CN105671003A - Infectious bronchitis low-virulent live vaccine YX10 D90 strain - Google Patents
Infectious bronchitis low-virulent live vaccine YX10 D90 strain Download PDFInfo
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Abstract
The invention belongs to the field of the biotechnology and discloses an infectious bronchitis low-virulent live vaccine YX10 D90 strain. The infectious bronchitis low-virulent live vaccine YX10 D90 strain is preserved in China Center for Type Culture Collection on 14th March, 2016, the preservation number is CCTCC NO:V201610, and the preservation address is Wuhan University, Wuhan, China. The low-virulent strain is safe for all chickens and has no side reaction, the low-virulent strain is prepared into a vaccine safely and effectively, toxin attacking on homological strong toxicity can be protected, and practical and wide application value is achieved.
Description
Technical field
The present invention relates to biological technical field, more specifically, it relates to a kind of chicken infectious bronchitis attenuated live vaccines YX10D90 strain.
Background technology
Infectious bronchitis of fowl (Infectiousbronchitis, IB) is the respiratory tract disease a kind of acute, highly contagious infection of the chicken caused by avian infectious bronchitis virus (Infectiousbronchitisvirus, IBV). Mainly it is characterized as disease chicken respiratory symptom, kidney enlargement, pale, there is a large amount of urate deposition, even outward appearance is in " piebald kidney "; The quality decline of laying hen egg yield and egg, children hen infects and can cause uterine tube permanent regression, causes producing " false hen ". In addition, the strain of part IBV poison can also cause the pathology of enteron aisle, glandular stomach, muscle. IBV can infect the chicken of all ages day, most kind, is especially that the chick in 1～5 week age is fairly felt, it is easy to causes that chick growth is slow, weightening finish and the price of deed reduces, even death; And easily cause the secondary infections such as Mycoplasma polyinfection and intestinal bacteria, thus cause chicken group's mortality ratio to increase, bring huge financial loss to whole world poultry husbandry, IB is one of serious infectious diseases seriously affecting world's aviculture.
IBV belongs to shell type virales (Nidovirales), coronaviridae (Coronaviridae), coronavirus genus (Coronavirus), is the representative poison strain of the 3rd group of fowl coronavirus. IBV be have capsule film, the single strand plus RNA virus of non-segmented negative. The most outstanding feature of IBV is that serotype is numerous, and different serotype does not have each other or only has very low cross immunity originality. Oneself serotype or genotype through reporting IBV has at least kind more than 30 at present; and new serotype, genotype and variant still constantly occur; cross-protection between different serotypes and genotype is very weak, and this brings very big difficulty to the diagnosis of IB, control.
Vaccine plays a part extremely important in the prevention of IBV, and at present, the vaccine used in China mainly contains deactivation vaccine, Attenuate vaccine. Deactivation vaccine mainly opens antenatal use kind of a chicken, laying hen. But it is big still to there is deactivation vaccine usage quantity, it is necessary to coordinate adjuvant, preparation is complicated, the more high great number of issues of cost. In addition, deactivation vaccine can't cause mucosal immunity and cellular immunization, and therefore tracheae can not provide effective protection. Paracox causes weak rear preparation by the malicious strain tamed by the continuous passage of chicken embryo.The immunity system of Attenuate vaccine energy effective stimulus body, it is possible to activated cell immunity simultaneously, mucosal immunity and humoral immunization, it may also be useful to convenient, production cost is low. Generally we are mainly used in broiler chicken, plant the head of chicken and laying hen and exempt from. Passing an attenuated live vaccines to use in Holland from 1956, H52, H120 are the most widely used living vaccines in the current whole world. Although China widely uses Mass type vaccine (H120, H52) always, Kidney infectious bronchitis is frequent quick-fried widely at home in recent years.
Summary of the invention
Technical problem to be solved by this invention be overcome existing vaccine strain not can the attack of effectively anti-avian infectious bronchitis virus epidemic isolates processed, the defect that effect of control is undesirable, it is provided that a kind of chicken infectious bronchitis attenuated live vaccines YX10D90 strain.
2nd object of the present invention is to provide the application of described chicken infectious bronchitis attenuated live vaccines YX10D90 strain.
3rd object of the present invention is to provide the medicine containing described chicken infectious bronchitis attenuated live vaccines YX10D90 strain.
It is an object of the invention to be achieved by the following technical programs:
A kind of chicken infectious bronchitis attenuated live vaccines YX10D90 strain, is preserved in China typical culture collection center March 14 in 2016, and deposit number is CCTCCNO:V201610, and preservation address is: China, Wuhan, Wuhan University.
