Publication date |
|---|---|
| MXPA94008605A (en) | 2004-11-11 |
| WO1995012682A3 (en) | 1995-07-06 |
| NZ276234A (en) | 1998-01-26 |
| AU688819B2 (en) | 1998-03-19 |
| AU1042395A (en) | 1995-05-23 |
| EP0725831A1 (en) | 1996-08-14 |
| CA2172815A1 (en) | 1995-05-11 |
| KR960705944A (en) | 1996-11-08 |
| WO1995012682A2 (en) | 1995-05-11 |
| JPH09504435A (en) | 1997-05-06 |
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