CN107287218A - Infectious bronchitis of chicken velogen strain S1 genes and its velogen strain and application - Google Patents
Infectious bronchitis of chicken velogen strain S1 genes and its velogen strain and application Download PDFInfo
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Abstract
A kind of velogen strain the present invention relates to infectious bronchitis of chicken velogen strain S1 genes and containing velogen strain S1 genes.Velogen strain S1 genes and the infectious bronchitis of chicken low virulent strain S1 DNA homologs of the present invention, available for infectivity resistant bronchitis vaccine or the efficacy test of subunit vaccine or vaccine combination.A kind of low virulent strain the invention further relates to infectious bronchitis of chicken low virulent strain S1 genes and containing low virulent strain S1 genes.The polypeptide of low virulent strain S1 gene codes can stimulate body to produce neutralizing antibody; YB160 plants of infective bronchitis containing low virulent strain S1 genes; available for preparing infectious bronchitis vaccines composition or subunit vaccine; can the effectively anti-popular strain attack of IB processed, there is preferable protection to Glandular Stomach Type Infections Bronchitis.The invention further relates to a kind of infectious bronchitis of chicken diagnostic medicament as made from the infectious bronchitis of chicken low virulent strain of the present invention.
Description
The application is the Application No. 201210519405.7 submitted on December 06th, 2012, and entitled " chicken is infected
The divisional application of the Chinese invention patent application of property bronchitis low virulent strain S1 genes and its low virulent strain and application ".
Technical field
The invention belongs to avian creature pharmaceutical technology field, be related to a kind of infectious bronchitis of chicken velogen strain S1 genes,
The application of infectious bronchitis of chicken velogen strain and the velogen strain containing velogen strain S1 genes in terms of vaccine potency inspection.
Background technology
Infectious bronchitis of chicken (Avian infectious bronchitis, IB) is a kind of acute of chicken, is highly connect
The viral disease of contagion.China begins with respiratory diseases modification IB morbidity report from nineteen fifty-three, and nineteen eighty-two is in Guangdong
Nephrosis modification IB is found first, there is IB prevalence all parts of the country in recent years, wherein many based on nephrosis modification.From nineteen ninety-five with
Come China Jiangsu, Shandong, Shanxi, Anhui etc. and save to there occurs that one kind is become thin with retarded growth, extremely, had loose bowels for cardinal symptom, with
Glandular stomach enlargement is the Proventriculus Disease IB of major pathologic features.The sick incidence of disease is 30%~50%, and case fatality rate is up to more than 30%.
There is negative growth in ill chicken body weight, and the price of deed is remarkably decreased, and heavy losses are caused to aviculture.
Wang Yudong did following report to Glandular Stomach Type Infections Bronchitis equal to 1997：In green grass or young crops since in Septembers, 1996
Island and in the neighbourhood the age in days Growing Chicken of chicken farm 25~70 recurred one kind to shed tears, eye it is swollen, with respiratory symptom, pole
Degree is become thin, had loose bowels, the dead infectious disease being characterized, and main cut open inspection symptom is：Glandular stomach enlargement is for example spherical, glandular stomach wall thickening, glandular stomach
Mucosal bleeding ulcer, pancreas enlargement bleeding.The incidence of disease is up to 100%, and the death rate is 3%~95%.Different cultivars chicken is all
There is morbidity.Research shows that cause of disease is coronavirus, and the disease is fixed tentatively entitled chicken Glandular Stomach Type Infections Bronchitis by us.
Immunity inoculation is the most effective means for preventing and treating avian infectious bronchitis virus.At present, in the market has had
Attenuated vaccine is clinically applied, and also has some new strains to be separated.Because IBV serotypes are numerous, and popular strain and epidemic disease
Seedling strain is compared to be existed compared with Big mutation rate on gene level, cause existing vaccine strain can not the effectively anti-popular strain of IB processed attack,
IB effect of control is unsatisfactory.For example, Chinese patent CN 101514334A disclose a kind of avian infectious bronchitis virus
Attenuated vaccine strain and its application；It is weak that Chinese patent CN 102220287A disclose a kind of infectious bronchitis of chicken acclimatization to cold cause
Vaccine strain and its application；Chinese patent CN 101100656A disclose a kind of avian nephropathogenic infectious bronchitis HN99 strain virus
Strain.Above patent all refers to some new infectious bronchitis of chicken attenuated vaccine strains, available for preparing live vaccine, but these
Strain does not show can there is good protective effect to Glandular Stomach Type avian infectious bronchitis virus.
Therefore, the problem of presently, there are be need research and development one kind can the effectively anti-popular strain of IB processed attack,
Passing brace to Glandular Stomach Type has the vaccine of preferable protection.
The content of the invention
The technical problems to be solved by the invention are that there is provided a kind of infectious bronchitis of chicken in view of the shortcomings of the prior art
Low virulent strain S1 genes, the polypeptide of low virulent strain S1 gene codes can stimulate body to produce neutralizing antibody.
The present invention still further provides a kind of low virulent strain containing the S1 genes, and the low virulent strain can be used for preparing anti-chicken biography
Metachromia bronchitis vaccine composition, can effectively prevent the attack of proventriculus disease avian infectious bronchitis virus, to Glandular Stomach Type infectious bronchitis
Inflammation has preferable protection.
Present invention also offers subunit vaccine made from a kind of infectious bronchitis of chicken low virulent strain.
Especially, the invention provides it is a kind of as the present invention infectious bronchitis of chicken low virulent strain made from it is avian infectious
Bronchitis diagnostic medicament.
The present invention still further provides a kind of and of the invention infectious bronchitis of chicken low virulent strain S1 DNA homologs
Infectious bronchitis of chicken velogen strain S1 genes and the infectious bronchitis of chicken velogen strain containing velogen strain S1 genes, this is strong
Strain can be used for the efficacy test of infectivity resistant bronchitis vaccine or subunit vaccine or vaccine combination.
Therefore, the invention provides a kind of infectious bronchitis of chicken low virulent strain S1 genes, its nucleotide sequence such as sequence
In table shown in SEQ ID NO.1.
Infectious bronchitis of chicken low virulent strain S1 genes are that virus is easiest to morph in evolutionary process in the present invention
Gene, its polypeptide encoded can stimulate body to produce neutralizing antibody, and its hypervariable region is predominantly located in S1 genes.By right
Infectious bronchitis of chicken low virulent strain S1 gene orders are analyzed in the present invention, it was found that infectious bronchitis of chicken low virulent strain
The sequence total length of S1 genes is 1562bp.
In the specific embodiment of the present invention, separation is carried out to infectious bronchitis of chicken low virulent strain S1 genes pure
Change.Nucleic acid is first extracted from infectious bronchitis of chicken low virulent strain virus liquid, and as template, after reverse transcription, with special
Property primer enter performing PCR amplification, reclaim purpose fragment with PCR primer purification kit afterwards, that is, obtain the avian infectious branch of purifying
Tracheitis low virulent strain S1 genes.
IBV H52, H120 and W93 vaccine strain S1 gene orders are downloaded from GenBank, with DNAStar softwares and this hair
Bright middle infectious bronchitis of chicken low virulent strain S1 gene orders carry out multiple alignment, and comparison method is：DNAStar softwares are opened,
From MegAlign functions, being loaded into " File " needs aligned sequences, and " By ClustalV method " enter in selection " Align "
Row compare, selection " view " under " Sequence Distances " be it can be seen that aligned sequences nucleotides genetic distance (see
Fig. 1).Infectious bronchitis of chicken low virulent strain S1 genes and common infectious bronchitis of chicken in the present invention are found by comparing
The homology of H52 plants, H120 plants and W93 plants S1 genes of virus vaccine strain is respectively 71.4%, 71.6% and 71.3%.
Present invention also offers a kind of infectious bronchitis of chicken low virulent strain containing low virulent strain S1 genes of the present invention.
The low virulent strain can be used for preparing anti-infectious bronchitis vaccines composition, can effectively prevent attacking for proventriculus disease avian infectious bronchitis virus
Hit, there is preferable protection to Glandular Stomach Type Infections Bronchitis.
Term " virus vaccine strain " used refers to the Strain for preparing vaccine in the present invention.
Term " S1 albumen " used refers to the expression product of S1 genes in the present invention.
So-called " expression of gene " refers to cell in life process, and the hereditary information being stored in DNA sequence dna is passed through
Transcription and translation, is transformed into the protein molecule with bioactivity.
