CN104640991B - Recombinate type bovine herpes virus (BoHV 1) vaccine carrier of low toxicity power 1 - Google Patents

Recombinate type bovine herpes virus (BoHV 1) vaccine carrier of low toxicity power 1 Download PDF

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CN104640991B
CN104640991B CN201280074502.8A CN201280074502A CN104640991B CN 104640991 B CN104640991 B CN 104640991B CN 201280074502 A CN201280074502 A CN 201280074502A CN 104640991 B CN104640991 B CN 104640991B
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bohv
antigen
vaccine
virus
bvdv
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CN104640991A (en
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蒂莫西·约翰·马奥尼
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STATE OF QUEENSLAND ACTING THROUGH Department OF AGRICULTURE FISHERIES AND FORESTRY
Meat and Livestock Autralia Ltd
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STATE OF QUEENSLAND ACTING THROUGH Department OF AGRICULTURE FISHERIES AND FORESTRY
Meat and Livestock Autralia Ltd
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Priority claimed from PCT/AU2012/000804 external-priority patent/WO2013003904A1/en
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Abstract

Present disclosure instructs vaccine inoculation and the disease control field for typically belonging to bovid.One or more of cattle disease substances cattle disease substance as related to cattle respiratory disease syndrome is effectively controlled there is provided 1 type bovine herpes virus (BoHV 1) vaccine carrier of restructuring, such as bovine viral diarrhea virus (BVDV), and which improve thus caused disease states.Also include being used for managing the limited or scheme of bovid in groups herein.

Description

Recombinate type bovine herpes virus (BoHV-1) vaccine carrier of low toxicity power 1
Request for data
The application be relevant to and require to submit on July 5th, 2011 entitled " A vaccine " Australia is interim special Profit application No.2011902660 priority, the full content of the temporary patent application is incorporated herein by reference.
Technical field
Present disclosure teaching typically belongs to vaccine inoculation and the disease control neck of bovid (bovine animals) Domain.One or more of cattle disease substances (such as cattle respiratory disease syndrome (bovine is effectively controlled there is provided vaccine carrier Respiratory disease complex) related those cattle disease substances) and improve thus caused disease states. Also include being used for managing the limited or scheme of bovid in groups herein.
Background technology
The publication documents catalogue details alphabet sequence that author quotes in this manual is summarized in the ending of specification Place.
It is not to be not construed as being to recognize that yet or point out in any form to the reference of any prior art in this specification The prior art constitutes a part for any national common knowledge.
Cattle respiratory disease syndrome (BRDC) infects for Australia feeding ox (feedlot cattle) is most significant Property disease.BRDC is because the incidence of disease, the death rate, the loss of feed resource, medicine purchase, increased feeding time and related work Power cost and cause economic loss.The BRDC cause of disease is complicated, and reason is at least four virus types and three kinds of bacterium classes and made The environmental condition of animal susceptible disease.
The four kind viruses related to BRDC are 1 type bovine herpes virus (bovine herpesvirus 1, BoHV-1), cattle disease Viral diarrhea virus (bovine viral diarrhea virus, BVDV or rinderpest virus (bovine pestivirus)), 3 Type bovine parainfluenza virus (bovine parainfluenza 3) and Bovine Respiratory Syncytial virus (bovine respiratory syncytial virus).Serological research is it has been shown that all these viruses can all infect Australia feeding ox.In BRDC Also relate to pasteurella multocida (Pasteurella mutocida), Mannheimia haemolytica (Manhiemia ) and Haemophilus somnus (Haemophilus somnus) these three bacteria cultures haemolytica.
In North America and Europe, the disease as caused by BoHV-1 is controlled using both live vaccine and inactivated vaccine.These Vaccine is based on the BoHV-1 genotype different from BoHV-1 genotype seen by Australia.North America and Europe BoHV-1 strains Generally it is included into subgroup 1.1 and Australian strain composition subgroup 1.2.Compared with BoHV-1.2 viruses, BoHV-1.1 viruses are led Cause more serious clinical disease.Still it is unaware of the accurate molecular mechanism of the phenotypic difference.
BoHV-1 is the virus of herpesviral (Herpesviridae) section, and it causes a variety of diseases of ox in world wide, Including rhinotracheitis, vaginitis, balanoposthitis, miscarriage, conjunctivitis and enteritis.The influence of BoHV-1 or shipping fever because Element.It is contacted by property, artificial insemination and infection through air (aerosol transmission) are propagated.Such as other herpesvirals Equally, BoHV-1 causes lifelong latent infection and releasing virus.Sciatic nerve and trigeminal neuralgia are latency site.
Breathing problem is commonly known as infectious bovine rhinotracheitis as caused by BoHV-1.Symptom includes heating, nose stream Go out thing (discharge from the nose), cough, expiratory dyspnea and loss of appetite.Ulcer generally occurs in mouth and nose.Extremely The rate of dying can reach 10%.Genital diseases cause cow contagious ecthyma vulvovaginitis and bull infectiousness glans penis foreskin It is scorching.Symptom include heating, depression, loss of appetite, urodynamic, the swelling of cow vaginal orifice and with warts and effluent and with Pain when bull property is contacted.In both cases, lesion generally disappeared in two weeks.After infection one to three month may hair Raw miscarriage and stillbirth.BoHV-1 also results in the systemic disease newly calved, is characterized as enteritis and death.
Similarly, BVDV is the disease for reducing the reproductive capacity of ox and improving its death rate.It is by from flaviviridae (Flaviviridae) pestivirus causes.Pestivirus can set up lasting infection in gestation.Persistent infection pestivirus Generally it is difficult to be noticeable.BVDV also usually carries out non-homogeneous RNA restructuring, and this, which causes to occur in that, makes host lethal only in heredity Special virus.
Mucosal erosion and the clinical sign (clinical sign) of the diarrhoea occurred with the bovine viral diarrhoea of acute form Those infected animals are had a significant impact, but it is bigger by the cost of the animal of persistent infection.Generally, such animal is not Its genetic potential can be realized, it shows increased weight reduction, disease susceptibility increase and fertility-rate reduction.They discharge disease Poison, so as to cause nonimmune animal in colony to lose fecundity.
Cow (45 to 125 days gestation) exposed to the BVDV variants of cytopathogenic effect generally will miscarriage.It is exposed to earlier Any variant causes body early embryo dead.Exposure number of days can cause inborn defect (such as vision between pregnant 125 to 175 days Defect and hydrocephalus) and exposure generally will cause calf to be immunized completely at birth more than 175 days.
Therefore, because BRDC seriousness and its significantly affecting, it is necessary to improve vaccine inoculation on animal husbandry.With inactivation disease Poison is compared, and attenuated virus provides more preferable protection, and reason is that attenuated virus is anti-to the more viruses of immune system presentation of host It is former.Another important advantage of attenuated virus is that it is applied to intranasal potentiality, i.e., the first of wild-type virus numerous after infection Grow position.
It has long been recognized that BVDV antigenic variability makes it difficult to determine which kind of virus to carry out vaccine for Inoculation.BVDV specificity vaccine inoculations can be carried out using two methods.A kind of is to preventing the neutralization of target virus infected cell The induction of antibody.Second is that induction targeting destroys the cell-mediated immunity (CMI) for being infected cell, thus reduces virus Infection effect.BVDV main neutralizing epitope is structural glycoprotein, and due to Immune Selection, the variability of these protein It is maximum.Therefore, vaccine of the design based on glycoprotein needs to include most common antigenic type.BVDV non-structural protein is generally more Plus conservative, the change that the reason protein sequence that has been its certain enzyme function restriction may occur.
For suitable BRDC item controlleds, it is necessary to effective and safe with what can mutually be distinguished with wild-type virus Vaccine.Before this missing glycoprotein is built as using the BoHV-1 vaccines developed and/or comprising thymidine kinase deletion mutant. These vaccines there is also some problems that such as thymidine kinase gene participation virus replication and duplication are fewer to be caused to protect smaller, Reason is that the level for the glycoprotein for participating in generation humoral immunity is relatively low.
Exploitation is needed to control the improved and more effective vaccine of BRDC and special pathogen related to this.
The content of the invention
Nucleotides and amino acid sequence are quoted by sequence identifier (SEQ ID NO).SEQ ID NO are with numeral correspondence sequence Column identifier<400>1(SEQ ID NO:1)、<400>2(SEQ ID NO:2) etc..Table 1 provides sequence identifier overview. Sequence table is provided after claims.
Cattle respiratory disease syndrome (BRDC) is that bovid (is especially raised in constrained environment such as feed lot and breast Those in products factory) a kind of notable disease risks.It can rapidly be propagated by the related pathogenic infections of BRDC and can cause to show The incidence of disease, the death rate and the reproductive capacity of reduction of work.The improved vaccine carrier of teachings herein, it is comprising being modified to take Carry coding cattle disease substance in one or more antigens inhereditary material come from BoHV-1 low toxicities power (virulence) strain Genome.
Therefore, this paper vaccine can be directed at least one antigen from cattle disease substance, and the vaccine is included from low Virulence BoHV-1 1 type bovine herpes virus (BoHV-1) genome, its have with BoHV-1 it is heterologous, insertion two convergence (converging) inhereditary material of at least one antigen of coding between BoHV-1 genes, wherein the insertion is not cut substantially The expression of BoHV-1 genes.
The vaccine is being stimulated to BoHV-1 and other cattle disease substance (such as BVDV, mycoplasma (Mycoplasma), bars This moral Salmonella (Pasteurella), Mannheimia (Manhiemia) and haemophilic bacterium (Haemophilus)) related antigen is immune The ability of the aspect of response is multivalence.The example of BVDV antigens includes glycoprotein E 0 and E2.
In one embodiment, Heterologous genetic material is introduced using induction type recombination system (such as GET restructuring).
What is instructed herein is that bovid is inoculated with least one antigen from cattle disease substance on the other hand The method of vaccine, methods described includes applying humoral immunity inductivity or cell-mediated immunity inductivity effective dose to bovid The BoHV-1 genomes from low toxicity power BoHV-1, the BoHV-1 genomes have with BoHV-1 it is heterologous, insertion two meetings The inhereditary material of at least one antigen of coding between poly- BoHV-1 genes, wherein the insertion does not cut BoHV-1 genes substantially Expression.
Methods herein can prepare the vaccine at least one antigen from cattle disease substance, and methods described includes：
(i) the BoHV-1 genomes from low toxicity power BoHV-1 are incorporated to bacterial artificial chromosome (BAC) carrier to be formed BoHV-1 precoded vectors (pre-vector) BAC constructs；
(ii) inhereditary material for encoding at least one antigen is inserted into the BoHV-1 via induction type recombination system to preload In body BAC constructs BoHV-1-BAC (rBoHV-1-BAC) carrier is recombinated to produce；
(iii) the rBoHV-1-BAC carriers are converted or expanded in bacterial host；And
(iv) purified from the bacterial host and separate the rBoHV-1-BAC carriers and be configured to the carrier Vaccine combination.
Additionally provide for cattle respiratory disease syndrome (BRDC) to the method for ox vaccine inoculation, methods described includes pair Ox applies the 1 type bovine herpes virus from low toxicity power BoHV-1 of humoral immunity inductivity or cell-mediated immunity inductivity effective dose Malicious (BoHV-1) genome, the BoHV-1 genomes have and BoHV-1 is heterologous, insertion two is assembled between BoHV-1 genes At least one antigen of coding inhereditary material, wherein described insert the expression for not cutting BoHV-1 genes substantially.
Present disclosure includes the BoHV-1 genomes from low toxicity power BoHV-1 and ox vaccine inoculation is directed in manufacture Purposes in the medicine of cattle disease substance, the BoHV-1 genomes have with BoHV-1 it is heterologous, insertion two convergence BoHV-1 bases Therefore the inhereditary material of at least one antigen of coding between, wherein described insert the expression for not cutting BoHV-1 genes substantially.
Methods herein can prepare the vaccine at least one antigen from cattle disease substance, and methods described includes：
(i) the BoHV-1 genomes from low toxicity power BoHV-1 are incorporated to bacterial artificial chromosome (BAC) carrier to be formed BoHV-1 precoded vector BAC constructs；
(ii) inhereditary material for encoding at least one antigen is inserted into the BoHV-1 via induction type recombination system to preload In body BAC constructs BoHV-1-BAC (rBoHV-1-BAC) carrier is recombinated to produce；
(iii) the rBoHV-1-BAC carriers are converted or expanded in bacterial host；And
(iv) purified from the bacterial host and separate the rBoHV-1-BAC carriers and be configured to the carrier Vaccine combination.
BoHV-1 genomes come from low toxicity power BoHV-1, and when it is expressed, generation can produce immune response to it and resist Original, the BoHV-1 genomes also include with BoHV-1 it is heterologous, insertion two assemble BoHV-1 genes between codings at least one The inhereditary material of other antigens is planted, wherein the insertion does not cut the expression of BoHV-1 genes substantially and wherein heterologous antigen is lured Lead immune response.
Therefore, what is instructed herein is the vaccine carrier for including the BoHV-1 genomes from low toxicity power BoHV-1, described The something lost that BoHV-1 genomes have with BoHV-1 is heterologous, at least one antigen of coding between BoHV-1 genes is assembled in insertion two Material is passed, wherein described insert the expression for not cutting BoHV-1 genes substantially.
Polyvaccine can be realized herein, it is included：
(1) the first valency of the BoHV-1 genomes from low toxicity power BoHV-1 is included；And
(2) the second valency of the inhereditary material for encoding at least one antigen is included, its heterologous with BoHV-1, two convergence of insertion Between BoHV-1 genes, wherein described insert the expression for not cutting BoHV-1 genes substantially；
Wherein described first valency and the second valency, which produce two or more and produce immune response to it in ox host, to be resisted It is former.
This paper BoHV-1 vaccine carriers also include the BoHV-1 genomes from BoHV-1 bacterial strains V155, and it, which has, comes From the Heterologous genetic material of at least one antigen of coding of cattle disease substance, what the inhereditary material was inserted on BoHV-1 genomes Site is selected from BoHV-1 reference sequences GenBank registration numbers AJ004801 nucleotides 16600 to 16700；22400 to 22500； 40700 to 40800；58000 to 59000；67000 to 68000；74000 to 76000；84000 to 85000；90000 to 91000 And the function equivalent site in 96000 to 97000 or other BoHV-1.For example, reference table 2.
It is taught that pharmaceutical composition, treatment and vaccination protocols, present disclosure is limited or in groups for managing The business method of bovid.
Table 2 provides the site overview that BoHV-1 genomes are inserted between meeting pcl gene.Sequence coordinate refers to deposit GenBank registration numbers AJ004801 BoHV-1 reference sequences.
Table 1
Sequence identifier overview
Serial ID NO： Description
1 The nucleotide sequence of the primers of Tkleft 5 '
2 The nucleotide sequence of the primers of Tkleft 3 '
3 The nucleotide sequence of the primers of Tkright 5 '
4 The nucleotide sequence of the primers of Tkright 3 '
5 The nucleotide sequence of ChloramF primers
6 The nucleotide sequence of ChloramR primers
7 The nucleotide sequence of gE-KanF primers
8 The nucleotide sequence of gE-KanR primers
9 The nucleotide sequence of BHV1.3 primers
10 The nucleotide sequence of BHV1.6 primers
11 The nucleotide sequence of KanR fwd primers
12 The nucleotide sequence of KanR rev primers
Table 2
Insertion point between the integrator gene of 1 type bovine herpes virus (BoHV-1) genome1List
Insertion point Starting Terminate Annotation
Insertion point 1 16600 16612 Integrator gene UL46＆UL44
Insertion point 2 22449 22493 Integrator gene UL41＆UL40
Insertion point 3 40734 40768 Integrator gene UL36＆UL35
Insertion point 4 58229 58563 Integrator gene UL27＆UL26
Insertion point 5 67037 67091 Integrator gene UL22＆UL21
Insertion point 6 74994 75041 Integrator gene UL19＆UL15
Insertion point 7 84496 84528 Integrator gene UL11＆UL10
Insertion point 8 90732 90760 Integrator gene UL8＆UL7
Insertion point 9 96870 96882 Integrator gene UL4＆UL3.6
1Sequence coordinate refers to BoHV-1 reference sequences or its equivalent with GenBank registration numbers AJ004801
Brief description of the drawings
When Figure 1A to Fig. 1 C is illustrated after infection 24 hours, bovine viral is come from by the type bovine herpes virus of parent 1 or carrying The viral yield of a variety of cell lines from mammal of the recombinant bovine herpesvirus infection of the glycoprotein E 2 of diarrhea virus Comparison.(A) cell in primate source；(B) Niu Laiyuan cell；(C) cell in rabbit and small ruminant source.Pass through reality When PCR amplifications determine the yield (carry out three times) of virus.
Detailed description of the invention
Through this specification, unless the context otherwise requires, otherwise word "comprising" or its version should be understood that for example " comprising " or " containing " mean include the key element or integer or method and step or key element or the group of integer or method and step, And it is not excluded for the group of any key element or integer or method and step or key element or integer or method and step.
In this specification using countless measure word modify noun include plural number for the use of, unless substantially separately had in context It is bright.Thus, for example, referring to that " virus " includes a kind of virus and two or more viruses；Refer to that " antigen " includes a kind of anti- Former and two or more antigens；Refer to that " disclosure " includes one or more aspects taught herein.
Disclosure teaches the recombinant vaccine vector of the BoHV-1 forms from viral low virulence strain.In a reality Apply in scheme, the low virulence strain refers to BoHV-1V155 (Snowden (1964) Australian Veterinary Journal 40:277-288).By the recombinant vaccine vector be used as supporting agent (vehicle) come express with from cattle disease substance Protein heterologous BoHV-1, immune response is sought to the protein.The genome portion of BoHV-1 carriers in itself can also be expressed Induce the protein of anti-BoHV-1 immune responses.In one embodiment, the immune response of bovid is considered as protection Property immune response, reason is the immune response targeting protein or produced by pathogen, and this advantageously reduces disease Infection, field planting (colonization) and/or symptom and/or pathogen propagation and/or infection result, for example the incidence of disease or The death rate.The immune response can be humoral immune response and/or cell-mediated immune response.
In one embodiment, BoHV-1 carriers are carried out genetic manipulation to insert the gene from cattle disease substance Between meeting pcl gene on BoHV-1 genomes.In one embodiment, the insertion does not make BoHV-1 two sides substantially The expression of wing gene or any other gene in BoHV-1 genomes is lowered.Carried in the cell infection recombinant vaccine of bovid After body, pathogen gene carries out expression to form proteantigen and trigger for one or more of pathogen antigens Immune response.As described above, BoHV-1 carriers sheet provides target as the immunostimulation for BoHV-1.Therefore, this public affairs The realization of teachings dual vaccine method is opened, methods described is based on stimulation for BoHV-1 immune response and for being lost Pass the immune response for transforming the heterologous protein by BoHV-1 vaccine carrier expression as.
Therefore, this paper vaccine can be directed at least one antigen from cattle disease substance, and the vaccine is included from low Virulence BoHV-1 BoHV-1 genomes, the BoHV-1 genomes have with BoHV-1 it is heterologous, insertion two convergence BoHV-1 The inhereditary material of at least one antigen of coding between gene, wherein described insert the expression for not cutting BoHV-1 genes substantially.
The vaccine can expressing heterologous antigen to promote the stimulation for the antigen immune response.In addition, BoHV-1 carriers Itself it can promote the immune response to BoHV-1 protein.Improved using low toxicity power BoHV-1 rather than inactivation strain or attenuation strain Its infection, the ability for replicating and producing non-pathogen infection and should for BoHV-1 and the effectively immune of any heterologous antigen Answer.
The polyvaccine for two or more antigens from cattle disease substance is taught herein, and the vaccine is comprising next From low toxicity power BoHV-1 BoHV-1 genomes, the antigen that immune response is produced to it, the BoHV-1 are produced when it is expressed The hereditary thing that genome is also included with BoHV-1 is heterologous, at least one antigen of coding between BoHV-1 genes is assembled in insertion two Matter, wherein described insert the expression for not cutting BoHV-1 genes substantially and wherein described heterologous antigen induction immune response.
Term " multivalence " and " multiple valencys " are used interchangeably to describe this paper this aspect.
Vaccine teaching herein is also regarded as vaccine carrier.
Therefore, this paper's is vaccine carrier on the other hand, and it includes the BoHV-1 genomes from low toxicity power BoHV-1, The BoHV-1 genomes have and BoHV-1 is heterologous, two at least one antigens of the codings assembled between BoHV-1 genes of insertion Inhereditary material, wherein described insert does not cut the expression of BoHV-1 genes substantially.
This paper another aspect is polyvaccine carrier, and it is included：
(1) the first valency of the BoHV-1 genomes from low toxicity power BoHV-1 is included；And
(2) the second valency of the inhereditary material for encoding at least one antigen is included, the inhereditary material is heterologous, slotting with BoHV-1 Enter two to assemble between BoHV-1 genes, wherein described insert does not cut the expression of BoHV-1 genes substantially；
Wherein described first valency and the second valency, which produce two or more and produce immune response to it in ox host, to be resisted It is former.
Term " basic " on downward means that expression does not cut or expressed only slight drop." small " means from function From the point of view of angle, any change of expression can not adversely influence the function of virus.
As described above, " immune response " can be humoral immune response and/or cell-mediated immune response.
This paper vaccine can be treated or prevented in cattle respiratory disease especially prevalent in the disease of ox in batch or in groups Syndrome (BRDC)." ox (lot cattle) in batch " include be used for feed, raise or dairy husbandry purpose limited ox.BRDC is Multi-factor disease.Generally, bovid is infected with BoHV-1, BVDV, 3 type bovine parainfluenza viruses and/or Bovine Respiratory Syncytial One or more in virus.This typically results in the second virus or microorganism infection and causes the illness of such as pneumonia.
