CN105802918B - Chicken's infectious bronchitis nephritis strain and its vaccine composition, preparation method and application - Google Patents
Chicken's infectious bronchitis nephritis strain and its vaccine composition, preparation method and application Download PDFInfo
- Publication number
- CN105802918B CN105802918B CN201410837138.7A CN201410837138A CN105802918B CN 105802918 B CN105802918 B CN 105802918B CN 201410837138 A CN201410837138 A CN 201410837138A CN 105802918 B CN105802918 B CN 105802918B
- Authority
- CN
- China
- Prior art keywords
- chicken
- strain
- infectious bronchitis
- vaccine composition
- albumen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention belongs to veterinary biologics fields, it is related to the chicken's infectious bronchitis nephritis strain and its attenuated IBDVs of the albumen that a kind of gene coded sequence is SEQ ID NO.2, attenuated IBDVs are as will be obtained by the strain continuous passage to 110-140 generation in chicken embryo.The invention further relates to the vaccine composition, the preparation method and application that use the strain, its attenuated IBDVs and its antigen protein preparation, the antigen containing inactivated whole virus of preparation, the attenuation totivirus antigen of work, the vaccine composition containing subunit antigen can effectively prevent avian nephropathogenic infectious bronchitis related disease.
Description
Technical field
The invention belongs to veterinary biologics fields, and in particular to chicken's infectious bronchitis nephritis strain and its epidemic disease
Seedling composition, preparation method and applications.
Background technique
Infectious bronchitis of chicken is that one kind as caused by coronavirus is acute, highly contagious disease, according to virus
Tissue tropism and caused diseased region be often divided into breathing pattern, kidney type, gonotype and Glandular Stomach Type etc..
1948, the U.S. reported kidney type infective bronchitis (Kidney infectious for the first time
bronehitis).1962, Winterfied etc. had found nephropathic type infectious bronchitis virus in the U.S., and will
It is named as (Winterfield R W, the Hitchner S such as Holte plants, Gray plants, CV56b plants, Wolgemuth plants
B.Etiology of an infectious nephritis-nephrosis syndrome of
Chickens.Am.J.Vet.Res.1962,23:1273~1279).The same year, Cumming isolate N1/62 (T in Australia
Strain), which is considered as representative strains (the Cumming R G B.Infectious of avian infectious bronchitis virus virus
avian nephrosis(uraemia)in Australia.Aust Vet J.1963,39:145-147).The 1990s
Since, kidney type infective bronchitis also outburst in succession at home is popular very extensive.Especially in recent years, chicken kidney type infects
Property bronchitis pandemic in China broiler chicken group, to poultry production bring serious loss.
Avian infectious bronchitis virus is typical coronavirus, and genome is single-stranded positive RNA, genome encoding
Structural proteins necessary to four kinds: spike protein (S protein), memebrane protein (M albumen), nucleocapsid protein (N protein) and small membrane gene
(E protein), wherein S protein is encoded by mRNA2, and the product after translation is cut into S1 albumen and S2 albumen in the cell.Due to
The variation of kidney type infective bronchitis virus and its strain is sufficiently complex, under certain condition biological characteristics and serotype water
Some variations malicious (strain) kind often occurs on flat, so that the diagnosis and treatment of this disease encounter very big difficulty.In clinic, normal immunological chicken group
The disease frequent occurrence, is mainly shown as following symptom: early period gas tube orifice rale, draw white loose stool, the later period is mainly under water sample
Dysentery, due to serious dehydration, claw is withered (being commonly called as " dry pawl disease "), and it is main that kidney enlargement and uric acid mineralization etc. is presented in the chicken that dies of illness
Characteristic symptoms.Kidney type infective can be diagnosed as in conjunction with epidemic characteristic, clinical symptoms and pathological anatomical change through laboratory diagnosis
Bronchitis.Therefore, a kind of new avian infectious branch that can effectively prevent instantly popular infective bronchitis is researched and developed
Asthma Vaccine is particularly significant.
Patent application CN101514334A is disclosed one plant of infectious bronchitis virus velogen strain tl/CH/ of separation
LDT3/03 plants, progress continuous passage causes weak in 9-10 age in days SPF chicken embryo, reaches for 120 generations, causes weak evaluation test qualified, makees
For attenuated vaccine strain use, it is named as LDT3-A, however the current IBV prevalence strain homology in its vaccine strain S1 gene order China
It is lower, complete protection cannot be provided current kidney type epidemic strain.
Research (the chicken such as Wang Wencheng, Wei Guangsen, Li Shangbo of infectious bronchitis of chicken (thermophilic kidney type) live vaccine (W93 plants)
The research China Preventive Veterinary Medicine report of infective bronchitis (thermophilic kidney type) live vaccine (W93 plants), 2001,23 (6): 436-
440) it discloses and weak one plant W93 plants of low virulent strain of acquisition is caused by chicken embryo continuous passage, be applied to low Japanese instar chickling immunoprophylaxis, so
And the isolation of strains Attenuation age is more early, and it is apart from each other with current popular strain gene level, effective protection can not be provided.
Summary of the invention
In order to solve the deficiencies in the prior art, it is high with current IBV prevalence strain genetic homology that the present invention provides a kind of
Chicken's infectious bronchitis nephritis strain and its preparation inactivated vaccine, cause its preparation of weak live vaccine, subunit vaccine
Method and application, the vaccine can effectively prevent current IBV prevalence virus strain infection and classics IBV virus strain infection simultaneously.
The first aspect of the present invention is related to a kind of chicken's infectious bronchitis nephritis strain, described avian nephropathogenic infectious
Bronchitis poison strain gene coded sequence is the albumen of SEQ ID NO.2.
Term " chicken's infectious bronchitis nephritis strain " refers to chicken's infectious bronchitis nephritis through ability
The field technique personnel strain isolated using conventional technical means, including but not limited to its S1 albumen have and SEQ ID
The chicken's infectious bronchitis nephritis of No.1 essentially identical nucleotide sequence.With SEQ ID No.1 is essentially identical refers to,
The nucleotide sequence of chicken's infectious bronchitis nephritis, which is preferably comprised, has the identical sequence of 85%-100% with SEQ ID No.1
Column, preferably in the condition that chicken's infectious bronchitis nephritis is not chicken's infectious bronchitis nephritis described herein
Under, such as compared with as the PLK of reference virus isolate strain (CCTCC NO.V201442), the nucleotide sequence of S1 albumen is same
Source property is lower than 91%, preferably shorter than 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.Avian nephropathogenic infectious branch
Bronchitis virus nucleotide sequence preferably have more than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or
89%, it is identical as SEQ ID No.1, also, it is preferred that being not that chicken kidney type described herein passes in chicken's infectious bronchitis nephritis
Under conditions of metachromia bronchitis virus, such as compared with as PLK plants of reference virus isolate, the nucleotide homology of S1 albumen
Property be lower than 91%, preferably shorter than 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
Term " homology " refers to the similarity degree of two amino acid sequences or two nucleotide sequences.Amino acid sequence or
The homology of nucleotide sequence can be calculated by any method appropriate well known in the art, for example, can be by target
Amino acid (or nucleotide) sequence and reference amino acid (or nucleotide) sequence carry out sequence alignment, it may be necessary to introduce empty
It lacks, so that identical amino acid (or nucleotide) being optimal of number between the sequence of two comparisons, and calculate on this basis
The percentage of same amino acid (or nucleotide) between two amino acid (or nucleotide) sequences.Amino acid (or nucleotide) sequence
Comparison and the calculating of homology can pass through software realization well known in the art, such as, but not limited to, BLAST software is (in beauty
It can get in the network address of state National Biotechnology Information Center NCBI: http://blast.ncbi.nlm.nih.gov/
Blast.cgi, Huo Zhejian, for example, Altschul S.F.et al, J.Mol.Biol., 215:403-410 (1990)；
Stephen F.et al, Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 software is (raw in Europe
It can get in object information research institute network address: http://www.eji.ac.uk/Toolsa/clustalw2/, see also, for example,
Higgins D.G.et al, Methods in Enzymology, 266:383-402 (1996)；Larkin M.A.et al,
Bioinformatics (Oxford, England), 23 (21): 2947-8 (2007))；It is (raw in Sweden with TCoffee software etc.