In the present invention, term " proventricular type infectious bronchitis virus, abbreviation Glandular Stomach Type passes branch " (Avian used
Infectious bronchitis, IB) refer to heretofore described infectious bronchitis virus for Glandular Stomach Type infectiousness branch gas
Pipe inflammation low virulent strain, it has following feature：
(1) morbidity chicken glandular stomach enlargement is for example spherical, glandular stomach wall thickening, glandular stomach mosucas teat bleeding ulcer；
(2) breeding of the virus isolates to NDV LaSota strain virus produces interference；
(3) virus isolates do not produce aggegation to 1% chicken red blood cell；
(4) specific serum neutralization test result is indicated, virus isolates and the YB160 positive serums of diseased chicken are positive instead
Should, and it is negative with LaSota plants of NDV, B87 plants of IBDV, H9N2 plants of AIV, EDS76 positive serum.
In the present invention, " infectious bronchitis of chicken low virulent strain " should be from broadly being understood, it is included containing SEQ
The low virulent strain S1 genes of ID NO.1 nucleotide sequences contain the low virulent strain S1 genes with SEQ ID NO.1 nucleotide sequence
Nucleotide sequence of the homology more than 80% S1 genes infectious bronchitis of chicken low virulent strain, preferably comprise and SEQ
The chicken of the S1 genes of nucleotide sequence of the homology of the low virulent strain S1 genes of ID NO.1 nucleotide sequence more than 90% passes
Metachromia bronchitis low virulent strain, it is more preferred to, containing with the low virulent strain S1 genes of SEQ ID NO.1 nucleotide sequence
The infectious bronchitis of chicken low virulent strain of the S1 genes of nucleotide sequence of the homology more than 95%~99%.
Term " homology " used refers to the similar journey of two amino acid sequences or two nucleotide sequences in the present invention
Degree.The homology of amino acid sequence or nucleotide sequence can be calculated by any appropriate method well known in the art and obtained,
For example, target amino acid (or nucleotides) sequence and reference amino acid (or nucleotides) sequence can be subjected to sequence alignment, must
Vacancy can be introduced when wanting so that identical amino acid (or nucleotides) being optimal of number between the sequence of two comparisons, and
On this basis calculate two amino acid (or nucleotides) sequences between same amino acid (or nucleotides) percentage.Amino acid
The comparison of (or nucleotides) sequence and the calculating of homology can be realized by software well known in the art, such as, but not limited to,
BLAST softwares (can be obtained in US National Biotechnology Information center (NCBI) network address：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 softwares (can be obtained) in European Bioinformatics research institute network address.
In a detailed embodiment, it is a kind of according to low virulent strain of the present invention, wherein, the low virulent strain is that chicken passes
YB160 plants of metachromia bronchitis virus (Avian infectious virus strain YB160), its deposit number is:
CCTCC NO：V201235, preservation date：On 08 29th, 2012, it is preserved in China typical culture collection center (referred to as：
CCTCC；Address：The Wuhan University of Wuhan City, Hubei Province Wuchang District Luo Jia Shan road 16).
Contain the anti-avian infectious of infectious bronchitis of chicken low virulent strain of the present invention present invention also offers a kind of
Bronchitis vaccine composition.
In a preferred embodiment, the vaccine combination includes YB160 plants of infectious bronchitis virus.
According to the present invention, the vaccine combination also further contains freeze drying protectant.The freeze drying protectant include but
It is not limited to containing gelatin and sucrose.
In one particular embodiment of the present invention, the vaccine combination is by YB160 plants of infectious bronchitis virus
With freeze drying protectant composition：The production seed culture of viruses of YB160 plants of infectious bronchitis virus is taken, 10 are diluted with sterile saline
Wan Bei, is inoculated in 9~10 age in days SPF (specific pathogen free, no-special pathogen) chick embryo allantois intracavitary, per embryo
0.1ml is inoculated with, pin hole is sealed after inoculation, is incubated under the conditions of being placed in 37 DEG C.24h dead germs are discarded, chicken embryo is observed to 72h, by 30~
72h chick embryo air sac upwards, cools down 4~24h under the conditions of being placed in 4 DEG C.Chick embryo allantoic liquid is harvested, 6~8 chick embryo allantoic liquids are mixed
One group is combined into, is placed in sterilizing bottle, is preserved under the conditions of 2~8 DEG C, while doing steriling test, is measured viral level and should be
107.0EID50/0.1ml.Protected again with sucrose gelatin after qualified chick embryo allantoic liquid mixing is determined through steriling test and viral level
Agent presses 1:1 (v/v) matches somebody with somebody seedling, and freeze drying protectant is with 8% (w/v) gelatin, 40% (w/v) sucrose protective agent, through 115 DEG C of autoclavings
40min, puts in 4 DEG C of preservations, 72h and is finished.Virus liquid should be constantly shaken in adding procedure, after fully mixing, as vaccine group
Compound stoste.Vaccine combination stoste is subjected to sterile quantitative separating, quick freeze vacuum drying is sealed.Institute of the present invention
Per plumage, IBV is 10 to the infectious bronchitis vaccines composition of preparation in part4.5EID50。
In a preferred embodiment of the invention, the vaccine combination also includes other attenuated vaccine strains.Wherein, institute
Stating other attenuated vaccine strains, to include but is not limited to the weak strain of newcastle disease virus and/or bursa of farbricius low virulent strain etc. such.It is preferred that
Other described attenuated vaccine strains are newcastle disease low virulent strain.
Present invention also offers one kind according to infectious bronchitis of chicken low virulent strain of the present invention in prevention and treatment
The application of chicken infectious bronchits.The application is construed as preparing for preventing and treating chicken infectious bronchits
Vaccine and combinations thereof in application.
The invention provides a kind of infectious bronchitis of chicken subunit vaccine, it is characterised in that：The subunit vaccine
The substantial amino acid sequence containing SEQ ID NO.3 in ordered list.
In one particular embodiment of the present invention, the preparation method of the subunit vaccine, comprises the steps：
(1) encoding gene of YB160 plants of S1 albumen is cloned using reverse transcriptase chain reaction (RT-PCR) method；
(2) the recombinant transfer plasmid pFASTYB160S1 for including S1 genes is built；
(3) recombinant baculovirus rAcYB160S1 is built；
(4) culture of recombinant baculovirus rAcYB160S1 infected insect cells and insect cell；
(5) the avian infectious bronchitis virus restructuring avian infectious branch of S1 gene expressions of recombinant baculovirus
Bronchitis virus recombinates S1 albumen, harvest and purifying infectious bronchitis of chicken restructuring S1 albumen；
(6) S1 albumen is recombinated using avian infectious bronchitis virus, is aided with adjuvant, through emulsification, prepared
Avian infectious bronchitis virus subunit vaccine.
The present invention still further provides a kind of avian infectious bronchus of the S1 DNA homologs with low virulent strain of the present invention
Scorching velogen strain S1 genes, its nucleotide sequence is as shown in SEQ ID NO.2 in sequence table.
The present invention also provides a kind of infectious bronchitis of chicken velogen strain containing velogen strain S1 genes of the present invention, its
It is characterised by：The velogen strain is YBX plants of avian infectious bronchitis virus (Avian infectious virus strain
YBX), its deposit number is:CCTCC NO：V201236, preservation date：On 08 29th, 2012, it is preserved in Chinese Typical Representative culture
Thing collection is (referred to as：CCTCC；Address：The Wuhan University of Wuhan City, Hubei Province Wuchang District Luo Jia Shan road 16).
The present invention still further provides it is a kind of according to velogen strain of the present invention in infectivity resistant bronchitis vaccine or
Application in the efficacy test of subunit vaccine or vaccine combination.It uses YBX plants of infectious bronchitis virus as strong
Strain carries out challenge test.
In the present invention, above-mentioned velogen strain, which is removed, is used for infectivity resistant bronchitis vaccine or subunit vaccine or vaccine combination
Outside the efficacy test of thing, infectious bronchitis of chicken low virulent strain and infectious bronchitis of chicken low virulent strain S1 in itself or the present invention
The important sources of gene.Infectious bronchitis of chicken low virulent strain is the chicken biography by velogen strain S1 genes of the present invention in the present invention
Metachromia bronchitis velogen strain is obtained through Attenuation.And infectious bronchitis of chicken low virulent strain S1 genes are then from the present invention
Infectious bronchitis of chicken low virulent strain through isolating and purifying acquisition.