The microbial pathogens covered herein includes mycoplasma (Mycoplasma) category, salmonella (Salmonella) Category, pasteurella (Pasteurella) category, Mannheimia (Manhiemia) category and haemophilic bacterium (Haemophilus) category, example Such as Mycoplasma bovis (Mycoplasma bovis), multocida (Pasteurella multocida), hemolytic Man Bacterium (Manhiemia haemolytica) and Haemophilus somnus (Haemophilus somnus).Can be by from any or all The inhereditary material of the coding for antigens of these or other bacterium is used in BoHV-1 vaccine carriers.BVDV antigens include the He of glycoprotein E 0 E2。
Therefore, present disclosure is related to connects at least one antigen from cattle disease substance to bovid progress vaccine Kind method, methods described includes applying humoral immunity inductivity or cell-mediated immunity inductivity effective dose to bovid BoHV-1 genomes from low toxicity power BoHV-1, the BoHV-1 genomes have with BoHV-1 it is heterologous, insertion two convergence The inhereditary material of at least one antigen of coding between BoHV-1 genes, wherein the insertion does not cut BoHV-1 genes substantially Expression.
Disclosure teaches for for methods of the BRDC to ox vaccine inoculation, methods described to include applying body to ox The BoHV-1 genomes from low toxicity power BoHV-1 of liquid immune induction or cell-mediated immunity inductivity effective dose, it is described The something lost that BoHV-1 genomes have with BoHV-1 is heterologous, at least one antigen of coding between BoHV-1 genes is assembled in insertion two Material is passed, wherein described insert the expression for not cutting BoHV-1 genes substantially.
BoHV-1 vaccine carriers genetic manipulation is typically carried out with inserting heterologous nucleic acids material by induction type recombination system. In one embodiment, induction type recombination system is GET restructuring, and it can be big using recE and recT transient expression Homologous recombination is (referring to the .Nucleic Acids such as Orford Research 28 in enterobacteria (Escherichia coli) (18):e84；The such as Mahoney (2002) Journal of Virology 76 (13):6660-6668；The such as Narayanan (1999)Gene therapy 6:442-447；The such as Schumacher (2000) Journal of Virology 74:11088- 11098)。
In one embodiment, the Heterologous genetic material is inserted into the poly- adenosine of two meeting pcl genes in following site Between polyadenylation signal, the site be selected from BoHV-1 reference sequences GenBank registration numbers AJ004801 16600 to 16700 and Function equivalent site in 22400 to 22500 or other BoHV-1.In one embodiment, by the Heterologous genetic material Insertion is selected from the site between nucleotides 16600 to 16700 and 22400 to 22493, and the site is based on BoHV-1 reference sequences The sequence coordinate (sequence coordinate) of GenBank registration numbers AJ004801 or its equivalent.Refer to " 16600 to 16700 " include：16600、16601、16602、16603、16604、16605、16606、16607、16608、16609、16610、 16611、16612、16613、16614、16615、16616、16617、16618、16619、16620、16621、16622、 16623、16624、16625、16626、16627、16628、16629、16630、16631、16632、16633、16634、 16635、16636、16637、16638、16639、16640、16641、16642、16643、16644、16645、16646、 16647、16648、16649、16650、16651、16652、16653、16654、16655、16656、16657、16658、 16659、16660、16661、16662、16663、16664、16665、16667、16668、16669、16670、16671、 16672、16673、16674、16675、16676、16677、16678、16679、16680、16681、16682、16683、 16684、16685、16686、16687、16688、16689、16690、16691、16692、16693、16694、16695、 16696th, 16697,16698,16699 and 16700.Refer to that " 22400 to 22500 " include：22400、22401、22402、 22403、22404、22405、22406、22407、22408、22409、22410、22411、22412、22413、22414、 22415、22416、22417、22418、22419、22420、22421、22422、22423、22424、22425、22426、 22427、22428、22429、22430、22431、22432、22433、22434、22435、22436、22437、22438、 22439、22440、22441、22442、22443、22444、22445、22446、22447、22448、22449、22450、 22451、22452、22453、22454、22455、22456、22457、22458、22459、22460、22461、22462、 22463、22464、22465、22466、22467、22468、22469、22470、22471、22472、22473、22474、 22475、22476、22477、22478、22479、22480、22481、22482、22483、22484、22485、22486、 22487、22488、22489、22490、22491、22492、22493、22494、22495、22496、22497、22498、22499 With 22500.
Other sites include 40700 to 40800 scope；It covers site：40,700、40,701、40,702、40, 703、40,704、40,705、40,706、40,707、40,708、40,709、40,710、40,711、40,712、40,713、40, 714、40,715、40,716、40,717、40,718、40,719、40,720、40,721、40,722、40,723、40,724、40, 725、40,726、40,727、40,728、40,729、40,730、40,731、40,732、40,733、40,734、40,735、40, 736、40,737、40,738、40,739、40,740、40,741、40,742、40,743、40,744、40,745、40,746、40, 747、40,748、40,749、40,750、40,751、40,752、40,753、40,754、40,755、40,756、40,757、40, 758、40,759、40,760、40,761、40,762、40,763、40,764、40,765、40,766、40,767、40,768、40, 769、40,770、40,771、40,772、40,773、40,774、40,775、40,776、40,777、40,778、40,779、40, 780、40,781、40,782、40,783、40,784、40,785、40,786、40,787、40,788、40,789、40,790、40, 791、40,792、40,793、40,794、40,795、40,796、40,797、40,798、40,799、40,800；58,000 to 59,000 include：58,001、58,002、58,003、58,004、58,005、58,006、58,007、58,008、58,009、58, 010、58,011、58,012、58,013、58,014、58,015、58,016、58,017、58,018、58,019、58,020、58, 021、58,022、58,023、58,024、58,025、58,026、58,027、58,028、58,029、58,030、58,031、58, 032、58,033、58,034、58,035、58,036、58,037、58,038、58,039、58,040、58,041、58,042、58, 043、58,044、58,045、58,046、58,047、58,048、58,049、58,050、58,051、58,052、58,053、58, 054、58,055、58,056、58,057、58,058、58,059、58,060、58,061、58,062、58,063、58,064、58, 065、58,066、58,067、58,068、58,069、58,070、58,071、58,072、58,073、58,074、58,075、58, 076、58,077、58,078、58,079、58,080、58,081、58,082、58,083、58,084、58,085、58,086、58, 087、58,088、58,089、58,090、58,091、58,092、58,093、58,094、58,095、58,096、58,097、58, 098、58,099、58,100、58,101、58,102、58,103、58,104、58,105、58,106、58,107、58,110、58, 111、58,112、58,113、58,114、58,115、58,116、58,117、58,118、58,119、58,120、58,121、58, 122、58,123、58,124、58,125、58,126、58,127、58,128、58,129、58,130、58,131、58,132、58, 133、58,134、58,135、58,136、58,137、58,138、58,139、58,140、58,141、58,142、58,143、58, 144、58,145、58,146、58,147、58,148、58,149、58,150、58,151、58,152、58,153、58,154、58, 155、58,156、58,157、58,158、58,159、58,160、58,161、58,162、58,163、58,164、58,165、58, 166、58,167、58,168、58,169、58,170、58,171、58,172、58,173、58,174、58,175、58,176、58, 177、58,178、58,179、58,180、58,181、58,182、58,183、58,184、58,185、58,186、58,187、58, 188、58,189、58,190、58,191、58,192、58,193、58,194、58,195、58,196、58,197、58,198、58, 199、58,200、58,201、58,202、58,203、58,204、58,205、58,206、58,207、58,208、58,209、58, 210、58,211、58,212、58,213、58,214、58,215、58,216、58,217、58,218、58,219、58,220、58, 221、58,222、58,223、58,224、58,225、58,226、58,227、58,228、58,229、58,230、58,231、58, 232、58,233、58,234、58,235、58,236、58,237、58,238、58,239、58,240、58,241、58,242、58, 243、58,244、58,245、58,246、58,247、58,248、58,249、58,250、58,251、58,252、58,253、58, 254、58,255、58,256、58,257、58,258、58,259、58,260、58,261、58,262、58,263、58,264、58, 265、58,266、58,267、58,268、58,269、58,270、58,271、58,272、58,273、58,274、58,275、58, 276、58,277、58,278、58,279、58,280、58,281、58,282、58,283、58,284、58,285、58,286、58, 287、58,288、58,289、58,290、58,291、58,292、58,293、58,294、58,295、58,296、58,297、58, 298、58,299、58,300、58,301、58,302、58,303、58,304、58,305、58,306、58,307、58,308、58, 309、58,310、58,311、58,312、58,313、58,314、58,315、58,316、58,317、58,318、58,319、58, 320、58,321、58,322、58,323、58,324、58,325、58,326、58,327、58,328、58,329、58,330、58, 331、58,332、58,333、58,334、58,335、58,336、58,337、58,338、58,339、58,340、58,341、58, 342、58,343、58,344、58,345、58,346、58,347、58,348、58,349、58,350、58,351、58,352、58, 353、58,354、58,355、58,356、58,357、58,358、58,359、58,360、58,361、58,362、58,363、58, 364、58,365、58,366、58,367、58,368、58,369、58,370、58,371、58,372、58,373、58,374、58, 375、58,376、58,377、58,378、58,379、58,380、58,381、58,382、58,383、58,384、58,385、58, 386、58,387、58,388、58,389、58,390、58,391、58,392、58,393、58,394、58,395、58,396、58, 397、58,398、58,399、58,400、58,401、58,402、58,403、58,404、58,405、58,406、58,407、58, 408、58,409、58,410、58、58,411、58,412、58,413、58,414、58,415、58,416、58,417、58,418、 58,419、58,420、58,421、58,422、58,423、58,424、58,425、58,426、58,427、58,428、58,429、 58,430、58,431、58,432、58,433、58,434、58,435、58,436、58,437、58,438、58,439、58,440、 58,441、58,442、58,443、58,444、58,445、58,446、58,447、58,448、58,449、58,450、58,451、 58,452、58,453、58,454、58,455、58,456、58,457、58,458、58,459、58,460、58,461、58,462、 58,463、58,464、58,465、58,466、58,467、58,468、58,469、58,470、58,471、58,472、58,473、 58,474、58,475、58,476、58,477、58,478、58,479、58,480、58,481、58,482、58,483、58,484、 58,485、58,486、58,487、58,488、58,489、58,490、58,491、58,492、58,493、58,494、58,495、 58,496、58,497、58,498、58,499、58,500、58,501、58,502、58,503、58,504、58,505、58,506、 58,507、58,508、58,509、58,510、58,511、58,512、58,513、58,514、58,515、58,516、58,517、 58,518、58,519、58,520、58,521、58,522、58,523、58,524、58,525、58,526、58,527、58,528、 58,529、58,530、58,531、58,532、58,533、58,534、58,535、58,536、58,537、58,538、58,539、 58,540、58,541、58,542、58,543、58,544、58,545、58,546、58,547、58,548、58,549、58,550、 58,551、58,552、58,553、58,554、58,555、58,556、58,557、58,558、58,559、58,560、58,561、 58,562、58,563、58,564、58,565、58,566、58,567、58,568、58,569、58,570、58,571、58,572、 58,573、58,574、58,575、58,576、58,577、58,578、58,579、58,580、58,581、58,582、58,583、 58,584、58,585、58,586、58,587、58,588、58,589、58,590、58,591、58,592、58,593、58,594、 58,595、58,596、58,597、58,598、58,599、58,600、58,601、58,602、58,603、58,604、58,605、 58,606、58,607、58,608、58,609、58,610、58,611、58,612、58,613、58,614、58,615、58,616、 58,617、58,618、58,619、58,620、58,621、58,622、58,623、58,624、58,625、58,626、58,627、 58,628、58,629、58,630、58,631、58,632、58,633、58,634、58,635、58,636、58,637、58,638、 58,639、58,640、58,641、58,642、58,643、58,644、58,645、58,646、58,647、58,648、58,649、 58,650、58,651、58,652、58,653、58,654、58,655、58,656、58,657、58,658、58,659、58,660、 58,661、58,662、58,663、58,664、58,665、58,666、58,667、58,668、58,669、58,670、58,671、 58,672、58,673、58,674、58,675、58,676、58,677、58,678、58,679、58,680、58,681、58,682、 58,683、58,684、58,685、58,686、58,687、58,688、58,689、58,690、58,691、58,692、58,693、 58,694、58,695、58,696、58,697、58,698、58,699、58,700、58,701、58,702、58,703、58,704、 58,705、58,706、58,707、58,708、58,709、58,710、58,711、58,712、58,713、58,714、58,715、 58,716、58,717、58,718、58,719、58,720、58,721、58,722、58,723、58,724、58,725、58,726、 58,727、58,728、58,729、58,730、58,731、58,732、58,733、58,734、58,735、58,736、58,737、 58,738、58,739、58,740、58,741、58,742、58,743、58,744、58,745、58,746、58,747、58,748、 58,749、58,750、58,751、58,752、58,753、58,754、58,755、58,756、58,757、58,758、58,759、 58,760、58,761、58,762、58,763、58,764、58,765、58,766、58,767、58,768、58,769、58,770、 58,771、58,772、58,773、58,774、58,775、58,776、58,777、58,778、58,779、58,780、58,781、 58,782、58,783、58,784、58,785、58,786、58,787、58,788、58,789、58,790、58,791、58,792、 58,793、58,794、58,795、58,796、58,797、58,798、58,799、58,800、58,801、58,802、58,803、 58,804、58,805、58,806、58,807、58,808、58,809、58,810、58,811、58,812、58,813、58,814、 58,815、58,816、58,817、58,818、58,819、58,820、58,821、58,822、58,823、58,824、58,825、 58,826、58,827、58,828、58,829、58,830、58,831、58,832、58,833、58,834、58,835、58,836、 58,837、58,838、58,839、58,840、58,841、58,842、58,843、58,844、58,845、58,846、58,847、 58,848、58,849、58,850、58,851、58,852、58,853、58,854、58,855、58,856、58,857、58,858、 58,859、58,860、58,861、58,862、58,863、58,864、58,865、58,866、58,867、58,868、58,869、 58,870、58,871、58,872、58,873、58,874、58,875、58,876、58,877、58,878、58,879、58,880、 58,881、58,882、58,883、58,884、58,885、58,886、58,887、58,888、58,889、58,890、58,891、 58,892、58,893、58,894、58,895、58,896、58,897、58,898、58,899、58,900、58,901、58,902、 58,903、58,904、58,905、58,906、58,907、58,908、58,909、58,910、58,911、58,912、58,913、 58,914、58,915、58,916、58,917、58,918、58,919、58,920、58,921、58,922、58,923、58,924、 58,925、58,926、58,927、58,928、58,929、58,930、58,931、58,932、58,933、58,934、58,935、 58,936、58,937、58,938、58,939、58,940、58,941、58,942、58,943、58,944、58,945、58,946、 58,947、58,948、58,949、58,950、58,951、58,952、58,953、58,954、58,955、58,956、58,957、 58,958、58,959、58,960、58,961、58,962、58,963、58,964、58,965、58,966、58,967、58,968、 58,969、58,970、58,971、58,972、58,973、58,974、58,975、58,976、58,977、58,978、58,979、 58,980、58,981、58,982、58,983、58,984、58,985、58,986、58,987、58,988、58,989、58,990、 58,991st, 58,992,58,993,58,994,58,995,58,996,58,997,58,998,58,999 or 59,000；67,000 Include to 68,000：67,001、67,002、67,003、67,004、67,005、67,006、67,007、67,008、67,009、 67,010、67,011、67,012、67,013、67,014、67,015、67,016、67,017、67,018、67,019、67,020、 67,021、67,022、67,023、67,024、67,025、67,026、67,027、67,028、67,029、67,030、67,031、 67,032、67,033、67,034、67,035、67,036、67,037、67,038、67,039、67,040、67,041、67,042、 67,043、67,044、67,045、67,046、67,047、67,048、67,049、67,050、67,051、67,052、67,053、 67,054、67,055、67,056、67,057、67,067、67,059、67,060、67,061、67,062、67,063、67,064、 67,065、67,066、67,067、67,068、67,069、67,070、67,071、67,072、67,073、67,074、67,075、 67,076、67,077、67,078、67,079、67,080、67,081、67,082、67,083、67,084、67,085、67,086、 67,087、67,088、67,089、67,090、67,091、67,092、67,093、67,094、67,095、67,096、67,097、 67,098、67,099、67,100、67,101、67,102、67,103、67,104、67,105、67,106、67,107、67,110、 67,111、67,112、67,113、67,114、67,115、67,116、67,117、67,118、67,119、67,120、67,121、 67,122、67,123、67,124、67,125、67,126、67,127、67,128、67,129、67,130、67,131、67,132、 67,133、67,134、67,135、67,136、67,137、67,138、67,139、67,140、67,141、67,142、67,143、 67,144、67,145、67,146、67,147、67,148、67,149、67,150、67,151、67,152、67,153、67,154、 67,155、67,156、67,157、67,167、67,159、67,160、67,161、67,162、67,163、67,164、67,165、 67,166、67,167、67,168、67,169、67,170、67,171、67,172、67,173、67,174、67,175、67,176、 67,177、67,178、67,179、67,180、67,181、67,182、67,183、67,184、67,185、67,186、67,187、 67,188、67,189、67,190、67,191、67,192、67,193、67,194、67,195、67,196、67,197、67,198、 67,199、67,200、67,201、67,202、67,203、67,204、67,205、67,206、67,207、67,208、67,209、 67,210、67,211、67,212、67,213、67,214、67,215、67,216、67,217、67,218、67,219、67,220、 67,221、67,222、67,223、67,224、67,225、67,226、67,227、67,228、67,229、67,230、67,231、 67,232、67,233、67,234、67,235、67,236、67,237、67,238、67,239、67,240、67,241、67,242、 67,243、67,244、67,245、67,246、67,247、67,248、67,249、67,250、67,251、67,252、67,253、 67,254、67,255、67,256、67,257、67,267、67,259、67,260、67,261、67,262、67,263、67,264、 67,265、67,266、67,267、67,268、67,269、67,270、67,271、67,272、67,273、67,274、67,275、 67,276、67,277、67,278、67,279、67,280、67,281、67,282、67,283、67,284、67,285、67,286、 67,287、67,288、67,289、67,290、67,291、67,292、67,293、67,294、67,295、67,296、67,297、 67,298、67,299、67,300、67,301、67,302、67,303、67,304、67,305、67,306、67,307、67,308、 67,309、67,310、67,311、67,312、67,313、67,314、67,315、67,316、67,317、67,318、67,319、 67,320、67,321、67,322、67,323、67,324、67,325、67,326、67,327、67,328、67,329、67,330、 67,331、67,332、67,333、67,334、67,335、67,336、67,337、67,338、67,339、67,340、67,341、 67,342、67,343、67,344、67,345、67,346、67,347、67,348、67,349、67,350、67,351、67,352、 67,353、67,354、67,355、67,356、67,357、67,367、67,359、67,360、67,361、67,362、67,363、 67,364、67,365、67,366、67,367、67,368、67,369、67,370、67,371、67,372、67,373、67,374、 67,375、67,376、67,377、67,378、67,379、67,380、67,381、67,382、67,383、67,384、67,385、 67,386、67,387、67,388、67,389、67,390、67,391、67,392、67,393、67,394、67,395、67,396、 67,397、67,398、67,399、67,400、67,401、67,402、67,403、67,404、67,405、67,406、67,407、 67,408、67,409、67,410、67,411、67,412、67,413、67,414、67,415、67,416、67,417、67,418、 67,419、67,420、67,421、67,422、67,423、67,424、67,425、67,426、67,427、67,428、67,429、 67,430、67,431、67,432、67,433、67,434、67,435、67,436、67,437、67,438、67,439、67,440、 67,441、67,442、67,443、67,444、67,445、67,446、67,447、67,448、67,449、67,450、67,451、 67,452、67,453、67,454、67,455、67,456、67,457、67,467、67,459、67,460、67,461、67,462、 67,463、67,464、67,465、67,466、67,467、67,468、67,469、67,470、67,471、67,472、67,473、 67,474、67,475、67,476、67,477、67,478、67,479、67,480、67,481、67,482、67,483、67,484、 67,485、67,486、67,487、67,488、67,489、67,490、67,491、67,492、67,493、67,494、67,495、 67,496、67,497、67,498、67,499、67,500、67,501、67,502、67,503、67,504、67,505、67,506、 67,507、67,508、67,509、67,510、67,511、67,512、67,513、67,514、67,515、67,516、67,517、 67,518、67,519、67,520、67,521、67,522、67,523、67,524、67,525、67,526、67,527、67,528、 67,529、67,530、67,531、67,532、67,533、67,534、67,535、67,536、67,537、67,538、67,539、 67,540、67,541、67,542、67,543、67,544、67,545、67,546、67,547、67,548、67,549、67,550、 67,551、67,552、67,553、67,554、67,555、67,556、67,557、67,567、67,559、67,560、67,561、 67,562、67,563、67,564、67,565、67,566、67,567、67,568、67,569、67,570、67,571、67,572、 67,573、67,574、67,575、67,576、67,577、67,578、67,579、67,580、67,581、67,582、67,583、 67,584、67,585、67,586、67,587、67,588、67,589、67,590、67,591、67,592、67,593、67,594、 67,595、67,596、67,597、67,598、67,599、67,600、67,601、67,602、67,603、67,604、67,605、 67,606、67,607、67,608、67,609、67,610、67,611、67,612、67,613、67,614、67,615、67,616、 67,617、67,618、67,619、67,620、67,621、67,622、67,623、67,624、67,625、67,626、67,627、 67,628、67,629、67,630、67,631、67,632、67,633、67,634、67,635、67,636、67,637、67,638、 67,639、67,640、67,641、67,642、67,643、67,644、67,645、67,646、67,647、67,648、67,649、 