2008～2015 years Chinese pathogenic strain S1 genes are carried out evolutionary analysis early stage by contriver, and result is consistent with domestic scholars report, and class QX type IBV has become the topmost IBV genotype of China, accounts for strain isolated about 60%. Class QX type and China conventional vaccine strain S1 gene similarity lower (70～85%) such as (H120, H52, LDT3A, 4/91), evolutionary distance is far away. Study discovery further, the domestic main attenuated vaccine of immunity for chickens (H120, H52, LDT3A, 4/91 etc.), then attack poison with the poison strain of class QX genotype, show the protection that current advantage epidemic isolates can not be provided enough by commercially available vaccine poison strain.
Attenuated live vaccines YX10D90 strain of the present invention is through repeatedly Attenuation by YX10 virulent strain, separation obtains, age on the 9th～11 SPF chicken embryo is specifically used YX10 strain is carried out continuous passage to cause weak, each generation inoculates 5 pieces of chicken embryos by allantoic cavity, every embryonic breeding kind 200 μ L, put 37 DEG C hatch 36～48h after, sterile collection chick embryo allantoic liquid, goes down to posterity next time. Reached for 90 generations continuously, called after YX10D90.
The biological property of YX10D90 strain is as follows: virus allantoic fluid, without coagulation, inoculate instar chicken embryo on the 9th～11 and can be caused chick embryo development and be obstructed or dead, and to SPF chicken no pathogenicity, morphological observation is typical coronavirus.
The present invention also provides a peptide species, and its aminoacid sequence is as shown in SEQIDNO:2.
The present invention also provides the nucleotide sequence of coding said polypeptide, and described sequence is as shown in SEQIDNO:1.
Through order-checking and analysis, the peptide sequence of the genes encoding of described vaccine YX10D90 strain is as shown in SEQIDNO:2; The nucleotides sequence of the gene of described vaccine YX10D90 strain is classified as shown in SEQIDNO:1, or through replacing, lack and/or add the gene order of the equal function of albumen described in one or more Nucleotide and coding SEQIDNO:2 in sequence basis described in SEQIDNO:1.
The present invention also provides the application of described chicken infectious bronchitis attenuated live vaccines YX10D90 strain in the medicine of preparation treatment chicken infectious bronchitis.
The present invention also provides the medicine containing described chicken infectious bronchitis attenuated live vaccines YX10D90 strain;Specifically, it is possible to be vaccine, the lyophilized vaccine that described vaccine comprises chicken infectious bronchitis attenuated live vaccines YX10D90 strain described in claim 1 or 4 or 5 and pharmaceutically can accept.
Compared with prior art, the present invention has following useful effect:
The present invention provides a kind of chicken infectious bronchitis attenuated live vaccines YX10D90 strain, is preserved in China typical culture collection center March 14 in 2016, and deposit number is CCTCCNO:V201610, and preservation address is: China, Wuhan, Wuhan University; Described low virulent strain is all safe to various chicken, and without side reaction, described low virulent strain is made vaccine safety effective, and that can protect same source strength poison attacks poison, has reality and using value widely.
Accompanying drawing explanation
Fig. 1 is the prevention effect of the attenuated vaccine of injection YX10D90.
Embodiment
Below by Figure of description and specific embodiment, the present invention is specifically described further. The design philosophy of the present invention or the simple replacement of allied substances belong to protection scope of the present invention. If following used experimental technique is without specified otherwise, it is the method for the existing routine of the art, the batching used or material, if no special instructions, it is by the available batching of commercial sources or material.
Embodiment 1 infectious bronchitis virus YX10 strain isolation identification
YX10 is by this laboratory 2010 isolation identification from Zhejiang Huzhou falls ill yellow-feathered broiler age on the 30th, and detailed process is as follows:
(1) separation and Culture of virus
Aseptic collection is died of illness chicken kidney, spleen, lungs, tracheae, by volume ratio (volume ratio) sterile saline of 1:5, grinding, freeze thawing 3 times, the centrifugal 20min of 5000rpm, the degerming rear allantoic cavity of supernatant liquid filtering inoculates SPF chicken embryo 10 pieces age on the 10th, every embryonic breeding kind 0.2ml, chicken embryo after inoculation 37 DEG C is hatched, every day shines embryo 2 times, abandons the dead embryo within 24 hours, within 48 hours, gets 5 pieces of results allantoic fluids, all the other continued to hatch to 144 hours, record chicken embryo pathology situation. Blind biography 5 generation like this. The virus of results is stored in-80 DEG C. To the 3rd generation chicken embryo present and typical pass a virus pathology, show as that chick embryo development is slow, idiosome rolls up dehydration etc. Measuring viral level to the 5th generation is 105.0EID50/0.1ml。
(2) viruses indentification
1, coagulation: 1～5 withholds the allantoic fluid obtained carries out hemagglutination test. All being there is not aggegation by each generation allantoic fluid of result in the chicken red blood cell of 1%, virus, without coagulation, is got rid of for orthomyxovirus, paramyxovirus, adenovirus etc. have the possibility of coagulation avian viral.