It can be obtained on the website of object informatics research institute: http://tcoffee.vital-it.ch/cgi-bin/Tcoffee/
Tcoffee_cgi/index.cgi, see also, for example, Poirot O.et al, Nucleic Acids Res., 31 (13):
3503-6(2003)；Notredame C.et al, J.Mol.Boil., 302 (1): 205-17 (2000)).It is carried out using software
When sequence alignment, the default parameters of software offer, or the parameter that can also be provided according to the actual situation software can be used
It is adjusted, these are all in the knowledge of those skilled in the range.
Preferably, the chicken's infectious bronchitis nephritis strain, M albumen have as shown in SEQ ID No.3
Gene order, encoding amino acid sequence SEQ ID No.4.
Term " M albumen " is one of structural proteins of chicken's infectious bronchitis nephritis, by 224~225 amino
Acid composition (grind by Zhang Youfeng, the building of avian infectious bronchitis virus S1, M, N gene VR1020 and pVAX1 and immunogenicity
Study carefully, Sichuan Agricultural University's Master's thesis, 2008).
It is further preferred that the chicken's infectious bronchitis nephritis strain, N protein has such as SEQ ID No.5
Shown in gene order, encoding amino acid sequence SEQ ID No.6.
Term " N protein " is one of structural proteins of chicken's infectious bronchitis nephritis, is made of 409 amino acid
(Zhang Youfeng, the building and immunogenicity research of avian infectious bronchitis virus S1, M, N gene VR1020 and pVAX1, Sichuan
Agriculture university's Master's thesis, 2008).
As one embodiment of the present invention, described chicken's infectious bronchitis nephritis PLK plants, deposit number is
CCTCC NO.V201442。
PLK plants of avian infectious bronchitis virus (Infectious Bronchitis Virus, strain PLK) preservation
In China typical culture collection center, deposit number is CCTCC NO.V201442, and preservation address: Wuhan, China Wuhan is big
It learns, the deposit date is on December 9th, 2014.
Another aspect of the present invention relates to a kind of low virulent strains of chicken's infectious bronchitis nephritis, wherein the chicken
The virus attenuated strain of kidney type infective bronchitis is the avian nephropathogenic infectious branch for the albumen that gene coded sequence is SEQ IDNO.2
The low virulent strain that bronchitis virus strain passes through continuous passage any generation into 110-140 generation in chicken embryo；Or it is described
Chicken's infectious bronchitis nephritis low virulent strain is described chicken's infectious bronchitis nephritis PLK plants by chicken embryo
The low virulent strain of continuous passage any generation into 110-140 generation.
Another aspect of the present invention relates to a kind of vaccine compositions, wherein the vaccine composition includes the institute of immune amount
The chicken's infectious bronchitis nephritis strain antigen and pharmaceutically acceptable carrier stated.
As one embodiment of the present invention, the chicken's infectious bronchitis nephritis strain antigen is inactivation
Totivirus antigen, the totivirus antigen of attenuation, subunit antigen.
As one embodiment of the present invention, the vaccine composition includes inactivation preceding 0.4 × 106.7EID50/
The totivirus antigen of the inactivation of 0.1ml.
Chicken's infectious bronchitis nephritis strain as a kind of preferred embodiment of the invention, after the inactivation
It is to be inactivated by the chicken embryo liquid that the chicken's infectious bronchitis nephritis strain is expanded acquisition in chicken embryo through inactivator
Gained.
Chicken's infectious bronchitis nephritis strain as a kind of preferred embodiment of the invention, after the inactivation
It is by the way that the chicken's infectious bronchitis nephritis strain to be cultivated to the culture of amplification acquisition on passage cell through going out
Agent inactivation gained living.
As one embodiment of the present invention, the vaccine composition includes 1.0~7.0log10EID50Attenuation
Totivirus antigen；Preferably, the vaccine composition includes 2.5~5.0log10EID50The totivirus antigen of attenuation；It is optimal
Selection of land, the vaccine composition include 3.0~4.0log10EID50The totivirus antigen of attenuation.
The present invention provides chicken's infectious bronchitis nephritis low virulent strain, the avian nephropathogenic infectious bronchitis disease
Malicious low virulent strain be by chicken embryo by the chicken's infectious bronchitis nephritis strain pass on 110-140 generation and get it is weak
Strain, so that the clinical sign of IBV disease can not be caused when the modified virus is given to the animal of easy infection IBV
As, but can induce the immune response for keeping the immune pathogenicity form of the animal to IBV immune.
The present invention provides avian nephropathogenic infectious bronchitis attenuated live vaccines, the attenuated live vaccines contain the chicken kidney
The infectivity bronchitis virus attenuated strain of type and/or veterinarily acceptable carrier.
As one embodiment of the present invention, the chicken's infectious bronchitis nephritis low virulent strain is by chicken kidney type
PLK plants of infectious bronchitis virus strain cause weak strain through chicken embryo continuous passage, by chicken embryo by the chicken kidney type
PLK plants of infectious bronchitis virus passage 110-140 generation and get low virulent strain.
As one embodiment of the present invention, the carrier includes freeze drying protectant, adjuvant, the freeze drying protectant packet
Include sucrose skim milk and gelatin and sucrose；The adjuvant includes aluminium glue adjuvant, saponin(e, water-in-oil emulsion, oil in water emulsion, water
Packet water-in-oil emulsion, the polymer of acrylic or methacrylic acid, maleic anhydride and alkenyl (alkenyl) derivative
It is copolymer, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, big
One or more of enterobacteria heat-labile toxin, cholera toxin, IMS 1314, muramyl dipeptide or Gel adjuvant.
As one embodiment of the present invention, chicken's infectious bronchitis nephritis is passaged in vaccine composition
The content of low virulent strain obtained by 110-140 generation is usually 1.0~7.0log10EID50, preferably 2.5~5.0log10EID50,
More preferably 3.0~4.0log10EID50。
The attenuated live vaccines also contain other compositions, and the other compositions include adhesive, colorant, desiccant, disappear
Toxic agent, stabilizer, excipient, adsorption enhancer, fatty acid, surfactant, water and buffer, preferably comprise buffer,
Disinfectant and surface reactive material.
As one embodiment of the present invention, the present invention provides a kind of avian nephropathogenic infectious bronchitis subunit epidemic disease
Seedling, the vaccine composition include the S1 albumen of 0.5~1.5 μ g/ml, the M albumen of 0.5~1.5 μ g/ml or N protein, or
The N protein of the S1 albumen of 0.5~1.5 μ g/ml, the M albumen of 0.25~0.75 μ g/ml and 0.25~0.75 μ g/ml.
As one embodiment of the present invention, the present invention provides a kind of avian nephropathogenic infectious bronchitis subunit epidemic disease
Seedling, the subunit vaccine include the M albumen and/or SEQ of the S1 albumen of SEQ ID No.2 coding, SEQ ID No.4 coding
The encoded N protein of ID No.6.
As a kind of preferred embodiment of the invention, the present invention provides a kind of avian nephropathogenic infectious bronchitis subunit
Vaccine, the subunit vaccine include S1 albumen, 0.5~1.5 μ g/ml that the SEQ ID No.2 of 0.5~1.5 μ g/ml is encoded
The M albumen or the encoded N protein of 0.5~1.5 μ g/ml SEQ ID No.6 of SEQ ID No.4 coding or 0.5~1.5 μ
G/ml SEQ ID No.2 coding S1 albumen, 0.25~0.75 μ g/ml SEQ ID No.4 coding M albumen and 0.25~
The encoded N protein of SEQ ID No.6 of 0.75 μ g/ml.
As one embodiment of the present invention, the vaccine composition of the invention further includes adjuvant.