67,650、67,651、67,652、67,653、67,654、67,655、67,656、67,657、67,667、67,659、67,660、 67,661、67,662、67,663、67,664、67,665、67,666、67,667、67,668、67,669、67,670、67,671、 67,672、67,673、67,674、67,675、67,676、67,677、67,678、67,679、67,680、67,681、67,682、 67,683、67,684、67,685、67,686、67,687、67,688、67,689、67,690、67,691、67,692、67,693、 67,694、67,695、67,696、67,697、67,698、67,699、67,700、67,701、67,702、67,703、67,704、 67,705、67,706、67,707、67,708、67,709、67,710、67,711、67,712、67,713、67,714、67,715、 67,716、67,717、67,718、67,719、67,720、67,721、67,722、67,723、67,724、67,725、67,726、 67,727、67,728、67,729、67,730、67,731、67,732、67,733、67,734、67,735、67,736、67,737、 67,738、67,739、67,740、67,741、67,742、67,743、67,744、67,745、67,746、67,747、67,748、 67,749、67,750、67,751、67,752、67,753、67,754、67,755、67,756、67,757、67,767、67,759、 67,760、67,761、67,762、67,763、67,764、67,765、67,766、67,767、67,768、67,769、67,770、 67,771、67,772、67,773、67,774、67,775、67,776、67,777、67,778、67,779、67,780、67,781、 67,782、67,783、67,784、67,785、67,786、67,787、67,788、67,789、67,790、67,791、67,792、 67,793、67,794、67,795、67,796、67,797、67,798、67,799、67,800、67,801、67,802、67,803、 67,804、67,805、67,806、67,807、67,808、67,809、67,810、67,811、67,812、67,813、67,814、 67,815、67,816、67,817、67,818、67,819、67,820、67,821、67,822、67,823、67,824、67,825、 67,826、67,827、67,828、67,829、67,830、67,831、67,832、67,833、67,834、67,835、67,836、 67,837、67,838、67,839、67,840、67,841、67,842、67,843、67,844、67,845、67,846、67,847、 67,848、67,849、67,850、67,851、67,852、67,853、67,854、67,855、67,856、67,857、67,867、 67,859、67,860、67,861、67,862、67,863、67,864、67,865、67,866、67,867、67,868、67,869、 67,870、67,871、67,872、67,873、67,874、67,875、67,876、67,877、67,878、67,879、67,880、 67,881、67,882、67,883、67,884、67,885、67,886、67,887、67,888、67,889、67,890、67,891、 67,892、67,893、67,894、67,895、67,896、67,897、67,898、67,899、67,900、67,901、67,902、 67,903、67,904、67,905、67,906、67,907、67,908、67,909、67,910、67,911、67,912、67,913、 67,914、67,915、67,916、67,917、67,918、67,919、67,920、67,921、67,922、67,923、67,924、 67,925、67,926、67,927、67,928、67,929、67,930、67,931、67,932、67,933、67,934、67,935、 67,936、67,937、67,938、67,939、67,940、67,941、67,942、67,943、67,944、67,945、67,946、 67,947、67,948、67,949、67,950、67,951、67,952、67,953、67,954、67,955、67,956、67,957、 67,967、67,959、67,960、67,961、67,962、67,963、67,964、67,965、67,966、67,967、67,968、 67,969、67,970、67,971、67,972、67,973、67,974、67,975、67,976、67,977、67,978、67,979、 67,980、67,981、67,982、67,983、67,984、67,985、67,986、67,987、67,988、67,989、67,990、 67,991st, 67,992,67,993,67,994,67,995,67,996,67,997,67,998,67,999 or 68,000；74,000 Include to 76,000：74,001、74,002、74,003、74,004、74,005、74,006、74,007、74,008、74,009、 74,010、74,011、74,012、74,013、74,014、74,015、74,016、74,017、74,018、74,019、74,020、 74,021、74,022、74,023、74,024、74,025、74,026、74,027、74,028、74,029、74,030、74,031、 74,032、74,033、74,034、74,035、74,036、74,037、74,038、74,039、74,040、74,041、74,042、 74,043、74,044、74,045、74,046、74,047、74,048、74,049、74,050、74,051、74,052、74,053、 74,054、74,055、74,056、74,057、74,074、74,059、74,060、74,061、74,062、74,063、74,064、 74,065、74,066、74,074、74,068、74,069、74,070、74,071、74,072、74,073、74,074、74,075、 74,076、74,077、74,078、74,079、74,080、74,081、74,082、74,083、74,084、74,085、74,086、 74,087、74,088、74,089、74,090、74,091、74,092、74,093、74,094、74,095、74,096、74,097、 74,098、74,099、74,100、74,101、74,102、74,103、74,104、74,105、74,106、74,107、74,110、 74,111、74,112、74,113、74,114、74,115、74,116、74,117、74,118、74,119、74,120、74,121、 74,122、74,123、74,124、74,125、74,126、74,127、74,128、74,129、74,130、74,131、74,132、 74,133、74,134、74,135、74,136、74,137、74,138、74,139、74,140、74,141、74,142、74,143、 74,144、74,145、74,146、74,147、74,148、74,149、74,150、74,151、74,152、74,153、74,154、 74,155、74,156、74,157、74,174、74,159、74,160、74,161、74,162、74,163、74,164、74,165、 74,166、74,174、74,168、74,169、74,170、74,171、74,172、74,173、74,174、74,175、74,176、 74,177、74,178、74,179、74,180、74,181、74,182、74,183、74,184、74,185、74,186、74,187、 74,188、74,189、74,190、74,191、74,192、74,193、74,194、74,195、74,196、74,197、74,198、 74,199、74,200、74,201、74,202、74,203、74,204、74,205、74,206、74,207、74,208、74,209、 74,210、74,211、74,212、74,213、74,214、74,215、74,216、74,217、74,218、74,219、74,220、 74,221、74,222、74,223、74,224、74,225、74,226、74,227、74,228、74,229、74,230、74,231、 74,232、74,233、74,234、74,235、74,236、74,237、74,238、74,239、74,240、74,241、74,242、 74,243、74,244、74,245、74,246、74,247、74,248、74,249、74,250、74,251、74,252、74,253、 74,254、74,255、74,256、74,257、74,274、74,259、74,260、74,261、74,262、74,263、74,264、 74,265、74,266、74,274、74,268、74,269、74,270、74,271、74,272、74,273、74,274、74,275、 74,276、74,277、74,278、74,279、74,280、74,281、74,282、74,283、74,284、74,285、74,286、 74,287、74,288、74,289、74,290、74,291、74,292、74,293、74,294、74,295、74,296、74,297、 74,298、74,299、74,300、74,301、74,302、74,303、74,304、74,305、74,306、74,307、74,308、 74,309、74,310、74,311、74,312、74,313、74,314、74,315、74,316、74,317、74,318、74,319、 74,320、74,321、74,322、74,323、74,324、74,325、74,326、74,327、74,328、74,329、74,330、 74,331、74,332、74,333、74,334、74,335、74,336、74,337、74,338、74,339、74,340、74,341、 74,342、74,343、74,344、74,345、74,346、74,347、74,348、74,349、74,350、74,351、74,352、 74,353、74,354、74,355、74,356、74,357、74,374、74,359、74,360、74,361、74,362、74,363、 74,364、74,365、74,366、74,374、74,368、74,369、74,370、74,371、74,372、74,373、74,374、 74,375、74,376、74,377、74,378、74,379、74,380、74,381、74,382、74,383、74,384、74,385、 74,386、74,387、74,388、74,389、74,390、74,391、74,392、74,393、74,394、74,395、74,396、 74,397、74,398、74,399、74,400、74,401、74,402、74,403、74,404、74,405、74,406、74,407、 74,408、74,409、74,410、74,411、74,412、74,413、74,414、74,415、74,416、74,417、74,418、 74,419、74,420、74,421、74,422、74,423、74,424、74,425、74,426、74,427、74,428、74,429、 74,430、74,431、74,432、74,433、74,434、74,435、74,436、74,437、74,438、74,439、74,440、 74,441、74,442、74,443、74,444、74,445、74,446、74,447、74,448、74,449、74,450、74,451、 74,452、74,453、74,454、74,455、74,456、74,457、74,474、74,459、74,460、74,461、74,462、 74,463、74,464、74,465、74,466、74,474、74,468、74,469、74,470、74,471、74,472、74,473、 74,474、74,475、74,476、74,477、74,478、74,479、74,480、74,481、74,482、74,483、74,484、 74,485、74,486、74,487、74,488、74,489、74,490、74,491、74,492、74,493、74,494、74,495、 74,496、74,497、74,498、74,499、74,500、74,501、74,502、74,503、74,504、74,505、74,506、 74,507、74,508、74,509、74,510、74,511、74,512、74,513、74,514、74,515、74,516、74,517、 74,518、74,519、74,520、74,521、74,522、74,523、74,524、74,525、74,526、74,527、74,528、 74,529、74,530、74,531、74,532、74,533、74,534、74,535、74,536、74,537、74,538、74,539、 74,540、74,541、74,542、74,543、74,544、74,545、74,546、74,547、74,548、74,549、74,550、 74,551、74,552、74,553、74,554、74,555、74,556、74,557、74,574、74,559、74,560、74,561、 74,562、74,563、74,564、74,565、74,566、74,574、74,568、74,569、74,570、74,571、74,572、 74,573、74,574、74,575、74,576、74,577、74,578、74,579、74,580、74,581、74,582、74,583、 74,584、74,585、74,586、74,587、74,588、74,589、74,590、74,591、74,592、74,593、74,594、 74,595、74,596、74,597、74,598、74,599、74,600、74,601、74,602、74,603、74,604、74,605、 74,606、74,607、74,608、74,609、74,610、74,611、74,612、74,613、74,614、74,615、74,616、 74,617、74,618、74,619、74,620、74,621、74,622、74,623、74,624、74,625、74,626、74,627、 74,628、74,629、74,630、74,631、74,632、74,633、74,634、74,635、74,636、74,637、74,638、 74,639、74,640、74,641、74,642、74,643、74,644、74,645、74,646、74,647、74,648、74,649、 74,650、74,651、74,652、74,653、74,654、74,655、74,656、74,657、74,674、74,659、74,660、 74,661、74,662、74,663、74,664、74,665、74,666、74,674、74,668、74,669、74,670、74,671、 74,672、74,673、74,674、74,675、74,676、74,677、74,678、74,679、74,680、74,681、74,682、 74,683、74,684、74,685、74,686、74,687、74,688、74,689、74,690、74,691、74,692、74,693、 74,694、74,695、74,696、74,697、74,698、74,699、74,700、74,701、74,702、74,703、74,704、 74,705、74,706、74,707、74,708、74,709、74,710、74,711、74,712、74,713、74,714、74,715、 74,716、74,717、74,718、74,719、74,720、74,721、74,722、74,723、74,724、74,725、74,726、 74,727、74,728、74,729、74,730、74,731、74,732、74,733、74,734、74,735、74,736、74,737、 74,738、74,739、74,740、74,741、74,742、74,743、74,744、74,745、74,746、74,747、74,748、 74,749、74,750、74,751、74,752、74,753、74,754、74,755、74,756、74,757、74,774、74,759、 74,760、74,761、74,762、74,763、74,764、74,765、74,766、74,774、74,768、74,769、74,770、 74,771、74,772、74,773、74,774、74,775、74,776、74,777、74,778、74,779、74,780、74,781、 74,782、74,783、74,784、74,785、74,786、74,787、74,788、74,789、74,790、74,791、74,792、 74,793、74,794、74,795、74,796、74,797、74,798、74,799、74,800、74,801、74,802、74,803、 74,804、74,805、74,806、74,807、74,808、74,809、74,810、74,811、74,812、74,813、74,814、 74,815、74,816、74,817、74,818、74,819、74,820、74,821、74,822、74,823、74,824、74,825、 74,826、74,827、74,828、74,829、74,830、74,831、74,832、74,833、74,834、74,835、74,836、 74,837、74,838、74,839、74,840、74,841、74,842、74,843、74,844、74,845、74,846、74,847、 74,848、74,849、74,850、74,851、74,852、74,853、74,854、74,855、74,856、74,857、74,874、 74,859、74,860、74,861、74,862、74,863、74,864、74,865、74,866、74,874、74,868、74,869、 74,870、74,871、74,872、74,873、74,874、74,875、74,876、74,877、74,878、74,879、74,880、 74,881、74,882、74,883、74,884、74,885、74,886、74,887、74,888、74,889、74,890、74,891、 74,892、74,893、74,894、74,895、74,896、74,897、74,898、74,899、74,900、74,901、74,902、 74,903、74,904、74,905、74,906、74,907、74,908、74,909、74,910、74,911、74,912、74,913、 74,914、74,915、74,916、74,917、74,918、74,919、74,920、74,921、74,922、74,923、74,924、 74,925、74,926、74,927、74,928、74,929、74,930、74,931、74,932、74,933、74,934、74,935、 74,936、74,937、74,938、74,939、74,940、74,941、74,942、74,943、74,944、74,945、74,946、 74,947、74,948、74,949、74,950、74,951、74,952、74,953、74,954、74,955、74,956、74,957、 74,974、74,959、74,960、74,961、74,962、74,963、74,964、74,965、74,966、74,974、74,968、 74,969、74,970、74,971、74,972、74,973、74,974、74,975、74,976、74,977、74,978、74,979、 74,980、74,981、74,982、74,983、74,984、74,985、74,986、74,987、74,988、74,989、74,990、 74,991、74,992、74,993、74,994、74,995、74,996、74,997、74,998、74,999、75,000、75,001、 75,002、75,003、75,004、75,005、75,006、75,007、75,008、75,009、75,010、75,011、75,012、 75,013、75,014、75,015、75,016、75,017、75,018、75,019、75,020、75,021、75,022、75,023、 75,024、75,025、75,026、75,027、75,028、75,029、75,030、75,031、75,032、75,033、75,034、 75,035、75,036、75,037、75,038、75,039、75,040、75,041、75,042、75,043、75,044、75,045、 75,046、75,047、75,048、75,049、75,050、75,051、75,052、75,053、75,054、75,055、75,056、 75,057、75,075、75,059、75,060、75,061、75,062、75,063、75,064、75,065、75,066、75,075、 75,068、75,069、75,070、75,071、75,072、75,073、75,075、75,075、75,076、75,077、75,078、 75,079、75,080、75,081、75,082、75,083、75,084、75,085、75,086、75,087、75,088、75,089、 75,090、75,091、75,092、75,093、75,094、75,095、75,096、75,097、75,098、75,099、75,100、 75,101、75,102、75,103、75,104、75,105、75,106、75,107、75,110、75,111、75,112、75,113、 75,114、75,115、75,116、75,117、75,118、75,119、75,120、75,121、75,122、75,123、75,124、 75,125、75,126、75,127、75,128、75,129、75,130、75,131、75,132、75,133、75,134、75,135、 75,136、75,137、75,138、75,139、75,140、75,141、75,142、75,143、75,144、75,145、75,146、 75,147、75,148、75,149、75,150、75,151、75,152、75,153、75,154、75,155、75,156、75,157、 75,175、75,159、75,160、75,161、75,162、75,163、75,164、75,165、75,166、75,175、75,168、 75,169、75,170、75,171、75,172、75,173、75,175、75,175、75,176、75,177、75,178、75,179、 75,180、75,181、75,182、75,183、75,184、75,185、75,186、75,187、75,188、75,189、75,190、 75,191、75,192、75,193、75,194、75,195、75,196、75,197、75,198、75,199、75,200、75,201、 75,202、75,203、75,204、75,205、75,206、75,207、75,208、75,209、75,210、75,211、75,212、 75,213、75,214、75,215、75,216、75,217、75,218、75,219、75,220、75,221、75,222、75,223、 75,224、75,225、75,226、75,227、75,228、75,229、75,230、75,231、75,232、75,233、75,234、 75,235、75,236、75,237、75,238、75,239、75,240、75,241、75,242、75,243、75,244、75,245、 75,246、75,247、75,248、75,249、75,250、75,251、75,252、75,253、75,254、75,255、75,256、 75,257、75,275、75,259、75,260、75,261、75,262、75,263、75,264、75,265、75,266、75,275、 75,268、75,269、75,270、75,271、75,272、75,273、75,275、75,275、75,276、75,277、75,278、 75,279、75,280、75,281、75,282、75,283、75,284、75,285、75,286、75,287、75,288、75,289、 75,290、75,291、75,292、75,293、75,294、75,295、75,296、75,297、75,298、75,299、75,300、 75,301、75,302、75,303、75,304、75,305、75,306、75,307、75,308、75,309、75,310、75,311、 75,312、75,313、75,314、75,315、75,316、75,317、75,318、75,319、75,320、75,321、75,322、 75,323、75,324、75,325、75,326、75,327、75,328、75,329、75,330、75,331、75,332、75,333、 75,334、75,335、75,336、75,337、75,338、75,339、75,340、75,341、75,342、75,343、75,344、 75,345、75,346、75,347、75,348、75,349、75,350、75,351、75,352、75,353、75,354、75,355、 75,356、75,357、75,375、75,359、75,360、75,361、75,362、75,363、75,364、75,365、75,366、 75,375、75,368、75,369、75,370、75,371、75,372、75,373、75,375、75,375、75,376、75,377、 75,378、75,379、75,380、75,381、75,382、75,383、75,384、75,385、75,386、75,387、75,388、 75,389、75,390、75,391、75,392、75,393、75,394、75,395、75,396、75,397、75,398、75,399、 75,400、75,401、75,402、75,403、75,404、75,405、75,406、75,407、75,408、75,409、75,410、 75,411、75,412、75,413、75,414、75,415、75,416、75,417、75,418、75,419、75,420、75,421、 75,422、75,423、75,424、75,425、75,426、75,427、75,428、75,429、75,430、75,431、75,432、 75,433、75,434、75,435、75,436、75,437、75,438、75,439、75,440、75,441、75,442、75,443、 75,444、75,445、75,446、75,447、75,448、75,449、75,450、75,451、75,452、75,453、75,454、 75,455、75,456、75,457、75,475、75,459、75,460、75,461、75,462、75,463、75,464、75,465、 75,466、75,475、75,468、75,469、75,470、75,471、75,472、75,473、75,475、75,475、75,476、 75,477、75,478、75,479、75,480、75,481、75,482、75,483、75,484、75,485、75,486、75,487、 75,488、75,489、75,490、75,491、75,492、75,493、75,494、75,495、75,496、75,497、75,498、 75,499、75,500、75,501、75,502、75,503、75,504、75,505、75,506、75,507、75,508、75,509、 75,510、75,511、75,512、75,513、75,514、75,515、75,516、75,517、75,518、75,519、75,520、 75,521、75,522、75,523、75,524、75,525、75,526、75,527、75,528、75,529、75,530、75,531、 75,532、75,533、75,534、75,535、75,536、75,537、75,538、75,539、75,540、75,541、75,542、 75,543、75,544、75,545、75,546、75,547、75,548、75,549、75,550、75,551、75,552、75,553、 75,554、75,555、75,556、75,557、75,575、75,559、75,560、75,561、75,562、75,563、75,564、 75,565、75,566、75,575、75,568、75,569、75,570、75,571、75,572、75,573、75,575、75,575、 75,576、75,577、75,578、75,579、75,580、75,581、75,582、75,583、75,584、75,585、75,586、 75,587、75,588、75,589、75,590、75,591、75,592、75,593、75,594、75,595、75,596、75,597、 75,598、75,599、75,600、75,601、75,602、75,603、75,604、75,605、75,606、75,607、75,608、 75,609、75,610、75,611、75,612、75,613、75,614、75,615、75,616、75,617、75,618、75,619、 75,620、75,621、75,622、75,623、75,624、75,625、75,626、75,627、75,628、75,629、75,630、 75,631、75,632、75,633、75,634、75,635、75,636、75,637、75,638、75,639、75,640、75,641、 75,642、75,643、75,644、75,645、75,646、75,647、75,648、75,649、75,650、75,651、75,652、 75,653、75,654、75,655、75,656、75,657、75,675、75,659、75,660、75,661、75,662、75,663、 75,664、75,665、75,666、75,675、75,668、75,669、75,670、75,671、75,672、75,673、75,675、 75,675、75,676、75,677、75,678、75,679、75,680、75,681、75,682、75,683、75,684、75,685、 75,686、75,687、75,688、75,689、75,690、75,691、75,692、75,693、75,694、75,695、75,696、 75,697、75,698、75,699、75,700、75,701、75,702、75,703、75,704、75,705、75,706、75,707、 75,708、75,709、75,710、75,711、75,712、75,713、75,714、75,715、75,716、75,717、75,718、 75,719、75,720、75,721、75,722、75,723、75,724、75,725、75,726、75,727、75,728、75,729、 75,730、75,731、75,732、75,733、75,734、75,735、75,736、75,737、75,738、75,739、75,740、 75,741、75,742、75,743、75,744、75,745、75,746、75,747、75,748、75,749、75,750、75,751、 75,752、75,753、75,754、75,755、75,756、75,757、75,775、75,759、75,760、75,761、75,762、 75,763、75,764、75,765、75,766、75,775、75,768、75,769、75,770、75,771、75,772、75,773、 75,775、75,775、75,776、75,777、75,778、75,779、75,780、75,781、75,782、75,783、75,784、 75,785、75,786、75,787、75,788、75,789、75,790、75,791、75,792、75,793、75,794、75,795、 75,796、75,797、75,798、75,799、75,800、75,801、75,802、75,803、75,804、75,805、75,806、 75,807、75,808、75,809、75,810、75,811、75,812、75,813、75,814、75,815、75,816、75,817、 75,818、75,819、75,820、75,821、75,822、75,823、75,824、75,825、75,826、75,827、75,828、 75,829、75,830、75,831、75,832、75,833、75,834、75,835、75,836、75,837、75,838、75,839、 75,840、75,841、75,842、75,843、75,844、75,845、75,846、75,847、75,848、75,849、75,850、 75,851、75,852、75,853、75,854、75,855、75,856、75,857、75,875、75,859、75,860、75,861、 75,862、75,863、75,864、75,865、75,866、75,875、75,868、75,869、75,870、75,871、75,872、 75,873、75,875、75,875、75,876、75,877、75,878、75,879、75,880、75,881、75,882、75,883、 75,884、75,885、75,886、75,887、75,888、75,889、75,890、75,891、75,892、75,893、75,894、 75,895、75,896、75,897、75,898、75,899、75,900、75,901、75,902、75,903、75,904、75,905、 75,906、75,907、75,908、75,909、75,910、75,911、75,912、75,913、75,914、75,915、75,916、 75,917、75,918、75,919、75,920、75,921、75,922、75,923、75,924、75,925、75,926、75,927、 75,928、75,929、75,930、75,931、75,932、75,933、75,934、75,935、75,936、75,937、75,938、 75,939、75,940、75,941、75,942、75,943、75,944、75,945、75,946、75,947、75,948、75,949、 75,950、75,951、75,952、75,953、75,954、75,955、75,956、75,957、75,975、75,959、75,960、 75,961、75,962、75,963、75,964、75,965、75,966、75,975、75,968、75,969、75,970、75,971、 75,972、75,973、75,975、75,975、75,976、75,977、75,978、75,979、75,980、75,981、75,982、 75,983、75,984、75,985、75,986、75,987、75,988、75,989、75,990、75,991、75,992、75,993、 75,994th, 75,995,75,996,75,997,75,998,75,999 or 76,000；84,000 to 85,000 include：84,001、 