The present invention also specifically provides a kind of infectious bronchitis of chicken as made from infectious bronchitis of chicken low virulent strain
Diagnostic medicament, it can be used for the measure of antibody level in infectious bronchitis vaccines efficacy test.
It is that virus is easiest to occur in evolutionary process according to the infectious bronchitis of chicken low virulent strain S1 genes of the present invention
The gene of variation, its polypeptide encoded can stimulate body to produce neutralizing antibody, and its hypervariable region is predominantly located in S1 genes.By
Infectious bronchitis vaccines composition obtained by infectious bronchitis of chicken low virulent strain containing low virulent strain S1 genes
Or subunit vaccine can effectively prevent and treat the attack of the popular viruses of IB, especially have to Glandular Stomach Type Infections Bronchitis preferable
Protection.
Infectious bronchitis of chicken diagnostic medicament can use according to made from the infectious bronchitis of chicken low virulent strain of the present invention
The measure of antibody level in infectious bronchitis vaccines efficacy test.The infective bronchitis velogen strain of the present invention can
For infectivity resistant bronchitis vaccine or the efficacy test of subunit vaccine or vaccine combination.
Brief description of the drawings
Fig. 1 be infectious bronchitis of chicken low virulent strain S1 genes of the present invention and the common vaccine strain H120 of IBV in embodiment 1,
The nucleotide sequence comparison result of H52 and W93 plants of S1 gene.
Fig. 2 be embodiment 2 in infectious bronchitis virus YB160 strains F115 of the present invention, F125, F145, F160,
Nucleotide sequence comparison results of the F170 for virus liquid S1 genes.
Fig. 3 is the pcr amplification product qualification result of IBV S1 genes in embodiment 5；The implication of reference in Fig. 3 is such as
Under：1IBV S1 gene amplification products；2DNA Marker2000.
Culture presevation
YB160 plants of avian infectious bronchitis virus (Avian infectious virus strain YB160), by general
The separation of Lai Ke bioengineering limited company, identification, in China typical culture collection center (referred to as：CCTCC；Address：
The Wuhan University of Wuhan City, Hubei Province Wuchang District Luo Jia Shan road 16) carry out preservation, preservation date：08 month 2012 29 days, preservation
Numbering：CCTCC NO：V201235.
YBX plants of avian infectious bronchitis virus (Avian infectious virus strain YBX), by Pu Laike
The separation of bioengineering limited company, identification, in China typical culture collection center (referred to as：CCTCC；Address：Hubei
Wuchang, wuhan area of province Luo Jia Shan road 16 Wuhan University) carry out preservation, preservation date：08 month 2012 29 days, deposit number：
CCTCC NO：V201236.
Embodiment
To make the present invention easier to understand, the present invention is described in detail below in conjunction with drawings and examples, these realities
Apply example only serve it is illustrative, it is not limited to NM specific experiment side in application of the invention, the following example
Method, is generally carried out according to normal experiment method.
Embodiment
Embodiment 1：The preparation of chicken infectivity bronchitis virus attenuated vaccine strain (IBV) of the present invention
IBV velogen strains are YBX plants of avian infectious bronchitis virus
Infectious bronchitis virus strain Attenuation, using 9~10 age in days SPF (specific pathogen free,
No-special pathogen) chicken embryo to strain carry out continuous passage cause it is weak, per generation by allantoic cavity be inoculated with 3 pieces of chicken embryos, continuously reach 170
In generation, carry out causing weak evaluation test.F30, F65, F90, F115, F125, F145, F160 and F170 are inoculated with 1 respectively for strain
Age SPF chicken, every chicken collunarium, oral, intramuscular injection 1ml, the wherein inoculation of F145, F160 and F170 generation poison chick, does not find
Morbidity and death, illustrate that the strain has been caused completely in F145, F160 and F170 generation poison weak, have to chicken preferable safe
Property.15 Japanese instar chicklings are immunized with low virulent strain of the present invention simultaneously, 15 days after immune, is attacked, found immune with same source strength poison
Group chicken is in good condition, no disease symptom.And control group morbidity is obvious, its about 30% (6-7/20) chicken death after poison is attacked.With
2 repetitions are passed through in upper experiment, obtain consistent result, illustrate that attenuated IBDVs have good immune protective, choose F160 generations
Malicious candidate's strain as vaccine development.
Embodiment 2：The cultivation of YB160 plants of attenuated vaccine strain avian infectious bronchitis virus of the present invention and its gene are stable
Property
1. materials and methods
1.1 Strain YBX Attenuations
YBX plants are carried out with continuous passage cause using 9~10 age in days SPF chicken embryos weak.Each generation poison is inoculated with 3 by allantoic cavity
Piece chicken embryo, per embryonic breeding kind 0.1ml, puts 37 DEG C and is incubated 72h (discarding dead germ in 24h), daily observation chicken embryo twice, if chicken embryo exists
24~72h is dead, then puts 4 DEG C of preservations.After 72h by it is all connect embryo and take out put 4 DEG C, 4~24h, observed and recorded chicken embryo lesion situation,
Sterile collection chick embryo allantoic liquid carries out passage of future generation.Select 72h not dead, but lesion typical case, the limpid chicken embryo progress of allantoic fluid
Passage.
1.2 virus titers are determined
The chick embryo allantoic liquid of F30, F65, F90, F115, F125, F145, F160 and F170 generation poison is taken, is made successively respectively
10 times are serially diluted；Select 4 extension rates (104~107Or 105~108) viral suspension be inoculated with 5 piece of 9~10 age in days respectively
Chicken embryo is put in 37 DEG C of incubators and is incubated 1 week by SPF chicken embryos, every piece of egg inoculation 0.1ml, daily according to embryo, records chicken embryo in 1 week
Number and existence number are infected, EID is calculated by Reed-Muench methods50。
1.3 steriling tests and mycoplasma are examined
By YB plants of F30, F65, F90, F115, F125, F145, F160 and F170 generation poison by existing《Chinese people's republicanism
State's veterinary drug allusion quotation》Annex carries out steriling test and mycoplasma is examined.
1.4 exogenous viruses are examined
YB160 plants of F30, F65, F90, F115, F125, F145, F160 and F170 generation poison are subjected to exogenous virus detection.
1.5 YB160 plants of cause is weak to be evaluated
180 1 age in days SPF chickens are randomly divided into 9 groups (every group 20), respectively by 9 groups of fowl raisings in 9 negative pressure isolators
In, chick free choice feeding drinking-water.In 15 age in days, 1~8 group of chick uses chicken embryo generation poison F30 (10 respectively6.5EID50)、F65
(107.2EID50)、F90(107.5EID50)、F115(107.8EID50)、F125(108.0EID50)、F145(108.2EID50)、F160
(108.3EID50) and F170 (108.2EID50) be inoculated with, every collunarium 0.1ml；As a control group, every drips 9th group of chick
The normal allantoic fluids of nose 0.1ml.From inoculation, daily observation, the incidence of record chicken group, death condition, to dead chicken
Carry out cut open inspection, observation IBV target organs, the pathological change of tissue and in 14 days after inoculation are attacked using same source strength poison YBX,
Every eye droppings collunarium 0.1ml；Attack after poison, daily observation, record chicken group incidence, death condition carry out cut open inspection to dead chicken,
Observe target organ, the pathological change of tissue.
1.6 YB160 plants of immune efficacy evaluation
1.6.1 SPF chicken immune potency tests of 15 ages in days
40 1 age in days SPF chickens are randomly divided into 2 groups (every group 20), raise respectively in negative pressure isolator, chick is freely adopted
Food drinking-water.During 15 age in days, one of which chick is inoculated with using YB160 plants, every collunarium 0.1ml；Another group of chick then makees
For control group, every normal allantoic fluid of collunarium 0.1ml.From inoculation, daily observation, record connect the morbidity feelings of breeder flock
Condition, death condition, and 15 days after immune, detection of specific antibody is carried out to every group of collection serum, specific method is with reference to infection
Property bronchitis virus antibody assay kit specification carry out；2 groups of chick are carried out into collunarium with same source strength poison YBX simultaneously to attack
Hit, every 0.1ml；Attack after poison, daily observation, the incidence of record chicken group, death condition.
1.6.2 3 age in days SPF chicken immune potency tests
The same 1.6.1 of method, SPF chickens are 3 ages in days.
1.7 YB160 pnca gene stability tests
By F115, F125, F145, F160, F170 plants of S1 gene sequencing, compare its gene stability.