Term " adjuvant " may include aluminium glue adjuvant；Saponin(e (saponin), such as Quil A, QS-21 (Cambridge
Biotech Incorporation,Cambridge MA)、GPI-0100(Galenica Pharmaceuticals
Incorporation, Birmingham AL)；Water-in-oil emulsion；Oil in water emulsion；W/O/W emulsion；Acrylic acid or first
The polymer of base acrylic acid；The compound that the copolymer of maleic anhydride and alkenyl (alkenyl) derivative is selected.
It is oily (European Pharmacopea type) that term " emulsion " can be particularly based on light liquid paraffin；Because of olefin oligomerisation
The isoprenoid of generation is oily (isoprenoid oil), such as saualane (squalane) or squalene oil (squalene
Oil), especially isobutene or certain herbaceous plants with big flowers alkene；The ester containing linear alkyl of acid or alcohol, more specifically vegetable oil, ethyl oleate, propylene glycol two-
(caprylate/certain herbaceous plants with big flowers acid esters), glycerol three-(caprylate/certain herbaceous plants with big flowers acid esters) or Rikemal PO 200；The ester of branched chain fatty acid or alcohol, especially
Its isostearate.Oil is applied in combination with emulsifier to form emulsion.Emulsifier preferred nonionic surfactants, especially mountain
The ester of pears glycan, the ester (such as anhydrous mannitol oleate) of mannide (mannide), aliphatic dihydroxy alcohol (glycol)
Ester, the ester of polyglycereol (polyglycerol), the ester of propylene glycol and the ester of oleic acid, the ester of isostearic acid, ricinoleic acid ester
Or the ester of hydroxy stearic acid, their optional ethoxylations, there are also Pluronic L121s, especially
Pluronic product, especially L121." the The theory and practical write referring to Hunter etc.
application of adjuvants》(Ed.by DES Stewart-Tull,John Wiley and Sons,New
York, 1995:51-94) and Todd etc. " Vaccine " (1997,15:564-570) that writes.For example, Powell M can be used
" Vaccine design, the Subunit and adiuvant the approach " (Plenum write with Newman M
Press, 1995) the SPT emulsion of page 147 description and the MF59 emulsion of the description of page 183.
Term " polymer of acrylic or methacrylic acid " is preferably the acrylic or methacrylic acid polymer being crosslinked,
Especially be crosslinked with the poly alkenyl ether of sugared (sugar) or polyalcohols, these compounds be known as carbomer (Carbomer,
Trade name Carbopol) (Phameuropa, 1996,8 (2)).Those skilled in the art are referring also to United States Patent (USP)
US2909462 and gathers hydroxylated compound crosslink which depict this kind of acrylate copolymer, and the compound has extremely
Few 3 hydroxyls, preferably more than 8, the hydrogen atom of wherein at least 3 hydroxyls is by the unsaturated lipid at least two carbon atom
Alkyl (aliphatic radical) replaces.Preferred group is the group that those contain 2-4 carbon atom, such as vinyl,
Allyl and other ethylenically unsaturated groups (ethylenically unsaturated group).The unsaturated group is certainly
Body may include other substituent groups, such as methyl.These products are sold with the name of carbopol, and (BF Goodrich, Ohio, USA) is special
It is unsuitable.They are crosslinked with allyl sucrose or with Allyl pentaerythritol (allyl pentaerythritol).Among these may be used
Refer to carbopol 974P, 934P and 971P, most preferably with carbopol 971P.
Term " copolymer of maleic anhydride and alkenyl derivative " is also it is contemplated that maleic anhydride and ethylene
Copolymer EMA (Monsanto), these polymer dissolve in water generates acid solution, neutralized, is preferably neutralized to physiological pH,
To generate assist agent solution, immunogenicity, immunogenicity or vaccinal composition itself can be mixed thereto.
Term " adjuvant " further includes, but is not limited to, RIBI adjuvant system (Ribi Incorporation), Block co-
Polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A
(monophosphoryl lipid A), Avridine lipid-amine adjuvant, E.coli LT (recombination or its
It), cholera toxin, IMS1314, muramyl dipeptide, Gel adjuvant etc..
Preferably, the adjuvant includes aluminium glue adjuvant, saponin(e, water-in-oil emulsion, oil in water emulsion, W/O/W cream
Agent, the polymer of acrylic or methacrylic acid, maleic anhydride and alkenyl (alkenyl) derivative copolymer,
RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli
One or more of heat-labile toxin, cholera toxin, IMS 1314, muramyl dipeptide or Gel adjuvant.
In some preferred embodiments, the inactivated vaccine includes one or more non-avian nephropathogenic infectious bronchuses
The inactivating pathogens of scorching virus, such as: non-chicken's infectious bronchitis nephritis inactivating pathogens can be selected from, newcastle disease virus
(newcastle disease virus, NDV), fowlpox virus (avian pox virus, APV), avian encephalomyclitis virus
(avian encephalomyelitis virus, AEV), infectious bursal disease virus (infectious bursal
Disease virus, IBDV), eggs crack detection (avian pasteurella multocida, Pm), avian flu
Poison (avian influenza virus, AIV), egg drop syndrome (egg drop syndrome-76 virus, EDS-
76 virus)。
In some embodiments, the inactivated vaccine further includes one or more other chickens selected from the following and infects
Property bronchitis virus inactivating pathogens, such as: M41 plants (be purchased from China Veterinery Drug Inspection Office).
It is another aspect of the invention to provide the antigen proteins of the chicken's infectious bronchitis nephritis strain.
As one embodiment of the present invention, chicken's infectious bronchitis nephritis strain antigen protein of the invention
For S1 albumen, the amino acid sequence of the encoding histone SEQ ID NO.2.
As one embodiment of the present invention, chicken's infectious bronchitis nephritis strain antigen protein of the invention
For M albumen, the amino acid sequence of the encoding histone SEQ ID NO.4.
As one embodiment of the present invention, chicken's infectious bronchitis nephritis strain antigen protein of the invention
For N protein, the amino acid sequence of the encoding histone SEQ ID NO.6.
It is another aspect of the invention to provide a kind of methods for preparing the vaccine composition.
As one embodiment of the present invention, which comprises (1) avian nephropathogenic infectious branch gas described in amplification cultivation
Pipe inflammation virus stain；(2) the chicken's infectious bronchitis nephritis strain of the amplification cultivation is inactivated, addition can pharmaceutically connect
The carrier received.
As one embodiment of the present invention, which comprises (1) chicken kidney type described in continuous passage passes in chicken embryo
Metachromia bronchitis poison strain, is passaged to 110-140 generation；(2) the weak chicken kidney type of cause in 110-140 generation is passed on described in amplification cultivation
Infectious bronchitis virus strain；(3) in the weak chicken's infectious bronchitis nephritis strain of the cause of the amplification cultivation
Pharmaceutically acceptable carrier is added.
As one embodiment of the present invention, which comprises (1) avian nephropathogenic infectious described in clonal expression respectively
S1 albumen, the M albumen, N protein of bronchitis poison strain；(2) be proportionally added into the S1 albumen of the expression, M albumen and/or
N protein；(3) pharmaceutically acceptable carrier is added in above-mentioned chicken's infectious bronchitis nephritis antigen protein.
It is avian nephropathogenic infectious in preparation prevention and treatment that it is another aspect of the invention to provide the vaccine compositions
Application in bronchitis related disease.
Term " prevention " refers to the symptom quilt by its infection relevant to chicken's infectious bronchitis nephritis or disease
It blocks or postpones；Term " treatment " refers to the symptom quilt by infection relevant to chicken's infectious bronchitis nephritis or disease
The process for mitigating or completely eliminating.
The present invention has the advantages that
One plant of chicken's infectious bronchitis nephritis strain is provided, it can not only be effective by inactivated vaccine prepared by the strain
The current domestic popular avian nephropathogenic infectious bronchitis of prevention and treatment, moreover it is possible to which effective protection breathing pattern IBV velogen strain attacks poison.
Provided subunit vaccine prepares after being expressed by S1, M and N gene of offer strain, the subunit
Vaccine attacks poison and can provide effective protection to avian nephropathogenic infectious bronchitis, breathing pattern IBV velogen strain.
