CN101906404A - Avian infectious bronchitis virus (IBV) as well as culture method and application thereof - Google Patents
Avian infectious bronchitis virus (IBV) as well as culture method and application thereof Download PDFInfo
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Abstract
The invention relates to an avian infectious bronchitis virus (IBV) ZZ2004 as well as a separating culture method and application thereof. The avian IBV ZZ2004 has the microorganism preservation number of CGMCC No.3842. The separating culture method comprises the following steps of: collecting infectious attacked poultry kidney under the aseptic condition; after adding sterilizing PBS (Phosphate Buffer Saline) for repeatedly washing, grinding to emulsion; after adding sterilizing PBS diluent, separately charging into a freezing storage tube; repeatedly freezing and melting for many times, filtering and sterilizing; vaccinating filter liquid to an SPF (Specific Pathogen Free) chick embryo at ages of 10 days by an allantoic cavity; culturing at a temperature of 37 DEG C for 72 hours, collecting allantoic fluid and carrying out continuous blind pass for more than five generations; and taking chick embryo allantoic fluid which is negative by a hemagglutination test. Compared with coronaviruses of other poultries, the virus separating strain derived from a culturing farm with chick and duck mixed culture has high variation degree and wider infection spectrum on animals; and the discovery and research of the virus have significance for disclosing an IBV variation rule, distinguishing different serotypes and preparing a novel IBV vaccine and a medicament for preventing and treating avian infectious bronchitis.
Description
Technical field
The present invention relates to bioengineering field, be specifically related to a kind of duck-origin coronavirus (Avian infectiousbronchitis virus, IBV) ZZ2004 and isolation cultivation method thereof and application.
Background technology
Nineteen thirty-seven, coronavirus (Coronaviruses) is at first separated on one's body from chicken.Nineteen sixty-five, isolate the first strain people's coronavirus.Owing under electron microscope, can be observed tangible rod-shaped particle projection arranged on its adventitia, its form is looked look like European emperor's in Middle Ages imperial crown, so called after " coronavirus ".According to the serology characteristics of virus and the difference of nucleotide sequence, coronaviridae is divided into coronavirus and two genus of Torovirus at present.The representative strains of coronaviridae be avian infectious bronchitis virus (Avian infectious bronchitis virus, IBV).This virus is for having cyst membrane, being the spherical or oval-shaped virus particle of medium polymorphism, and there is rod-like protrusions on the surface.
Infectious bronchitis of fowl (IB) is the acute height contagious disease of a kind of common pilosity of causing of the avian infectious bronchitis virus (IBV) by coronaviridae, shows as multiple sick types such as breathing pattern, kidney type, visible peristalsis visible intestinal peristalsis, glandular stomach type.This disease can infect the chicken of all ages in days, causes growth retardation, death, weightening finish and the price of deed to reduce.IBV often causes multiplicity of infection, as newcastle disease (Newcastle Disease, ND), chronic respiratory tract disease (CRD) etc., but and the secondary bacteriosis, as colibacillosis, salmonellosis etc., thereby increased the weight of harm to the chicken group.According to " world poultry " data in 1999, the most serious with respiratory system disease in the global poultry diease, wherein IB is the principal disease of the most of area harm in world poultry husbandry.Since 1988, IB is popular in succession in the most of area of China, and the sickness rate in epidemic-stricken area can reach 100%, and mortality ratio can reach 10～30%.The IBV that has reported both at home and abroad has serotype more than 26 kinds, and is numerous because of serotype and new modification, lacks cross protection between the different serotypes, and new variant constantly occurs and be popular, is the reason that causes chicken group immuning failure.This threat that makes IB cause poultry husbandry continues to exist.And biological characteristics and the epidemic characteristic of grasping epidemic isolates are correctly to use the prerequisite of vaccine.
Summary of the invention
The technical problem to be solved in the present invention is to have isolated big, wider to the pattern of infection of animal the duck-origin coronavirus ZZ2004 of a kind of degree of variation, and has provided its isolation cultivation method and application.