2, PCR qualification: the 5th generation allantoic fluid 200 μ l, TAKARA company RNA extracts reagent RNAisoPlus and extracts viral RNA, carries out RT-PCR with N gene primer.
Upstream 5 '-CGGAGCAATAGCAAGAAAAGC-3 '
Downstream: 5 '-GCAGCAACCCACACTATACCATC-3 '.
PCR primer has object band through 1.5% agarose gel electrophoresis, result at about 500bp, proves that isolated viral is IBV.
3, S1 gene sequencing and an analysis is passed
In 5th generation allantoic fluid 200 μ l, TAKARA company RNA extracts reagent RNAisoPlus and extracts viral RNA, amplification S1 gene, IBVS1 upstream: 5 '-AAGACTGAACAAAAGACCGACT-3 '; Downstream: 5 '-CAAAACCTGCCATAACTAACATA-3 '; Product about length 1700bp. PCR primer is through 1.5% agarose gel electrophoresis, and the assorted band of the clear nothing of object band, directly delivers to the order-checking of Hua Da genome company. The splicing of S1 gene order arranges rear total length 1620bp.
YX10 strain S1 gene order submits Genebank, AccessionNo:JX840411 to. With H120(EU822341), H52(AF352315), W93(AY842862), M41(DQ664534), 4/91(JN192154), LDT3-A(AY702975) the S1 gene of vaccine strain carries out multiple comparison, homology lower (all lower than 85%), is respectively 77.9%, 78.3%, 77.5%, 77.8%, 78.6%, 84.8%. The spike protein of S1 genes encoding is positioned at the surface of virus; induction body produces neutralizing antibody; closely related with the protection of body reaction; S1 gene is the gene the most easily morphed in virus evolutionary process, and its sequence differs by more than 5% and just may can not produce or can not produce cross protection effect completely.
4, morphological observation
YX10D90 strain virus allantoic fluid can see the crown poison that diameter is about 80～120nm after centrifugal, phospho-wolframic acid negative staining under Electronic Speculum, and virus particle is polymorphism, and majority is circular, has capsule film, and surface has in loose, evenly distributed coronal process. There is coronavirus representative configuration.
5, animal returns test
Age on the 7th, SPF chicken 20, dripped nose eye droppings approach inoculation the 5th generation virus, and 100 μ l are every only, and chicken group starts to occur facing to examine symptom attacking poison for latter 2 days, within 3 days, starts have chicken dead, and sickness rate is 100%(10/10), mortality ratio is 50%(5/10). Morbidity chicken shows slight respiratory symptom, and part chicken lassitude, has loose bowels, and drinking-water increases, and food consumption declines. The chicken that dies of illness extremely becomes thin, and the chicken that dies of illness all has slight tracheae mucus, hemorrhage, has airsacculitis, the obvious enlargement of kidney, in piebald, has a large amount of white urate deposition. After the 14th day, clinical symptom engenders improvement, attacks poison latter 20 days, substantially recovers normal. Prop up virus infection by the biography being separated to easily to feel chick and can copy clinical case.
6, serum neutralization test
Prepared by YX10 strain positive serum: age on the 14th, SPF chicken 20 was raised in negative pressure isolator, drip nose eye droppings approach inoculation YX10 strain the 5th generation virus, dosage 0.1ml/ is only, dead chicken neck subcutaneous injection YX10 strain oil-emulsion inactivated vaccine 0.3ml will not be had after 10 days, neck subcutaneous injection YX10 strain oil-emulsion inactivated vaccine Culling heart blood after 0.5m1,10 days again after 14 days, aseptic separation of serum, adding mycillin by every milliliter of 2000 units, packing is for subsequent use.
Test with the cross neutralization of class disease positive serum: by YX10 strain the 5th generation poison dilution 1000 times, respectively with ND, IBD, ITL, IB(M41 of equivalent) after positive serum (purchased from veterinary medicament supervision institute of China) and YX10 positive serum mix, 60min is made 37 DEG C of senses, each inoculation SPF chicken embryo age on the 10th 5 pieces, 0.2mL in every embryo allantoic cavity. Virus control group is YX10 strain 10-4Dilution poison mixes with equivalent salt solution. 37 DEG C of hatchings, observe 6d, the results are shown in Table 1.
With the neutralization test of IB positive serum: YX10 strain is diluted to every 0.1ml containing 100 EID with physiological saline50; IBVM41 strain, 4/91 strain, LDT3-A strain and YX10 strain positive serum, do 16 times of dilutions respectively, the virus of dilution and serum balanced mix, 60min is made in 37 DEG C of senses, inoculates SPF chicken embryo through allantoic cavity respectively, every part of serum 5 pieces, every embryo 0.2mL, 37 DEG C of hatching 168h, with the chicken embryo of death in 24～168h and in the embryo alive of 144 hours, its fetus has dehydration, rolls up, grows the specific lesions such as little, as the infection index whether with IB specific lesions.The results are shown in Table 2.
Result shows, the positive serum of ND, IBD, ILT can not neutralize IBVYX10 strain virus. IBV, YX10 strain can be neutralized completely by YX10 strain positive serum, and all chick embryo developments are normal, and IBVM41 strain, 4/91 strain, LDT3-A strain positive serum can not neutralize YX10 strain virus.
So far return the Pathogen Biology characteristic research such as experiment by the isolation identification of virus, PCR qualification, morphological observation, serum chicken embryo neutralization test, animal, obtain a strain class QX type avian infectious bronchitis virus, called after YX10 strain. YX10 complete genome sequence has checked order and has uploaded GenBank, AccessionNo:JX840411.
The cultivation of embodiment 2YX10D90 low virulent strain
1, poison strain chicken embryo continuous passage causes weak
Age on the 9th～11 SPF chicken embryo is used YX10 strain is carried out continuous passage to cause weak, every generation inoculates 10 pieces of chicken embryos by allantoic cavity, 0.1ml/ piece, hatch for 37 DEG C, abandon dead chicken embryo in 24 hours, 36 hours gather in the crops 5 pieces of embryos alive, and sterile collection blastochyle goes down to posterity next time, and all the other are left to 144 hours observes chicken embryo with or without biography pathology. Continuous passage like this is to the 120th generation. The certain generation in period interval carries out viral level, aseptic and detection of mycoplasma, exogenous virus detection and the safety testing of easily sense chick is weak to determine whether virulence causes.
2, viral level measures
The process that goes down to posterity measures the EID of D5, D25, D40, D58, D75, D90, D120 generation virus respectively50. Virus, with sterile saline 10 times of doubling dilutions, chooses 5 extent of dilution (10-4～10-8) inoculate respectively 5 pieces age on the 10th SPF chicken embryo, every embryo 0.2m1,37 DEG C was hatched to 144 hours, and in 24 hours, dead chicken embryo is disregarded, and record infected chicken embryo number, calculates EID by Reed-Muench method50. Measurement result is in table 3.
The poison strain YX10 poison that goes down to posterity can adapt to chicken embryo very soon, and YX10D5 generation poison valency is up to 106.3EID50/ 0.2ml, after going down to posterity, virus titer continues to raise, but amplitude is little, is up to YX10D75 for reaching 107.8EID50/ 0.2ml, major part all maintains 106.5EID50About/0.2ml.
3, aseptic and detection of mycoplasma
Respectively D5, D25, D58, D75, D90, D120 generation virus is carried out aseptic and mycoplasma inspection according to existing " People's Republic of China's veterinary drug allusion quotation " annex. Each generation detected result is feminine gender.
4, exogenous virus detection
Respectively D5, D25, D58, D75, D90, D120 generation virus is carried out exogenous virus detection according to existing " People's Republic of China's veterinary drug allusion quotation " annex. Each generation detected result is feminine gender.
5, to the safety testing of easily sense chick
Carrying out the safety testing of D5, D25, D58, D75, D90, D120 generation virus in continuous passage process respectively, age on the 1st, SPF chick 30 was divided into two groups at random, and test group is dripped nose eye droppings approach and inoculated each generation virus, every only dripped nose 105.5EID50/ 200 μ l, another group does not process in contrast. Negative pressure isolator is raised, freely drinks water and search for food. After inoculation, every day observes, the morbidity of record chicken group, death condition, cuts open the dead chicken of inspection in time. After 2 weeks, cuing open inspection survival chicken, the pathology situation of the organs such as record kidney tracheae, the results are shown in Table 4.
Along with virus progressively adapts to chicken embryo, the virulence of chicken is progressively being declined by it, chick sickness rate 100%, 100% after D5, D25, D58 inoculation, 75%(9/12), mortality ratio is respectively 50%(6/12), 25%(3/12), 16.7%(2/12), the existing decline of D75 virulence, only has 16.7%(2/12) show transient respiratory symptom, without dead.D90, D120 for chicken group after inoculation spirit search for food completely normal, No respiratory signs, without dead, cuts open inspection tracheae and kidney without pathology, is passaged to D90 and causes a disease property for virus.
6, the immunogenicity test of YX10D90
Immunogenicity determining is carried out to causing weak YX10D90 virus. Age on the 1st, SPF chick 30 was divided into 4 groups at random, and test group is with 106.0EID50/ only, 105.0EID50/ only, 104.0EID50/ three various dose drip nose eye droppings approach immunity YX10D90 virus, and only, the 4th group does not process 200 μ l/ in contrast. Negative pressure isolator is raised, freely drinks water and search for food. After inoculation every day observed and recorded chicken group situation, carry out attacking poison with same source strength poison YX10D5 after 2 weeks, drip nose eye droppings approach, every only 105.5EID50/ 200 μ l, attack morbidity, the death condition of poison observed and recorded chicken group rear every day, cut open the dead chicken of inspection in time. Observe 2 weeks, cut open inspection survival chicken, the pathology situation of the record organ such as kidney, tracheae. After immunity, experimental group and control group chicken group are all normal, attack the rear control group of poison the 3rd day chicken performance respiratory symptom, part chicken lassitude, draining just, sickness rate 100%, totally 5 death, survival chicken has the pathologies such as tracheorrhagia in various degree, tracheae have mucus, and kidney enlargement is pale. And immune group is all without morbidity, without dead; The results are shown in Table 5.
Immunizing dose >=10 of conclusion: YX10D904.0EID50/ time can reach satisfied protected effect, protection ratio 100%.
7, attenuated vaccine strain YX10D90 full genome order-checking
YX10D90 has completed full genome order-checking, and YX10D90S1 sequence is as shown in SEQIDNO:1, and YX10D903 '-UTR is as shown in SEQIDNO:3.
YX10 is through SPF chicken embryo continuous passage 120 times, and virus adapts to good on SPF chicken embryo, and after D40, poison valency is stabilized in 107.0EID50/ more than 0.2ml, to the 90th generation virus cause weak completely and remain with good immunogenicity, and bacterium, mycoplasma, other exogenous virus detected results are feminine gender, and YX10D90 can as biography attenuated vaccine original species poison.
Submit gained poison strain to preservation, the preservation name of described poison strain is called avian infectious bronchitis virus YX10D90 strain, the preservation time is on March 14th, 2016, deposit number is CCTCCNO:V201610, depositary institution is China typical culture collection center, preservation address is: China, Wuhan, Wuhan University.
The nucleotide sequence of the S1 gene of described poison strain is as shown in SEQIDNO:1, and the peptide sequence of its coding is as shown in SEQIDNO:2.
Prepared by embodiment 3YX10D90 attenuated vaccine
1, the preparation of IBV virus liquid
YX10D90 kind poison sterile saline embodiment 2 obtained dilutes 1000 times, allantoic cavity inoculate 100 pieces age on the 10th SPF chicken embryo, every embryo 0.2ml, sealing pin hole, puts 37 DEG C and continues to hatch, it is not necessary to egg-turning, inoculate in latter 24 hours according to egg 1 time, abandon dead embryo, later every 4 hours photograph eggs 1 time, the chicken embryo of death is taken out at any time, to 36 hours, no matter whole chicken embryo whether, is taken out by death, air chamber is upwards upright, is placed in 4 DEG C of coolings 12 hours. Being taken out by the chicken embryo of cooling, sterilization air chamber position, then with aseptic operation stripping except air chamber portion chorion, throw off shell membrane, break chorioallantoic membrane and amnion (not making yolk break), draw chicken blastochyle, sampling is checked respectively, is placed in-20 DEG C and saves backup.
2, the inspection of IBV venom
The virus liquid of results does steriling test and hemagglutination test (HA test) by existing " Chinese veterinary pharmacopoeia " annex method by bottle, and samples one part and measure viral level (EID50).The virus liquid asepsis growth of result results, should be negative to 1% chicken erythrocyte suspension agglutination test, and every 0.1ml viral level is 107.0EID50Above.
3, seedling and packing is joined
The virus liquid being up to the standards is filtered with 4 layers of sterilizing gauze, is mixed in same container, add 5% sucrose skimmed milk used as stabilizers of equivalent, add mycillin by every milliliter of 1000 units simultaneously, fully shake even, quantitative separating, make every plumage part viral level 104.0EID50, every bottle of 1000 plumage parts.
4, lyophilize, gland labeling, less than-15 DEG C preservations.