2 results
2.1 YB160 plants of Attenuation
We utilize IBV-YBX velogen strains generation of continuous passage 170 in SPF chicken embryos, have cultivated one plant of virulence and have substantially subtracted
It is weak, and the IBV low virulent strains with good immunogenicity.Experiment confirms that IBV-YB160F160 is decreased obviously for virulence, with
105.5EID501 age in days SPF chickens 5/5 are inoculated with to have no adverse reaction.IBV-YB160F160 continues through the generation of 1 age in days SPF chickens body 5 for poison,
Have no that virulence is returned by force.With 103EID50Immune 1 age in days SPF chickens, attack 10/10 protection after poison, and control 10/10 is fallen ill.
2.2 virus titer measurement results
YB160 plants can highly adapt to chicken embryo, the EID in F30 generations50For 106.5/ 0.1ml, passage restrovirus titre gradually rises,
F65~F125 generations >=107.0EID50In/0.1ml, F145~F160 generations, tend towards stability, and titre is about 108.2EID50/0.1ml。
The YB160 plants of malicious titer determination results of chicken embryo passage of table 1
2.3 steriling tests and mycoplasma are examined
F30, F65, F90, F115, F125, F145, F160 and F170 generation poisons in chicken embryo succeeding generations are examined without thin
Bacterium, mould and mycoplasma contamination.
2.4 exogenous viruses are examined
F30, F65, F90, F115, F125, F145, F160 and F170 generation poisons in chicken embryo succeeding generations are examined without outer
Source virus pollution.
The cause of 2.5 YB160 plants of different generation poison is weak to be evaluated
YB160 plants are gradually weakened in chicken embryo after continuous passage to the pathogenicity of SPF chick.It the results are shown in Table 2.
Pathogenicity of the YB160 of the table 2 difference generation poison to SPF chickens
2.6 YB160 plants of immune efficacy preliminary assessment
The immune group of 2 different days (is immunized, 10 using YB160 plants of F160 generations5.0EID50), 15 days after immune,
Chicken blood serum sample is gathered, detects and finds using indirect ELISA reagent kit, inoculated YB160 plants blood serum sample all switchs to
IBV antibody positives, and control group blood serum sample is feminine gender.Using same source strength poison YBX to being seen after immune group and control group attack
Examine 5 days, it is found that immune group chicken is in good condition, no disease symptom；And the morbidity of control group chicken is obvious, show as spiritual depressed, hogback,
The symptoms such as hair thick unrest, mouth breathing, and about 30% (6-7/20) death (being shown in Table 3) after poison is attacked.
The immune efficacy evaluation of YB160 plants of table 3
2.7 YB160 pnca gene stability：
F115, F125, F145, F160, F170 plants of S1 gene nucleotide series homologys more than 97.5% (see Fig. 2),
Illustrate its stabilization that gene order is maintained in succeeding generations.
Embodiment 3：It is prepared by vaccine
The preparation of 2.1 infectious bronchitis of chicken live vaccines (YB160 plants)
YB160 plants of seeds culture of viruses (preserving number is CCTCC.V201235) are taken, 100,000 times is diluted with sterile saline, is inoculated in 9
~10 age in days SPF chick embryo allantois intracavitary, per embryonic breeding kind 0.1ml, seal pin hole after inoculation, put 37 DEG C of incubations.24h dead germs are discarded,
Chicken embryo is observed to 72h, by 30~72h chick embryo air sac upwards, puts 4 DEG C of 4~24h of cooling.Chick embryo allantoic liquid is harvested, by several
Chick embryo allantoic liquid is mixed into one group, puts in sterilizing bottle, and in 2~8 DEG C of preservations, while doing steriling test, measuring viral level is
107.0EID50/0.1ml.Protected again with sucrose gelatin after qualified chick embryo allantoic liquid mixing is determined through steriling test and viral level
Agent presses 1:1 (volume ratio) matches somebody with somebody seedling, and freeze drying protectant is with 8% (W/W) gelatin, 40% (W/W) sucrose protective agent, through 115 DEG C of high pressures
Sterilize 40min, puts in 4 DEG C of preservations, 72h and is finished.Virus liquid should be constantly shaken in adding procedure, after fully mixing, as epidemic disease
Seedling stoste.By the sterile quantitative separating of vaccinogen liquid, quick freeze vacuum drying is sealed.Obtain infectious bronchitis of chicken
Per plumage, IBV viral levels are 10 to vaccine in part4.5EID50.Determine lot number to test for 07012.2 active immunity
1~3 group of 3 age in days SPF chick is inoculated with 3 batches of vaccines respectively, every collunarium is inoculated with 1 dosage (i.e.
104.5EID50).Sterile saline is dripped in the every collunarium inoculation 1 of 4th group of 3 age in days SPF chick.14 days after immune, 4 groups of chicken drops
Respectively the strong poison (10 of (about 0.03ml) YBX is dripped in inoculation 1 for nose, eye droppings method6.0EID50), while intramuscular injection 1.5ml.Observation 14 days, note
Record morbidity and death condition.
2.3 passive immunitys are tested
1~3 group of 18 week old AA breeder is inoculated with 3 batches of vaccines respectively, every collunarium is inoculated with 1 dosage and is
104.5EID5).Sterile saline is dripped in the every collunarium inoculation 1 of 4th group of 18 week old AA breeder.After immune after 14 days, every group random
Take the hatching of 100 pieces of chicken embryo, every group take hatch chicken 60 only determined respectively at 7 days, 9 days and 14 days maternal antibodies and with collunarium,
Respectively the drop of inoculation 1 YBX is malicious by force for eye droppings method, while intramuscular injection 1.5ml.Observation 14 days, record morbidity and death condition.
3 results
3.1 active immunities are tested
After each batch of vaccine immunity group is immune 14 days, with strong virus attack, wherein saline control group is all fallen ill, and essence occurs
The clinical symptoms such as refreshing depressed, loose random, the dissection of dead chicken finds the specific lesions of glandular stomach enlargement within the observation period.Batch
Numbers 201113 be the infectious bronchitis of chicken live vaccine (W93 plants) of Liaoning Yikang Biological Co., Ltd., lot number S111101
For the infectious bronchitis of chicken live vaccine (H52) of Qian Yuanhao companies, detailed results are shown in Table 4.
The infectious bronchitis of chicken live vaccine active immunity potency test data of table 4
3.2 passive immunitys are tested
The chick that YB160 plants of infectious bronchitis vaccine immune health AA chickens produce egg hatching can be in 9 days
Protective effect is obtained, malicious protective rate is attacked more than 90%.Antibody test result is shown, although testing result remains in that the positive,
But maternal antibody is gradually being reduced during persistent infection to the protective rate of strong virus attack.This may one side and infectiousness
Bronchitis is main based on upper respiratory tract local immunity, on the other hand in terms of immunization type based on cellular immunity, with body
Relevant supplemented by liquid is immune, testing result the results are shown in Table 5 with attacking poison protection.
The infectious bronchitis vaccines of table 5 (YB160 plants) passive immunity result of the test
Test result indicates that, active immunity protective rate is 80%, and passive immunity can obtain more than 90% guarantor in 9 days
Shield, illustrates that strain of the present invention has good Vaccine effectiveness to infectious bronchitis of chicken.
Embodiment 4：The immune efficacy experiment of new branch (YB160 plants of+IBV of Sota plants of NDV La) bigeminy live seedling
1 material
Usage and consumption：By label mark plumage part, with normal saline dilution, vaccine is drawn with drop bottle, every chicken collunarium 1 drips
(about 0.03ml).
2 methods
The preparation of 2.1 new branch (YB160 plants of+IBV of Sota plants of NDV La) bigeminy live seedlings
The preparation of NDV virus liquids：Sota plants of seed culture of viruses sterile salines of La are taken to make appropriate dilution (such as 10-4), per embryo
Inoculation 0.1m1 in allantoic cavity.Pin hole is sealed after inoculation, 36~37 DEG C is put and continues to be incubated, it is not necessary to egg-turning.After egg inoculation, daily
According to egg 1 time, dead chicken embryo before 60 hours is discarded.After 60 hours, every 4~8 hours photograph eggs 1 time, dead chicken embryo is at any time
Take out.Until 96 hours, no matter whether dead, all take out, air chamber is upright upwards, be placed in 2~8 DEG C and cool down 4~24 hours.Receive
Chick embryo allantoic liquid is obtained, one group is mixed into per several chick embryo allantoic liquids, is placed in sterilizing bottle.The measure that keeps sample HA, HA<1：256
It should discard.Make steriling test simultaneously, should be without bacterial growth.Measure viral level and should be 108.0EID50/0.1ml.