For solving the problems of the technologies described above, the technical solution used in the present invention is:
Isolate a kind of duck-origin coronavirus (Avian infectious bronchitis virus) ZZ2004, its microbial preservation is numbered CGMCC NO.3842.
A kind of isolation cultivation method of above-mentioned duck-origin coronavirus may further comprise the steps:
(1) under aseptic condition, the kidney of the poultry of collection infection IBV intercurrent disease, segmental bronchus, the fabricius bursa etc. are organized internal organs, add a small amount of sterilization PBS damping fluid repetitive scrubbing 2～3 times, are ground to emulsus and add a small amount of sterilization PBS diluent again;
(2) divide the frozen pipe of packing into, multigelation 2～4 times, filtration sterilization;
(3) filtrate is inoculated in 9～11 age in days SPF chicken embryo numbers piece through allantoic cavity, cultivates 72h for 37 ℃, discard the dead germ in the 24h, aseptic condition down the results allantoic fluid and continuously blind passage be cultured to chicken embryo performance dysplasia more than 5 generations, be roll up embryo, idiosome is hemorrhage;
(4) the different generation chick embryo allantoic liquids of isolated strain are done the chicken red blood cell agglutination test, get the chick embryo allantoic liquid that pathology is obvious, hemagglutination test (HA test) is negative;
(4) the different generation chick embryo allantoic liquids of isolated strain are done the chicken red blood cell agglutination test, get the negative chick embryo allantoic liquid of the obvious hemagglutination test (HA test) of pathology;
(5) get negative allantoic fluid and do dilution in 1: 10, get in the SPF chicken embryo fine hair allantoic cavity that 0.2ml is inoculated in 9 ages in days with sterilization PBS, 37 ℃ of hatching 36～48h, put 4 ℃ of following 12h after the results chick embryo allantoic liquid get final product.
Also comprise enrichment step afterwards in above-mentioned steps (5): get centrifuge tube 3 pipes of the above-mentioned chick embryo allantoic liquid of 50ml, the centrifugal 20min of 5000rpm in sterilization; Abandon precipitation respectively, get supernatant in the centrifuge tube of another sterilization, the centrifugal 20min of 12000rpm; Abandon precipitation respectively, get the ultracentrifugation pipe that supernatant adds 50ml, insufficiently supply centrifugal 4 hours of 40000rpm with sterilization PBS; Abandon supernatant liquor, it is standby with 1ml sterilization PBS-20 ℃ of preservations in back that suspend to get precipitation.
The application of above-mentioned duck-origin coronavirus in preparation IBV vaccine.
Duck-origin coronavirus ZZ2004 can be used for preparing conventional vaccines such as inactivated vaccine, attenuated vaccine, live vector vaccine, also can its S1, M, eukaryon expression plasmid pIBVS1, the pIBVM of N gene, pIBVN, preparation IBV plasmid DNA vaccine, liposome/dna vaccination, chitosan/dna vaccination etc.
The application of above-mentioned duck-origin coronavirus in preparation control chicken infectious bronchitis medicine.
The present invention has actively useful effect:
Also not relevant before this tame duck infects the report of IBV, this virus (duck-origin coronavirus ZZ2004) strain isolated derives from the plant that chicken and duck is raised together with, this virus not only can cause large quantities of morbidities and the death of chicken, also can have a strong impact on growing of tame duck, causes the death of part duck simultaneously; This virus compare with coronavirus other bird, its degree of variation is big, pattern of infection to animal is wider, carry out research to this virus isolated strain, for disclosing the IBV variation law, distinguish different serotype, prepare the novel I BV vaccine and the medicine of control chicken infectious bronchitis, and then thoroughly prevent and treat IBV and have great significance.
Duck-origin coronavirus of the present invention (Avian infectious bronchitis virus, IBV) ZZ2004, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 29th, 2010, it abbreviates CGMCC as, the address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, the deposit number of this duck-origin coronavirus is CGMCC NO.3842.