5, the safety testing of vaccine
With 10 times of immunizing dose immunity SPF chickens age on the 1st, the security of inspection vaccine. Vaccine sterile saline suitably dilutes immunity SPF chicken age on the 1st 10, and every nose 200 μ l(that only drips is containing 10 plumage part vaccines), establish 10 not immunized controls simultaneously, negative pressure isolator is raised, freely drink water and search for food, every day observed and recorded chicken health condition, observe 21 days. Result immune group and control group chicken are without morbidity, and weightening finish, without difference, shows that vaccine is safe.
6, the potency test of vaccine
Age on the 1st, SPF chicken 20 was only divided into 2 groups, and one group of immunity above-mentioned YX10D90 living vaccine prepared of one plumage part, another group does not process in contrast, and in negative pressure isolator, raising, freely drinks water and search for food. After inoculation every day observed and recorded chicken group situation, carry out attacking poison with strong poison YX10D5 after 2 weeks, drip nose eye droppings approach often 105.5EID50/ 0.2ml, attacks morbidity, the death condition of poison observed and recorded chicken group rear every day, cuts open the dead chicken of inspection in time. Observe 2 weeks, cut open inspection survival chicken, the pathology situation of the record organ such as kidney, tracheae.
After immunity, experimental group and control group chicken group are all normal, after attacking poison, control group the 2nd day existing chicken shows respiratory symptom, part chicken lassitude, draining is just, totally 5 death, survival chicken has the pathologies such as tracheorrhagia in various degree, tracheae have mucus, and kidney enlargement is pale, sickness rate 100%(10/10), mortality ratio 50%(5/10). And acomia the dying of illness of immune group is died, chicken health (Fig. 1) without exception, shows that the living vaccine with causing weak D90 virus preparation is safe and effective, it is possible to protection attacks poison with source strength poison.
SEQUENCELISTING
<110>Agricultural University Of South China
<120>a kind of chicken infectious bronchitis attenuated live vaccines YX10D90 strain
<130>
<160>7
<170>PatentInversion3.3
<210>1
<211>1620
<212>DNA
<213>nucleotide sequence of the S1 gene of YX10D90
<400>1
atgttggggaagtcactgtttttagtgaccattttgtgtgcactatgtagtgcaaatttg60
tttgattctgccaataattatgtgtactactaccaaagtgcctttaggcctccaaatgga120
tggcatctgcaagggggtgcttatgcagtagtgaattctactaattatactaataatgcc180
ggttctgcaagtgggtgcactgttggtgttattaaggacgtctataatcaaagtgcggct240
tccatagctatgacagcacctcctcagggtatggcttggtctaagtcacaattttgtagt300
gcacactgtaacttttctgaaattacagtttttgtcacacattgttatagtagtggtgca360
ggttcttgtcctataacaggcatgattgcacgtgatcatattcgtatttctgcaatgaaa420
aatggttctttattttataatttaacagttagcgtatctaaataccctacgtttaaatct480
tttcaatgtgttaacaattccacatctgtttatctaaatggtgatcttgtttttacttct540
aacaaaactgctgatgttacgtcagcaggtgtgtattataaagcaggtggacccgtaaat600
tatagtgttatgaaagaatttaaggttcttgcttactttgttaatggtacagcacaagat660
gtaattttgtgcgacaattcccccaagggtttgctggcttgtcaatataatactggcaat720
ttttcagatggcttttatccttttactaatagtactttagttagggacaagttcattgtc780
tatcgtgaaagtagtgtcaatactactttgacgttaactaatttcacttttactaatgta840
agtactgcacagcctaatagtggtggtgttagtacttttcatttatatcaaacacaaaca900
gctcagagtggttattataattttaatttgtcatttctgagtcagtttgtgtataaggca960
agtgattttatgtatgggtcttaccaccctaggtgttcttttagaccagaaaccattaat1020
agtggtttatggtttaattccttgtcagtttctcttacttatggacccctacagggaggg1080
tgtaagcaatctgtttttaatggtaaggcaacgtgttgttatgcctactcttataatggc1140
ccaagggcatgtaaaggtgtttattcaggtgaattaagcaagacttttgaatgtggattg1200