The preparation of IBV virus liquids：Production seed culture of viruses of the present invention is taken, 100,000 times is diluted with sterile saline, is inoculated in 10
Age in days SPF chick embryo allantois intracavitary, per embryonic breeding kind 0.1nl, seals pin hole after inoculation, puts 37 DEG C of incubations.Discard 24h dead germs, chicken embryo
Observation, puts 4 DEG C of 4~24h of cooling by 30~72h chick embryo air sac upwards to 72h.Chick embryo allantoic liquid is harvested, by several chicken embryos
Allantoic fluid is mixed into one group, puts in sterilizing bottle, in 2~8 DEG C of preservations, while doing steriling test, measures viral level and should be
107.0EID50/0.1ml。
The preparation of new branch (YB160 plants of+IBV of Sota plants of NDV La) bigeminy live seedling：Surveyed through steriling test and viral level
Fixed qualified NDV and IBV chick embryo allantoic liquids, 1:After 1 (volume ratio) mixing, then with sucrose gelatin protective agent by 1:1 (volume ratio)
With seedling, freeze drying protectant is with 8% gelatin, 40% sucrose protective agent, through 115 DEG C of autoclaving 40min, puts in 4 DEG C of preservations, 72h
It is finished.Virus liquid should be constantly shaken in adding procedure, after fully mixing, as vaccinogen liquid.Vaccinogen liquid is sterile quantitative
Packing, quick freeze vacuum drying, is sealed.In prepared new branch Combined vaccine of the invention, infectious bronchitis vaccines
Per plumage, IBV is 10 in part4.5EID50, newcastle disease virus La Sota strain virus content is 106.5EID50.It is 0901 to determine lot number.
Lot number for 201118 newcastle disease, infectiousness bronchitis bigeminy live vaccine (LaSota plants+H120 plants) is purchased from
Harbin Pharmaceutical Group Biological Vaccine Co., Ltd., newcastle disease, infectiousness bronchitis bigeminy live vaccine that lot number is 110516
(LaSota plants+H52 plants) are purchased from Guangdong Dahuanong Animal Health Products Co., Ltd..
2.2 potency test
2.2.1 examined with chicken embryo
By vaccine normal saline dilution to l plumage part/0.5ml, it is respectively charged into two test tubes, often pipe lml.First pipe adds
Enter the anti-newcastle disease virus specific serum of equivalent, the second pipe adds the anti-avian infectious bronchitis virus YB160 of equivalent
Strain specific serum.At room temperature with 1 hour (shaking 1~2 time centre), now viral level is 0.1 plumage part/0.1ml.First pipe
Continue 10 times to be serially diluted, 10 age in days SPF chicken embryos 5 of inoculation in 3 each allantoic cavities of acceptable diluent degree are taken, per embryo 0.1ml, in 37
, there is dehydration in the chicken embryo survived during according to 24~144 hours dead germs after inoculation and 6 days, rolls up, develops small (connect in DEG C observation 6 days
Kind of fetus is than compareing most light low more than the 2g of fetal weight) etc. specific lesions embryo summation, calculating EID50.Second pipe continues 10 times
It is serially diluted, takes each inoculated into chick embryo of 3 acceptable diluent degree 5, it is dead before 48 hours to disregard per embryo 0.1m1,48~120
The hour chicken embryo of death is taken out at any time, harvests chicken embryo liquid, with dilution factor mixed in equal amounts, embryo living was taken out to 120 hours, is harvested one by one
Blastochyle, determines HA-HI test, HA-HI test >=1 respectively:128 (micromethods) are judged to infection, calculate EID50.
2.2.2 examined with chicken
(1) newcastle disease part
With the SPF chickens 10 of 30~60 ages in days, every collunarium is inoculated with l/100 dosages, after 10~14 days, together with condition
The non-immunized controls chicken of identical 3, each intramuscular injection contains 105.0ELD50The strong poison lml of newcastle disease Beijing Strain, observation 14 days.
(2) infectious bronchitis of chicken part
With the age in days SPF chickens 10 of l~3, the vaccine of 1 dosage of every collunarium, after 10~14 days, together with control chicken 10
Only, with YBX plants strong toxogen liquid eye droppings, each 1 drop of collunarium, while intramuscular injection 1.5ml, is observed 14.
3 results
3.1 chicken embryo inspection techniques
3 batches of Combined vaccines every batch take out 1 bottle and are diluted to l plumage part/0.5ml with sterile saline respectively, are respectively charged into two
In test tube, every pipe lml.First pipe adds the anti-newcastle disease virus specific serum of equivalent, and the second pipe adds the anti-chicken of equivalent
YB160 plants of specific serums of infectious bronchitis virus.At room temperature with 1 hour (shaking 1~2 time centre), now virus contains
Measure as 0.1 plumage part/0.1ml.First pipe continues 10 times and is serially diluted, and takes 10 ages in days of inoculation in 3 each allantoic cavities of acceptable diluent degree
SPF chicken embryos 5, per embryo 0.1ml, are observed 6, the chicken embryo survived during according to 24~144 hours dead germs after inoculation and 6 days in 37 DEG C
It is middle dehydration occur, roll up, develop the total of specific lesions embryos such as small (inoculation fetus are than compareing most light low more than the 2g of fetal weight)
With calculating EID50.Second pipe continues 10 times and is serially diluted, and takes each inoculated into chick embryo of 3 acceptable diluent degree 5, per embryo 0.1m1,48
Dead before hour to disregard, dead chicken embryo is taken out at any time within 48~120 hours, harvests chicken embryo liquid, with dilution factor mixed in equal amounts,
Embryo living was taken out to 120 hours, blastochyle is harvested one by one, HA-HI test, HA-HI test >=1 are determined respectively:128 (micromethods) are judged to infection,
Calculate EID50.Examine the every plumage part viral level in newcastle disease part >=106.5EID50, infectious bronchitis of chicken part is per plumage
Part viral level is >=104.0EID50.New branch Combined vaccine efficacy test (chick embryo method) examines qualified.It the results are shown in Table 6.
Table 6 Combined vaccine efficacy test (chick embryo method) result
3.2 Combined vaccines every batch times of chicken inspection technique 3 batches are taken out 1 bottle and diluted respectively with sterile saline.
(1) newcastle disease part
With the SPF chickens 10 of 30 ages in days, every collunarium is inoculated with l/100 dosages, identical together with condition after 10~14 days
Non- immunized controls chicken 3, the strong poison lml of newcastle disease Beijing Strain of each intramuscular injection containing 104.0ELD50, observation 14 days.Examine
Compare chicken 3/3 in chicken to fall ill death, immune chicken protective rate reaches 90%.
(2) infectious bronchitis of chicken part
With l age in days SPF chickens 10, the vaccine of 1 dosage of every collunarium, another 15 are used as control with age in days SPF chickens
Under equal conditions isolated rearing.After 14 days, together with control chicken 10, dripped with YBX plants strong toxogen liquid eye droppings, collunarium each 1, simultaneously
Intramuscular injection 1.5ml, is observed 14.Attack poison control chicken 8/10 in inspection chicken to fall ill, immune chicken protective rate reaches 80%, just
Often control chicken is without any inner clinical symptoms.New branch Combined vaccine efficacy test (chicken inspection technique) examines qualified, the results are shown in Table 7,8.
The Combined vaccine efficacy test of table 7 (chicken inspection technique) ewcastle disease partial results
The Combined vaccine efficacy test of table 8 (chicken inspection technique) passes branch partial results
Test result indicates that, it is immunized with the new branch bigeminal live vaccine of infectious bronchitis of chicken YB160 plants of the present invention preparations
Chicken, to the shield rate of NDV and IBV (YBX is malicious by force) strong virus attack more than 80%, illustrate the new of use strain of the present invention preparation
Branch bigeminal live vaccine has good Vaccine effectiveness to newcastle disease and Glandular Stomach Type infectious bronchitis of chicken.