Description of drawings
Fig. 1 inoculates 10 age in days SPF chicken embryos in the observed result of 18 ages in days for IBV ZZ2004 strain, a among the figure: connect malicious chicken embryo, b: contrast chicken embryo.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment, but protection content of the present invention is not limited to following specific embodiment.Test method among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment, if no special instructions, all available from routine biochemistry reagent shop.
Embodiment 1 viral separation and Culture
In May, 2004, it is the doubtful IB disease of cardinal symptom that Zhengzhou, Henan Province plant has taken place with diarrhoea and growth retardation, it is depressed that infected chicken mainly shows spirit, diarrhea, poor growth and resistibility descend, sickness rate is 100%, and mortality ratio reaches more than 10%, and chicken infected production performance significantly reduces.Show in the investigation, only locate one 1700 meat duck group apart about 150m, gave birth to the disease of similar symptom in first 10 days left and right sides of chicken morbidity precontract duck mass-sending, but do not cause mass mortality with hen house.Dead chicken pathology is mainly enteric hemorrhage, the attenuation of intestines wall, have have to burst swung, and intestinal villi comes off, and the enlargement of glandular stomach nipple, mucous membrane come off, muscular stomach that has and glandular stomach intersection have hemorrhage, muscular stomach has hemorrhage or routed swinging, and the kidney enlargement is " piebald kidney ", lungs do not have considerable change, atrophy of immune organ such as the fabricius bursa, thymus gland; Sick duck is cutd open inspection with the kidney enlargement, palely has that hemorrhage, leg flesh and liver are hemorrhage to be principal character concurrently.In the disease that same symptom has also taken place with geographic other chicken houses, though be not effectively controlled through the immunity epidemic situation of multiple IBV vaccine.Animal science institute of Henan Science and Technology College has carried out the separation and Culture research of this kind virus in the Preventive Veterinary Medicine laboratory immediately, under aseptic condition, gather the tissues such as kidney of morbidity duck, add a small amount of sterilization PBS damping fluid repetitive scrubbing 2～3 times, be ground to emulsus and add a small amount of sterilization PBS damping fluid again, divide the frozen pipe of packing into, multigelation 3 times (70 ℃/30 ℃), with 0.22 μ m disposable filter filtration sterilization, filtrate is inoculated 5 pieces of 10 age in days SPF chicken embryos through allantoic cavity, cultivate 72h (discarding the dead germ in the 24h) for 37 ℃, aseptic results allantoic fluid and continuous blind passage are more than 5 generations, to chicken embryo performance dysplasia, be and roll up embryo, idiosome is hemorrhage etc.The different generation chick embryo allantoic liquids of isolated strain are done the chicken red blood cell agglutination test.Getting the negative chick embryo allantoic liquid of the obvious hemagglutination test (HA test) of pathology does experiment with kind of a poison.The result is separated to a strain new virus, called after duck-origin coronavirus ZZ2004 strain in morbidity duck body.
Embodiment 2 viruses are identified
(1) chicken embryo medium lethal dose (ELD
50) measure
Above-mentioned gained seed poison (duck-origin coronavirus ZZ2004) is increased progressively dilution by 10 times, get 10
-3～10
-7The diluent inoculated into chick embryo, 5 piece of 9～11 age in days SPF chicken embryo of each extent of dilution inoculation, 37 ℃ of cultivations discard dead chicken embryo in the 24h, every day, observed and recorded dead germ number was observed to 18 embryo ages.Press the Reed-Muench method and calculate ELD
50(Reed and Muench, 1938).
(2) RT-PCR of viral ZZ2004 strain identifies
The design primer sequence is as follows: upstream primer 5 '-GACCGCTTGTCAAGCAAATT-3 ', downstream primer 5 '-TGAGTACTAAGAGTGCAATT-3 '.