Detailed description of the invention
Fig. 1 is IBV PLK plants of S1 gene PCR amplified production electroresis appraisal, wherein left lane is DL2000 Marker,
The right swimming lane is IBV S1 gene PCR amplified production.
Specific embodiment
The invention will now be further described with reference to specific embodiments, and the advantages and features of the present invention will be with description more
It is clear.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art
It should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form carry out
Modifications or substitutions, but these modifications and replacement are fallen within the protection scope of the present invention.
Infectious bronchitis virus PLK strain employed in the embodiment of the present invention, deposit number CCTCC
NO.V201442, the deposit date is on December 09th, 2014, and depositary institution is China typical culture collection center.
Used chemical reagent is that analysis is pure in the embodiment of the present invention, is purchased from Chinese medicines group.
The embodiment of the present invention is prepared with volume ratio for the freeze drying protectant (8% (W/W) gelatin, 40% (W/W) sucrose) of 1:1
It is avian infectious for 46:54 (antigen: ISA206 oil adjuvant) to prepare kidney type with volume ratio for kidney type infectious bronchitis of chicken live vaccine
It is illustrated respectively for bronchitis subunit vaccine, but no matter the embodiment is not constituted to this hair under any circumstance
Bright restriction.
To keep the present invention easier to understand with reference to specific embodiments the present invention is further explained.It is of the present invention
Experimental method, if being conventional method without specified otherwise；The biomaterial, if without specified otherwise, it can be from business way
Diameter obtains.
Separation identification, preservation and the gene sequencing of 1 chicken's infectious bronchitis nephritis of embodiment
Separation identification, the preservation of 1.1 chicken's infectious bronchitis nephritis
Chicken's infectious bronchitis nephritis is in 2013 beginning of the years by Pulaike Biological Engineering Co., Ltd.'s technology
Personnel separate from the obvious diseased kidneys tissue of doubtful outburst infective bronchitis chicken house illness chicken (enlargement is in piebald shape)
Arrive, egg inoculation expand culture obtain virus liquid, acquired virus liquid can cause inoculated into chick embryo occur dehydration, roll up, develop it is small
Etc. infectious bronchitis virus typical cytopathic；Confirm that obtaining virus is avian infectious branch through negative staining Electronic Speculum, RT-PCR detection
Bronchitis virus.Animal returns experiments have shown that the strain can cause chicken to occur with kidney enlargement, serious visible piebald kidney sample typical case
Avian nephropathogenic infectious bronchitis clinical symptoms.It thus is classified as chicken's infectious bronchitis nephritis strain, is ordered
It is PLK plants entitled.
PLK plants of preservation information are as follows: deposit number is CCTCC NO.V201442, the deposit date is on December 09th, 2014,
Depositary institution is China typical culture collection center.
1.2 chicken's infectious bronchitis nephritis S1 gene sequencing
By RT-PCR reaction amplification obtain PLK plant of S1 gene (see SEQ ID No.1), then progress gene cloning and
Sequencing, and carry out S1 gene order with H120, W93 and LDT3-A vaccine strain S1 gene to PLK plants and compare and analyze.
PLK plants of S1 gene order sequencing result details SEQ ID No.1, with IBV H120, H52 and W93 vaccine strain S1 base
Because sequence carries out multiple alignment, the homology of PLK plants of discovery and H120, W93 and LDT3-A vaccine strain S1 gene is respectively 78%,
77.8% and 85.2%, show: for PLK plants of S1 genes compared with above-mentioned traditional vaccine strain, there are many places nucleotide diversities, and lead
The change of corresponding amino acid sequence is caused.
The preparation of 2 avian nephropathogenic infectious bronchitis inactivated vaccine of embodiment
2.1 PLK plants of avian infectious bronchitis virus production kind of a foundation for malicious seed lot
The PLK strain for taking separation dilutes 100 times with sterile saline, and it is intracavitary to be inoculated in 9~10 age in days SPF chick embryo allantois,
Every embryonic breeding kind 0.1ml, seals pin hole after inoculation, sets 37 DEG C of incubations.Dead germ for 24 hours is discarded, chicken embryo is observed to 48h, by 30~72h's
Chick embryo air sac is upward, sets 4 DEG C of coolings 4~for 24 hours.Chick embryo allantoic liquid is harvested, several chick embryo allantoic liquids are mixed into one group, sets and goes out
It in bacterium bottle, is saved at 2~8 DEG C, while doing steriling test, method is using SPF chicken embryo to avian infectious bronchitis virus according to this
PLK plants of velogen strain have carried out continuous 13 generation passage.According to " Republic of China Veterinary Pharmacopoeia " (version in 2010) to E2~E13 generation
Kind poison is identified, the results showed that does not find bacterium, mould, mycoplasma and exogenous virus pollution；Viral level be 106.0~
107.3EID50/0.1ml；Inactivated vaccine is made in the virus liquid in E3, E7 and E13 generation, can produce complete guarantor within 28 days after immune chicken
Shield.According to test result, PLK plants of productions kind of seed lot for poison is established.It regard PLK plants of P2 generations as primordial seed, PLK P3
~P7 was no more than for 3 generations with seed subculture as basic seed, production.PLK plants of simultaneous selection, as effect inspection strain, virus are used
Sterile saline is diluted to 106.0ELD50/ 0.1ml, by intramuscular injection 1ml/ plumage, while the agent of collunarium eye droppings 0.2ml/ plumage
Amount attacks poison to 10 2 monthly age SPF chickens, observe 14 after attacking poison, and dead chicken record in time, dissect, cuts open after surviving chicken observation 14 days
Inspection, should at least 8 chickens swollen, piebald kidney occurs using kidney as the symptom of typical cytopathic.
The preparation of 2.2 avian infectious bronchitis virus inactivated vaccines
2.2.1 the preparation of production seed and inspection
After the PLK plants of malicious rehydrations in P3, P4, P7 freeze-drying basis kind, it is diluted using sterile saline, is connect through allantoic cavity
Kind SPF chicken embryo, 0.1ml/ embryo, 37 DEG C of cultures are observed 48 hours.The allantoic fluid of sterile collection 24 hours~48 hours dead chicken embryos,
Mixing is as production seed.Malicious valence measurement, disease are carried out according to " Republic of China Veterinary Pharmacopoeia " (version in 2010) the method
Malicious assay, specificity identification and pure property are examined.
1 batch of production seed, every batch of 100ml are respectively prepared for using PLK plants of P3, P4, P7 generation basic kind of poison.Inspection result is 3
It criticizes and is polluted without bacterium, mould, mycoplasma and exogenous virus, viral level is respectively 106.6、106.8With 107.0EID50/0.1ml。
2.2.2 the preparation of seedling venom
The production seed of step 2.2.1 is subjected to 100 times of dilutions using sterile saline, through allantoic cavity inoculation 10~11
The susceptible chicken embryo of age in days, 0.1ml/ embryo, 37 DEG C of cultures are observed 48 hours, dead embryo and embryo living, sterile collection after collecting 24 hours
Chicken embryo liquid, as seedling venom.
It is respectively prepared for 1 batch of virus liquid using above-mentioned 3 batches of production seed, lot number is 1001,1002 and 1003 respectively, and every batch of is about
200ml, viral level are 10 respectively6.7、106.5、106.8EID50/0.1ml。
2.2.3 inactivation of virus and inspection
In the three batches of virus liquids prepared to step 2.2.2,10% formalin is added, makes the final concentration of of formalin
0.2% (volumn concentration), after mixing well, 37 DEG C are inactivated 16~18 hours, during which primary every shaking in 2~4 hours.It goes out
After the completion of work, sampling is done inactivation and is examined.Using 10 10 pieces of age in days SPF chicken embryos, 0.2ml stoste, 37 DEG C of cultures are inoculated with through allantoic cavity
Observation 5 days, and method like this, take a blastochyle blind passage generation, and chicken embryo is that inactivation is complete without IBV typical cytopathic.The result shows that: 3 batches go out
Virus liquid after work connects in SPF chicken embryo passed for 2 generations, did not caused chicken embryo death, IBV typical cytopathic.Show that inactivation of virus is thorough.