Embodiment 5：Pass the preparation and efficacy test of YB160 plants of subunit vaccines of branch
1. materials and methods
The structure of 1.1 S1 gene sequencing and pMDYB160S1 recombinant plasmids
Design primer I BV-87 first (5 '-TAT TGA TTA GAG ATG TTG GG-3 ') and primer S1Oligo3 ' (5
'-CAT AAC TAA CAT AAG GGC AA-3 '), using preserving number as CCTCC.V201235 IBV-YB160 strains genome
RNA is template, and IBVS1 genes are expanded by RT-PCR, and the S1 gene orders of amplification are cloned into pMD-18T carriers, is obtained
Obtain recombinant plasmid pMDYB160S1.
The structure of 1.2 restructuring swivel base plasmids
By recombinant plasmid pMDYB160S1 and transfer vector pFASTBac HTa respectively with BamH I and Sal I digestions after,
S1 genetic fragments and carrier segments are reclaimed, after T4DNA Ligase connections, escherichia coli DH5a are converted, coating is big mould containing celebrating
Cultivate, chosen after bacterium Zengjing Granule on the LB flat boards of element and ampicillin, extract DNA, by electrophoresis, preliminary screening goes out to divide
The higher plasmid of son amount, single, double digestion identification is then carried out to it, insertion and the closure of target gene is determined, is weighed
Group transferring plasmid pFASTYB160S1.
1.3 recombinant baculovirus rAcYB160S1 acquisition
PFASTYB160S1 is converted into the Escherichia coli DH10Bac competent cells containing viral shuttle plasmid Bacmid
In (being purchased from Invitrogen companies), recombinant shuttle plasmid rBacmidYB160S1 is obtained；PCR is screened, and extracts positive plasmid, system
Standby positive recombinant plasmid rBacmidYB160S1 DNA.UsingReagent transfection Sf9 cells (are purchased from
Invitrogen companies), after lesion occurs in cell, recombinant virus plaque purification and virus amplification are carried out, then by general
Primer M13F and M13R enter performing PCR verifying purpose restructuring S1 genes.Purifying obtains recombinant baculovirus, is named as
rAcYB160S1.Recombinant baculovirus rAcYB160S1 is expanded as kind of a poison, potency titration is carried out after 4 DEG C of guarantors by plaque ethods
Deposit standby.
The expression and identification of 1.4 restructuring S1 albumen
The insect High FiveTM cultivated that rAcYB160S1 recombinate shape virus infections are suspended (are purchased from Invitrogen
Company).The sterile suspension culture High in the 500ml rolling bottles of the EXPRESS FIVE SFM culture mediums containing 200~350ml
FiveTM cells, inoculating cell density is 0.3~0.6 × 106 cell/ml.When cell density reaches 1 × 106Individual cell/ml
When, each rolling bottle is inoculated with recombinant virus rAcYB160S1, the recombinant baculovirus being inoculated into each rolling bottle has different infection
Plural (M.O.I), is that with 26~28 DEG C, rotating speed is 100rpm, culture 4 after 0.01,0.1,1. inoculation recombinant baculovirus respectively
My god.
Each bottle was sampled in every 12 hours after inoculation, i.e., 48h, 60h, 72h, 84h and 96h are sampled.Each sample with 10000g from
Heart 20min, separation supernatant and precipitation.Supernatant is filtered by 1um filter membrane, after filtering carry out SDS-PAGE electrophoresis and
Ultraviolet specrophotometer determines avian infectious bronchitis virus restructuring S1 albumen and its content in supernatant.
The avian infectious bronchitis virus for identifying expression using Western blotting recombinates S1 albumen.Collect respectively
The High FiveTM cells of recombinant virus infection, take supernatant through 3000r/min centrifugations 10min, carry out SDS-PAGE electrophoresis.
1.5 harvests and purifying avian infectious bronchitis virus restructuring S1 albumen
Culture is centrifuged into 20min, precipitation and separation and supernatant through 10000g, supernatant was carried out by 1um filter membrane
Filter, is then concentrated by ultrafiltration purifying, and ultrafiltration membrane aperture is 10000 (molecular weight cut offs), and sample solution after purification is through 7mM bis-
Aziridine (BEI) inactivates recombination bacillary viral vector, and 36~48 hours, the sodium thiosulfate for adding equivalent was neutralized.
The preparation of 1.6 infectious bronchitis of chicken subunit vaccines
Prepare the antigen diluent thing of various concentrations：Restructuring S1 albumen is diluted to 0.5 mcg/ml, 1 with PBS (PH7.4)
Mcg/ml, 2 mcg/mls, 4 mcg/mls.
By the antigen diluent thing of above-mentioned various concentrations, with oily adjuvant ISA206 mixing and emulsifyings.ISA206 adjuvants through 121 DEG C,
60Min high pressure moist heat sterilizations, according to antigen：Adjuvant volume ratio 46:54 mixing.IKA2000/4 type mulsers are produced using Germany,
First adjuvant is added in mulser adjuvant barrel, (100r/min) slowly instills antigen aqueous phase in adjuvant under stirring,
After dripping off, continue to stir 5min.Mulser 5000r/min, circulating emulsion 4min are used, subunit vaccine is obtained.
The effect detection of 1.7 IBV subunit vaccines
A. subunit vaccine is in the immune response of SPF chickens and the detection of antibody level
Test chicken is 21 ages in days without specified pathogen (SPF) chicken 15, immune group 10, control group 5, collunarium inoculation
Infectious bronchitis of chicken live vaccine (H120 plants) 1 plumage part, 21 days after inoculation, takes a blood sample respectively, respectively subcutaneously or intramuscularly injects sub- single
Position vaccine 0.3ml.SPF chickens are raised in isolator.28 days after injection, then take a blood sample respectively, separate serum, serum will distinguish twice
Determine HI antibody titers.Two geometrical means for exempting from Serum HI antibody potency should be not less than first exempt from serum 4 times.
HI antibody detection methods：
(1) infectious bronchitis of chicken HI antigens HA-HI test determines and takes 96 1 piece of hole V-type micro-reaction plates, adds 25 μ l per hole
PBS (0.01mol/L, pH value 7.0~7.4), the 1st row adds 25 μ l antigens, and makees 2~4 repeating holes, and antigen then is carried out into 2
It is serially diluted again, per hole adding 25 μ l PBS after dilution, (0.01mol/L, pH value 7.0~7.4) finally add 1% chicken red
The μ l of cell suspension 25, are mixed with micro oscillator, 2~8 DEG C of standings, 40 minutes result of determination, so that 100% erythrocyte agglutination
Antigen highest extension rate is used as judgement terminal.
(2) preparation of 4HA antigens is according to the HI antigen HA potency of measure, with PBS (0.01mol/L, pH value 7.0~7.4)
Prepare 4HA unit antigens.By the 4HA unit antigens PBS prepared, (0.01mol/L, pH value 7.0~7.4) dilute, makes its dilute
Degree of releasing is 1:2、1:3、1:4、1:5、1:6、1:7.Add 25 μ l PBS (0.01mol/L, pH in 25 μ l antigens of each dilution factor
Value 7.0~7.4), the μ l of 1% chicken erythrocyte suspension 25 are added, are mixed, 2~8 DEG C stand 40 minutes, result of determination.If 1:4
100% erythrocyte agglutination terminal is diluted to, show preparation is 4HA unit antigens；If 100% erythrocyte agglutination terminal is 1:
5,1:6, it is actually higher than 4 units to show the 4HA units antigen prepared；If 100% erythrocyte agglutination terminal is 1:2,
1:3, it is actually less than 4 units to show the 4HA units antigen prepared.It should be appropriately adjusted according to assay, make antigen
Working solution is defined as 4HA units.
(3) hemagglutination-inhibition test (HI)
1. 96 hole V-type micro-reaction plates are taken, and per the μ l PBS of Kong Zhongjia 25 (0.01mol/L, pH value 7.0~7.4).
2. 25 μ l serum to be checked are drawn respectively, and in the 1st each respective aperture of row for adding to every block of plate, and bidding is accurate on every block of plate
Positive serum and negative serum control, are then serially diluted for 2 times.
3. the antigen 25 μ l of 4HA units are added into each hole respectively, 2~8 DEG C stand 30 minutes.
4. the μ l of 1% (V/V) chicken erythrocyte suspension 25 are added in every hole, gently mixed, 2~8 DEG C stand 40 minutes.
5. result judgement tilts reaction plate, and red blood cell is at the same rate in all seroreaction holes and red blood cell control wells
Blood clotting is judged to from bottom hole trickling person to suppress.When negative serum HI potency is not higher than 1:8th, positive serum HI potency and regulation potency phase
Ratio error is not higher than 1:When 2, experiment can be set up.Imitated using the serum highest dilution that completely inhibits 4HA unit antigens as HI
Valency.