ctggtttatgttactaagagtgatggctctcgtatacagactagaacggagcccttagta1260
ttaatgcaacacaattataataatattactttagataagtgtgttgactataatatatat1320
ggcagagtaggccaaggttttattactaatgtgactgattctgctgctaattttagttat1380
ttagcagatggtgggttagctattttagatacttcgggtgccatagatgtctttgttgta1440
cagggcagctatggtcttaattattacaaggtcaatccttgtgaagatgttaaccaacag1500
tttgtagtgtctggtggcaatatagttggcattcttacttctagaaatgaaacaggttct1560
gaacaggttgagaaccagttttatgttaagttaaccaatagctcacatcgtcgcaggcgt1620
<210>2
<211>540
<212>PRT
<213>aminoacid sequence of the S1 genes encoding of YX10D90
<400>2
MetLeuGlyLysSerLeuPheLeuValThrIleLeuCysAlaLeuCys
151015
SerAlaAsnLeuPheAspSerAlaAsnAsnTyrValTyrTyrTyrGln
202530
SerAlaPheArgProProAsnGlyTrpHisLeuGlnGlyGlyAlaTyr
354045
AlaValValAsnSerThrAsnTyrThrAsnAsnAlaGlySerAlaSer
505560
GlyCysThrValGlyValIleLysAspValTyrAsnGlnSerAlaAla
65707580
SerIleAlaMetThrAlaProProGlnGlyMetAlaTrpSerLysSer
859095
GlnPheCysSerAlaHisCysAsnPheSerGluIleThrValPheVal
100105110
ThrHisCysTyrSerSerGlyAlaGlySerCysProIleThrGlyMet
115120125
IleAlaArgAspHisIleArgIleSerAlaMetLysAsnGlySerLeu
130135140
PheTyrAsnLeuThrValSerValSerLysTyrProThrPheLysSer
145150155160
PheGlnCysValAsnAsnSerThrSerValTyrLeuAsnGlyAspLeu
165170175
ValPheThrSerAsnLysThrAlaAspValThrSerAlaGlyValTyr
180185190
TyrLysAlaGlyGlyProValAsnTyrSerValMetLysGluPheLys
195200205
ValLeuAlaTyrPheValAsnGlyThrAlaGlnAspValIleLeuCys
210215220
AspAsnSerProLysGlyLeuLeuAlaCysGlnTyrAsnThrGlyAsn
225230235240
PheSerAspGlyPheTyrProPheThrAsnSerThrLeuValArgAsp
245250255
LysPheIleValTyrArgGluSerSerValAsnThrThrLeuThrLeu
260265270
ThrAsnPheThrPheThrAsnValSerThrAlaGlnProAsnSerGly
275280285
GlyValSerThrPheHisLeuTyrGlnThrGlnThrAlaGlnSerGly
290295300
TyrTyrAsnPheAsnLeuSerPheLeuSerGlnPheValTyrLysAla
305310315320
SerAspPheMetTyrGlySerTyrHisProArgCysSerPheArgPro
325330335
GluThrIleAsnSerGlyLeuTrpPheAsnSerLeuSerValSerLeu
340345350
ThrTyrGlyProLeuGlnGlyGlyCysLysGlnSerValPheAsnGly
355360365
LysAlaThrCysCysTyrAlaTyrSerTyrAsnGlyProArgAlaCys
370375380
LysGlyValTyrSerGlyGluLeuSerLysThrPheGluCysGlyLeu
385390395400
LeuValTyrValThrLysSerAspGlySerArgIleGlnThrArgThr
405410415
GluProLeuValLeuMetGlnHisAsnTyrAsnAsnIleThrLeuAsp
420425430
LysCysValAspTyrAsnIleTyrGlyArgValGlyGlnGlyPheIle
435440445
ThrAsnValThrAspSerAlaAlaAsnPheSerTyrLeuAlaAspGly
450455460
GlyLeuAlaIleLeuAspThrSerGlyAlaIleAspValPheValVal
465470475480
GlnGlySerTyrGlyLeuAsnTyrTyrLysValAsnProCysGluAsp
485490495
ValAsnGlnGlnPheValValSerGlyGlyAsnIleValGlyIleLeu
500505510
ThrSerArgAsnGluThrGlySerGluGlnValGluAsnGlnPheTyr
515520525
ValLysLeuThrAsnSerSerHisArgArgArgArg
530535540
<210>3
<211>391
<212>DNA
<213>YX10D903'-UTR sequence
<400>3
cataattgttgattgcagtttgctttctttctgtgcttattgtttttattctgctttatt60
ctttgttatatcgtagaagtttattaaaggttaaggaagataggcatgtagcttgattac120
ctacatgtctatcgccagggaaatgtctaatctgtctacttagtagcctggaaacgaacg180
gtagacccttagattttaatttagtttaatttttagtttagtttaagttagtttagagta240
ggtataaagatgccagtgccggggccacgcggagtacgatcgagggtacagcactaggac300
gcccactaggggaagagctaaattttagtttaagttaagtttaattgtctaagtatagtt360
aaaatttataggctagtatagagttagagca391
<210>4
<211>21
<212>DNA
<213>N upstream region of gene primer
<400>4
cggagcaatagcaagaaaagc21
<210>5
<211>23
<212>DNA
<213>N gene downstream primer
<400>5
gcagcaacccacactataccatc23
<210>6
<211>22
<212>DNA
<213>amplification passes the upstream primer of a S1 gene
<400>6
aagactgaacaaaagaccgact22
<210>7
<211>23
<212>DNA
<213>amplification passes the downstream primer of a S1 gene
<400>7
caaaacctgccataactaacata23
Claims (7)
1. a chicken infectious bronchitis attenuated live vaccines YX10D90 strain, it is characterised in that, it is preserved in China typical culture collection center March 14 in 2016, deposit number is CCTCCNO:V201610.