2.2.4 vaccine formulation
Three batches of vaccines 1001,1002 and 1003 are prepared for the virus liquid through 2.2.3 inactivation treatments of step.
2.2.5 oily mutually prepare
Match liquefaction phase in the ratio of 1.5 capacity part of 94 capacity part of injection white oil, Si Ben -806 capacity part and aluminum stearate.
Take a small amount of injection white oil to mix with aluminum stearate, heating dissolve to it is translucent again with full dose Si Ben -80 and note
It penetrates and is uniformly mixed with white oil, sterilize 30 minutes, be cooled to room temperature spare through 116 DEG C.
2.2.6 prepared by water phase
It is 10 by the viral level of 96 capacity part steps 2.2.26.7EID501001 batches of diseases of IBV of/0.1ml (before inactivation)
After the assay was approved through step 2.2.3 inactivation sterilizing 4 parts by weight of Tween-80 are added in venom, and sufficiently oscillation mixes, until tween-
80 are completely dissolved, and obtain water phase.
According to oily mutually with the capacity ratio of water phase 1.5:1, oil is added in homogenate cup, when slowly running, is slowly added to water
Phase.It is homogenized 2~3 minutes after adding water phase, stops 0.01% thimerosal of addition emulsion total weight before emulsification, obtaining lot number is
1001 500ml vaccine.
The method for repeating 2.2.2~2.2.6, is prepared for a collection of inactivated vaccine using 1002 and 1003 batches of virus liquids respectively,
Lot number is 1002 and 1003 respectively.
It is respectively 1001,1002 and 1003 500ml vaccine by lot number, quantitative separating, after labeling, 2~8 DEG C of preservations.Together
When sampling according to the regulation of " Republic of China Veterinary Pharmacopoeia " (version in 2010) do steriling test, physical behavior examine, formaldehyde
Content and thiomersal measurement.As a result, 3 batches of equal asepsis growths of vaccine；Physical behavior, formaldehyde and thiomersal meet rule
It is fixed.
The test of 2.3 minimum immune dosages
Using 1001 and 1002 two batches vaccines, respectively with 0.5,0.25 and 0.13ml, tri- dosage, each subcutaneous injection is immune
The SPF chicken of 10 40 ages in days (after H120 fundamental immunity 21 days).After 28 days, with PLK plants of P2 generations with 106.0ELD50/ 0.1ml, passes through
Intramuscular injection 1ml/ plumage, while the dosage of collunarium eye droppings 0.2ml/ plumage carries out attacking poison, 10/group, observes 14 after attacking poison, it is dead
Chicken records in time, dissect, and dissect after survival chicken is observed 14 determines whether to fall ill.Reach the minimum for the HI effect standard worked out
Dosage determines dosage as minimum immune dosage.Concrete outcome is shown in Table 2.
The result shows that 28 days after immune, the effect for meeting the protection of 8/10 chicken examines mark when with the dose immunization chicken of 0.25ml
It is quasi-.Illustrate, the dosage of 0.25ml is minimum immune dosage, for safety, determines that dosage is 0.5ml.
1 IBV inactivated vaccine minimum immune dosage measurement result of table
2.4 immuning effect test
Using 1001,1002 and 1003 totally 3 batches of products carry out the test, with dosage 0.5ml, each subcutaneous injection is exempted from
The SPF chicken of 10 40 ages in days of epidemic disease (after H120 fundamental immunity 21 days), while control group is set up, subcutaneously with M41 plants of inactivated vaccines
Furthermore 20 40 ages in days of injecting immune set up saline control group 20,0.5ml physiological saline is subcutaneously injected (through H120 base
Plinth is immune).After 28 days, respectively with PLK plants of P2 generations and M41 plants with 106.0ELD50/ 0.1ml, by intramuscular injection 1ml/ plumage, simultaneously
The dosage of collunarium eye droppings 0.2ml/ plumage carries out attacking poison, observes 14 after attacking poison, and dead chicken records in time, dissect, survival chicken observation
Dissect after 14 days, PLK attack malicious group and have seen whether the classical symptoms such as swollen, the piebald kidney of kidney, M41 plant attack poison organize tracheae whether there is or not bleeding and
Stickiness, catarrh sexual secretion determine whether to fall ill.Concrete outcome is shown in Table 2.The result shows that using PLK plants of inactivated vaccines to 40 days
The sheldrake in age carries out after being immunized 28 days, and the attack to M41 plants and PLK plants can generate 8/10 or more protection.
The immuning effect test result of 23 batches of IBV inactivated vaccines of table
2.5 safety testing
1001,1002 and 1003 totally 3 batches of vaccines are taken, SPF chicken nonimmune to 14 ages in days has carried out single dose, single dose repeats
With overdose safety testing.It the results are shown in Table 3~table 5.
The single-dose safety test result of 3 IBV inactivated vaccine of table
The single dose of 4 IBV inactivated vaccine of table repeats safety testing result
The overdose safety testing result of 5 IBV inactivated vaccine of table
The safety testings of 3 batches of vaccines the result shows that, except the injection same day slightly subtracts food, in 7 days locally slightly outside tubercle, not
See systemic adverse reactions.
The preparation of 3 chicken's infectious bronchitis nephritis strain low virulent strain of embodiment
3.1 test processes
3.1.1 viral passages
The separated PLK plants of progress continuous passages causes of chicken's infectious bronchitis nephritis of embodiment 1 are weak, specifically
Are as follows: by the PLK plants of continuous passages in 9~10 age in days SPF chicken embryos, each generation poisons allantoic cavity is inoculated with 5 pieces of SPF chicken embryos, every time
Previous generation virus liquid is made into 100 times of dilutions, every 100 μ L of embryonic breeding kind sets 37 DEG C of incubators and is incubated for 48h (discarding dead germ for 24 hours), daily
Twice according to embryo, dead germ taking-up is placed in 4 DEG C, and the taking-up of all chicken embryos is set 4 DEG C after 48h, for 24 hours, observes and records chicken embryo lesion situation, nothing
Bacterium collection chick embryo allantoic liquid is passed on next time.Select lesion typical, the limpid chicken embryo of allantoic fluid is passed on.
3.1.2 virus titer measures
Take PLK plants of P5, P10, P25, P50, P80, P100, P110, the chick embryo allantoic liquid of P120 and P130 generation poison, difference
Successively make 10 times with physiological saline to be serially diluted；Select 5 extension rates 10-4~10-8Viral dilution suspension be inoculated with 5 respectively
Chicken embryo is set and is incubated in 37 DEG C of incubators 6 days, daily according to embryo, note by the SPF chicken embryo of piece 9~10 ages in days, every piece of 100 μ L of egg inoculation
The death condition of chicken embryo in record 6 days, by embryo dissect, observation chicken embryo lesion situation is calculated by Reed-Muench method within 6 days after inoculation
EID50。
3.1.3 steriling test and mycoplasma are examined
Respectively by PLK plants of P5, P10, P25, P50, P80, P100, P110, P120 and P130 generation poison is by existing " middle Chinese
People republic veterinary drug allusion quotation " annex carries out steriling test and mycoplasma is examined.
3.1.4 exogenous virus is examined
Respectively by PLK plants of P5, P10, P25, P50, P80, P100, P110, P120 and P130 generation poison progress exogenous virus
Detection.
3.1.4.1 chick embryo method carries out exogenous virus detection
PLK plants of viral sample each generation poison are taken, appropriate dilution are done, with infectivity resistant bronchitis virus specific antisera
Mixing in room temperature and 1 hour, takes 9~10 20 pieces of age in days SPF chicken embryos, 10 pieces of chick embryo allantoic cavities inoculations, every piece of virus inoculation serum
Mixed liquor 0.2m1, another 10 pieces of chicken embryos urinate allantocherion vaccination serum virus mixed liquor 0.2m1, while setting Normal group, incubate
Change 7, every sunshine embryo, the dead chicken embryo after inoculation in 24 hours is nonspecific death, discards and disregards, every group at least 8/10 is deposited
It is living, dissect is carried out to the chicken embryo survived after all inoculations 24 hours, observation has without exception.Chorioallantoic membrane is observed, is examined
Looked into it is without exception, and to allantoic fluid carry out antigen hemagglutinating antigen detection.