B. the IBV virus challenges after SPF chickens are immunized in subunit vaccine
The immune chicken and nonimmune chicken of above-mentioned " a " item, two exempt from 28 days afterwards, are dripped with YBX plants strong toxogen liquid eye droppings, collunarium each 1,
Intramuscular injection 1.5ml, is observed 14 simultaneously.
2 results
The structure of 2.1 recombinant plasmids and identification PCR primer size about 1700bp, are as a result shown in accompanying drawing 3, wherein, the 1st hole is
IBV S1 amplified productions；2nd hole is DNA Marker2000.PCR primer is sequenced, from electrophoresis and sequencing result,
Recombinant vector pMDYB160S1 is containing the SEQ ID NO.1 sequences in sequence table.
The identification of 2.2 restructuring swivel base plasmids identifies that the direction of insertion of S1 genes is correct, the size of endonuclease bamhi through digestion
It is consistent with expected results.
2.3 transfer vectors comprising avian infectious bronchitis virus S1 genes are transformed into the large intestine containing shuttle vector
In bacillus competent cell, obtain and include the shuttle vectors of avian infectious bronchitis virus S1 genes, then by the shuttle vector
Be transfected into insect cell (be purchased from Invitrogen companies), after transfection 24h begin with cell occur it is dead, hereafter cell have after
The trend of continuous propagation.Cell starts lesion occur after 72h, and nucleus enlargement, lesion gradually increases, and cell starts death after 96h,
Substantially all death after 1 week.
The expression and identification of 2.4 restructuring S1 albumen
Recombinant protein content reaches 60 mcg/mls in supernatant after recombinant virus-infected cell 72h.Illustrate recombinant virus
After infection cell, culture supernatant is reclaimed after culture at least 72h, avian infectious bronchitis virus restructuring S1 eggs can be significantly improved
White yield.
The specific band that SDS-PAGE protein electrophoreses gel observation is about 56KDa to size, as recombination chicken are infected
Property bronchitis virus S1 albumen.Destination protein is sequenced, from electrophoresis and sequencing result, avian infectious branch gas is recombinated
Pipe inflammation virus S1 protein sequences are containing the SEQ ID NO.3 sequences in sequence table.
2.5 recombinant proteins are harvested and purifying：Assay is carried out to purifying protein by ultraviolet specrophotometer, reached
68 mcg/mls.
The inspection of 2.6 subunit vaccines：The vaccine prepared is through viscosity measurements, centrifugation detection, formulation detection, stability
After detection, indices meet regulation.Vaccine is stored in 4 DEG C.
The proteantigen of the inventive method production can also be mixed with other oil adjuvants, aqueous adjuvants or other adjuvants
Use, white-oil adjuvant as common in the art, aluminium glue adjuvant, Freund's adjuvant etc..
The efficacy test result of 2.7 subunit vaccines
Antibody test result is shown in Table 9 after subunit vaccine is immune, and thus table result understands that two exempt from rear Serum HI antibody potency
Geometrical mean be not less than first exempt from serum 4 times.
Anti- IBVHI antibody test results are produced in the SPF chicken bodies that table 9 is immunized with subunit vaccine
In the protest test of subunit vaccine, poison control chicken 5/5 is attacked in inspection chicken and is fallen ill, chicken protective rate is immunized equal
90% is reached, normal control chicken is shown in Table 10 without any clinical symptoms, concrete outcome.
The IBV virus challenge results after SPF chickens are immunized with subunit vaccine for table 10
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc. should be included in the scope of the protection.
<110>Pulaike Biological Engineering Co., Ltd.
<120>Infectious bronchitis of chicken velogen strain S1 genes and its velogen strain and application
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1562
<212> DNA
<213>Infectious bronchitis of chicken low virulent strain S1 genes
<400> 1
ctgtgtagtg ccgctttgtt tgataataat gaaaccgttt actactacca aagtgccttt 60
agaccggctg atggatggca tttgcatggt ggtgcttatg cagtagtaaa cgtttcttta 120
gaaactaata atgcaggcac agcttcacaa tgcattgcag gggctatttc ttggagtaaa 180
aatttctctg cttctgctgt agccatgact gcacctgagt tagggatgac gtggtcaact 240
gggcaattct gcacggctca ctgtaacttc tcggatttta cagtgttcgt tacgcattgt 300
tttaaacacg gtgacggtct atgcccgcta acagggctta ttccaagtgg atttattcgt 360
gtttctgcta tgaggaaggg aagtaattcc ttgttttata atttaacagt ttctgtgact 420
aaatatccta gatttaagtc gcttcaatgt gttaataatt atacatctgt gtacttaaat 480
ggtgatcttg tgttcacttc taatgaaact aaacctgtta gtgcagcagg tgtttctttt 540
acagctggtg gacctataac ttacaagact atgagtgaag ttaaagtcct agcttatttt 600
gccaatggaa ccgcacaaac tgttattcct tgtgatggtt cacctagagg cttgttagct 660
tgtcagtata atacaggcaa tttttcagat ggtttctacc cttacactaa tagtagttta 720
gttaaggaaa ggtttattgt ttatagagaa agtagtgtta acactacctt agtgttaact 780
aattttactt tctcaaatgt tagtaacgcc cctcctaata caggtggtgt tcatagtatt 840
gttctacatc aaacacaaac aactcagagt ggttattata attttaattt ctcctttctg 900
agtagtttcc gttatgtaga atcagatttt atgtatgggt cataccaccc aaaatgttca 960
tttagactag aaactattaa taatggtttg tggtttaatt cactttcggt ttctcttggt 1020
tacggtccac tacagggtgg ttgtaagcaa tctgtgttta ataatatggc aacttgttgt 1080
tatgcttatt catatagtgg tcccacacta tgtaaaggtg tttatagtgg tgagttacaa 1140
aaaacttttg agtgtgggtt gctggttttt gtgactaaga gcgatggctc tcgtatacaa 1200
actagaaatg aaccacttgt gttaactcag cacaattaca ataatattac tttaaataag 1260
tgtgttgagt ataatatata tggcagagtt ggccaaggtc ttattactaa cataacagat 1320
tcagctgcta atcatggcta tttggcagat ggcgggttgg ctgttttaga tacttcaggt 1380
gccatagacg tttttgttgt acaaggtgtt tatggcctaa cttactataa ggttaatccc 1440
tgtgaagatg ttaaccaaca atttgtagtc tctggtggac agttagttgg catacttaca 1500
tctcgtaatg aaactggttc tcaacctatt gagaaccggt tttatgttaa atttcctaat 1560
ag 1562
<210> 2
<211> 1530
<212> DNA
<213>Infectious bronchitis of chicken velogen strain S1 genes
<400> 2
ggctcgagcg gtggtaataa gggaaccgtt tactactacc aaagtgcctt tagaccggct 60
gatggatggc atttgcatgg tggtgcttat gcagtagtaa acgtttcttt agaaactaat 120
aatgcaggca cagcttcaca atgcattgca ggggctattt cttggagtaa aaatttctct 180
gcttctgctg tagccatgac tgcacctgag ttagggatga cgtggtcaac tgggcaattc 240
tgcacggctc actgtaactt ctcggatttt acagtgttcg ttacgcattg ttttaaacac 300
ggtaacggtc tatgcccgct aacagggctt attccaagtg gatttattcg tgtttctgct 360
atgaggaagg gaagtaattc cttgttttat aatttaacag tttctgtgac taaatatcct 420
agatttaagt cgcttcaatg tgttaataat tatacatctg tgtacttaaa tggtgatctt 480
gtgttcactt ctaatgaaac taaacctgtt agtgcagcag gtgtttcttt tacagctggt 540
ggacctataa cttacaagac tatgagtgaa gttaaagtcc tagcttattt tgtcaatgga 600
accgcacaaa ctgttattcc ttgtgatggt tcacctagag gcttgttagc ttgtcagtat 660
aatacaggca atttttcaga tggtttctac ccttacacta atagtagttt agttaaggaa 720
aggtttattg tttatagaga aagtagtgtt aacactacct tagtgttaac taattttact 780
ttctcaaatg ttagtaacgc cccctcccta atacaggtgg tgttcatagt attgttctac 840
atcaaacaca aacaactcag agtggttatt ataattttaa tttctccttt ctgagtagtt 900
tccgttatgt agaatcagat tttatgtatg ggtcatacca ccccaaaatg ttcatttaga 960
ctagaaacta ttaataatgg tttgtggttt aattcacttt cggtttctct tggttacggt 1020
ccactacagg gtggttgtaa gcaatctgtg tttaataata tggcaacttg ttgttatgct 1080
tattcatata gtggtcccac actatgtaaa ggtgtttata gtggtcagtt acaaaaaact 1140
tttgagtgtg ggttgctggt ttttgtgact aagagcgatg gctctcgtat acaaactaga 1200
aatgaaccac ttgtgttaac tcagcacaat tacaataata ttactttaaa taagtgtgtt 1260
gagtataata tatatggcag agttggccaa ggtcttatta ctaacataac agattcagct 1320
gctaatcatg gctatttggc agatggcggg ttggctgttt tagatacttc aggtgccata 1380
gacgtttttg ttgtacaagg tgtttatggc ctaacttact ataaggttaa tccctgtgaa 1440
gatgttaacc aacaatttgt agtctctggt ggacagttag ttggcatact tacatctcgt 1500
aatgaaactg gttctcaacc tattgagaac 1530
<170>By hand
<210> 3
<211> 520
<212> PRT
<213>Infectious bronchitis of chicken subunit
<400> 3
Leu Cys Ser Ala Ala Leu Phe Asp Asn Asn Glu Thr Val Tyr Tyr Tyr Gln
1 5 10 15
Ser Ala Phe Arg Pro Ala Asp Gly Trp His Leu His Gly Gly Ala Tyr Ala
20 25 30
Val Val Asn Val Ser Leu Glu Thr Asn Asn Ala Gly Thr Ala Ser Gln Cys
35 40 45 50
Ile Ala Gly Ala Ile Ser Trp Ser Lys Asn Phe Ser Ala Ser Ala Val Ala
55 60 65
Met Thr Ala Pro Glu Leu Gly Met Thr Trp Ser Thr Gly Gln Phe Cys Thr
70 75 80 85
Ala His Cys Asn Phe Ser Asp Phe Thr Val Phe Val Thr His Cys Phe Lys
90 95 100
His Gly Asp Gly Leu Cys Pro Leu Thr Gly Leu Ile Pro Ser Gly Phe Ile
105 110 115
Arg Val Ser Ala Met Arg Lys Gly Ser Asn Ser Leu Phe Tyr Asn Leu Thr
120 125 130 135
Val Ser Val Thr Lys Tyr Pro Arg Phe Lys Ser Leu Gln Cys Val Asn Asn
140 145 150
Tyr Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Phe Thr Ser Asn Glu Thr
155 160 165 170
Lys Pro Val Ser Ala Ala Gly Val Ser Phe Thr Ala Gly Gly Pro Ile Thr
175 180 185
Tyr Lys Thr Met Ser Glu Val Lys Val Leu Ala Tyr Phe Ala Asn Gly Thr
190 195 200
Ala Gln Thr Val Ile Pro Cys Asp Gly Ser Pro Arg Gly Leu Leu Ala Cys
205 210 215 220
Gln Tyr Asn Thr Gly Asn Phe Ser Asp Gly Phe Tyr Pro Tyr Thr Asn Ser
225 230 235
Ser Leu Val Lys Glu Arg Phe Ile Val Tyr Arg Glu Ser Ser Val Asn Thr
240 245 250 255
Thr Leu Val Leu Thr Asn Phe Thr Phe Ser Asn Val Ser Asn Ala Pro Pro
260 265 270
Asn Thr Gly Gly Val His Ser Ile Val Leu His Gln Thr Gln Thr Thr Gln
275 280 285
Ser Gly Tyr Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Arg Tyr Val
290 295 300 305
Glu Ser Asp Phe Met Tyr Gly Ser Tyr His Pro Lys Cys Ser Phe Arg Leu
310 315 320
Glu Thr Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Leu Gly
325 330 335 340
Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Asn Asn Met Ala
345 350 355
Thr Cys Cys Tyr Ala Tyr Ser Tyr Ser Gly Pro Thr Leu Cys Lys Gly Val
360 365 370
Tyr Ser Gly Glu Leu Gln Lys Thr Phe Glu Cys Gly Leu Leu Val Phe Val
375 380 385 390
Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Arg Asn Glu Pro Leu Val Leu
395 400 405
Thr Gln His Asn Tyr Asn Asn Ile Thr Leu Asn Lys Cys Val Glu Tyr Asn
410 415 420 425
Ile Tyr Gly Arg Val Gly Gln Gly Leu Ile Thr Asn Ile Thr Asp Ser Ala
430 435 440
Ala Asn His Gly Tyr Leu Ala Asp Gly Gly Leu Ala Val Leu Asp Thr Ser
445 450 455
Gly Ala Ile Asp Val Phe Val Val Gln Gly Val Tyr Gly Leu Thr Tyr Tyr
460 465 470 475
Lys Val Asn Pro Cys Glu Asp Val Asn Gln Gln Phe Val Val Ser Gly Gly
480 485 490
Gln Leu Val Gly Ile Leu Thr Ser Arg Asn Glu Thr Gly Ser Gln Pro Ile
495 500 505 510
Glu Asn Arg Phe Tyr Val Lys Phe Pro Asn
515 520
Claims (7)
1. a kind of infectious bronchitis of chicken velogen strain S1 genes, its nucleotide sequence is as shown in SEQ ID NO.2 in sequence table.
2. velogen strain S1 genes according to claim 1, it is characterised in that the velogen strain S1 genes and nucleotide sequence
Infectious bronchitis of chicken low virulent strain S1 DNA homologs in table shown in SEQ ID NO.1.
3. velogen strain S1 genes according to claim 1 or 2, it is characterised in that the velogen strain S1 genes can be used in
The efficacy test of infectivity resistant bronchitis vaccine or subunit vaccine or vaccine combination.
4. velogen strain S1 genes according to claim 3, it is characterised in that the infectivity resistant bronchitis vaccine or Asia
Subunit vaccine or vaccine combination can prevent the attack of proventricular type infectious bronchitis virus.
5. velogen strain S1 genes according to claim 4, it is characterised in that the proventricular type infectious bronchitis virus
With following feature：Chicken glandular stomach enlargement of falling ill is for example spherical, glandular stomach wall thickening, glandular stomach mosucas teat bleeding ulcer.
6. the infectious bronchitis of chicken velogen strain of velogen strain S1 genes described in any one in a kind of 1-5 containing claim,
It is characterized in that：The velogen strain is YBX plants of avian infectious bronchitis virus, and its deposit number is：CCTCC NO：
V201236, is preserved in China typical culture collection center.
7. a kind of velogen strain according to claim 6 preparing for infectivity resistant bronchitis vaccine or subunit vaccine or
The application in medicament in the efficacy test of vaccine combination.
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| CN109293769A (en) * | 2018-10-29 | 2019-02-01 | 中崇信诺生物科技泰州有限公司 | A kind of QX type avian infectious bronchitis virus positive serum and preparation method thereof |
| CN109628412A (en) * | 2018-12-27 | 2019-04-16 | 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） | One plant of avian infectious bronchitis virus strain and its application |
| WO2019205753A1 (en) * | 2018-04-27 | 2019-10-31 | Shanghai Veterinary Research Institute, Chinese Academy Of Agricultural Sciences (National Center For Animal Health, Shanghai) | A composite multi-epitope expression cassette, a recombinant virus composed thereof and application thereof |
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| CN104762271A (en) * | 2015-01-26 | 2015-07-08 | 河南科技学院 | Preparation method and use of duck-derived coronavirus attenuated strain DCV35 |
| CN105622760A (en) * | 2016-01-22 | 2016-06-01 | 青岛明勤生物科技有限公司 | Avian infectious bronchitis multi-epitope mucosa immune vaccine and application thereof |
| CN105671003A (en) * | 2016-03-18 | 2016-06-15 | 华南农业大学 | Infectious bronchitis low-virulent live vaccine YX10 D90 strain |
| CN108203707A (en) * | 2017-12-28 | 2018-06-26 | 华南农业大学 | F80 plants of infectious bronchitis of chicken attenuated live vaccines GZ14 |
| CN110551696A (en) * | 2019-09-06 | 2019-12-10 | 扬州大学 | Natural low virulent strain of avian infectious bronchitis virus and application thereof |
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| CN103849632A (en) | 2014-06-11 |
| CN107287218B (en) | 2021-08-27 |
| CN103849632B (en) | 2017-05-17 |
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