2. a peptide species, it is characterised in that, its aminoacid sequence is as shown in SEQIDNO:2.
3. encode the nucleotide sequence of polypeptide described in claim 2, it is characterised in that, described sequence is as shown in SEQIDNO:1.
4. chicken infectious bronchitis attenuated live vaccines YX10D90 strain according to claim 1, it is characterised in that, the peptide sequence of the genes encoding of described vaccine YX10D90 strain is as shown in SEQIDNO:2.
5. chicken infectious bronchitis attenuated live vaccines YX10D90 strain according to claim 4, it is characterized in that, the nucleotides sequence of the gene of described vaccine YX10D90 strain is classified as shown in SEQIDNO:1, or through replacing, lack and/or add the gene order of the equal function of albumen described in one or more Nucleotide and coding SEQIDNO:2 in sequence basis described in SEQIDNO:1.
6. the application in the medicine of chicken infectious bronchitis is treated in chicken infectious bronchitis attenuated live vaccines YX10D90 strain described in claim 1 or 4 or 5 in preparation.
7. prevent and treat the vaccine of chicken infectious bronchitis for one kind, it is characterised in that, the lyophilized vaccine comprising chicken infectious bronchitis attenuated live vaccines YX10D90 strain described in claim 1 or 4 or 5 and pharmaceutically can accepting.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610155509.2A CN105671003A (en) | 2016-03-18 | 2016-03-18 | Infectious bronchitis low-virulent live vaccine YX10 D90 strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610155509.2A CN105671003A (en) | 2016-03-18 | 2016-03-18 | Infectious bronchitis low-virulent live vaccine YX10 D90 strain |
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| Publication Number | Publication Date |
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| CN105671003A true CN105671003A (en) | 2016-06-15 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610155509.2A Pending CN105671003A (en) | 2016-03-18 | 2016-03-18 | Infectious bronchitis low-virulent live vaccine YX10 D90 strain |
Country Status (1)
| Country | Link |
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| CN (1) | CN105671003A (en) |
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| CN106754746A (en) * | 2016-11-30 | 2017-05-31 | 中山大学 | Avian infectious bronchitis attenuated vaccine strain, vaccine and its application |
| CN107893057A (en) * | 2017-12-27 | 2018-04-10 | 北京华都诗华生物制品有限公司 | Chicken infectivity bronchitis virus attenuated strain and its construction method and application |
| CN108066757A (en) * | 2017-12-27 | 2018-05-25 | 北京华都诗华生物制品有限公司 | Infectious bronchitis of chicken live vaccine and preparation method thereof |
| 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 |
| CN108330109A (en) * | 2017-12-28 | 2018-07-27 | 华南农业大学 | GDt29 plants of avian encephalomyelitis attenuated live vaccines |
| CN110358740A (en) * | 2019-08-12 | 2019-10-22 | 江苏农牧科技职业学院 | A kind of QX type avian infectious bronchitis virus strain and its application |
| CN110885795A (en) * | 2019-12-09 | 2020-03-17 | 江苏省家禽科学研究所 | Infectious bronchitis virus IBYZ and application thereof |
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| CN108066757A (en) * | 2017-12-27 | 2018-05-25 | 北京华都诗华生物制品有限公司 | Infectious bronchitis of chicken live vaccine and preparation method thereof |
| 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 |
| CN108330109A (en) * | 2017-12-28 | 2018-07-27 | 华南农业大学 | GDt29 plants of avian encephalomyelitis attenuated live vaccines |
| CN110358740A (en) * | 2019-08-12 | 2019-10-22 | 江苏农牧科技职业学院 | A kind of QX type avian infectious bronchitis virus strain and its application |
| CN110885795A (en) * | 2019-12-09 | 2020-03-17 | 江苏省家禽科学研究所 | Infectious bronchitis virus IBYZ and application thereof |
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