3.1.4.2 carrying out exogenous virus detection with cell culture
It is carried out by existing " Republic of China Veterinary Pharmacopoeia " annex, sees whether CPE and hemadsorption phenomenon occur.
3.1.4.3COFAL testing (avian leukosis virus inspection)
It is carried out by existing " Republic of China Veterinary Pharmacopoeia " annex.
3.1.4.5PLK the weak evaluation of the cause of strain
The SPF chicken of 100 14 ages in days is randomly divided into 9 groups (every group 10), and 10 groups of fowl raisings are isolated in 9 negative pressure respectively
In device, chicken uses PLK plants of P5 respectively, and P10, P25, P50, P80, P100, P110, P120 and P130 generation poison makes suitable of physiological saline
When dilution is inoculated with, every 100 μ L of collunarium (contains 103.5EID50)；As a control group, every 100 μ L of collunarium is just for 10th group of chick
Normal allantoic fluid.From inoculation, daily observation, record connect incidence, the death condition of breeder flock, cut open to dead chicken
The pathological change of IBV target organ, tissue is observed in inspection.
3.1.4.6PLK-130 the immune efficacy of strain
40 3 age in days SPF chickens are randomly divided into 2 groups (every group 10), raise in negative pressure isolator respectively, wherein one group young
Chicken is inoculated with using PLK-130 plants (P130), and every 100 μ L of collunarium (contains 103.5EID50)；Another group of chick is then as control
Group, every 100 normal allantoic fluid of μ L of collunarium.From inoculation, daily observation, record connect incidence, the death of breeder flock
Situation, and 14 days after immune take a blood sample, and test infective bronchitis HI prepared by this laboratory of every group of acquisition serum anti-
Original is measured, while two groups of chick are carried out collunarium attack with homologous virulent PLK plants of P5 generation, and every 100 μ L (contain
103.0EID50)；After attacking poison, daily observation, the incidence of record chicken group, death condition, and the acquisition tracheae cotton on the 5th after attacking poison
It wipes, processing is followed by embryo, and 5 pieces of chicken embryos of each cotton swab sample inoculation, 37 DEG C hatch 6, and every sunshine embryo is dead in 24 hours after inoculation
Dying chicken embryo is nonspecific death, discards and disregards, and 144h dissect observes chicken embryo lesion after inoculated into chick embryo.
3.2 result
3.2.1PLK the Attenuation of strain
More and more stronger to the adaptability of chicken embryo with the increase to PLK strain virus passage number, Embryo mortality is gradually
It increases, the death time is gradually shortened, and chicken embryo lesion is also more obvious.Obvious developmental disorder (dwarf is presented in the chicken embryo of virus inoculation
Embryo), curl embryo, the variation of the typical infectious bronchitis virus infection such as idiosome dehydration, bleeding.The chicken embryo of each generation is urinated
Cyst fluid cannot be aggregated chicken peripheral red blood cells, illustrate in chick embryo allantoic liquid without the fowl that chicken peripheral red blood cells can be made to be aggregated
Other viruses.
3.2.2 virus titer measurement result
The PLK plants of malicious titer determinations of chicken embryos passage, which the results are shown in Table 6, PLK strain, can highly adapt to chicken embryo, the EID in P5 generation50Up to
106.5EID50/ 0.1m1, passage restrovirus titre gradually rise, and P100~P130 generation tends towards stability, and titre is about 106.90EID50/
0.1m1。
6 PLK plants of chicken embryo of table passes on malicious titer determination result
| Generation | P5 | P10 | P25 | P50 | P80 | P100 | P110 | P120 | P130 |
| EID50/0.1m1 | 106.50 | 106.63 | 107.22 | 107.16 | 106.85 | 106.80 | 106.78 | 107.0 | 106.90 |
3.2.3 steriling test and mycoplasma are examined
P5, P10, P25, P50, P80, P100, P110, P120 and P130 generation poisons in chicken embryo succeeding generations examine nothing
Bacterium, mould and mycoplasma contamination.
3.2.4 exogenous virus is examined
P5, P10, P25, P50, P80, P100, P110, P120 and P130 generation poisons in chicken embryo succeeding generations examine nothing
The pollution of exogenous virus.
3.2.5PLK-130 the weak evaluation of the cause of strain
PLK-130 plants are gradually weakened to the pathogenicity of SPF chick after continuous passage in chicken embryo and (are shown in Table 7).P5, P10,
P25, P50 generation poison are 100% to SPF chick disease incidence, and the death rate is gradually decrease to 1/10, P80, P100 for malicious right by 5/10
SPF chick disease incidence is respectively 100% and 80%, but the death rate is 0%.It is thick that morbidity chicken shows as depressed spirit, contracting neck, coat
The symptoms such as unrest, the sagging, mouth breathing of two wings.The visible tracheorrhagia of chicken dissect of dying of illness, two sides kidney enlargement, clean mark, appearance
Typical " piebald kidney " is presented, there is uric acid mineralization.All control group chickens are in good condition, no morbidity or dead.And P110, P120
It is then 0% to SPF chick morbidity and mortality with P130 generation poison, dissect has no tracheorrhagia, the specificity such as kidney enlargement
Pathological change, it is consistent with control group SPF chicken.
Pathogenicity of 7 PLK plants of the table different generation poison to SPF chick
| Group | Generation | Disease incidence | The death rate |
| 1 | P5 | 100% | 50% |
| 2 | P10 | 100% | 40% |
| 3 | P25 | 100% | 30% |
| 4 | P50 | 100% | 10% |
| 5 | P80 | 100% | 0% |
| 6 | P100 | 80% | 0% |
| 7 | P110 | 0% | 0% |
| 8 | P120 | 0% | 0% |
| 9 | P130 | 0% | 0% |
| Control group | - | 0% | 0% |
3.2.6PLK-110, PLK-120, PLK-130 plants of immune efficacy
Respectively use PLK-110, PLK-120, PLK-130 plant, be immunized 3 age in days SPF chickens, immunizing dose for containing
3.5log10EID50/ 30 microlitres, 14 days after immune, immune group and control group chicken is attacked using PLK plants, observe 5 after attack
It, acquires 144h dissect after tracheae cotton swab inoculated into chick embryo, and 10/10 morbidity of poison group, immune group (PLK-110, PLK- are attacked in discovery control
120, PLK-130) it is 10/10 protection.It is observed 14 days after attacking poison, discovery immune group chicken is in good condition, no disease symptom；And it is right
It is obvious according to group morbidity, the symptoms such as spirit is depressed, coat thick unrest, the sagging, mouth breathing of two wings are shown as, and 30% after attacking poison
(7/10) chicken death (being shown in Table 8).
The immune efficacy that 8 PLK-110, PLK-120, PLK-130 plants of table
| Group | Attack disease incidence after poison | Attack the death rate after poison | Attack dissect lesion after poison |
| PLK-110 | 0/10 | 0/10 | Without specific lesions |
| PLK-120 | 0/10 | 0/10 | Without specific lesions |
| PLK-130 | 0/10 | 0/10 | Without specific lesions |
| Control group | 10/10 | 3/10 | Dead chicken piebald kidney |
The preparation of 4 chicken's infectious bronchitis nephritis subunit vaccine of embodiment
The building of 4.1 recombination rhabdovirus expression vectors
The S1 albumen of IBV is prepared according to surveyed PLK plants of S1 albumen of embodiment 1 (successively seeing SEQ ID No.1).Design is drawn
Object sequence is as follows:
S1 gene cloning primer:
SF1:5 '-TAT TGA TTA GAG ATG TTG GG-3 '
SR1:5 '-CAT AAC TAA CAT AAG GGC AA-3 '
The chicken's infectious bronchitis nephritis popularity variant M protein sequence according to disclosed in NCBI, and part is replaced
Change nucleotide residue, the artificial synthesized gene order of the M albumen of SEQ ID No.3.