84,002、84,003、84,004、84,005、84,006、84,007、84,008、84,009、84,010、84,011、84,012、 84,013、84,014、84,015、84,016、84,017、84,018、84,019、84,020、84,021、84,022、84,023、 84,024、84,025、84,026、84,027、84,028、84,029、84,030、84,031、84,032、84,033、84,034、 84,035、84,036、84,037、84,038、84,039、84,040、84,041、84,042、84,043、84,044、84,045、 84,046、84,047、84,048、84,049、84,050、84,051、84,052、84,053、84,054、84,055、84,056、 84,057、84,084、84,059、84,060、84,061、84,062、84,063、84,064、84,065、84,066、84,084、 84,068、84,069、84,070、84,071、84,072、84,073、84,084、84,084、84,076、84,077、84,078、 84,079、84,080、84,081、84,082、84,083、84,084、84,085、84,086、84,087、84,088、84,089、 84,090、84,091、84,092、84,093、84,094、84,095、84,096、84,097、84,098、84,099、84,100、 84,101、84,102、84,103、84,104、84,105、84,106、84,107、84,110、84,111、84,112、84,113、 84,114、84,115、84,116、84,117、84,118、84,119、84,120、84,121、84,122、84,123、84,124、 84,125、84,126、84,127、84,128、84,129、84,130、84,131、84,132、84,133、84,134、84,135、 84,136、84,137、84,138、84,139、84,140、84,141、84,142、84,143、84,144、84,145、84,146、 84,147、84,148、84,149、84,150、84,151、84,152、84,153、84,154、84,155、84,156、84,157、 84,184、84,159、84,160、84,161、84,162、84,163、84,164、84,165、84,166、84,184、84,168、 84,169、84,170、84,171、84,172、84,173、84,184、84,184、84,176、84,177、84,178、84,179、 84,180、84,181、84,182、84,183、84,184、84,185、84,186、84,187、84,188、84,189、84,190、 84,191、84,192、84,193、84,194、84,195、84,196、84,197、84,198、84,199、84,200、84,201、 84,202、84,203、84,204、84,205、84,206、84,207、84,208、84,209、84,210、84,211、84,212、 84,213、84,214、84,215、84,216、84,217、84,218、84,219、84,220、84,221、84,222、84,223、 84,224、84,225、84,226、84,227、84,228、84,229、84,230、84,231、84,232、84,233、84,234、 84,235、84,236、84,237、84,238、84,239、84,240、84,241、84,242、84,243、84,244、84,245、 84,246、84,247、84,248、84,249、84,250、84,251、84,252、84,253、84,254、84,255、84,256、 84,257、84,284、84,259、84,260、84,261、84,262、84,263、84,264、84,265、84,266、84,284、 84,268、84,269、84,270、84,271、84,272、84,273、84,284、84,284、84,276、84,277、84,278、 84,279、84,280、84,281、84,282、84,283、84,284、84,285、84,286、84,287、84,288、84,289、 84,290、84,291、84,292、84,293、84,294、84,295、84,296、84,297、84,298、84,299、84,300、 84,301、84,302、84,303、84,304、84,305、84,306、84,307、84,308、84,309、84,310、84,311、 84,312、84,313、84,314、84,315、84,316、84,317、84,318、84,319、84,320、84,321、84,322、 84,323、84,324、84,325、84,326、84,327、84,328、84,329、84,330、84,331、84,332、84,333、 84,334、84,335、84,336、84,337、84,338、84,339、84,340、84,341、84,342、84,343、84,344、 84,345、84,346、84,347、84,348、84,349、84,350、84,351、84,352、84,353、84,354、84,355、 84,356、84,357、84,384、84,359、84,360、84,361、84,362、84,363、84,364、84,365、84,366、 84,384、84,368、84,369、84,370、84,371、84,372、84,373、84,384、84,384、84,376、84,377、 84,378、84,379、84,380、84,381、84,382、84,383、84,384、84,385、84,386、84,387、84,388、 84,389、84,390、84,391、84,392、84,393、84,394、84,395、84,396、84,397、84,398、84,399、 84,400、84,401、84,402、84,403、84,404、84,405、84,406、84,407、84,408、84,409、84,410、 84,411、84,412、84,413、84,414、84,415、84,416、84,417、84,418、84,419、84,420、84,421、 84,422、84,423、84,424、84,425、84,426、84,427、84,428、84,429、84,430、84,431、84,432、 84,433、84,434、84,435、84,436、84,437、84,438、84,439、84,440、84,441、84,442、84,443、 84,444、84,445、84,446、84,447、84,448、84,449、84,450、84,451、84,452、84,453、84,454、 84,455、84,456、84,457、84,484、84,459、84,460、84,461、84,462、84,463、84,464、84,465、 84,466、84,484、84,468、84,469、84,470、84,471、84,472、84,473、84,484、84,484、84,476、 84,477、84,478、84,479、84,480、84,481、84,482、84,483、84,484、84,485、84,486、84,487、 84,488、84,489、84,490、84,491、84,492、84,493、84,494、84,495、84,496、84,497、84,498、 84,499、84,500、84,501、84,502、84,503、84,504、84,505、84,506、84,507、84,508、84,509、 84,510、84,511、84,512、84,513、84,514、84,515、84,516、84,517、84,518、84,519、84,520、 84,521、84,522、84,523、84,524、84,525、84,526、84,527、84,528、84,529、84,530、84,531、 84,532、84,533、84,534、84,535、84,536、84,537、84,538、84,539、84,540、84,541、84,542、 84,543、84,544、84,545、84,546、84,547、84,548、84,549、84,550、84,551、84,552、84,553、 84,554、84,555、84,556、84,557、84,584、84,559、84,560、84,561、84,562、84,563、84,564、 84,565、84,566、84,584、84,568、84,569、84,570、84,571、84,572、84,573、84,584、84,584、 84,576、84,577、84,578、84,579、84,580、84,581、84,582、84,583、84,584、84,585、84,586、 84,587、84,588、84,589、84,590、84,591、84,592、84,593、84,594、84,595、84,596、84,597、 84,598、84,599、84,600、84,601、84,602、84,603、84,604、84,605、84,606、84,607、84,608、 84,609、84,610、84,611、84,612、84,613、84,614、84,615、84,616、84,617、84,618、84,619、 84,620、84,621、84,622、84,623、84,624、84,625、84,626、84,627、84,628、84,629、84,630、 84,631、84,632、84,633、84,634、84,635、84,636、84,637、84,638、84,639、84,640、84,641、 84,642、84,643、84,644、84,645、84,646、84,647、84,648、84,649、84,650、84,651、84,652、 84,653、84,654、84,655、84,656、84,657、84,684、84,659、84,660、84,661、84,662、84,663、 84,664、84,665、84,666、84,684、84,668、84,669、84,670、84,671、84,672、84,673、84,684、 84,684、84,676、84,677、84,678、84,679、84,680、84,681、84,682、84,683、84,684、84,685、 84,686、84,687、84,688、84,689、84,690、84,691、84,692、84,693、84,694、84,695、84,696、 84,697、84,698、84,699、84,700、84,701、84,702、84,703、84,704、84,705、84,706、84,707、 84,708、84,709、84,710、84,711、84,712、84,713、84,714、84,715、84,716、84,717、84,718、 84,719、84,720、84,721、84,722、84,723、84,724、84,725、84,726、84,727、84,728、84,729、 84,730、84,731、84,732、84,733、84,734、84,735、84,736、84,737、84,738、84,739、84,740、 84,741、84,742、84,743、84,744、84,745、84,746、84,747、84,748、84,749、84,750、84,751、 84,752、84,753、84,754、84,755、84,756、84,757、84,784、84,759、84,760、84,761、84,762、 84,763、84,764、84,765、84,766、84,784、84,768、84,769、84,770、84,771、84,772、84,773、 84,784、84,784、84,776、84,777、84,778、84,779、84,780、84,781、84,782、84,783、84,784、 84,785、84,786、84,787、84,788、84,789、84,790、84,791、84,792、84,793、84,794、84,795、 84,796、84,797、84,798、84,799、84,800、84,801、84,802、84,803、84,804、84,805、84,806、 84,807、84,808、84,809、84,810、84,811、84,812、84,813、84,814、84,815、84,816、84,817、 84,818、84,819、84,820、84,821、84,822、84,823、84,824、84,825、84,826、84,827、84,828、 84,829、84,830、84,831、84,832、84,833、84,834、84,835、84,836、84,837、84,838、84,839、 84,840、84,841、84,842、84,843、84,844、84,845、84,846、84,847、84,848、84,849、84,850、 84,851、84,852、84,853、84,854、84,855、84,856、84,857、84,884、84,859、84,860、84,861、 84,862、84,863、84,864、84,865、84,866、84,884、84,868、84,869、84,870、84,871、84,872、 84,873、84,884、84,884、84,876、84,877、84,878、84,879、84,880、84,881、84,882、84,883、 84,884、84,885、84,886、84,887、84,888、84,889、84,890、84,891、84,892、84,893、84,894、 84,895、84,896、84,897、84,898、84,899、84,900、84,901、84,902、84,903、84,904、84,905、 84,906、84,907、84,908、84,909、84,910、84,911、84,912、84,913、84,914、84,915、84,916、 84,917、84,918、84,919、84,920、84,921、84,922、84,923、84,924、84,925、84,926、84,927、 84,928、84,929、84,930、84,931、84,932、84,933、84,934、84,935、84,936、84,937、84,938、 84,939、84,940、84,941、84,942、84,943、84,944、84,945、84,946、84,947、84,948、84,949、 84,950、84,951、84,952、84,953、84,954、84,955、84,956、84,957、84,984、84,959、84,960、 84,961、84,962、84,963、84,964、84,965、84,966、84,984、84,968、84,969、84,970、84,971、 84,972、84,973、84,984、84,984、84,976、84,977、84,978、84,979、84,980、84,981、84,982、 84,983、84,984、84,985、84,986、84,987、84,988、84,989、84,990、84,991、84,992、84,993、 84,994th, 84,995,84,996,84,997,84,998,84,999 or 85,000；90,000 to 91,000 include：90,001、 90,002、90,003、90,004、90,005、90,006、90,007、90,008、90,009、90,010、90,011、90,012、 90,013、90,014、90,015、90,016、90,017、90,018、90,019、90,020、90,021、90,022、90,023、 90,024、90,025、90,026、90,027、90,028、90,029、90,030、90,031、90,032、90,033、90,034、 90,035、90,036、90,037、90,038、90,039、90,040、90,041、90,042、90,043、90,044、90,045、 90,046、90,047、90,048、90,049、90,050、90,051、90,052、90,053、90,054、90,055、90,056、 90,057、90,090、90,059、90,060、90,061、90,062、90,063、90,064、90,065、90,066、90,090、 90,068、90,069、90,070、90,071、90,072、90,073、90,090、90,090、90,076、90,077、90,078、 90,079、90,080、90,081、90,082、90,083、90,090、90,085、90,086、90,087、90,088、90,089、 90,090、90,091、90,092、90,093、90,094、90,095、90,096、90,097、90,098、90,099、90,100、 90,101、90,102、90,103、90,104、90,105、90,106、90,107、90,110、90,111、90,112、90,113、 90,114、90,115、90,116、90,117、90,118、90,119、90,120、90,121、90,122、90,123、90,124、 90,125、90,126、90,127、90,128、90,129、90,130、90,131、90,132、90,133、90,134、90,135、 90,136、90,137、90,138、90,139、90,140、90,141、90,142、90,143、90,144、90,145、90,146、 90,147、90,148、90,149、90,150、90,151、90,152、90,153、90,154、90,155、90,156、90,157、 90,190、90,159、90,160、90,161、90,162、90,163、90,164、90,165、90,166、90,190、90,168、 90,169、90,170、90,171、90,172、90,173、90,190、90,190、90,176、90,177、90,178、90,179、 90,180、90,181、90,182、90,183、90,190、90,185、90,186、90,187、90,188、90,189、90,190、 90,191、90,192、90,193、90,194、90,195、90,196、90,197、90,198、90,199、90,200、90,201、 90,202、90,203、90,204、90,205、90,206、90,207、90,208、90,209、90,210、90,211、90,212、 90,213、90,214、90,215、90,216、90,217、90,218、90,219、90,220、90,221、90,222、90,223、 90,224、90,225、90,226、90,227、90,228、90,229、90,230、90,231、90,232、90,233、90,234、 90,235、90,236、90,237、90,238、90,239、90,240、90,241、90,242、90,243、90,244、90,245、 90,246、90,247、90,248、90,249、90,250、90,251、90,252、90,253、90,254、90,255、90,256、 90,257、90,290、90,259、90,260、90,261、90,262、90,263、90,264、90,265、90,266、90,290、 90,268、90,269、90,270、90,271、90,272、90,273、90,290、90,290、90,276、90,277、90,278、 90,279、90,280、90,281、90,282、90,283、90,290、90,285、90,286、90,287、90,288、90,289、 90,290、90,291、90,292、90,293、90,294、90,295、90,296、90,297、90,298、90,299、90,300、 90,301、90,302、90,303、90,304、90,305、90,306、90,307、90,308、90,309、90,310、90,311、 90,312、90,313、90,314、90,315、90,316、90,317、90,318、90,319、90,320、90,321、90,322、 90,323、90,324、90,325、90,326、90,327、90,328、90,329、90,330、90,331、90,332、90,333、 90,334、90,335、90,336、90,337、90,338、90,339、90,340、90,341、90,342、90,343、90,344、 90,345、90,346、90,347、90,348、90,349、90,350、90,351、90,352、90,353、90,354、90,355、 90,356、90,357、90,390、90,359、90,360、90,361、90,362、90,363、90,364、90,365、90,366、 90,390、90,368、90,369、90,370、90,371、90,372、90,373、90,390、90,390、90,376、90,377、 90,378、90,379、90,380、90,381、90,382、90,383、90,390、90,385、90,386、90,387、90,388、 90,389、90,390、90,391、90,392、90,393、90,394、90,395、90,396、90,397、90,398、90,399、 90,400、90,401、90,402、90,403、90,404、90,405、90,406、90,407、90,408、90,409、90,410、 90,411、90,412、90,413、90,414、90,415、90,416、90,417、90,418、90,419、90,420、90,421、 90,422、90,423、90,424、90,425、90,426、90,427、90,428、90,429、90,430、90,431、90,432、 90,433、90,434、90,435、90,436、90,437、90,438、90,439、90,440、90,441、90,442、90,443、 90,444、90,445、90,446、90,447、90,448、90,449、90,450、90,451、90,452、90,453、90,454、 90,455、90,456、90,457、90,490、90,459、90,460、90,461、90,462、90,463、90,464、90,465、 90,466、90,490、90,468、90,469、90,470、90,471、90,472、90,473、90,490、90,490、90,476、 90,477、90,478、90,479、90,480、90,481、90,482、90,483、90,490、90,485、90,486、90,487、 90,488、90,489、90,490、90,491、90,492、90,493、90,494、90,495、90,496、90,497、90,498、 90,499、90,500、90,501、90,502、90,503、90,504、90,505、90,506、90,507、90,508、90,509、 90,510、90,511、90,512、90,513、90,514、90,515、90,516、90,517、90,518、90,519、90,520、 90,521、90,522、90,523、90,524、90,525、90,526、90,527、90,528、90,529、90,530、90,531、 90,532、90,533、90,534、90,535、90,536、90,537、90,538、90,539、90,540、90,541、90,542、 90,543、90,544、90,545、90,546、90,547、90,548、90,549、90,550、90,551、90,552、90,553、 90,554、90,555、90,556、90,557、90,590、90,559、90,560、90,561、90,562、90,563、90,564、 90,565、90,566、90,590、90,568、90,569、90,570、90,571、90,572、90,573、90,590、90,590、 90,576、90,577、90,578、90,579、90,580、90,581、90,582、90,583、90,590、90,585、90,586、 90,587、90,588、90,589、90,590、90,591、90,592、90,593、90,594、90,595、90,596、90,597、 90,598、90,599、90,600、90,601、90,602、90,603、90,604、90,605、90,606、90,607、90,608、 90,609、90,610、90,611、90,612、90,613、90,614、90,615、90,616、90,617、90,618、90,619、 90,620、90,621、90,622、90,623、90,624、90,625、90,626、90,627、90,628、90,629、90,630、 90,631、90,632、90,633、90,634、90,635、90,636、90,637、90,638、90,639、90,640、90,641、 90,642、90,643、90,644、90,645、90,646、90,647、90,648、90,649、90,650、90,651、90,652、 90,653、90,654、90,655、90,656、90,657、90,690、90,659、90,660、90,661、90,662、90,663、 90,664、90,665、90,666、90,690、90,668、90,669、90,670、90,671、90,672、90,673、90,690、 90,690、90,676、90,677、90,678、90,679、90,680、90,681、90,682、90,683、90,690、90,685、 90,686、90,687、90,688、90,689、90,690、90,691、90,692、90,693、90,694、90,695、90,696、 90,697、90,698、90,699、90,700、90,701、90,702、90,703、90,704、90,705、90,706、90,707、 90,708、90,709、90,710、90,711、90,712、90,713、90,714、90,715、90,716、90,717、90,718、 90,719、90,720、90,721、90,722、90,723、90,724、90,725、90,726、90,727、90,728、90,729、 90,730、90,731、90,732、90,733、90,734、90,735、90,736、90,737、90,738、90,739、90,740、 90,741、90,742、90,743、90,744、90,745、90,746、90,747、90,748、90,749、90,750、90,751、 90,752、90,753、90,754、90,755、90,756、90,757、90,790、90,759、90,760、90,761、90,762、 90,763、90,764、90,765、90,766、90,790、90,768、90,769、90,770、90,771、90,772、90,773、 90,790、90,790、90,776、90,777、90,778、90,779、90,780、90,781、90,782、90,783、90,790、 90,785、90,786、90,787、90,788、90,789、90,790、90,791、90,792、90,793、90,794、90,795、 90,796、90,797、90,798、90,799、90,800、90,801、90,802、90,803、90,804、90,805、90,806、 90,807、90,808、90,809、90,810、90,811、90,812、90,813、90,814、90,815、90,816、90,817、 90,818、90,819、90,820、90,821、90,822、90,823、90,824、90,825、90,826、90,827、90,828、 90,829、90,830、90,831、90,832、90,833、90,834、90,835、90,836、90,837、90,838、90,839、 90,840、90,841、90,842、90,843、90,844、90,845、90,846、90,847、90,848、90,849、90,850、 90,851、90,852、90,853、90,854、90,855、90,856、90,857、90,890、90,859、90,860、90,861、 90,862、90,863、90,864、90,865、90,866、90,890、90,868、90,869、90,870、90,871、90,872、 90,873、90,890、90,890、90,876、90,877、90,878、90,879、90,880、90,881、90,882、90,883、 90,890、90,885、90,886、90,887、90,888、90,889、90,890、90,891、90,892、90,893、90,894、 90,895、90,896、90,897、90,898、90,899、90,900、90,901、90,902、90,903、90,904、90,905、 90,906、90,907、90,908、90,909、90,910、90,911、90,912、90,913、90,914、90,915、90,916、 90,917、90,918、90,919、90,920、90,921、90,922、90,923、90,924、90,925、90,926、90,927、 90,928、90,929、90,930、90,931、90,932、90,933、90,934、90,935、90,936、90,937、90,938、 90,939、90,940、90,941、90,942、90,943、90,944、90,945、90,946、90,947、90,948、90,949、 90,950、90,951、90,952、90,953、90,954、90,955、90,956、90,957、90,990、90,959、90,960、 90,961、90,962、90,963、90,964、90,965、90,966、90,990、90,968、90,969、90,970、90,971、 90,972、90,973、90,990、90,990、90,976、90,977、90,978、90,979、90,980、90,981、90,982、 90,983、90,990、90,985、90,986、90,987、90,988、90,989、90,990、90,991、90,992、90,993、 90,994th, 90,995,90,996,90,997,90,998,90,999 or 91,000；And 96,000 to 97,000 include：96, 001、96,002、96,003、96,004、96,005、96,006、96,007、96,008、96,009、96,010、96,011、96, 012、96,013、96,014、96,015、96,016、96,017、96,018、96,019、96,020、96,021、96,022、96, 023、96,024、96,025、96,026、96,027、96,028、96,029、96,030、96,031、96,032、96,033、96, 034、96,035、96,036、96,037、96,038、96,039、96,040、96,041、96,042、96,043、96,044、96, 045、96,046、96,047、96,048、96,049、96,050、96,051、96,052、96,053、96,054、96,055、96, 056、96,057、96,096、96,059、96,060、96,061、96,062、96,063、96,064、96,065、96,066、96, 096、96,068、96,069、96,070、96,071、96,072、96,073、96,096、96,096、96,076、96,077、96, 078、96,079、96,080、96,081、96,082、96,083、96,096、96,085、96,086、96,087、96,088、96, 089、96,096、96,091、96,092、96,093、96,094、96,095、96,096、96,097、96,098、96,099、96, 100、96,101、96,102、96,103、96,104、96,105、96,106、96,107、96,110、96,111、96,112、96, 113、96,114、96,115、96,116、96,117、96,118、96,119、96,120、96,121、96,122、96,123、96, 124、96,125、96,126、96,127、96,128、96,129、96,130、96,131、96,132、96,133、96,134、96, 135、96,136、96,137、96,138、96,139、96,140、96,141、96,142、96,143、96,144、96,145、96, 146、96,147、96,148、96,149、96,150、96,151、96,152、96,153、96,154、96,155、96,156、96, 157、96,196、96,159、96,160、96,161、96,162、96,163、96,164、96,165、96,166、96,196、96, 168、96,169、96,170、96,171、96,172、96,173、96,196、96,196、96,176、96,177、96,178、96, 179、96,180、96,181、96,182、96,183、96,196、96,185、96,186、96,187、96,188、96,189、96, 196、96,191、96,192、96,193、96,194、96,195、96,196、96,197、96,198、96,199、96,200、96, 201、96,202、96,203、96,204、96,205、96,206、96,207、96,208、96,209、96,210、96,211、96, 212、96,213、96,214、96,215、96,216、96,217、96,218、96,219、96,220、96,221、96,222、96, 223、96,224、96,225、96,226、96,227、96,228、96,229、96,230、96,231、96,232、96,233、96, 234、96,235、96,236、96,237、96,238、96,239、96,240、96,241、96,242、96,243、96,244、96, 245、96,246、96,247、96,248、96,249、96,250、96,251、96,252、96,253、96,254、96,255、96, 256、96,257、96,296、96,259、96,260、96,261、96,262、96,263、96,264、96,265、96,266、96, 296、96,268、96,269、96,270、96,271、96,272、96,273、96,296、96,296、96,276、96,277、96, 278、96,279、96,280、96,281、96,282、96,283、96,296、96,285、96,286、96,287、96,288、96, 289、96,296、96,291、96,292、96,293、96,294、96,295、96,296、96,297、96,298、96,299、96, 300、96,301、96,302、96,303、96,304、96,305、96,306、96,307、96,308、96,309、96,310、96, 311、96,312、96,313、96,314、96,315、96,316、96,317、96,318、96,319、96,320、96,321、96, 322、96,323、96,324、96,325、96,326、96,327、96,328、96,329、96,330、96,331、96,332、96, 333、96,334、96,335、96,336、96,337、96,338、96,339、96,340、96,341、96,342、96,343、96, 344、96,345、96,346、96,347、96,348、96,349、96,350、96,351、96,352、96,353、96,354、96, 355、96,356、96,357、96,396、96,359、96,360、96,361、96,362、96,363、96,364、96,365、96, 366、96,396、96,368、96,369、96,370、96,371、96,372、96,373、96,396、96,396、96,376、96, 377、96,378、96,379、96,380、96,381、96,382、96,383、96,396、96,385、96,386、96,387、96, 388、96,389、96,396、96,391、96,392、96,393、96,394、96,395、96,396、96,397、96,398、96, 399、96,400、96,401、96,402、96,403、96,404、96,405、96,406、96,407、96,408、96,409、96, 410、96,411、96,412、96,413、96,414、96,415、96,416、96,417、96,418、96,419、96,420、96, 421、96,422、96,423、96,424、96,425、96,426、96,427、96,428、96,429、96,430、96,431、96, 432、96,433、96,434、96,435、96,436、96,437、96,438、96,439、96,440、96,441、96,442、96, 443、96,444、96,445、96,446、96,447、96,448、96,449、96,450、96,451、96,452、96,453、96, 454、96,455、96,456、96,457、96,496、96,459、96,460、96,461、96,462、96,463、96,464、96, 465、96,466、96,496、96,468、96,469、96,470、96,471、96,472、96,473、96,496、96,496、96, 476、96,477、96,478、96,479、96,480、96,481、96,482、96,483、96,496、96,485、96,486、96, 487、96,488、96,489、96,496、96,491、96,492、96,493、96,494、96,495、96,496、96,497、96, 498、96,499、96,500、96,501、96,502、96,503、96,504、96,505、96,506、96,507、96,508、96, 509、96,510、96,511、96,512、96,513、96,514、96,515、96,516、96,517、96,518、96,519、96, 520、96,521、96,522、96,523、96,524、96,525、96,526、96,527、96,528、96,529、96,530、96, 531、96,532、96,533、96,534、96,535、96,536、96,537、96,538、96,539、96,540、96,541、96, 542、96,543、96,544、96,545、96,546、96,547、96,548、96,549、96,550、96,551、96,552、96, 553、96,554、96,555、96,556、96,557、96,596、96,559、96,560、96,561、96,562、96,563、96, 564、96,565、96,566、96,596、96,568、96,569、96,570、96,571、96,572、96,573、96,596、96, 596、96,576、96,577、96,578、96,579、96,580、96,581、96,582、96,583、96,596、96,585、96, 586、96,587、96,588、96,589、96,596、96,591、96,592、96,593、96,594、96,595、96,596、96, 597、96,598、96,599、96,600、96,601、96,602、96,603、96,604、96,605、96,606、96,607、96, 608、96,609、96,610、96,611、96,612、96,613、96,614、96,615、96,616、96,617、96,618、96, 619、96,620、96,621、96,622、96,623、96,624、96,625、96,626、96,627、96,628、96,629、96, 630、96,631、96,632、96,633、96,634、96,635、96,636、96,637、96,638、96,639、96,640、96, 641、96,642、96,643、96,644、96,645、96,646、96,647、96,648、96,649、96,650、96,651、96, 652、96,653、96,654、96,655、96,656、96,657、96,696、96,659、96,660、96,661、96,662、96, 663、96,664、96,665、96,666、96,696、96,668、96,669、96,670、96,671、96,672、96,673、96, 696、96,696、96,676、96,677、96,678、96,679、96,680、96,681、96,682、96,683、96,696、96, 685、96,686、96,687、96,688、96,689、96,696、96,691、96,692、96,693、96,694、96,695、96, 696、96,697、96,698、96,699、96,700、96,701、96,702、96,703、96,704、96,705、96,706、96, 707、96,708、96,709、96,710、96,711、96,712、96,713、96,714、96,715、96,716、96,717、96, 718、96,719、96,720、96,721、96,722、96,723、96,724、96,725、96,726、96,727、96,728、96, 729、96,730、96,731、96,732、96,733、96,734、96,735、96,736、96,737、96,738、96,739、96, 740、96,741、96,742、96,743、96,744、96,745、96,746、96,747、96,748、96,749、96,750、96, 751、96,752、96,753、96,754、96,755、96,756、96,757、96,796、96,759、96,760、96,761、96, 762、96,763、96,764、96,765、96,766、96,796、96,768、96,769、96,770、96,771、96,772、96, 773、96,796、96,796、96,776、96,777、96,778、96,779、96,780、96,781、96,782、96,783、96, 796、96,785、96,786、96,787、96,788、96,789、96,796、96,791、96,792、96,793、96,794、96, 795、96,796、96,797、96,798、96,799、96,800、96,801、96,802、96,803、96,804、96,805、96, 806、96,807、96,808、96,809、96,810、96,811、96,812、96,813、96,814、96,815、96,816、96, 817、96,818、96,819、96,820、96,821、96,822、96,823、96,824、96,825、96,826、96,827、96, 828、96,829、96,830、96,831、96,832、96,833、96,834、96,835、96,836、96,837、96,838、96, 839、96,840、96,841、96,842、96,843、96,844、96,845、96,846、96,847、96,848、96,849、96, 850、96,851、96,852、96,853、96,854、96,855、96,856、96,857、96,896、96,859、96,860、96, 861、96,862、96,863、96,864、96,865、96,866、96,896、96,868、96,869、96,870、96,871、96, 872、96,873、96,896、96,896、96,876、96,877、96,878、96,879、96,880、96,881、96,882、96, 883、96,896、96,885、96,886、96,887、96,888、96,889、96,896、96,891、96,892、96,893、96, 894、96,895、96,896、96,897、96,898、96,899、96,900、96,901、96,902、96,903、96,904、96, 905、96,906、96,907、96,908、96,909、96,910、96,911、96,912、96,913、96,914、96,915、96, 916、96,917、96,918、96,919、96,920、96,921、96,922、96,923、96,924、96,925、96,926、96, 927、96,928、96,929、96,930、96,931、96,932、96,933、96,934、96,935、96,936、96,937、96, 938、96,939、96,940、96,941、96,942、96,943、96,944、96,945、96,946、96,947、96,948、96, 949、96,950、96,951、96,952、96,953、96,954、96,955、96,956、96,957、96,996、96,959、96, 960、96,961、96,962、96,963、96,964、96,965、96,966、96,996、96,968、96,969、96,970、96, 971、96,972、96,973、96,996、96,996、96,976、96,977、96,978、96,979、96,980、96,981、96, 982、96,983、96,996、96,985、96,986、96,987、96,988、96,989、96,996、96,991、96,992、96, 993rd, 96,994,96,995,96,996,96,997,96,998,96,999 or 97,000.
The example of insertion point is provided in table 2.As described above, the site is based on GenBank registration numbers AJ004801 or its equivalent.
Disclosure teaches the load of the BoHV-1 vaccines comprising the BoHV-1 genomes from BoHV-1 strains V155 Body, it has the Heterologous genetic material of at least one antigen of the coding from cattle disease substance, and the inhereditary material is in BoHV-1 bases Because of the site insertion in group, the site is selected from BoHV-1 reference sequences GenBank registration numbers AJ004801 nucleotides 16600 To 16700,22400 to 22500；40700 to 40800；58000 to 59000；67000 to 68000；74000 to 76000； 84000 to 85000；Function equivalent site in 90000 to 91000 and 96000 to 97000 or other BoHV-1.Example bag Include site listed in table 2.
There are a series of Heterologous genetic materials and can be inserted into other sites therein.Include all such sites herein.
The another aspect instructed herein is the method for preparing the vaccine at least one antigen from cattle disease substance, institute The method of stating includes：
(i) the BoHV-1 genomes from low toxicity power BoHV-1 are incorporated to bacterial artificial chromosome (BAC) carrier to be formed BoHV-1 precoded vector BAC constructs；
(ii) inhereditary material for encoding at least one antigen is inserted into the BoHV-1 via induction type recombination system to preload In body BAC constructs BoHV-1-BAC (rBoHV-1-BAC) carrier is recombinated to produce；
(iii) converted in bacterial host and expand the rBoHV-1-BAC carriers；And
(iv) purified from the bacterial host and separate the rBoHV-1-BAC carriers and be configured to the carrier Vaccine combination.
Disclosure teaches bovid (ox of such as feedlot, the ox of Milk Products Plant and other confinement feedings Ox) vaccination protocols.The administration of bacterin preparation can be carried out by a series of locally and systemically schemes, such as intranasal, oral, Intramuscular, sublingual, intravenous, subcutaneous or intra-arterial injection, skin spraying or including other in being applied in intravaginal and rectum Conventional route.Intra-nasal route is especially effective.The preparation can be standard pharmaceutical formulation.In one embodiment, the system Agent is lyophilized and using preceding reconstruct.
Therefore, generally by this paper vaccine be prepared as or suitable for be reconstructed into injectable or can nasal administration liquid solution or Supensoid agent or lyophilized formulations.The vaccine can also be emulsification.Before use, pharmaceutically acceptable diluent, carrier or excipient.Epidemic disease Seedling preparation can also include auxiliary substance such as wetting agent or emulsifying agent, pH buffer and/or adjuvant.
Therefore, the bacterin preparation for including the BoHV-1 genomes from low toxicity power BoHV-1, the BoHV- are taught herein The hereditary thing that 1 genome has with BoHV-1 is heterologous, at least one antigen of coding between BoHV-1 genes is assembled in insertion two Matter, wherein described insert the expression for not cutting BoHV-1 genes substantially；The preparation is for lyophilized form or also comprising one kind or more A variety of pharmaceutical acceptable carrier, diluent and/or excipient.
The preparation can also include the BoHV-1 genomes from low toxicity power BoHV-1, and being produced when it is expressed can be to it Produce immune response antigen, the BoHV-1 genomes also include with BoHV-1 it is heterologous, insertion two convergence BoHV-1 genes Between at least one antigen of coding inhereditary material, wherein described insert the expression and its for not cutting BoHV-1 genes substantially Middle heterologous antigen induces immune response；The preparation is lyophilized form or also includes one or more of pharmaceutical acceptable carrier, dilution Agent and/or excipient.
The present invention also allows for diagnostic assay to distinguish the ox of vaccine inoculation and non-vaccine inoculation in serology Section animal.Generally, the antibody for carrying out standard antibody measure to produce after detecting it is contemplated that being inoculated with BoHV-1 recombinant vaccines. It is label albumen, such as green fluorescent protein by one of heterologous antigen of BoHV-1 vector expressions in one embodiment. It facilitates label and as proprietary label for the effect for vaccine inoculation.
Present document relates to the business method for managing bovid in limited place, the business method is included for coming from Thus the propagation probability that at least one antigen of cattle disease substance reduces BRDC to bovid vaccine inoculation maintains bovid Economic feasibility, methods described include to bovid apply humoral immunity inductivity or cell-mediated immunity inductivity it is effective Amount the BoHV-1 genomes from low toxicity power BoHV-1, the BoHV-1 genomes have with BoHV-1 it is heterologous, insertion two The inhereditary material of at least one antigen of coding between BoHV-1 genes is assembled, wherein the insertion does not cut BoHV-1 bases substantially The expression of cause.
The business method incorporates the Managed Solution for raising bovid and may include to be used for practitioner's test BRDC, maintain to bovid vaccine inoculation and afterwards herd animals serological analysis service charge.
Performing the cost of the business method can be undertaken or be shared to consumption by the owner of bovid in groups Person.
Serology test also enables epidemiological study to carry out.
Therefore, there is provided herein the vaccine at least one antigen from cattle disease substance, the vaccine is included and come from Low toxicity power BoHV-1 1 type bovine herpes virus (BoHV-1) genome, the BoHV-1 genomes have heterologous, slotting with BoHV-1 Enter the inhereditary material of the two at least one antigen of the coding assembled between BoHV-1 genes, wherein the insertion is not cut substantially The expression of BoHV-1 genes.In one embodiment, it will be encoded extremely via induction type recombination system (such as being recombinated via GET) In a kind of inhereditary material insertion BoHV-1 genomes of few antigen.
The inhereditary material for encoding at least one antigen is routinely inserted to the poly- adenosine of two meeting pcl genes in following site Between polyadenylation signal, the site be selected from BoHV-1 reference sequences GenBank registration numbers AJ004801 16600 to 16700, 22400 to 22500；40700 to 40800；58000 to 59000；67000 to 68000；74000 to 76000；84000 to 85000；90000 to 91000；And the function equivalent site in 96000 to 97000 or other BoHV-1.In an embodiment party In case, at least one antigen is inserted between two meeting pcl genes selected from the site listed by table 2.In another embodiment In, at least one antigen is inserted between two meeting pcl genes selected from 16602 to 16603 and 22421 to 22470 site.
Routinely, the vaccine, which is provided, produces BoHV-1 antigens and/or the BoHV-1 selected from following antigen：From cattle disease The antigen of viral diarrhea virus (BVDV) and the antigen from microorganism.
The example of BVDV antigens is glycoprotein E 0 and glycoprotein E 2.Example as the microorganism of antigenic source includes：Ox Mycoplasma (Mycoplasma bovis), salmonella (Salmonella) class, multocida (Pateurella Multocida), Mannheimia haemolytica (Manhiemia haemolytica) and Haemophilus somnus (Haemophilus somnus)。
In one embodiment, low toxicity power BoHV-1 strains are strain V155.This paper vaccine can be also included via limit Property endonuclease digestion processed and insert coding other antigens other inhereditary material.Also can be medicine group by the vaccine formulation Compound, is for example suitable for the pharmaceutical composition of nasal administration.
Cover herein for for method of at least one antigen from cattle disease substance to bovid vaccine inoculation, its 1 type from low toxicity power BoHV-1 of humoral immunity inductivity or cell-mediated immunity inductivity effective dose is applied to bovid Bovine herpes virus (BoHV-1) genome, the BoHV-1 genomes have with BoHV-1 it is heterologous, insertion two convergence BoHV-1 The inhereditary material of at least one antigen of coding between gene, wherein described insert the expression for not cutting BoHV-1 genes substantially. The inhereditary material insertion BoHV-1 bases of at least one antigen can will be encoded via induction type recombination system (such as being recombinated via GET) Because in group.
As described above, poly- gland of the inhereditary material in two meeting pcl genes of following site insertion that at least one antigen will be encoded Between nucleotide signal, the site be selected from BoHV-1 reference sequences GenBank registration numbers AJ004801 16600 to 16700, 22400 to 22500；40700 to 40800；58000 to 59000；67000 to 68000；74000 to 76000；84000 to 85000；90000 to 91000；And the function equivalent site in 96000 to 97000 or other BoHV-1, it is included in two Can between pcl gene selected from 16600 to 16612 and 22400 to 22493 site, be included between two meeting pcl genes and be selected from 16602 to 16603 and 22421 to 22470 site, it is included between two meeting pcl genes selected from the site listed by table 2.
According to this method, BoHV-1 produces BoHV-1 antigens and/or selected from following at least one antigen：From bovine viral The antigen of property diarrhea virus (BVDV) and the antigen from microorganism.
The example of BVDV antigens is glycoprotein E 0 or glycoprotein E 2.The example of microorganism be Mycoplasma bovis, Salmonella mushroom, Multocida, Mannheimia haemolytica or Haemophilus somnus.
As described above, low toxicity power BoHV-1 strains include strain V155.Also it can be inserted via digestion with restriction enzyme Encode the other inhereditary material of other antigens.
The method for preparing vaccine at least one antigen from cattle disease substance, it is achieved by the following way：
(i) the BoHV-1 genomes from low toxicity power BoHV-1 are incorporated to bacterial artificial chromosome (BAC) carrier to be formed BoHV-1 precoded vector BAC constructs；
(ii) inhereditary material for encoding at least one antigen is inserted into the BoHV-1 via induction type recombination system to preload In body BAC constructs BoHV-1-BAC (rBoHV-1-BAC) carrier is recombinated to produce；
(iii) converted in bacterial host and expand the rBoHV-1-BAC carriers；And
(iv) purified from the bacterial host and separate the rBoHV-1-BAC carriers and be configured to the carrier Vaccine combination.
Induction type recombination system and insertion point, antigen and its source is disclosed as above.
Also relate to transfect the culture cell of rBoHV-1-BAC carriers.
The method to ox vaccine inoculation for cattle respiratory disease syndrome (BRDC) is taught herein, and methods described includes The 1 type ox bleb from low toxicity power BoHV-1 of humoral immunity inductivity or cell-mediated immunity inductivity effective dose is applied to ox Viral (BoHV-1) genome, the BoHV-1 genomes have with BoHV-1 is heterologous, insertion two assemble BoHV-1 genes it Between at least one antigen of coding inhereditary material, wherein described insert the expression for not cutting BoHV-1 genes substantially.
1 type bovine herpes virus (BoHV-1) genome from low toxicity power BoHV-1 is also contemplated by herein is directed to cattle disease in manufacture Substance is to the purposes in the medicine of ox vaccine inoculation, and the BoHV-1 genomes have and BoHV-1 is heterologous, two convergences of insertion The inhereditary material of at least one antigen of coding between BoHV-1 genes, wherein the insertion does not cut BoHV-1 genes substantially Expression.
This paper some aspects are described by following non-limiting example.
Embodiment 1
BoHV-1 strains
Low toxicity power BoHV-1 strains used are strain V155, are initially described by foregoing Snowden (1964).Nucleotides Position Number is based on GenBank registration numbers AJ004801.
Embodiment 2
Recombinate the structure of 1 type bovine herpes virus (BoHV-1)
Making BoHV-1 strains V155, (the anti-pestivirus of ATCC CRL-11883, MDBK cells derives in CRIB-1 cells Thing) middle breeding.At 37 DEG C, by CRIB-1 cells maintain comprising antibiotic/antifungal agent, nonessential amino acid, Han Keshi essential essential mediums (Hank ' the s minimal of glutaMAX, 25mM Hepes and 5%v/v donor calf serums Essential medium, H-MEM) in.Unless otherwise indicated, all reagents otherwise for cell and virus breeding are all obtained from Invitrogen Australia。
Before transfection 24h by CRIB-1 cells with 5 × 105Individual cells/well is put into six orifice plates (Corning), and In 5%v/v CO at 37 DEG C2Atmosphere in be incubated.For transfection every time, 1 μ g to 2 μ g DNA are diluted to using OptiMEM 100 μ l are simultaneously mixed with 8 μ l Lipofectamine that 100 μ l are diluted to using OptiMEM.By gained mixture at room temperature 45 minutes are incubated to form lipid/DNA compounds.Reaction volume is increased to 1mL and is added to using OptiMEM and has been used The twice-washed cell monolayers of OptiMEM.Then 5%v/v CO are used at 37 DEG C2It is incubated after transfection individual layer 16h to 18h, Addition includes the 1ml OptiMEM of 10%v/v donor calf serums.After 24h, remove transfection liquid and change maintenance culture medium. 7 days individual layers are reached after transfection produces CPE.
In order to purify BoHV-1 genomic DNAs, body cell is become with BoHV-1 strains V155 infection MDBK for 5 times in MOI CRIB-1 simultaneously makes infection carry out to completion.Then, by clarifying cell culture supernatant within 10 minutes with 5000g centrifugations.Pass through 2 hours BoHV-1 virion to precipitate maturation are centrifuged with 120000g.It is basic using Qiagen genome DNA extracting reagent kits On according to come described in supplier from precipitation virus in reclaim BoHV-1 genomic DNAs.Viral pellet is set to be resuspended in originating Supernatant volume 1:In the extracting genome DNA buffer solution of 65 ratios.After being eluted from post, BoHV-1DNA is stored up with aliquot It is stored at -20 DEG C.1 μ g to 2 μ g DNA is digested using HindIII to be used for compared with known BoHV-1 digestion spectrum.
In order to be conducive to inserting transgenosis using GET homologous recombinations in BoHV-1 TK genes, build missing/insertion and carry Body pTK del.The carrier includes two sections (TKleft and TKright) of BOHV-1 thymidine kinase genes, for use as restructuring Arm.
Use Taq polymerase buffer solution (10mM Tris-HCl, 1.5mM MgCl2、50mM KCl、pH 8.5)、dATP、 Each 1.25mM of dCTP, dGTP and dTTP, 12.5 μM of every kind of primer, 1U Taq archaeal dna polymerases, 10ng to 20ng genome DNA5%v/v DMSO, 10%v/v glycerine enter performing PCR to BoHV-1 genomes.The end reaction volume of these components is 20 μ l.
The PCR cycle condition used is：94 DEG C are denatured 4 minutes；94 DEG C 20 seconds, 60 DEG C 20 seconds and 72 DEG C 120 seconds, circulation 35 times；10 minutes and 4 DEG C are then held in followed by 72 DEG C.Circulate in Hybaid Sprint thermal cyclers and carry out.Following After ring, dissociate PCR primer on 1%w/v Ago-Gels, cleaved products and using Qiagen gel extraction kits according to The explanation of manufacturer reclaims DNA.
By PCR by purified BoHV-1 genomic DNA amplifications TKleft and TKright.After amplification, use Qiagen PCR purification columns are according to the explanation purified product of manufacturer.With KpnI and SalI digestion TKleft PCR primers, through solidifying Glue purification is simultaneously connected into the pBluescript-SK+ (Stratagene) also digested by KpnI/SalI.Confirmed by being sequenced The presence of TKleft products.After EcoRI and SpeI digestion, TKright PCR primers are cloned into using Standard cloning methods In plasmid containing TKleft products.Gained plasmid is referred to as pTKdel (referring to Mahoney etc. (2002), ibid).Show in table 3 The primer expanded for TK target areas PCR is gone out.NsiI integrations are entered in Tkleft5' and Tkright3' primers so that Transgene product can be cut off for reconstruction experiment from pTKdel carriers by obtaining.Exist between two TK intersection regions to permit Perhaps four unique restriction endonuclease sites of BoHV-1 genomes are inserted and be transferred to transgenosis.
In order to which bacterial artificial chromosome (BAC) to be transferred to BoHV-1 genome, BAC carrier is digested with HindIII PBello-BAC II and through gel-purified.BAC carrier through digestion is connected to and digested and dephosphorylized by HindIII In pTKdel.After E.Coli strain X L1-Blue cells are transformed into, transformant is layered on comprising 12.5 μ g/ml chloramphenicol (CAP) and on the selective agar of 100 μ g/ml ampicillins.The DNA for being recovered from gained bacterium colony is cut off by using HindIII To confirm the insertion of BAC carrier.TK deletion fragments (TK-BAC) are cut off from pTKdel-BAC by using NsiI digestion, the TK lacks Fragment is lost comprising flank for TKleft and TKright BAC carrier and through gel-purified.
In order to promote the homologous recombination between BAC-TK fragments and BoHV-1 genomic DNAs, digested with NsiI purified BoHV-1DNA and with bacterial alkaline phosphatase (Pharmacia) dephosphorylation.Digested by BAC-TK fragments and through NsiI BoHV-1 genomic DNA cotransfections enter in above-mentioned CRIB-1 cells.After 18h to 24h, remove transfection mixture and be replaced by bag The complete H-MEM of N containing 2mM, N'- Vitro By Hexamethylene Bisacetamide (ICN) promotes viral gene to transcribe.Gained vial supernatant In the intracellular passages of CRIB-1 once.It is specific by chloramphenicol resistance gene using primer pair ChloramF and ChloramR PCR determines to confirm in BAC carrier insertion BoHV-1 genomes.
(0.45%v/v Triton X- are included by using 10 μ l lysis buffers of the 10mg/ml Proteinase Ks containing 2 μ l The 10mM Tris-HCl pH 8.0 of 100 and 0.45%v/v polysorbas20s) 10 μ l vial supernatants are incubated, then incubated at 60 DEG C 2h is educated to prepare pcr template.Proteinase K is set to be inactivated 15 minutes at 95 DEG C.The prepared product using 1 μ l to 2 μ l is used as template To enter performing PCR reaction.CAP resistant genes are carried out after RCR detections in BoHV-1 genomes, are reclaimed from above-mentioned virion Most gene group DNA.In order to promote conversion and growth of the purified BoHV-1 genomic DNAs in bacterial host, mark is used Quasi- connection method is cyclized to it.By the connection mixture electroporation of equal portions into E.coli DH10B cells (1.5kV, 100 Ω, 25 μ F, Electroporator II；Invitrogen,San Diego).After electroporation, DH10B cells are recovered in 5h to 6h is incubated in 960 μ l SOB and under gentle concussion in 37 DEG C.Equal portions electroporation mix is layered on comprising 12.5 μ g/ On ml CAP LB flat boards.Bacterium colony is set to grow 24h to 48h at 37 DEG C.
CAP resistant clones are inoculated into the 5mL LB nutrient solutions comprising 12.5 μ g/ml CAP and grown at 37 DEG C 16h.BAC DNA are reclaimed using standard alkaline lysis protocol.HindIII digestion spectrum and the BoHV-1 genes that these BAC are cloned Group DNA HindIII spectrums compare.The BAC clone similar with the HindIII of genomic DNA spectrums is transfected into above-mentioned CRIB-1 In cell.Daily to transfection is considered as that typical BoHV-1 CPE is monitored.
To be perforated by common-battery, (pGETrec passes through the Murdoch Childrens of Melbourne, AUS to plasmid Research Institute are obtained) BoHV-1BAC that is produced in DH10B recombinates electroporation to carrying pBACBHV37 In the DH10B cells of (BoHV-1 infection clones).In the agar comprising 12.5 μ g/ml CAP and 100 μ g/ml ampicillins Upper selection includes the DH10B cells of two kinds of plasmids.During exponential phase cells using 0.2%w/v arabinoses induction come The Electrocompetent cells for preparing Double DH10B cells enable to homologous recombination as discussed previously.Then, PCR is expanded Purpose transgenosis electroporation into these cells.From electroporation container reclaim after, make under earthquake cell in 37 DEG C Recover in SOC nutrient solutions at least 6 hours.The cell of all recoveries and appropriate antibiotic are put on chloramphenicol selection flat board To select transgenosis.
In order to confirm that BoHV-1 genomes can be modified using GET restructuring, two kinds of different rBoHV-1 viruses are set up.Build Two kinds of different rBOHV-1 have been found, wherein：
(i) missing coding gE gene.This is used by PCR by transposons EZ::TN<KAN-1>(Epicentre Technologies) minimum kanamycin resistance cassette (the Kan of amplificationR) come carry out.PCR primer (gE-KanF and the gE- used KanR) comprising 50bp and the 5' ends (the base number of the 121671st to 121720 of AJ004801) of gE genes and 3' ends (the base number of the 123371st to 123420 of AJ004801) homologous region.Products therefrom gE-KanRLength be 1300bp And it is reclaimed after according to the explanation of manufacturer using the separation of gel extraction kit (Qiagen) Ago-Gel.
(ii) heterologous gene of encoding green fluorescent protein (GFP) is inserted in the Nsil sites REMR of V155 genomes. This is by the way that GFP expression cassettes are inserted positioned at UL46 (prion body envelope protein) and the UL44 (restructuring promoted using pGETrec GC) between genome in carry out.Gel extraction kit (Qiagen) agar is being used according to the explanation of manufacturer Products therefrom is reclaimed after sugared gel separation.
Other sites are selected from those sites listed by table 2.
By the about 200ng PCR primer electroporation through gel-purified to above-mentioned Electrocompetent DH10B (pBACBHV37, PGETrec in).By will restructuring bacterium colony be layered on the LB flat boards comprising 12.5 μ g/ml CAP and 50 μ g/ml kanamycins come pair It is identified.PCR and Southern engram analysis confirm gE genes by KanRBox is substituted.
The virus genom DNA for Southern traces is purified as described below.BoHV-1 is used in the case where MOI is 5 Strain V155 infection cells individual layer and infection is carried out until about 40% cell shows the form of " bunched (rounding up) " Type.Growth medium is removed, then the individual layer is gently washed twice with PBS at 0 DEG C.To each flask (4ml/ 175cm2) add 0 DEG C of cell lysis buffer solution (pH 7.3,10mM sodium phosphate contain 1%v/v Nonidet P-40) and shake Dynamic flask so that lysis buffer is contacted with whole individual layer.Remove lysis buffer and be put on ice, it is lower to each gently shaking Individual flask adds other 4mL, removes it and is added to initial cracking solution.Cracking is made in 10 minutes with 4300g centrifugations at 4 DEG C Liquid is clarified.Collect supernatant and centrifuged 100 minutes with 112700g at 5 DEG C.Viral pellet is set to be resuspended in 500 μ l G2 buffer solutions (Qiagen).To the viral pellet addition Proteinase K (25 μ l, 10mg/ml) of resuspension, then 1h is incubated at 50 DEG C.It is used as described below Genomic tip 20/G (Qiagen) reclaim genomic viral DNA.With 1ml QBT buffer solutions balance genomic tip 20/G.The material handled through Proteinase K is diluted with isometric QBT buffer solutions and it is carried in genomic tip (660 μ l/ Tip on).Tip is washed twice with 7.5ml QC buffer solutions.At 50 DEG C virus is eluted with 2 × 1.5ml QF buffer solutions from tip DNA.DNA is precipitated, washed once and be resuspended in 50 μ l 10mM Tris-HCl (pH is 8.5) with 75%v/v ethanol.
Field inversion gel electrophoresis (field inversion gel electrophoresis, FIGE) is used at 5 DEG C Equipment (Biorad) makes the electrophoresis 13.5h on 1%w/v gels in 0.5X tbe buffer liquids of the DNA sample through limiting enzymic digestion. Under 180V forward voltage and 120V backward voltage, the switching time gradient is linear for 0.1s's to 2s.Made by capillary With DNA fragmentation is transferred into the uncharged films of Hybond-N (Amersham) and DNA is fixed on film using ultraviolet.With DIG-II-dUTP (Roche Molecular Biosystems) label probe.Pass through using comprising following reactant mixture PCR carrys out synthesising probing needle：Taq polymerase buffer solution (10mM Tris-HCl, 1.5mM MgCl in 20 μ l final volumes2、50mM KCl, pH8.5), each 1.25mM of dATP, dCTP, dGTP and dTTP, 1.25 μM of DIG-II-dUTP, 1U Taq polymerase (Roche Molecular Biosystems) and 5ng template DNAs.PCR reaction conditions are：94 DEG C 3 minutes；94 DEG C 30 seconds, 55 DEG C 30 seconds, 68 DEG C 1 minute, circulate 35 times；68 DEG C 6 minutes.After gel-purified, PCR probes are made to hybridize 12h to 16h with film at 55 DEG C (being used for the DIG system users Guides of filter hybridization, Roche Molecular Biosystems).In hybrid heater (Hybaid) Hybridized in revolving bottle.At room temperature, film is washed in culture dish on earthquake platform.
A variety of rBoHV-1 duplicating dynamics are determined using standard virus technology.In brief, in 5%v/v CO2 Atmosphere in making 1TCID at 37 DEG C50Virus to be laid in 24 hole plates 1 × 105CRIB-1 cell infections 90 minutes.So Extracellular virus is inactivated by adding sodium citrate solution (40mM sodium citrates, 10mM KCl, 135mM NaCl, pH 3.0) afterwards, Then cellular layer is washed twice with PBS and 1ml maintenance culture medium is added, and in 5%CO2Cultivated in atmosphere at 37 DEG C. 2h, 4h, 6h, 12h, 24h, 48h and 72h collect vial supernatant and cell precipitation and are frozen in -70 DEG C until making after infection With.Then the TCID of each supernatant at each time point is determined50, in triplicate.After a freeze/thaw, also survey The TCID of the intracellular virus at fixed each time point50, in triplicate.
Table 3
Embodiment 3
RBoHV-1 can infectiousness (transmissibility) evaluation
The purpose of the experiment be to determine restructuring (genetically modified, GM) virus whether can by vaccine inoculation Niu Chuanran To positioned at from other oxen at vaccine inoculation ox different distance.Other ruminants (sheep and goat) also are located at relative inoculation epidemic disease At the diverse location of the ox of seedling, so that it is determined that whether GM viruses can be infected to these ruminant species.
Before zoopery is started, all ox, sheep and goat are to BoHV-1 and bovine viral diarrhea virus (BVDV) specific antibodies are negative.The animal of each species is randomly assigned to following group：
Sentry post group (sentinel group) A：[sheep and goat is housed in by attention for ox (4), sheep (4) and goat (4) It is located at together] at about 23 meters from animal house；
Sentry post group B：Ox (2 × 2), sheep (4) and goat (4), in from the western end fence of animal house；
Sentry post group C：Ox (2 × 2), sheep (4) and goat (4), the fence positioned at animal house the east Yu vaccine inoculation ox opposite In；And
Vaccine contact group：The ox (4) and contact ox (4) of vaccine inoculation, positioned at animal house the east.
Environment swab is derived from animal house and neutralizes the multiple places located around it, for testing long-term existence in its natural host The GM viruses of (ox) outside, are collecting animal sample on the same day.
Before vaccine inoculation (0 day), blood (20ml) and nasal swab are collected from all animals.It is also recorded for all animals Rectal temperature.Then intranasal vaccination is carried out to ox (94) with 2ml prototype vaccine (BoHV-1TK-E2+).Epidemic disease will be inoculated with The cattle pen of the Niu Yuwei vaccine inoculations of seedling is supported together.After vaccine inoculation (the 1st day to the 14th day) 14 days, received from all animals Collection nasal swab and the rectal temperature for recording all animals.Terminate experiment after vaccine inoculation the 28th day.Now, from all animals Collect blood (20ml) and nasal swab.In addition, recording the rectal temperature of all animals.After these samples are collected, to all Euthanizing animals simultaneously collect tissue (heart, lung, kidney, spleen, muscle, liver, brain and trigeminal ganglia) sample.Via high temperature Incinerate to handle corpse.
Embodiment 4
The comparison of wet gmBoHV-1 preparations or lyophilized gmBoHV-1 preparations
The purpose of the experiment is to provide the effect of gmBoHV-1 as wet preparation and lyophilized formulations with Q-Vax Pty Ltd Rhinogard (trade mark) effect be compared.
Before zoopery is started, all oxen are negative to BoHV-1 and BVDV specific antibodies.By Niu Suiji points It is assigned to following group：
Group 1：Non- vaccine inoculation；
Group 2：1ml to 2ml vaccine (gmBoHV-1) intranasal vaccination is entered in a nostril；
Group 3：1ml to 2ml vaccine (FD-gmBoHV-1) intranasal vaccination is entered in a nostril；
Group 4：1ml to 2ml vaccine (Rhinogard) intranasal vaccination is entered in a nostril.
Environment swab is derived from animal house and neutralizes the multiple places located around it, so as to test long-term existence in its natural host The GM viruses of (ox) outside, are collecting animal sample on the same day.
Before vaccine inoculation (0 day), blood (20ml) and nasal swab and nose tampon swab are collected from all oxen.In addition, note The rectal temperature and body weight (combine to weight) of all oxen are recorded.Then intranasal vaccine is carried out to group 2 and group 4 with appropriate formulation Inoculation.Facing the lyophilized gmBoHV-1 of preceding quick reconfiguration that instils.Plan applies epidemic disease using business medicator (applicator) Seedling, yet with realizing that its normal operating has some problems, so as previous research are implemented, inoculation is delivered using syringe Vaccine.
After vaccine inoculation (the 1st to the 7th day) 7 days, nasal swab is collected from all animals and all animals are recorded Clinical sign.
Such as following the 14th day after vaccine inoculation, all oxen are attacked with BoHV-1 strains Q3932.Before attack, record The nasal swab and clinical sign of all animals.Then using intranasal instillation 107TCID50BoHV-1 strains Q3932 attacks ox. After BoHV-1 attacks, nasal swab is collected and using the methods of marking the daily (the 15th to the 18th described in table 4 from all oxen My god) carry out clinical evaluation.
Table 4
Clinical sign used and parameter during animal clinical is evaluated before and after attack.By by the breathing in 15 seconds Number of times is multiplied by 4 to calculate respiratory rate.Respiratory rate in observation fence before daily sampling.The morning determine rectal temperature and if Observe the then later duplicate measurements on the day of if significantly rise.If necessary to also record other clinical characters, for example, exhale The sound of suction.Numerical score (S) is assigned with for clinical sign.By the body weight of ox1Be recorded as combined measure because when in group into During to processing, the sampling routine of animal can be more stable (settled).
The 18th day after vaccine inoculation, all oxen are attacked with Mannheimia haemolytica (M.haemolytica).In attack Before, collect nasal swab for all animals and record clinical sign.Then intranasal instillation about 5 × 109Cfu hemolytic Man Bacillus attacks ox.After second attack, nasal swab is collected from all oxen and (the 19th day extremely daily using methods of marking 26th day) carry out clinical evaluation.
Terminate experiment within the 35th day after vaccine inoculation.Now, blood (20ml), nasal swab and nose are collected from every animal Tampon swab.In addition, recording the rectal temperature of all animals.After these samples are collected, to all euthanizing animals simultaneously Collect tissue sample (heart, lung, kidney, spleen, muscle, liver, brain and trigeminal ganglia).Corpse is handled via buried.
Embodiment 5
The influence to vaccine potency has been immunized
The purpose of the experiment is to determine existing whether can influence gmBoHV-1 vaccines to the immune of BoHV-1 or BVDV Effect.If for example, antibodies positive of the animal to BoHV-1, it is immune (referring to table 5) that gmBoHV-1 still is able to induction BVDV.
Table 5
The evaluation on restructuring BoHV-1 vaccine potencies influence has been immunized
1Serologic during vaccine inoculation is indicated, BoHV-1 is positive and the BVDV positives are designated as being exposed to BoHV-1 before this Or BVDV, as by determined by for the presence of the antibody of various viruses in serum collected by before vaccine inoculation.
2The 14th day after vaccine inoculation, attacked with via the BoHV-1 wild strains Q3932 of Aerosol delivery, then after 5 days Attacked with Mannheimia haemolytica, as described in Example 4.
3In addition to replacing BoHV-1 with BVDV wild strains, carried out according to 2.
Environment swab is derived from animal house and neutralizes the multiple places located around it, and its natural host (ox) is maintained at outside so as to test GM virus presence, animal sample is being collected on the same day.
Before vaccine inoculation (0 day), blood (20ml) and nasal swab and nose tampon swab are collected from all oxen.This Outside, the rectal temperature and body weight of all oxen are recorded (combination is to weight).Then using the lyophilized gmBoHV-1 for facing preceding reconstruct of instiling Intranasal vaccination is carried out to group.Plan applies vaccine using business medicator, yet with realizing that its normal operating has some to ask Topic, so as previous research are implemented, vaccine inoculation is delivered using syringe.
After vaccine inoculation (the 1st day to the 7th day) 7 days, nasal swab is collected from all animals and records all dynamic The clinical sign of thing.
The 14th day after such as following vaccine inoculations, all oxen are attacked with BoHV-1 strains Q3932.Before attack, for institute There is animal to collect nasal swab and record clinical evaluation.Then using intranasal instillation 107TCID50BoHV-1 strains Q3932 is attacked Hit ox.After BoHV-1 attacks, collect nasal swab from all oxen and (the 15th day extremely daily using the methods of marking described in table 3 18th day) carry out clinical evaluation.
The 18th day after vaccine inoculation, all oxen are attacked with Mannheimia haemolytica.Before attack, all animals are collected Nasal swab and record the rectal temperature, respiratory rate and body weight of all animals.Then intranasal instillation about 5 × 109Cfu's is molten Courageous and upright Mannheimia attacks ox.After this second is attacked, nasal swab is collected and using the scoring described in table 3 from all oxen Method carries out clinical evaluation in (the 19th to the 26th day) daily.
Terminate experiment within the 35th day after vaccine inoculation.Now, blood (20ml), nasal swab and nose are collected from every animal Tampon swab.After these samples are collected, to all euthanizing animals and tissue sample (heart, lung, kidney, spleen, flesh are collected Meat, liver, brain and trigeminal ganglia).Corpse is handled via buried.
Embodiment 6
Virulence replys (reversion to virulence)
The purpose of the experiment is to determine whether gmBoHV-1 can show that virulence strengthens mark by the continuous passage of multiple groups of ox As.
Environment swab is derived from animal house and neutralizes the multiple places located around it, so as to test long-term existence in its natural host The GM viruses of (ox) outside, are collecting animal sample on the same day.
Before vaccine inoculation (0 day), blood (20ml) and nasal swab and nose tampon swab are collected from all oxen.This Outside, the rectal temperature and body weight of all oxen are recorded (combination is to weight).
Then intranasal vaccination is carried out to ox (2) using the lyophilized gmBoHV-1 for facing the preceding quick reconfiguration that instils.In inoculation After vaccine (the 1st day to the 7th day), nasal swab is collected from these animals and the clinical evaluation of animal is recorded daily.
Terminate each passage assays within the 14th day after vaccine inoculation.Now, blood (20ml), nose is collected from every animal to wipe Son and nose tampon swab.Collect these samples after, to all euthanizing animals and collect tissue sample (heart, lung, Kidney, spleen, muscle, liver, brain and trigeminal ganglia).Corpse is handled via buried.
In order to complete gmBoHV-1 virus interior generation, from the nasal swab through vaccine inoculation ox isolate virus and For infecting two again not in contact with the ox for crossing BoHV-1.The process is repeated four times as described above.It should be noted that only passage makes first Completed with lyophilized gmBoHV-1.
Embodiment 7
Cross multiple dose
Whether the purpose of the experiment was to determine the gmBoHV-1 of multiple dose (ED) to the animal pest of vaccine inoculation.
Environment swab is derived from animal house and neutralizes the multiple places located around it, so as to test long-term existence in its natural host The GM viruses of (ox) outside, are collecting animal sample on the same day.
Before vaccine inoculation (0 day), blood (20ml) and nasal swab and nose tampon swab are collected from all oxen.Also remember The rectal temperature and body weight of all oxen have been recorded (combination is to weight).Then as described below use faces the lyophilized of preceding quick reconfiguration of instiling GmBoHV-1 carries out intranasal vaccination to Ed groups 1 to 3.Plan applies vaccine using business medicator, yet with realization Its normal operating has some problems, so as previous research are implemented, vaccine inoculation is delivered using syringe.
ED groups 1：Use intranasal instillation (105-6TCID50) 1ml vaccines/nostril carrys out vaccine inoculation ox (4)；
ED groups 2:Use intranasal instillation (106-7TCID50) 1ml vaccines/nostril carrys out vaccine inoculation ox (4)；
ED groups 3:Use intranasal instillation (107-8TCID50) 1ml vaccines/nostril carrys out vaccine inoculation ox (4).
After vaccine inoculation (the 1st day to the 7th day) 7 days, nasal swab is collected from all oxen and all animals are recorded Clinical evaluation.Terminate experiment within the 14th day after vaccine inoculation.Now, blood (20ml), nasal swab are collected from every animal With nose tampon swab.Collect these samples after, to all euthanizing animals and collect tissue sample (heart, lung, kidney, Spleen, muscle, liver, brain and trigeminal ganglia).
Embodiment 8
GmBoHV-1 genetic stability
For the restriction enzyme characteristic of inverse passage gmBoHV-1 prototype vaccine
By testing the nasal swab collected in whole process in fence, reflected via mammalian cell cultures (CRIB-1 cells) Determine the virus purification for the treatment of group representative.The bacterial artificial chromosome (BAC) existed in gmBoHV-1 skeleton enables DNA Separation and purifying, the DNA increase DNA output via bacterium duplication.
Cell culture
The confluent monolayer (70%) of the CRIB-1 cells for infection is prepared in six hole plates.Remove culture medium and use nothing Bacterium phosphate buffered saline (PBS) (PBS) washs the individual layer.(in PBS and it is to scrubbed individual layer addition 1mL nasal swab Five times of PSF) and at 27 DEG C in 5%v/v CO2It is middle to be incubated 1 hour.Remove inoculum and wash cell with 1mL PBS Once, it is then made in 37 DEG C and 5%CO2Under recover 5h in the 3mL fresh cultures with PSF.
6h, individual layer is washed with 1mL PBS after infection, then adds the full DNA lysis buffers of 1mL (tool to the individual layer Have fresh protein enzyme K) and cultivated 4 hours at 37 DEG C.This makes cell in-situ crack and discharge STb gene from CrIB-1 cells And replicate pBACBHV1E2s viral vaccine candidates.The STb gene of infection early stage is harvested to ensure that some BAC DNA will be in ring Shape and suitable for be transformed into bacterium for clone replicate.
The extraction and conversion of STb gene
STb gene is purified by using phenol/chloroform extraction and is precipitated using absolute ethyl alcohol.Then make to do at room temperature DNA be deposited in 50 μ l sterile high purity water (18M Ω) 2h be resuspended.Each volume of extracting is according to the viscous of DNA initial suspension Spend and change.By the DH10B ElectroMax competent cells that 10 μ l STb gene electroporation is entered to 20 μ l (Invitrogen) realized in and convert and selected on the Bacterial Plate comprising 12.5 μ g/ml chloramphenicol.From each flat board Three bacterium colonies of picking are into the 50mL LB nutrient solutions comprising 12.5 μ g/ml chloramphenicol and in the case where gently shaking in 37 DEG C of growths 18h.Using improved alkaline lysis scheme (based on the Roche high-purities Plasmid Isolation Kit for small volume preparation) come from these BAC DNA are extracted in culture.Make bacterium cell pellet to remove nutrient solution and be allowed to be resuspended in Tris buffer solutions.Make these Cell cracking removes the protein and salt of residual to discharge DNA to be purified.The DNA of precipitation is dried and is resuspended at 4 DEG C In 60 μ l 10mM Tris-HCl (pH 8.5) overnight.
Restriction enzyme characteristic
Via the RE characteristics for comparing candidate vaccine (not via the inverse passage of animal) and parental virus carrier (pBACHBV137) To analyze total mutation of multiple clones from each isolate.5 μ l alkaline bleach liquor cleavage agent is prepared, (NEB) is according to manufacturer Illustrate to make BAC DNA digest 4h with enzyme Hindlll and Sall in 20 μ l cumulative volumes.
Separated using field inversion gel electrophoresis (FIGE) through digesting BAC DNA and visualizing band spectrum.By 20 μ l warp Digest on 0.7% Ago-Gel (both of which has ethidium bromide) that BAC DNA are carried in 0.5 times of tbe buffer liquid and transport Row condition is the program 3 of FIGE equipment (BioRad), and its targeted molecular amount magnitude range is 5Kb to 100Kb, and run time is 16h.
Embodiment 9
GmBoHV-1 infects in mammal cell line, replicate and express transgenic external ability
For in terms of CPE outward appearance, indistinction sex differernce between parent BoHV-1 and GM BoHV-1.Intuitively CPE is Two kinds of viral typical cases of BoHV-1.Generally, amount viral in the cell line infected through both BoHV-1 or gmBoHV-1 is (by CT Value inference) after infection 24h be similar (Figure 1A to Fig. 1 C).This is that statistics shows for HaCaT and CRIB-1 cell lines Write, that is, the viral amount detected is between parent and GM without significant difference (P<The unpaired t of 0.05 pair of tail is examined).For it Remaining cell line, the difference between the CT values of parent and the GM cell infected is not more than 2.5, and in each cell line all the time Any virus of higher level is not detected.
Have confirmed compared with parental virus, will will not change in transgenosis (synthesis BVDV E2) insertion parent BoHV-1 The ability that virus infects and replicated in a variety of mammal cell lines of test.Interestingly be, test it is all thin Born of the same parents system is easily by BoHV-1 (both BoHV-1 and unmodified BoHV-1 through modification) infection.
Embodiment 10
Infection of the gmBoHV-1 from ox to other ruminants
Fence experiment is carried out to determine whether to change due to the genetic modification made BoHV-1 host range or BoHV- 1 ability infected to other ruminants.Fence experiment includes a series of hilllocks being placed at the ox different distance from vaccine inoculation Whistle ox, sheep and goat.
When starting fence experiment, it should be noted that some oxen (ox in particularly sentry post group A) have nasal discharge.Also mark As showing that some oxen have diarrhoea.Although not being most preferably to be tested, experiment is postponed until it is not that these phenomenons, which disappear, Feasible.It is that obvious-some animals exist in most of ox groups in sometimes these clinical signs through whole experiment Some signs are showed in whole experiment.
Using BRD multiple assays (FLOT.219) to extracting from the test of the nasal swab of all animals since the 0th day It is fubaritic to include 1 type bovine herpes virus, Bovine Respiratory Syncytial virus, 3 type bovine parainfluenza viruses or any sample of rinderpest virus Product.Paramyxovirus, adenovirus and the enterovirus for four category that standard PCR is determined also are negative.Without BoHV1 and pestivirus to the reality It is most important for testing, because any viral presence causes the experimental result is extremely difficult to explain and experiment can be caused to terminate.
Interestingly begun attempt to from the 0th day from nasal swab isolated viral, reason is to seem to have in cell Some induced cytopathic effects (CPE), show there is virus.However, the trial and unsuccessful of these supernatants passage.It is made that Other steps are attempted to determine whether that infectious agent is related to these signs, and it passes through to by from moving with lasting clinical sign The cell of the nasal swab infection of thing (animal 377 from sentry post A), which is dyed, carrys out fluorescence labeling for following bovine viral Antibody：Adenovirus 3, coronavirus, pestivirus, parainfluenza virus, Bovine Respiratory Syncytial virus, parvovirus and exhale the lonely disease of intestines Poison.Then extracted from the nasal swab of animal 377 at the 0th day using standard PCR tests and acquisition is consistent with desired size Amplicon.However, the sequencing of the amplicon shows that it is non-specific amplification product.And antibody staining is seemingly specific, The identification of pathogen is still unknown.These samples are analyzed using the available all molecular testings in our laboratories, this nothing The disease causing agents that method identification is worked to observed clinical sign.Previous application based on these tests, it is believed that the examination worked Agent extremely can not possibly be closely related with BoHV-1 or BVDV, thus should without interference with these virus serology data explanation.
GmBoHV-1 is not detected by the virus purification in any sheep or goat of RT-PCR or three sentry post group. Similarly, during testing whenever, pass through the virus point for the ox that RT-PCR or any is contained in sentry post group A, B or C It is viral from GM is not detected.
All through being inoculated with detection GM viruses in ox.Generally, if be positive for virus isolates (culture) sample, Then also it is positive for RT-PCR.Virus is most preferably recovered from being positive and the by RT-PCR nasal swabs for the 1st day to the 6th day The animal that 3 days and the 4th day culture are positive.
There is the contagion that GM viruses are detected in two in four pairs of animals.
Based on the RT-PCR amplifications of the GM viruses from nasal swab, virus is being 3 through being inoculated with the peak period expanded in ox My god.At the 3rd day GM viruses were detected in four-head through being inoculated with three of ox.
In order to determine whether GM viruses can be maintained at host (ox) outside, in whole experiment, from a variety of of Test sites Surface collection environment swab.Then utilize and separate with RT-PCR to test in these nasal swab extracts from cell culture The presence of GM viruses.Through whole experiment, GM viruses are not detected by the environment swab of collection.
As a result detection showed in 28 days samples only in the 0th day and the 28th day serum collected from all experimental animals The ox of inoculation produces the detectable antibody for BoHV-1..
Based on the result from the experiment, GM viruses are not transmitted to other category (sheep and goat).Similarly, GM viruses do not have Have with distance change (>2m) infect nigh through being inoculated with ox to poultry sample.It there occurs GM viruses to being raiseeed through being inoculated with Niu Yiqi Foster Niu Chuanran, but frequency is not high.
The result of experiment does not support any change of the host range of GM viruses.Although for natural host (ox) Some propagation are there occurs, but frequency is not high and may depend in the levels of replication through being inoculated with animal.In addition, whole GM viruses are not detected by the environment swab that individual experiment is collected outside viral natural host.In a word, these as shown by data GM viruses The risk discharged into environment is extremely low.
Embodiment 11
GmBoHV-1 reactivation
After each experiment terminates, nasal swab is collected with ox is not inoculated with from through inoculation.Extract total from these nasal swabs Nucleic acid and the tested gmBoHV-1 of the real-time PCR measure for targeting BoHV-1 and E2 transgenosis in the presence of use.Based on this, gained The rational conclusion gone out is not occur reactivation before experiment terminates.
Embodiment 12
GM BoHV-1 persistence and stability
The stability difference between the parental virus of GM BoHV-1 and wild condition is not detected.License regulation experiment exists It can not possibly implement in the PCI animal houses of direct sunlight.Under the conditions of reality is wild, exposed to sunlight meeting probably due to ultraviolet Line and the unstability for increasing both GM and parental virus.
Embodiment 13
Remaining gene outcome
GmBoHV-1 is not detected by any tissue of animal tested using real-time PCR.Although it is believed that can not possibly exist Any gene outcome, but still carry out immunoblotting assay for the gross protein for extracting from the animal tissue.Based on these results, GM viruses or the gene outcome of its expression can not possibly be persisted in the tissue of inoculation/infected animal.
The presence for both GM viruses and transgene product is not expected, and the tissue sample of animal is negative.Surveyed In the tissue of examination, it is contemplated that gmBoHV-1 is existed only in gasserian ganglion (TG), because it is expected that this, which is parental virus, forms latent The position of sexuality dye.Fail to detect virus in TG and show that gmBoHV-1 can not set up latent infection.Or, it may be difficult to GmBoHV-1 is detected in TG, because only that the cell body in a small number of neuromeres may carry virus-therefore succeed what is detected Possibility depends on the amount and detection sensitivity of handled tissue.
Embodiment 14
The effect of lyophilized gmBoHV-1 preparations
In vaccine inoculation/attack experiment, using gmBoHV-1 prototype vaccine as lyophilized formulations (FD-gmBoHV-1) effect Power is compared with gmBoHV-1 as the effect of wet preparation.In this experiment include be inoculated with Rhinogard ox group with For comparing.Table 3 was illustrated for all from the 0th day (date of vaccine inoculation) to the 7th day disease through being inoculated with and not being inoculated with ox Malicious testing result.Generally, FD-gmBoHV-1 with gmBoHV-1 vaccines were arranged with extremely consistent speed at the 3rd day and rush down, wherein having There is the high level virus detected by PCR the virus (table 6) consistent with separation.Determined and known through gmBoHV-1 inoculations three by PCR Most oxen in it or more day are positive.In addition to only in the animal (the 581 of design) being positive after being inoculated with 3 days.
Do not observe bad clinical sign in through inoculation or nonvaccinated group.During the experiment in the stage, three times BoHV-1 is detected in nonvaccinated three animals.Detected virus is not gmBoHV-1, because special to E2 transgenosis Different PCR, which is determined, knows that all animals are negative.In addition, BoHV-1 PCR results are in weakly positive, this shows that the result is attributable to The pollution of sample.This is most likely to occur for the sample being positive at the 0th day during the post processing of laboratory sample.
At the 0th day, some samples were positive to gmBoHV-1.This is probably the pollution because in inoculation group, because in logic On can not possibly pollute all surface between the animal for receiving same treatment.Similarly, animal is made to squeeze into receiving in pairs, reason is This generally causes its more stable, therefore before to second animal swab, however not excluded that it is transferred to by described couple of initial member Described couple of another member.
In order to minimize the possibility of cross pollution between group, treatment group in the following order all the time：It is nonvaccinated right According to, be inoculated with and be inoculated with through gmBoHV-1 through Rhinogard.In addition after to group inoculation Rhinogard, purification includes squeezing The animal processing region of pressure.
The BoHV-1 positive findingses for not being inoculated with animal 549 at the 5th day are attributable to come the biography for Rhinogard inoculation groups of hanging oneself Dye.Animal 549 is placed in the neighbouring fence through Rhinogard inoculation groups, and therefore these animals must be sent to extruding Region.However, animal is closely monitored to prevent directly contact in this process, animal 549 is when by Rhinogard fences The inhalable material containing virus is also feasible, and reason is that animal tends to detect environment in the moving process.Virus is uninfected by 549 and only detected at the 1st day, this supports it for environmental pollution rather than the viral biography from infected/inoculation animal Dye.Data are shown in Table 6.
Table 6
The vaccine inoculation stage：Through the Viral diagnosis and virus purification result that are inoculated with and are not inoculated with ox.Tested as fence A part, gmBoHV-1 (wet GM), lyophilized gmBoHV-1 (FD-GM), Rhinogard (RG) is inoculated with to ox or without inoculation To evaluate effect of the FD-GM compared to wet GM.After DNA is extracted from nasal swab, using to gmBoHV-1 carriers (BHV) and The special real-time PCR of BVDV E2 transgenosis (E2) determines (P) and carrys out test sample.PCR results be expressed as it is very strong (++++, Ct values <20), strong (+++, Ct values>20 but<30), weak (++, Ct values>30 but<35), very it is weak (+, Ct values>35 but<40) it is or negative (Pos), do not reclaim viral (Neg) or be not attempt to (NA).0 to 7 day each animal temperature (DEG C) scope is illustrated in animal volume Under number.7 days after vaccine inoculation, all temperature recorded are less than 39.5 DEG C, except the 1st day of 581 be 39.7 DEG C, 596 It is respectively 39.7 DEG C and 39.6 DEG C to be within 7th day 39.7 DEG C, the 3rd day and the 5th day of 565.
After the vaccine inoculation first 14 days for the treatment of group, ox is attacked with BoHV-1 strain Q3932 or BVDV strains MD74. BoHV-1 is detected using challenge virus (table 7) in the nose for collecting two animals wipes thing before.Nose wipes thing and turns base to E2 Because being negative, this shows that the virus of detection is not gmBoHV-1.The virus is not substantially originated, and reason is to be inoculated with Rhinogard all animals are negative to the virus, and this shows that possible source is the collection post processing of sample.In attack Three to four days afterwards (the 17th day and 18 days), in all animals attacked by BoHV-1 detecting high-caliber BoHV-1 attacks Hit strain Q3932 (table 7).
Implement bio-security to prevent the group that BoHV-1 is infected to the BVDV groups attacked or BVDV is attacked to BoHV-1 Infect.Nevertheless, BoHV-1 has also significantly infected the group (table 7) of BVDV attacks.The source of the cross-infection is not aobvious and easy See.Specific evaluation Australia's BoHV-1 strains are directed to the ability infected between ox however, not studying, and can generally be connect What is received is to need to be in close contact to infect.May support most of experiments that the conclusion is implemented be vaccination experiments simultaneously And the infection of vaccine strain (BoHV-1 strain V155) is not therefore observed.However, all dynamic in the phase of the attack of these experiments Thing is under attack, therefore has no chance to evaluate attack strain to the infection for the animal for being used to test first.On this basis, it appears that Strain Q3932 is attacked more easily by the method in addition to close contact to other Niu Chuanran.
Table 7
To phase of the attack Viral diagnosis, virus purification and the Mannheimia haemolytica after being inoculated with ox and control ox attack (M.haemolytica) testing result.After vaccine inoculation 14 days, with BoHV-1 (107TCID50) or BVDV attack oxen, then Implemented Mannheimia haemolytica (M.haemolytica, 6.8 × 10 at the 18th day9CFU) to the attack of all oxen.From nose Wipe in thing and extract after DNA, strain is attacked using strain Q3932 (HBV), BVDV E2 transgenosis (E2), BVDV is attacked to BoHV-1 Or the special real-time PCR of Mannheimia haemolytica (M.haemolytica, Mh) determines (P) and carrys out test sample (BVDV).PCR is tied Fruit be expressed as it is very strong (++++, Ct values<20), strong (+++, Ct values>20 but<30), weak (++, Ct values>30 but<35) it is, very weak (+, Ct values>35 but<40) it is or negative (-).Each animal temperature (DEG C) scope is illustrated under number of animals within 14 to 25 days.Only move Thing 546 is 36.7 to BHV Ct values at the end of experiment.BHV PCR have detected gmBoHV-1 and BoHV-1 attack strain two Person.
After two kinds of attack pathogen are applied, the clinical sign of animal is evaluated daily.Virus attack in 14 to 18 days it It is preceding not recorded any clinical sign.After the second stage attacked by Mannheimia haemolytica, the clinical sign in many groups Clearly.Generally, it was observed that clinical sign it is more slight.At any moment during phase of the attack, rise is not recorded Temperature, the forfeiture of appetite, the change of respiratory rate or cough.
From all before the 0th day (vaccine inoculation), the 14th day (virus attack) and experiment terminate to face implementation euthanasia Blood serum sample is collected in animal.Tested using commercially available ELISA and come in test sera to whether there is for the anti-of BoHV-1 and BVDV Body.The result of these tests is illustrated in table 8.The 0th day, antibodies positive of six oxen to BoHV-1.Ox (60) derives from The same owner (a single property) and the age is all almost identical.Test collected from the serum of all oxen and its The special antibody for BVDV is negative.However, have in 60 oxen from same drove the serum of 23 oxen to for BoHV-1 antibodies positive, the previous level of the antibody is considered as more normal (positive to BoHV-1 is less than 10%).Cause High seroprevalence and relatively small age (weaning before arrival about 8 weeks) for ox, it is believed that the high morbidity for the BoHV-1 that is positive Rate is probably to be caused by maternal antibody.If reason is maternal antibody, it is expected that be present in Serum Antibody amount will with when Between decline.Therefore, the presence weekly to the BoHV-1 antibody of ox carries out re-test.Between first time test and third time are tested Period in, three oxen are changed into negative from the positive, and one is changed into uncertain from the positive, and 15 show antibody level reduction simultaneously And four-head antibody level is stable, it is still the positive.One animal seems to generate the antibody (numbering 598) for BoHV-1, so And it is seronegativity when being tested after.