The concentration method of ZZ2004 strain
Get the allantoic fluid that the ZZ2004 strain infects and do dilution in 1: 10 with sterilization PBS, get in the SPF chicken embryo fine hair allantoic cavity that 0.2ml is inoculated in 9 ages in days, 37 ℃ of hatching 36～48h gather in the crops chick embryo allantoic liquid after putting 4 ℃ of 12h.
Get centrifuge tube 3 pipes of the SPF chick embryo allantoic liquid of 50ml ZZ2004 strain infection respectively, the centrifugal 20min of 5000rpm in sterilization; Abandon precipitation respectively, get supernatant liquor in the centrifuge tube of another sterilization, the centrifugal 20min of 12000rpm; Abandon precipitation respectively, get the ultracentrifugation pipe that supernatant adds 50ml, insufficiently supply centrifugal 4 hours of 40000rpm with sterilization PBS; Abandon supernatant, it is standby with 1ml sterilization PBS-20 ℃ of preservations in back that suspend to get precipitation.
The extraction of the total RNA of ZZ2004 strain is extracted test kit (MiniBEST Viral RNA/DNAExtraction Kit Ver.3.0) with DNA/RNA and is extracted RNA, and reference reagent box specification sheets carries out.
The segmental RT-PCR amplification of ZZ2004 pnca gene
1. reverse transcription reaction
Preparation following mixed solution (table 1) is established blank simultaneously in 0.2ml Microtube pipe.
The mixed solution preparation of table 1 template ribonucleic acid/primer etc.
On the PCR instrument, carry out sex change, annealing reaction: 65 ℃ of 5min, 4 ℃ of preservations.
The centrifugal several seconds makes the mixed solution of template ribonucleic acid/primer etc. be gathered in the Microtube pipe end.
The following inverse transcription reaction liquid of preparation is as shown in table 2 in above-mentioned Microtube pipe.
Table 2 inverse transcription reaction liquid
On the PCR instrument, carry out reverse transcription reaction by following reaction conditions.
30 ℃ of 10min, 42 ℃ of 30min, 95 ℃ of 5min, 4 ℃ of preservations.
2. PCR reaction
In the Microtube pipe, prepare PCR reaction solution (table 3) by following composition, establish blank simultaneously
Table 3PCR reaction solution
Instantaneous centrifugal after, put on the PCR instrument and increase.Reaction conditions is: 94 ℃, and 5min; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, totally 35 circulations, in 72 ℃ of extension 10min, reaction product is connected with pMD-18-T at last.
Result: occur significantly rolling up embryo behind the ZZ2004 strain virus inoculation SPF chicken embryo, see Fig. 3, so called after duck-origin coronavirus ZZ2004 strain.
Poison valency measurement result ELD
50=10
-5.68/ 0.2ml
The physics and chemistry test of embodiment 3 viruses
(1) virus is to the sensitivity test of ether and chloroform
1. viral sensitivity test: get ZZ2004 virus 800ul in an aseptic ep pipe to 20% ether, add the 200ul ether, put in 4 ℃ of refrigerators and handle 18～24h, frequently fully concussion, after 4 ℃ of centrifugal 20min of 2500rpm, take off layer liquid and blow and beat repeatedly and make the ether volatilization clean.With its EID of stroke-physiological saline solution dilution metering
50, establish the virus control group simultaneously.Test-results sees Table 4.
2. viral to 0.5% chloroform sensitivity test: as to get CSISV virus 995ul in an aseptic ep pipe, add the 5ul chloroform, put in 4 ℃ of refrigerators and handle 20min, frequently fully concussion, after 4 ℃ of centrifugal 20min of following 2500rpm get supernatant liquid its EID of stroke-physiological saline solution dilution metering
50, establish the virus control group simultaneously.Test-results sees Table 4.
(2) virus is tested temperature sensitivity
Get 1ml CSISV virus in an aseptic ep pipe, put to feel in 56 ℃ of water-baths and make 30min, with its EID of stroke-physiological saline solution dilution metering
50, establish the virus control group simultaneously.
With stroke-physiological saline solution the CSISV viral dilution is become 100EID
50Be sub-packed in the ep pipe, put respectively to feel in 60 ℃, 70 ℃, 80 ℃, the 90 ℃ water-baths and make 30min, inoculate 10 piece of 10 age in days SPF chicken embryo respectively, 0.2ml/ piece, 37 ℃ of hatchings are cultivated, and shine egg 1 every day, write down dead embryo quantity, discard dead embryo in the 24h, until 18 ages in days.All open residue chicken embryo during 18 ages in days, observe the chicken embryo and downgrade quantity, establish simultaneously with each 10 pieces of dosage virus control group and physiological saline control groups.Test-results sees Table 5.
(3) virus is to the soda acid sensitivity test
1. viral acid labile test
After getting the centrifugal 20min of 10mLCSISV viral suspension 3000r/min, supernatant liquor equivalent is sub-packed in 7 small test tubes 2mL in each small test tube.With 1mol/L HCl will be wherein in 3 small test tubes the pH value of viral liquid transfer to 3.0,2.5 and 2.0 respectively, with 1mol/L HCl the pH value of same sterile saline is transferred to 3.0,2.5 and 2.0 respectively in contrast simultaneously.Put water-bath 2h in 37 ℃ of thermostat water baths, use 5.6%NaHCO again
3Solution transfers to the pH value about 7.2.With sample and contrast liquid gradient dilution to 10
-2, 10-
310
-7Inoculate 6 piece of 10 age in days SPF chicken embryo then respectively, 0.2mL/ piece.37 ℃ of hatchings discard dead embryo in the 24h, and every 24h shines egg 1 time, writes down dead embryo, all opens the chicken embryo when 18 ages in days, observe the chicken embryo and downgrade quantity.Establish the virus control group simultaneously, triplicate is with virus virulence (EID
50) whether deactivation is a judgement criteria for decline titre and virus.Test-results sees Table 6.
2. virus is to the alkali sensitivity test
After getting the centrifugal 20min of 10mL CSISV viral suspension 3000r/min, supernatant liquor equivalent is sub-packed in 7 small test tubes, 2mL in each small test tube, with 1mol/L NaOH will be wherein in 3 small test tubes the pH value of viral liquid transfer to 10.0,11.0 and 11.5.And the pH value of same sterile saline is transferred to 11 and 11.5 in contrast with 1mol/L NaOH.Put to feel in 37 ℃ of thermostat water baths and make 2h, with 1mol/L HCl solution the pH value is transferred to about 7.2 again.With sample and contrast liquid gradient dilution to 10
-2, 10
-310
-7Inoculate 6 piece of 10 age in days SPF chicken embryo then respectively, 0.2mL/ piece.37 ℃ of hatchings discard dead embryo in the 24h, and every 24h shines egg 1 time, writes down dead embryo, all opens the chicken embryo when 18 ages in days, observe the chicken embryo and downgrade quantity.Establish the virus control group simultaneously, triplicate reaches more than 99.9% as the standard of judging complete inactivation with inactivation ratio.Test-results sees Table 6.
Table 4 ZZ2004 physicochemical property result
Table 5 ZZ2004 is to the susceptibility of temperature
Annotate: infect embryo and comprise dead embryo and downgrade embryo
Table 6 ZZ2004 EID behind acid-alkali treatment
50Measurement result
Embodiment 4 IBV ZZ2004 strain virus are pathogenic to the SPF chicken
The SPF chicken of getting 2 ages in days is divided into two groups at random, 30 of test group (male and female half and half, I group) wherein, and 20 of control groups (male and female half and half, II group), the I group is with the inoculation of ZZ2004 strain chick embryo allantois poisons collunarium, dosage 10
6ELD
50/ 0.5ml/ only.The II group is done same the processing with sterile saline.Infect back routine observation chicken and mass-send sick and deadly situation, the morbidity chicken is observed its disease symptom, and deadly chicken is cutd open inspection and observes and write down organ lesion, analyzes the cause of death.Liver, spleen, lung, the fabricius bursa and the kidney etc. of collecting test group and control group chicken are used for histological observation and RT-PCR detection.Infect the back in 25d to experimental group and control group chicken by only weighing, test-results sees Table 7, table 8.
The incidence of SPF chicken after the table 7 artificial challenge ZZ2004 strain
The body weight situation of 25 age in days SPF chickens after the table 8 artificial challenge ZZ2004 strain
Annotate: variance rate (%)=[(control group mean body weight-experimental group mean body weight) ÷ control group mean body weight] * 100%
The amino acid sequence homology comparison of the specific proteins that embodiment 5 IBV ZZ2004 are main
The IBV particle contains 3 specific specificity albumen, i.e. nucleocapsid protein (N), membranin (M) and spike protein (S); S albumen is positioned at the virus particle surface, is made up of equimolar S1 and two subunits of S2.S albumen is the most important protective antigen of IBV, can stimulate body to produce neutralizing antibody, plays a role in the virus absorption onto cell process, plays a decisive role on serological classification.IBV ORF1a protein gene coding IBV Nonstructural Protein, its corresponding expressed products size is 440KD, according to the ribosomal frameshift rule that is prevalent in coronavirus, the fusion polypeptide of 1a～1b of a 1a and an about 741KD of 1b protein gene co expression is after intracellular protease is cracked into the albumen of corresponding function.
With DNAstar software 7 of IBV ZZ2004 strain M gene deduced amino acid and GenBank login are shown with reference to the strain comparative result: ZZ2004 strain M aminopeptidase gene acid derivation sequence be respectively 93.9～91.1% with reference to the strain homology; Its variation zone of ZZ2004 strain M albumen and classical strains Beaudette strain comparison mainly concentrates on 3～16aa, in two zones of 211～222aa, and the point mutation that other zones still exist part to be dispersed in; Compare with the SAIBK strain that homology is the highest, its variation section mainly concentrates on 3～17aa zone, and other zones also have some point mutation that are dispersed in.
Between 73.7%～85.9%, the aminoacid sequence of its cracking site is RRHRR, obviously is different from other with reference to strain (RRF/SRR) with reference to the homology of strain for the proteic aminoacid sequence of IBV ZZ2004 strain S1 and 6; Mainly concentrate on 3～26aa, 53～57aa, 62～81aa with its variation zone of classical strains Beaudette strain comparison, 88～136aa, 199～209aa, 270～294aa, 382～395aa, in the zones such as 487～538aa, still there is the more point mutation that is dispersed in other zones; Compare with the SAIBK strain that homology is the highest, its variation section mainly concentrates on 23～25aa, 62～82aa, 89～131aa, 175～202aa, zones such as 455～538aa, other zones also have some point mutation that are dispersed in, and the great majority variation is positioned at preceding 4 zones, and promptly in preceding 300 amino-acid residues, this meets the feature of IBV S1 genovariation.
IBV ZZ2004 strain 1a gene and IBV compare homology 85.6%～93.1% with reference to strain.Compare with classical strains Beaudette strain, its variation section mainly concentrates on 698～703nt, 1079～1100nt, 1220～1233nt, 1730～2254nt, 2900～2930nt, 2944～2948nt, 3231～3542nt, 3840～3865nt, 3970～3991nt, 4988～4996nt, 5370～5412nt, 5503～5520nt, zones such as 7325～7400nt, other zones still have the point mutation that much is dispersed in; Compare with the SAIBK strain that homology is the highest, its variation section mainly concentrates on 2888～2889nt, 2911～2942nt, 3280～3300nt, 7918～7919nt, 7930～7980nt, 9907～9927nt, zones such as 9941～9943nt, other zones also have some point mutation that are dispersed in.
With DNAstar software 7 of IBV ZZ2004 strain N gene deduced amino acid and GenBank login are shown with reference to the strain comparative result: ZZ2004 strain N aminopeptidase gene acid derivation sequence be respectively 98.5～90.1% with reference to the strain homology; Its variation zone of strain isolated N albumen and classical strains Beaudette strain comparison mainly concentrates on 5～8aa, 184～202aa, and 220～247aa, in the zones such as 322～336aa, the point mutation that other zones also exist part to be dispersed in.
Claims (5)
1. a duck-origin coronavirus (Avian infectious bronchitis virus) ZZ2004, its microbial preservation is numbered CGMCC NO.3842.
2. the isolation cultivation method of the described duck-origin coronavirus of claim 1 may further comprise the steps:
(1) under aseptic condition, gather the renal tissue 1g of the poultry that infects described IBV intercurrent disease, add 3～5ml sterilization PBS damping fluid repetitive scrubbing 2～3 times, be ground to emulsus and add 3～5ml sterilization PBS damping fluid again;
(2) divide the frozen pipe of packing into, multigelation 2～4 times, filtration sterilization;
(3) filtrate is inoculated in 9～11 age in days SPF chicken embryo numbers piece through allantoic cavity, cultivates 72h for 37 ℃, discard the dead germ in the 24h, aseptic condition down the results allantoic fluid and continuously blind passage be cultured to chicken embryo performance dysplasia more than 5 generations, be roll up embryo, idiosome is hemorrhage;
(4) the different generation chick embryo allantoic liquids of isolated strain are done the chicken red blood cell agglutination test, get the negative chick embryo allantoic liquid of the obvious hemagglutination test (HA test) of pathology;
(5) get negative allantoic fluid and do dilution in 1: 10, get in the SPF chicken embryo fine hair allantoic cavity that 0.2ml is inoculated in 9 ages in days with sterilization PBS, 37 ℃ of hatching 36～48h, put 4 ℃ of following 12h after the results chick embryo allantoic liquid get final product.
3. the isolation cultivation method of duck-origin coronavirus according to claim 2 is characterized in that, also comprises enrichment step afterwards in described step (5): get centrifuge tube 3 pipes of the above-mentioned chick embryo allantoic liquid of 50ml in sterilization, the centrifugal 20min of 5000rpm; Abandon precipitation respectively, get supernatant liquor in the centrifuge tube of another sterilization, the centrifugal 20min of 12000rpm; Abandon precipitation respectively, get the ultracentrifugation pipe that supernatant liquor adds 50ml, insufficiently supply centrifugal 4 hours of 40000rpm with sterilization PBS; Abandon supernatant liquor, it is standby with 1ml sterilization PBS-20 ℃ of preservations in back that suspend to get precipitation.
4. the application of the described duck-origin coronavirus of claim 1 in preparation control chicken infectious bronchitis vaccine.
5. the application of the described duck-origin coronavirus of claim 1 in preparation control chicken infectious bronchitis medicine.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1784422A (en) * | 2003-04-08 | 2006-06-07 | 科罗诺瓦蒂夫股份有限公司 | Severe acute respiratory syndrome (SARS) causing coronavirus |
| CN101100656A (en) * | 2006-07-03 | 2008-01-09 | 河南农业大学 | Nephrotropic avian infectious bronchitis HN99 strain virus |
-
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1784422A (en) * | 2003-04-08 | 2006-06-07 | 科罗诺瓦蒂夫股份有限公司 | Severe acute respiratory syndrome (SARS) causing coronavirus |
| CN101100656A (en) * | 2006-07-03 | 2008-01-09 | 河南农业大学 | Nephrotropic avian infectious bronchitis HN99 strain virus |
Non-Patent Citations (1)
| Title |
|---|
| 任涛等: "鸡传染性支气管炎病毒中国地方流行株的分离与鉴定", 《畜牧兽医学报》, vol. 33, no. 4, 31 December 2002 (2002-12-31), pages 362 - 365 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102221611A (en) * | 2011-04-25 | 2011-10-19 | 河南科技学院 | Duck-origin coronavirus sandwich ELISA kit and its application |
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