According to (Yu Juan etc., the expression and table of the property IBVZZ2004 plants of S1 and M genes in duck source in sf9 insect cell such as Yu Juan
Up to the antigen Journal of Sex Research of albumen, Agricultural University Of He'nan, 2012.06) building of the operating method of document expresses avian infectious bronchus
The recombinant baculovirus expression system of inflammation virus S1 albumen, M albumen, expression and purification obtain S1 albumen, M albumen.Finally weighed
S1 protein concentration is 3.1mg/ml to group after purification, and M protein concentration is 1.8mg/ml after purification for recombination.
The chicken's infectious bronchitis nephritis popularity variant N protein sequence according to disclosed in NCBI, and part is replaced
Change nucleotide residue, the artificial synthesized gene order of the N protein of SEQ ID No.5.
According to (Liu Shengwang etc., the clone of avian infectious bronchitis virus nucleoprotein gene and in rod-shaped disease such as Liu Shengwang
Expression in malicious system, Chinese Preventive Veterinary Medicine report, 2001.06) the operating method building of document expresses avian infectious bronchus
The recombinant baculovirus expression system of scorching viral N proteins, expression and purification obtain N protein.N protein is dense after purification for final acquisition recombination
Degree is 2.2mg/ml.
The preparation of 4.2 subunit vaccines
S1, M and N protein are recombinated using avian infectious bronchitis virus, is aided with adjuvant, prepares infectious bronchitis of chicken
Subunit vaccine.
3 kinds of albumen of S1, M and N are diluted to after 2~6 mcg/mls with PBS (pH7.4) respectively and are prepared into comprising difference
The antigen diluent object (being shown in Table 9) of component:
The different group antigen diluent objects of table 9
| Group | Antigen protein composition | Antigen proportion |
| 1 | S1 | - |
| 2 | S1、M | 1:1 |
| 3 | S1、N | 1:1 |
| 4 | S1、M、N | 1:0.5:0.5 |
By the above-mentioned other antigen diluent object of 3 groups, with oil adjuvant ISA206 mixing and emulsifying.ISA206 adjuvant through 121 DEG C,
60min high pressure moist heat sterilization, according to antigen: adjuvant volume ratio 46:54 mixing.IKA2000/4 type mulser is produced using Germany,
First adjuvant is added in mulser adjuvant barrel, (100r/min) slowly instills antigen water phase in adjuvant under stirring,
After dripping off, continue to stir 5min.With mulser 5000r/min, circulating emulsion 4min, subunit vaccine, each group antigen are obtained
The subunit vaccine lot number of preparation is shown in Table 10.
10 subunit vaccine batch of table
| Group | Antigen protein composition | Batch |
| 1 | S1 | 1402 |
| 2 | S1、M | 1403 |
| 3 | S1、N | 1404 |
| 4 | S1、M、N | 1405 |
After viscosity measurements, centrifugation detection, dosage form detection, Detection of Stability, indices meet the vaccine prepared
Regulation.Vaccine is stored in 4 DEG C.
The effect detection of 4.3IBV subunit vaccine (the IBV virus challenge after SPF chicken is immunized in subunit vaccine)
Test chicken is 21 ages in days without specified pathogen (SPF) chicken 70, immune group 60, control group 10, and immune group
60 collunariums are inoculated with infectious bronchitis of chicken live vaccine (H120 plants) 1 plumage part, and 21 days after inoculation, each group randomly selects 10 respectively
1402,1403,1404 and 14050.5ml of subunit vaccine is subcutaneously injected respectively, 10 are subcutaneously injected immune M41 plants of inactivated vaccines
0.5ml, 10 subcutaneous injection sterile saline 0.5ml of residue.SPF chicken is raised in isolator.28 days after injection, 4 groups sub-
Subunit vaccine and M41 inactivated vaccine immune group and control group are with PLK plants with 103.5EID50Collunarium approach carries out attacking poison, attacks 5 after poison
Day, acquisition tracheae cotton swab is inoculated with 10 age in days SPF chicken embryos, and whether there is or not infective bronchitis allusion quotations for 144 hours observation chicken embryos after inoculation
Type lesion determines that test chicken whether there is or not infection infective bronchitis, the results are shown in Table 11.
The active immunity of 11 different component IBV subunit vaccine of table is tested
The result shows that it is compared to the existing M41 plants of inactivated vaccine in market, the Asia prepared by S1 gene, M gene, N gene
Subunit vaccine can provide more effective immunoprotection to the poison of attacking of kidney type infective bronchitis velogen strain.In contrast, three kinds
It in the subunit vaccine of different antigen component preparations, while including the subunit vaccine of S1, M, the preparation of N protein antigen component,
Immune effect be it is best, 10/10 protection can reach to the poison of attacking of velogen strain.
The above is only the preferred embodiment of the present invention, not does limitation in any form to the present invention, though
So the present invention is disclosed above with preferred embodiment, and however, it is not intended to limit the invention, any technology people for being familiar with this profession
Member, in the range of not departing from technical solution of the present invention, when the technology contents using the disclosure above make a little change or repair
Decorations are the equivalent embodiment of equivalent variations, but anything that does not depart from the technical scheme of the invention content, technology according to the present invention are real
Matter any simple modification, equivalent change and modification to the above embodiments, still fall within the range of technical solution of the present invention
It is interior.
Claims (12)
1. a kind of chicken's infectious bronchitis nephritis strain, the chicken's infectious bronchitis nephritis strain S1 gene
Coded sequence is the albumen of SEQ ID NO.2, and M gene coded sequence is the albumen of SEQ ID NO.4, and N gene coded sequence is
The albumen of SEQ ID NO.6.
2. a kind of chicken's infectious bronchitis nephritis PLK strain, deposit number is CCTCC NO.V201442.
3. a kind of low virulent strain of chicken's infectious bronchitis nephritis, wherein the low virulent strain is by that will weigh in chicken embryo
Benefit requires the low virulent strain of 1 or 2 strain continuous passage any generations into 110-140 generation.
4. a kind of vaccine composition, wherein the vaccine composition includes that the chicken kidney type described in claim 1 of immune amount infects
Property bronchitis poison strain antigen and pharmaceutically acceptable carrier, wherein avian nephropathogenic infectious bronchitis disease
The antigen of poison strain includes the totivirus antigen or subunit antigen S1 and M and N of the totivirus antigen of inactivation, attenuation.
5. vaccine composition according to claim 4, wherein the vaccine composition further includes adjuvant.
6. vaccine composition according to claim 4, wherein the vaccine composition include inactivate it is preceding 0.4 ×
106.7EID50The totivirus antigen of the inactivation of/0.1ml；Or
The vaccine composition includes 1.0~7.0log10EID50The totivirus antigen of the attenuation；Or
The vaccine composition include the S1 albumen of 0.5~1.5 μ g/ml, the M albumen of 0.25~0.75 μ g/ml and 0.25~
The N protein of 0.75 μ g/ml.
7. vaccine composition according to claim 6, wherein the vaccine composition include 2.5~
5.0log10EID50The totivirus antigen of the attenuation.
8. vaccine composition according to claim 6, wherein the vaccine composition include 3.0~
4.0log10EID50The totivirus antigen of the attenuation.
9. a kind of method for preparing vaccine composition as claimed in claim 4, wherein the described method includes:
(1) chicken's infectious bronchitis nephritis strain described in amplification cultivation；And
(2) the chicken's infectious bronchitis nephritis strain for inactivating the amplification cultivation, is added pharmaceutically acceptable carrier.
10. a kind of method for preparing vaccine composition as claimed in claim 4, wherein the described method includes:
(1) the chicken's infectious bronchitis nephritis strain described in continuous passage in chicken embryo, is passaged to 110-140 generation；
(2) the weak chicken's infectious bronchitis nephritis strain of cause in 110-140 generation is passaged to described in amplification cultivation；And
(3) pharmaceutically acceptable load is added in the weak chicken's infectious bronchitis nephritis strain of the cause of the amplification cultivation
Body.