When testing beginning, compared with test before this, all ox (table 8) level drops to BoHV-1 antibodies positives Low, this is supported again has antibody materials in these animals.
As expected, at the end of experiment, all oxen being inoculated with through gmBOHV-1 have carried out serum for gmBOHV-1 Conversion.
Through whole experiment, all oxen remain reacts (table 8) to BVDV seronegativity.It is not expected that in this way, because It is expected that those animals attacked by BVDV can carry out seroconversion for BVDV.However, not unexpected in Viral diagnosis result Seroconversion is not detected by context, reason is that BVDV strains used do not appear to replicate in nonvaccinated animal.It is dynamic Thing 546 is unique to animals of the BVDV in PCR positive (the 18th day) after being attacked four days with BVDV, however be believed that virus after Renew and be that the long period is without infecting and replicating in nasal cavity, in the event of replicating, then BVDV levels must be very low, former Because being that animal does not carry out seroconversion or in other random times do not detect virus.Serological supports the disease to BVDV Malicious testing result, it shows that BVDV strains infect or replicated not in these animals.
Table 8
Tested ox is directed to bovine herpes virus-1 (BHV) or bovine viral in the different phase of whole vaccination experiments Property diarrhea virus (BVDV) serology state experiment.Use the Pourquier (notes for detecting the serum antibody for HBV Volume trade mark) ELISA IBR-IPVSerum and Milk and for detect for BVDV serum antibody Pouriquier (note Volume trade mark) ELISA BVD-MD-BD P80Antibodies test the blood serum sample from all oxen.BHV specificity survey Examination will confirm that previously to be infected by wild type BoHV-1 or gmBoHV-1.BVDV specific tests will confirm that previously by wild type BVDV Infection, it is impossible to which detect has specific antibody to BVDV E2.
Embodiment 15
The influence to vaccine potency has been immunized
The possibility risk for carrying out combination-vaccine using genetic engineering is that immune can reduce already present to carrier or transgenosis connects Plant the validity of vaccine.For example, in existing research, if ox is existing to vaccine carrier BoHV-1 immune, the vaccine is carried Body can prevent replicating and preventing or reduce appointing for the BVDV E2 protein by transgenes encoding for gmBoHV-1 vaccines The stimulation of what immunological response.If this there occurs, the BVDV components of vaccine are not benefited from through being inoculated with ox, i.e. ox will be easy Infected by BVDV and produce disease.
In order to determine that the effect that whether may interfere with prototype vaccine has been immunized, it is in using the antibody for BoHV-1 or BVDV Positive animal is tested.In two stage challenge models previously used, the response being inoculated with gmBoHV-1 have rated.
For the BoHV-1 influence being immunized to vaccine potency
The Niu Jinhang for being defined as the antibodies positive special to BoHV-1 is inoculated with using gmBoHV-1.Using to BoHV-1 Determine to test the DNA isolated from these animal nasal swabs with the special real-time PCR of E2 transgenosis.As expected, All it is negative in two kinds of measure of the 0th day all animals.In the animal being inoculated with through gmBoHV-1,1 to 6 after vaccine inoculation My god, determined via two kinds of PCR and unanimously detect virus in all animals in addition to animal 552.
After vaccine inoculation seven days, bad clinical sign is not being observed through being inoculated with animal.
Two kinds of PCR of one of the 7th day nonvaccinated animal (animal 557) are determined and are positive.Disease is separated from nasal swab The trial of poison fails, and this shows PCR results by sample in sub- aliquot (sub-aliquoting) or DNA extraction process The cross-infection of period sample causes.
For BoHV-1, the PCR of one of inoculation animal (animal 556) is only in the 7th the heaven pertaining to YANG.Because to E2 transgenosis PCR is determined determines more sensitive than BoHV-1, so the BoHV-1 seems to be BoHV-1 non-recombinant or wild type strains.Do not make Go out the trial from isolated viral in the sample.Because BoHV-1 nucleic acid is widely used in a variety of applications in laboratory, Asia etc. The cross pollution of sample is the reason for seemingly most probable causes the result during point sample or DNA extractions.
The fortnight after vaccine inoculation, with BVDV attacks are through inoculation and are not inoculated with both oxen.Drawn by PCR the 14th My god, all oxen are negative to BoHV-1, E2 transgenosis and BVDV.With BVDV attacks from two groups of ox and at other 5 days Swab afterwards.The 18th day after vaccine inoculation, all oxen are attacked with Mannheimia haemolytica.Using BVDV strains, have in eight oxen Three being positive at least one day in phase of the attack.First animal was positive at the 15th day and last animal is the 23rd It is positive.The nonvaccinated animal of the other three carries out seroconversion for BVDV.These results show that BVDV attacks are successes 's.
After BVDV attacks, the clinical sign of all oxen is evaluated daily.Do not observed between the 14th day and the 18th day To clinical sign.Clinical sign was observed from the 19th day to the 25th day.
The Serological of these groups is illustrated in table 9.It can be seen that in the 0th day all animals in addition to 539 all to BoHV- 1 is positive, and the maternal antibody that the possible animal includes dissipates (dissipated) before the experiment starts.The animal is being ground Keep being negative to BoHV-1 antibody during studying carefully.As shown in table 8, there is three animals (non-vaccine inoculation) pin in eight animals Seroconversion has been carried out to BVDV.At the end of experiment, other three animals (a non-vaccine inoculation, two in eight animals Only it is vaccinated with vaccine) substantially show the rise of BVDV antibody levels.These results support concluding for BVDV successful attacks.
Table 9
Tested ox is directed to bovine herpes virus-1 (BHV) or bovine viral in the different phase of whole vaccination experiments The serology state of property diarrhea virus (BVDV).Use the Pourquier (registrars for detecting the serum antibody for HBV Mark) ELISA IBR-IPVSerum and Milk and for detect for BVDV serum antibody Pouriquier (registrars Mark) ELISABVD-MD-BD P80Antibodies test the sample from all oxen.BHV specific test will confirm that elder generation It is preceding to be infected by wild type BoHV-1 or gmBoHV-1.BVDV specific tests will confirm that previously to be infected by wild type BVDV, it is impossible to Detect has specific antibody to BVDV E2.* antibody level rise trend is obvious.
These result collectively show thats carry out inoculation to ox with gmBoHV-1 and provide protection for these oxen.For BVDV There is the immune influence (BoHV-1 attacks) to vaccine potency
The Niu Jinhang being defined as the specific antibodies positives of BVDV is inoculated with gmBoHV-1.Then using pair The specific real-time PCR of BoHV-1 and E2 transgenosis determines to test the DNA separated from the nasal swab of these animals.These The result that PCR is determined is illustrated in table 10.As expected, it is negative in two kinds of analyses of the 0th day all animals.In warp In the animal of gmBoHV-1 inoculations, after to all animal inoculation pvaccinations 2 to 6 days, determined via two kinds of PCR and unanimously detect disease Poison.In the 3rd day isolated viral from all animals.These data support the good absorption of vaccine jointly.It is not inoculated with Vaccine virus is detected in the animal (table 10) of vaccine.
After vaccine inoculation during 7 days, bad clinical sign is not being observed through being inoculated with animal.
Table 10
There is the Viral diagnosis and separating resulting of immune ox to BVDV.The vaccine inoculation stage：Exist to BVDV and exempt from The Viral diagnosis and virus purification result through being inoculated with and not being inoculated with ox of epidemic disease.Using lyophilized gmBoHV-1 (FD-GM) be inoculated with ox or Keep not vaccine inoculation.After DNA is extracted from nasal swab, using to gmBoHV-1 carriers (BHV) and BVDV E2 transgenosis (E2) specific real-time PCR measure (P) carrys out test sample.PCR results be expressed as it is very strong (++++, Ct values<20), strong (++ +, Ct values>20 but<30), weak (++, Ct values>30 but<35), very it is weak (+, Ct values>35 but<40) it is or negative (-).It has also been attempted Virus purification is carried out to chosen sample (VI), separating resulting is shown as the virus (Pos, positive) reclaimed, does not reclaim virus (Neg, negative) or it is not attempt to (NA).Each animal temperature (DEG C) scope is illustrated under number of animals within 0 to 7 day.* the disease attempted Poison, which is separated, does not isolate virus, and the result of BoHV-1 and E2 PCR ratios is also inconsistent with Previous results.
The 14th day after vaccine inoculation, all oxen are attacked with BoHV-1 strains Q3932 as discussed previously.Nose is collected daily Portion's swab and record clinical sign.At the 18th day, all animals are attacked with Mannheimia haemolytica.25 days after attack, often It is collected nasal swab and records clinical sign.DNA is extracted from all nasal swabs.
The fortnight after vaccine inoculation, with BoHV-1 attacks through inoculation and both nonvaccinated group.Drawn by PCR 14 days, all oxen were negative to BoHV-1 and E2 transgenosis.With BoHV-1 strains Q3932 attack from two groups of ox and Swab after other 5 days.The 18th day after vaccine inoculation, all oxen are attacked with Mannheimia haemolytica.As expected, pass through All animals of PCR are negative to E2 transgenosis.Be consistently detected Q2932 in all animals, however, with non-vaccine inoculation Animal is compared, and has the trend that a small amount of virus is discharged in shorter time through vaccinated animals.Mannheimia haemolytica PCR is determined There is substantially similar trend.
After BoHV-1 attacks, the clinical sign of all oxen is evaluated daily.Do not observed at the 14th to the 18th day Clinical sign.
The serology test of ox shows as expected, to be directed at the end of experiment by the BoHV-1 all oxen attacked BoHV-1 has carried out seroconversion (table 11).
Table 11
Tested ox is directed to bovine herpes virus-1 (BHV) or bovine viral in the different phase of whole vaccination experiments The serology state of property diarrhea virus (BVDV).Use the Pourquier (registrars for detecting the serum antibody for HBV Mark) ELISA IBR-IPVSerum and Milk and for detect for BVDV serum antibody Pouriquier (registrars Mark) ELISA BVD-MD-BD P80Antibodies test the blood serum sample from all oxen.BHV specific test will Confirm previously to be infected by wild type BoHV-1 or gmBoHV-1.BVDV specific tests will confirm that previously to be felt by wild type BVDV Dye, it is impossible to detect the antibody special to BVDV E2.
It can not prevent to replicate or reclaim from the animal through inoculation gmBoHV- for the already present antibody of BVDV or BoHV-1 1 vaccine virus
Whole result is supported to deliver multiple antigens from other pathogens using live vector.In addition, result shows host Pin will negatively affect vaccine performance in the immune state of vaccine carrier.
Embodiment 16
Virulence is replied
The inherent risk that parental virus virulence increases is brought using live-virus vaccine carrier, if viral from an animal If traveling to another susceptible animal.In order to examine whether its may have gmBoHV-1 and also for evaluate carry out The stability of genetic modification, is passed on prototype vaccine four times by (the being directed to BoHV-1 and BVDV) ox for carrying out immune first.With Parallel these passage assays of progress of immunity inoculation experiment.
Because the 3rd day after vaccine inoculation has been consistently detected gmBoHV-1 and it is separated, now separate Virus be used for later passages.The first generation is from No. 598 animal.
Then use and the special real-time PCR of BoHV-1 and E2 transgenosis is determined to test from these animal nasal swabs The DNA isolated, is negative in two kinds of analyses of the 0th day all animals.In the animal being inoculated with through gmBoHV-1, to institute There are animal vaccinations to be passed through after 2 to 7 days and unanimously detect virus by two kinds of PCR measure.Separated at the 3rd day from all animals Virus.
After vaccine inoculation during 7 days, bad clinical sign is observed not in the animal through inoculation.Examined during passing on Measured the slight elevated temperature of some animals (>40 DEG C), however, these are sporadic and not shown and virus In the presence of relevant.
As expected, most of animal has carried out seroconversion for BoHV-1 at the end of experiment.
In a word, do not find to support the evidence that gmBoHV-1 virulence increases during passage assays.In addition, transgenosis seems Highly stable and do not find by it is any passage loss evidence.
The result collectively show that E2 transgenosis of passage assays is stable in BoHV-1 genomes.Similarly, do not find GmBoHV-1 back mutations to the prototype of more virulence evidence.Also by examining the genomic DNA from isolated viral again Digestion with restriction enzyme pattern and the gene stability that have studied mBoHV-1.
Embodiment 17
Cross multiple dose
For feasibility economically, vaccine is generally provided as multi-dose formulation.If to use epidemic disease higher than recommended dose Seedling, then the possible withdrawal (drawn back) of these preparations is potential negatively affects through being inoculated with animal.In order to study it is possible not Profit influence, if to apply gmBoHV-1 higher than recommended dose, be wherein inoculated with the vaccine of a variety of concentration the reality of ox Test.
10 are inoculated with each nostril of the ox (four-head/group) of three groups6.5TCID50GmBoHV-1 (10 × dosage), 105.5TCID50GmBoHV-1 (desired effective dose) or 104.5TCID50GmBoHV-1 (0.1 × desired effective agents Amount).After vaccine inoculation, detect the clinical sign of ox and gather nasal swab daily.Then use and BoHV-1 and E2 is turned The real-time PCR of gene specific determines to test the DNA isolated from these animal nasal swabs.
In the animal being inoculated with through gmBoHV-1, pass through and determined by two kinds of PCR after to all animal vaccinations 2 to 7 days It has been consistently detected virus.Attempt to continue isolated viral from all animals at the 3rd day.
After vaccine inoculation during 7 days, bad clinical sign is not observed in the animal through inoculation.Detect The slight elevated temperature of animal (>40 DEG C), however, these are sporadic and not shown relevant with the presence of virus.
As expected, most of animal has carried out seroconversion for BoHV-1 at the end of experiment.As it can be desirable to , more animals through seroconversion have the tendency of to receive a large amount of viruses in processes.
It still there be no evidence to suggest animal inoculation pvaccination high dose gmBoHV-1 adverse effect.Under the relatively low-dose of vaccine, it is based on Detected by PCR and/or virus purification detects ability viral in nasal swab, it appears that it is less efficient that vaccine absorbs.
Embodiment 18
GmBoHV-1 genetic stability
In this embodiment, the vaccine strain of animal is isolated from by detection during prototype vaccine is by ox continuous passage Genetic characteristics evaluate gmBoHV-1 genetic stability.
The evaluation is carried out by separating gmBoHV-1 again for the first time in the nasal swab collected from infected ox.In order to demonstrate,prove Repetition passage in bright ox can not adversely influence the genetic stability of prototype vaccine, continuous biography of these analyses in the form of recovery Carried out in generation virus.Detected by digestion with restriction enzyme random selection clone through separation and through colone genome, this It is the method for being generally accepted the genetic stability for evaluating herpesviral.It is used in two restriction enzymes First enzyme is the Hindlll of estimation cutting BoHV-1 genomes 12 times and therefore provides any extensive genome rearrangement or restructuring The measurement of event.Second enzyme used for estimation cutting BoHV-1 genomes 45 times Sall and therefore provide any small-scale base Because of group rearrangement or the measurement of recombination event.
The 3rd day and the 7th day after vaccine inoculation, virus is reclaimed from the nasal swab of collection and limited characteristic is determined. The obvious extensive or more small-scale of Hindll and Sall characteristics is not based respectively on to reset.
Based on the characteristic of the viral restriction enzyme separated after being passed in ox, the card without any gene variability According to.These data are supported to draw a conclusion：The gmBoHV-1 for being used to be inoculated with ox in this study is highly stable.
It should be appreciated by those skilled in the art that unless specifically described, otherwise this paper some aspects are easy to be changed and repaiied Change.It should be understood that these aspects include all such changes and changed.Include separately or cooperatively referring in this manual herein Or all steps, feature, composition and the compound pointed out, and any including any two or more step or feature and All combinations.
Bibliography
Mahoneyet al.(2002)Journal of Virology76(13)：6660-6668
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Snowden(1964)Australian Veterinary Journal40：277-288

Claims (18)

1. a kind of vaccine at least one antigen from cattle disease substance, the vaccine is included from low toxicity power BoHV-1's 1 type bovine herpes virus (BoHV-1) genome, the BoHV-1 genomes have with BoHV-1 it is heterologous, insertion two convergence The inhereditary material of coding at least one antigen between BoHV-1 genes, wherein the insertion does not cut the BoHV-1 bases The expression of cause, and the inhereditary material of at least one antigen is wherein encoded in the poly- of two meeting pcl genes of following site insertion Between polyadenylation signal, the site be selected from BoHV-1 reference sequences GenBank registration numbers AJ004801 16600 to 16612nd, 22449 to 22493,40734 to 40768,58229 to 58563,67037 to 67091,74994 to 75041,84496 To 84528,90732 to 90760 and 96870 to 96882.
2. vaccine according to claim 1, wherein the inhereditary material for encoding at least one antigen is recombinated via GET and inserted Enter in the BoHV-1 genomes.
3. vaccine according to claim 1, wherein at least one antigen is inserted selected from 16600 to 16612 site Enter between two meeting pcl genes.
4. vaccine according to any one of claim 1 to 3, wherein at least one antigen, which is selected from, comes from bovine viral The antigen of diarrhea virus (BVDV), the antigen from 3 type bovine parainfluenza viruses, from Bovine Respiratory Syncytial virus antigen and Antigen from microorganism.
5. vaccine according to claim 4, wherein the BVDV antigens are selected from glycoprotein E 0 and glycoprotein E 2.
6. vaccine according to claim 4, wherein the microorganism is selected from：Mycoplasma bovis (Mycoplasma bovis), Salmonella (Salmonella) kind, multocida (Pasteurella multocida), Mannheimia haemolytica (Mannhiemia haemolytica) and Haemophilus somnus (Haemophilus somnus).
7. vaccine according to claim 4, wherein the low toxicity power BoHV-1 strains are strain V155.
8. veterinary medical composition, it includes vaccine according to any one of claim 1 to 7 and one or more Pharmaceutical acceptable carrier, excipient and/or diluent.
9. pharmaceutical composition according to claim 8, it is configured to be suitable to nasal administration.
10. 1 type bovine herpes virus (BoHV-1) genome from low toxicity power BoHV-1 is being prepared for being directed to from cattle disease original At least one antigen of body to the purposes in the medicine of bovid vaccine inoculation, wherein the BoHV-1 genomes have with The inhereditary material for coding at least one antigen that BoHV-1 is heterologous, insert between two convergence BoHV-1 genes, wherein institute State insertion and do not cut the expression of the BoHV-1 genes, and wherein encode the inhereditary material of at least one antigen following Site is inserted between the polyadenylation signal of two meeting pcl genes, and the site is stepped on selected from BoHV-1 reference sequences GenBank 16600 to 16612,22449 to 22493,40734 to 40768,58229 to 58563, the 67037 of mark AJ004801 to 67091st, 74994 to 75041,84496 to 84528,90732 to 90760 and 96870 to 96882.
11. purposes according to claim 10, wherein the medicine be configured to be used for through it is intranasal, oral, intramuscular, sublingual, Intravenous, subcutaneous, intra-arterial, skin spraying, intravaginal or intrarectal route are applied.
12. a kind of method for preparing the vaccine at least one antigen from cattle disease substance, methods described includes：
(i) the BoHV-1 genomes from low toxicity power BoHV-1 are incorporated in bacterial artificial chromosome (BAC) carrier to be formed BoHV-1 precoded vector BAC constructs；
(ii) recombinated via GET and the inhereditary material for encoding at least one antigen is inserted into the BoHV-1 precoded vectors BAC structures Build to produce restructuring BoHV-1-BAC (rBoHV-1-BAC) carrier in body, the heredity of at least one antigen will be encoded Material is inserted in following site between the polyadenylation signal of two meeting pcl genes, and the site is selected from BoHV-1 reference sequences GenBank registration numbers AJ004801 16600 to 16612,22449 to 22493,40734 to 40768,58229 to 58563, 67037 to 67091,74994 to 75041,84496 to 84528,90732 to 90760 and 96870 to 96882；
(iii) the rBoHV-1-BAC carriers are converted and expanded in bacterial host；And
(iv) purified from the bacterial host and separate the rBoHV-1-BAC carriers and the carrier is configured to vaccine Composition.
13. method according to claim 12, wherein by least one antigen selected from 16600 to 16612 position Between point two meeting pcl genes of insertion.
14. method according to claim 12, wherein at least one antigen, which is selected from, comes from bovine viral diarrhea virus (BVDV) antigen, the antigen from 3 type bovine parainfluenza viruses, from Bovine Respiratory Syncytial virus antigen and from micro- life The antigen of thing.
15. method according to claim 14, wherein the BVDV antigens are selected from glycoprotein E 0 and glycoprotein E 2.
16. method according to claim 14, wherein the microorganism is selected from：Mycoplasma bovis, salmonella strain, kill bar more This moral Salmonella, Mannheimia haemolytica and Haemophilus somnus.
17. method according to claim 12, wherein the low toxicity power BoHV-1 strains are strain V155.
18. the culture cell transfected with rBoHV-1-BAC carriers according to claim 12.
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CN102238961A (en) * 2008-10-03 2011-11-09 诺华公司 Bovine herpes virus -1 compositions, vaccines and methods

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