11. a kind of method for preparing vaccine composition as claimed in claim 4, wherein the described method includes:
(1) S1 albumen, the M albumen, N protein of chicken's infectious bronchitis nephritis strain described in difference clonal expression；
(2) the S1 albumen, M albumen and N protein of the expression are proportionally added into；And
(3) pharmaceutically acceptable carrier is added in above-mentioned chicken's infectious bronchitis nephritis antigen protein.
12. preventing and treating avian nephropathogenic infectious branch gas in preparation according to the described in any item vaccine compositions of claim 4~8
Application in the drug of pipe inflammation related disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410837138.7A CN105802918B (en) | 2014-12-29 | 2014-12-29 | Chicken's infectious bronchitis nephritis strain and its vaccine composition, preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410837138.7A CN105802918B (en) | 2014-12-29 | 2014-12-29 | Chicken's infectious bronchitis nephritis strain and its vaccine composition, preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105802918A CN105802918A (en) | 2016-07-27 |
| CN105802918B true CN105802918B (en) | 2019-11-19 |
Family
ID=56980544
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410837138.7A Active CN105802918B (en) | 2014-12-29 | 2014-12-29 | Chicken's infectious bronchitis nephritis strain and its vaccine composition, preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105802918B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754746B (en) * | 2016-11-30 | 2020-07-28 | 中山大学 | Avian infectious bronchitis virus vaccine strain, vaccine and application thereof |
| CN112553376B (en) * | 2020-12-23 | 2023-06-02 | 四川大学 | Primer group, kit, multiple PCR detection method for distinguishing different IBV vaccine strains and application of multiple PCR detection method |
| CN113105531B (en) * | 2021-03-24 | 2022-09-23 | 中国农业大学 | T cell epitope polypeptide of avian infectious bronchitis virus N protein and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001009290A2 (en) * | 1999-07-30 | 2001-02-08 | Laboratorio Avi-Mex, S.A. De C.V. | Avian infectious bronchitis virus serotype |
| KR20030059954A (en) * | 2002-01-04 | 2003-07-12 | 학교법인 건국대학교 | New infectious bronchitis viruses and vaccines for preventing infectious bronchitis using the same |
| CN101514334A (en) * | 2009-03-23 | 2009-08-26 | 中国农业科学院哈尔滨兽医研究所 | Chicken infectivity bronchitis virus attenuated vaccine strain and application thereof |
| CN101948812A (en) * | 2010-08-26 | 2011-01-19 | 国家兽用生物制品工程技术研究中心 | Eosinophilic kidney type avian infectious bronchitis virus (IBV) virulent vaccine strain and application thereof |
| CN102851257A (en) * | 2012-08-27 | 2013-01-02 | 上海启盛生物科技有限公司 | Attenuated vaccine strain for avian infectious bronchitis virus and application thereof |
| CN102994460A (en) * | 2012-12-31 | 2013-03-27 | 青岛易邦生物工程有限公司 | Infectious bronchitis virus attenuated vaccine strain |
| CN104130981A (en) * | 2014-07-10 | 2014-11-05 | 中国农业科学院哈尔滨兽医研究所 | Application of avian infectious bronchitis virus vaccine strain in preparation of inactivated vaccine |
-
2014
- 2014-12-29 CN CN201410837138.7A patent/CN105802918B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001009290A2 (en) * | 1999-07-30 | 2001-02-08 | Laboratorio Avi-Mex, S.A. De C.V. | Avian infectious bronchitis virus serotype |
| KR20030059954A (en) * | 2002-01-04 | 2003-07-12 | 학교법인 건국대학교 | New infectious bronchitis viruses and vaccines for preventing infectious bronchitis using the same |
| CN101514334A (en) * | 2009-03-23 | 2009-08-26 | 中国农业科学院哈尔滨兽医研究所 | Chicken infectivity bronchitis virus attenuated vaccine strain and application thereof |
| CN101948812A (en) * | 2010-08-26 | 2011-01-19 | 国家兽用生物制品工程技术研究中心 | Eosinophilic kidney type avian infectious bronchitis virus (IBV) virulent vaccine strain and application thereof |
| CN102851257A (en) * | 2012-08-27 | 2013-01-02 | 上海启盛生物科技有限公司 | Attenuated vaccine strain for avian infectious bronchitis virus and application thereof |
| CN102994460A (en) * | 2012-12-31 | 2013-03-27 | 青岛易邦生物工程有限公司 | Infectious bronchitis virus attenuated vaccine strain |
| CN104130981A (en) * | 2014-07-10 | 2014-11-05 | 中国农业科学院哈尔滨兽医研究所 | Application of avian infectious bronchitis virus vaccine strain in preparation of inactivated vaccine |
Non-Patent Citations (4)
| Title |
|---|
| Development of attenuated vaccines from Taiwanese infectious bronchitis virus strains;Huang YP 等;《Vaccine》;20060206;第24卷(第6期);第785-791页 * |
| S1 protein, partial [Infectious bronchitis virus];Ma, H. 等;《GenBank Database》;20120516;Accession No. ADY62565 * |
| 鸡传染性支气管炎病毒S1蛋白的研究进展;贺秀媛 等;《动物科学与动物医学》;20010331;第18卷(第2期);第36-38页 * |
| 鸡肾型传染性支气管炎病毒QG株的分离与鉴定;赵光伟 等;《兽医研究》;20070605;第8-10页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105802918A (en) | 2016-07-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108126191A (en) | A kind of vaccine composition and its preparation method and application | |
| CN108653724A (en) | It is a kind of for prevent fowl egg drop syndrome vaccine composition, and its preparation method and application | |
| CN103849632B (en) | S1 gene of low virulent strain of infectious bronchitis and low virulent strain and application thereof | |
| CN110872578B (en) | Variant infectious bursal disease virus, subunit vaccine, preparation method and application thereof | |
| CN108653725A (en) | It is a kind of for prevent fowl egg drop syndrome vaccine composition, and its preparation method and application | |
| CN103497934B (en) | Avian infectious bronchitis virus vaccine strain (HF2 strain) and application thereof | |
| CN113491767A (en) | Triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck viral hepatitis and preparation method thereof | |
| CN111154730B (en) | Goose astrovirus variant strain | |
| CN105802918B (en) | Chicken's infectious bronchitis nephritis strain and its vaccine composition, preparation method and application | |
| CN112574958B (en) | H9 subtype avian influenza virus isolate and application thereof | |
| CN107523556B (en) | Avian adenovirus strain, vaccine composition and application thereof | |
| CN113384692A (en) | Duck reovirus and duck circovirus bivalent inactivated vaccine and preparation method thereof | |
| CN108939063B (en) | Muscovy duck triple inactivated vaccine | |
| CN109055320B (en) | Infectious bronchitis virus isolate and application thereof in vaccine preparation | |
| RU2443429C2 (en) | Associated inactivated newcastle disease, infectious bronchitis, egg drop syndrome 76, infectious bursal disease and reovirus tenosynovitis emulsion vaccine | |
| KR101102271B1 (en) | Attenuated Avian Infectious Bronchitis Virus and Vaccine for Avian Infectious Bronchitis Comprising the Same | |
| CN114395536B (en) | Avian adenovirus type 4, 8 and 11 trivalent vaccine and preparation method and application thereof | |
| CN113832113B (en) | Novel duck reovirus attenuated strain and application thereof | |
| CN114573708B (en) | Avian bacillus paragallinarum HA fusion protein and trimer thereof, vaccine composition prepared by using same, preparation method and application | |
| CN110713987B (en) | Recombinant gene VII type Newcastle disease virus strain and vaccine composition, preparation method and application thereof | |
| CN108126192A (en) | A kind of vaccine composition and its application | |
| KR101073991B1 (en) | Avian metapneumovirus type B SC1509 strain isolated from chicken in Korea and the use thereof | |
| CN112063596A (en) | Pigeon paramyxovirus type 1 PPMV-1/BJ-C strain and application thereof | |
| CN106754744B (en) | Group I avian adenovirus strain of serum type 4 and application thereof | |
| CN111000995A (en) | Triple inactivated vaccine for pigs and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |