CN102337250A - Infectious bovine rhinotracheitis virus strain and preparation method and application thereof - Google Patents
Infectious bovine rhinotracheitis virus strain and preparation method and application thereof Download PDFInfo
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- CN102337250A CN102337250A CN2011101372991A CN201110137299A CN102337250A CN 102337250 A CN102337250 A CN 102337250A CN 2011101372991 A CN2011101372991 A CN 2011101372991A CN 201110137299 A CN201110137299 A CN 201110137299A CN 102337250 A CN102337250 A CN 102337250A
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Abstract
The invention discloses an infectious bovine rhinotracheitis virus strain and a preparation method and application thereof. A wild strain of infectious bovine rhinotracheitis virus is found. The preparation method comprises the following steps of: firstly, identifying a sample by a PCR (Polymerase Chain Reaction) and a fluorescence PCR; then adding bovine serum which is detected to be free of IBRV (Infectious Bovine Rhinotracheitis Virus) to MDBK (Madindarby Bovine Kidney) cells by means of a culture medium, cultivating for a certain period of time so as to grow single-layer cells; after inoculating the single-layer cells with the processed sample, observing the cytopathic effect; after taking a pathological cell sample and identifying the pathological cell to be positive by the PCR, measuring TCID50 (Tissue Culture Infective Dose) as 106.2, and carrying out the virus neutralization test, wherein the TCID is 27; then inoculating the sample into the single layer cells; and when 70 percent to 80 percent of cells undergo the cytopathic effect and storing the cells into a refrigerator at a temperature of -40 DEG C. The infectious bovine rhinotracheitis virus strain and the preparation method disclosed by the invention can be applied to preparation of an engineered vaccine for preventing the infectious bovine rhinotracheitis.
Description
Technical field
The present invention relates to a kind of infectious bovine rhinotracheitis virus, be specifically related to a kind of sick cell engineering vaccine of infectious bovine rhinotracheitis of preventing ox.
Background technology
The infectious bovine rhinotracheitis disease is a kind of important viral infectious of serious harm world cattle-raising.This sick main pathogen is an infectious bovine rhinotracheitis virus, and anti-system should disease mainly be with virus drugs and chemicals, but because of there being deficiencies such as susceptibility and drug residue, developing novel vaccine is the task of top priority.
Infectious bovine rhinotrachetis virus has all morphological specificitys of herpetoviridae member; The mature virion diameter that has cyst membrane is about 150nm～220nm; Virus is spherical in shape, and nucleocapsid is 20 bodies of three-dimensional symmetry, and 162 capsomeres are arranged on it; Contain the cyst membrane of lipid on every side for one deck, and contain a bigger distrand DNA molecule.
Infectious bovine rhinotrachetis (infectious bovine rhinotracheitis; IBR) be by I type BHV (bovine herpesvirus-1; BHV-1) more than one respiratory inflammations that cause ox are main contagious disease; Clinical manifestation is varied, is the principal character ox with rhinotracheitis, eye conjunctivitis, high heat, miscarriage, infectivity warts.There is the problem of latent infection in infectious bovine rhinotracheitis virus, the ox of recovering or infect after can be with poison for a long time, even lifelong band poison, but toxin expelling not.But, when condition was suitable, the virus of latent infection possibly be activated and the toxin expelling phenomenon occur, becomes the source of infection.The mechanism of latent infection is that because the viral genome fusion gets in the non-reproductive ability cell, particularly the neurocyte of peripheral nerve joint mainly is among the trigeminal nerve ganglion cell.Infectious bovine rhinotrachetis virus can hide in neurocyte for a long time in other words.Find so far that in the U.S. this disease has spread to all over the world from the fifties.The maximum characteristics of this virus are that tissue tropism is broad, except that frequent infringement respiratory system, can also encroach on reproductive system, neural system, eye conjunctiva and fetus.In addition, during natural infection, the difference of occurring degree is also very big, and the lightest is the inapparent infection of clinical symptom, or only shows body temperature and slightly raise; Weight person then encroaches on whole nasal cavity (nasal sinus), throat and tracheae, secondary acute upper respiratory tract inflammation.Animal and plant quarantine bureau of the People's Republic of China clearly stipulates at present: the ox of every import and export and goods thereof, must detect IBRV, so that control this sick generation and popular.The traditional method that detects IBRV is a serological method, like neutralization test, indirect hemagglutination test, EUSA and nucleic probe etc.These methods are the shortcoming of various degrees all, or detection sensitivity is not high, or time-consuming, effort, or false positive is arranged; And PCR is simple to operation and highly sensitive.
Summary of the invention
The problem that the present invention will solve provides infectious bovine rhinotrachetis virus strain and preparation method and application.
For solving the above problems, the present invention realizes through following technical scheme: infectious bovine rhinotrachetis virus strain and preparation method and application comprise:
A, extract morbidity nose of an ox solinocrine thing with aseptic cotton carrier and send the laboratory, chamber cotton swab sample;
B, being primer with gB-1 and gB-2, detecting amplification with PCR method, amplify specific fragment in the pathological material of disease, is primer with gB-F, gB-R, detects amplification with PCR and fluorescence PCR method, identifies infectious bovine rhinotracheitis virus, and detected result is all positive;
C, add the Ox blood serum that does not have IBRV through detection through substratum at the MDBK cell; Certain hour is cultivated in the back; Grow up to individual layer MDBK cell, the chamber cotton swab sample that detects with PCR and fluorescence PCR method is inoculated in individual layer MDBK cell, observation of cell pathology; When treating that viral pathology appears in the 70%-80% cell ,-40 ℃ of refrigerators of cell income are preserved.
Isolated infectious bovine rhinotrachetis virus strain, having registered on the books in DSMZ is numbered: CGMCC No.4655, name is called GD0109.
Adopt the Reed-Muench method, isolated strain is added the MDBK cell suspension, observe day by day, the TCID50 that calculates isolated strain tires, packing after the isolated strain freeze thawing 3 times, and the TCID50 of virus is 106.2.
With isolated strain with after IBRV mixes with reference to positive serum, the observation of cell pathology, IBRV is 27 with reference to positive serum to the NAT of isolated strain.
In application as the sick engineered vaccine of the infectious bovine rhinotracheitis of prevention ox.
Infectious bovine rhinotrachetis virus strain of the present invention and preparation method and application have following advantage and application prospect:
1, PCR detection method of the present invention is identified the simple operation easily of infectious bovine rhinotrachetis virus strain, and highly sensitive, accuracy is high.
2, the present invention can prevent the sick cell engineering vaccine of infectious bovine rhinotrachetis.
Embodiment
Observe the cows incidence and get the tentative diagnosis of disease appearance, get the clinical manifestation of disease ox and flow out slurry property, mucus or purulent secretion, fervescence for the eye nose; Spirit is depressed, appetite stimulator, and milk production reduces; A small amount of ox hydrostomia is arranged, cough, clinical principium is diagnosed as infectious bovine rhinotracheitis; With the aseptic cotton carrier nose of an ox solinocrine thing of taking to fall ill, send laboratory diagnosis.
Embodiment 1: utilize PCR to detect and identify infectious bovine rhinotracheitis virus; In the cotton swab of nose of an ox chamber, add 0.01mol/L PBS; Fully after the vibration flushing; Get washing fluid 200 μ L and extract DNA with Viral Nucleic AcidExtraction Kit II DNA extraction test kit, IBRV PCR method and IBRV fluorescence PCR method detect, and the PCR primer is:
gB-1：5’-TAC?GAC?TCG?TTC?GCG?CTC?TC-3’；
gB-2：5’-GGT?ACG?TCT?CCA?AGC?TGC?CC-3’；
The amplified fragments size is 478bp, adds 2 * Master Mix, 12.5 μ L (TAKARA Company products), sample nucleic acid 5 μ L, ddH2O to 25 μ L in the 25 μ LPCR reaction systems.Amplification condition is: 95 ℃ of preparatory sex change 5min, 95 ℃ of 1min, 65 ℃ of 45s, 72 ℃ of 45s, 10 circulations; 95 ℃ of 1min, 54 ℃ of 45s, 72 ℃ of 45s, 15 circulations; 95 ℃ of 1min, 60 ℃ of 45s, 72 ℃ of 45s, 10 circulations, last 72 ℃ are extended 10min.The PCR product with 1.6% agarose gel electrophoresis after observations, conclusion is to amplify specific fragment in the pathological material of disease.
Embodiment two: detect with fluorescent PCR and identify infectious bovine rhinotracheitis virus, fluorescence PCR primer is:
gB-F：5’-TGT?GGA?CCT?AAA?CCT?CAC?GGT?3’；
gB-R：5’-GTA?GTC?GAG?CAG?ACC?CGT?GTC-3’；
TaqMan probe: 5 '-FAM-AGG ACC GCG AGT TCT TGC CGC-TAMRA-3 '.
Adopt TaqMan fluorescent PCR kit (TAKARA Company products), add 2 * Master Mix, 12.5 μ L, sample nucleic acid 5 μ L, ddH2O to 25 μ L in the 25 μ L reaction systems.Amplification condition is: 95 ℃ of preparatory sex change 5min, and 95 ℃ of 15s, 60 ℃ of 45s (collection signal), amplification curve and Ct value are observed in 45 circulations.Adopt the TaqMan fluorescence RT-PCR test kit of BVDV to carry out the BVDV detection simultaneously; Ox blue tongue virus (BTV) shell type RT-PCR method detect; Bovine leukemia virus (BLV) shell type RT-PCR method detect, foot and mouth disease virus (FMDV) fluorescence RT-PCR method detect.Reaction system all adopts RT-PCR test kit or TaqMan fluorescence RT-PCR test kit; Add corresponding 2 * Master Mix, 12.5 μ L, sample nucleic acid 5 μ L, primer or probe, ddH2O to 25 μ L in the 25 μ L reaction systems, the reaction conditions that requires according to document increases and result's judgement.Nose of an ox chamber cotton swab sample detects with IBRV PCR and fluorescent PCR, and the PCR and the fluorescent PCR detected result of 3,9, No. 11 samples of morbidity ox are all positive, and all the other sample standard deviations are negative.All nose of an ox chamber cotton swab samples detect with shell type RT-PCR or the fluorescence RT-PCR of BTV, BLV, FMDV, and the result is all negative.
Embodiment three: virus is separated, and it is positive to get IBRV PCR and fluorescent PCR, after chamber cotton swab washing fluid mixes; Add two anti-penicillium mould 1000IU/mL, Streptomycin sulphate 1000 μ g/mL; To 37 ℃ of effect 2h, the centrifugal 10min of 2000g gets supernatant and is inoculated on 96 orifice plates that cover with individual layer MDBK cell; 100 μ L/ holes add 6 holes.37 ℃ of absorption 1h, the supernatant that inclines adds and keeps liquid (MEM that contains penicillium mould 200IU/mL, Streptomycin sulphate 200 μ g/mL and 2% calf serum); 37 ℃ of 5%CO2 cultivate; Observation of cell pathology every day (CPE) is received poison when treating that CPE appears in the 70%-80% cell, if when cell CPE do not occur; Blind passage after 5 days, isolate reached for the 5th generation.Begin to find the dead cell showed increased at the 48h in the 1st generation, to the 4th day, the cell death that comes off basically, and control cells well-grown; Reached for the 2nd generation, observe the cytopathy (CPE) that is similar to the ibr virus characteristic, show as the cell circle and contract at 48h; Be gathered into grape cluster appearance group, on monolayer, form the cavity, have to contain several nuclear giant cells on a small quantity; CPE appears in the 70-80% cell behind the 72h, receives poison.Sample reached for the 5th generation, and it is similar basically with characteristics of lesion and the 2nd generation that time of CPE appears in per generation; (4) PCR of isolate identifies: get the 1st, 3,5 generation isolate 200 μ L respectively and extract DNA with Viral Nucleic Acid Extraction Kit II DNA extraction test kit; PCR or fluorescence PCR method with IBRV, BTV, BLV, FMDV in 1.3 detect; 1st, 3,5 generation isolate carry out the pcr amplification of IBRV; Find that separation and Culture thing and ibr virus all can amplify and target gene fragment specific band of the same size with reference to strain, amplification is clear, does not have assorted band.1st, 3,5 generation isolate carry out the fluorescent PCR amplification of IBRV; Separation and Culture thing and ibr virus all can amplify shell type RT-PCR or the fluorescence RT-PCR detection of specificity curve the 1st, 3,5 generation isolate with BTV, BLV, FMDV with reference to strain, and the result is all negative.
Embodiment four, and: TCID50 measures, and adopts the Reed-Muench method, with the 5th generation isolate make continuous 10 times of doubling dilutions, add in the 96 porocyte plates, each extent of dilution adds 8 holes, 100 μ L/ holes add the MDBK cell suspension, 100 μ L/ holes; 37 ℃ of 5%CO2 cultivate, observe day by day, and the 72h result of determination, the TCID50 that calculates isolate tires.Packing after the 5th generation isolate freeze thawing 3 times, the TCID50 of virus is 106.2.
Embodiment five: virus neutralization tests, IBRV is made continuous 2 times of doubling dilutions with reference to positive serum, add equal-volume be diluted to 200TCID50/100 μ L the 5th generation isolate, behind 37 ℃ of effect 1h.Serum and virus mixture are added in the 96 porocyte plate holes, and each serum dilution is inoculated 8 holes, 100 μ L/ holes.Add MDBK cell suspension 100 μ L/ holes at last; 37 ℃ of 5%CO2 cultivate; Day by day the observation of cell pathology is carried out neutralization test with specific IBRV with reference to positive serum and isolated viral behind the 3d, and IBRV is 27 with reference to positive serum to the NAT of strain isolated; Is 27.5 to IBRV with reference to the NAT of strain, and strain isolated and IBRV differ less than a titre with reference to the corresponding antibody titers of strain.
Claims (5)
1. infectious bovine rhinotrachetis virus strain and preparation method and application is characterized in that may further comprise the steps:
A, extract morbidity nose of an ox solinocrine thing with aseptic cotton carrier and send the laboratory, chamber cotton swab sample;
B, be primer, detect amplification, amplify specific fragment in the pathological material of disease with PCR method with gB-1 and gB-2; With gB-F, gB-R is primer and probe; Detect amplification with PCR and fluorescence PCR method, identify infectious bovine rhinotracheitis virus, detected result is all positive;
C, add the Ox blood serum that does not have IBRV through detection through substratum at the MDBK cell; Certain hour is cultivated in the back; Grow up to individual layer MDBK cell, the chamber cotton swab sample that detects with PCR and fluorescence PCR method is inoculated in individual layer MDBK cell, observation of cell pathology; When treating that viral pathology appears in the 70%-80% cell ,-40 ℃ of refrigerators of cell income are preserved.
2. infectious bovine rhinotrachetis virus strain according to claim 1 and preparation method and application; It is characterized in that: isolated infectious bovine rhinotrachetis virus strain; Registered on the books in DSMZ and be numbered CGMCC No.4655, name is called GD0109.
3. infectious bovine rhinotrachetis virus strain according to claim 1 and preparation method and application; It is characterized in that: adopt the Reed-Muench method; Isolated strain is added the MDBK cell suspension, observe day by day, the TCID50 that calculates isolated strain tires; Packing after the isolated strain freeze thawing 3 times, the TCID50 of virus is 106.2.
4. infectious bovine rhinotrachetis virus strain according to claim 1 and preparation method and application; It is characterized in that: with isolated strain with after IBRV mixes with reference to positive serum; The observation of cell pathology, IBRV is 27 with reference to positive serum to the NAT of isolated strain.
5. infectious bovine rhinotrachetis virus strain according to claim 1 and preparation method and application is characterized in that: in the application as the sick engineered vaccine of the infectious bovine rhinotracheitis of prevention ox.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104928260A (en) * | 2015-06-01 | 2015-09-23 | 何洪彬 | Infectious bovine rhinotracheitis virus IBRV-JN03 isolate and application thereof |
| CN107299088A (en) * | 2017-07-10 | 2017-10-27 | 中国农业科学院北京畜牧兽医研究所 | B plants of infectious bovine rhinotrachetis virus IBRV and its application |
| CN108467902A (en) * | 2018-04-25 | 2018-08-31 | 江南大学 | A kind of method of quick detection Bovine Rhinotracheitis Virus |
| CN117070476A (en) * | 2023-10-13 | 2023-11-17 | 金宇保灵生物药品有限公司 | Bovine herpesvirus 4 strain and application thereof in preparation of inactivated vaccine |
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2011
- 2011-05-24 CN CN201110137299.1A patent/CN102337250B/en active Active
Non-Patent Citations (2)
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| 唐泰山等: "1株牛传染性鼻气管炎病毒的分离鉴定", 《动物医学进展》 * |
| 唐泰山等: "牛传染性鼻气管炎病毒实时荧光定量PCR检测方法的建立", 《动物医学进展》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104928260A (en) * | 2015-06-01 | 2015-09-23 | 何洪彬 | Infectious bovine rhinotracheitis virus IBRV-JN03 isolate and application thereof |
| CN104928260B (en) * | 2015-06-01 | 2018-10-23 | 山东师范大学 | A kind of infectious bovine rhinotrachetis virus IBRV-JN03 separation strains and its application |
| CN107299088A (en) * | 2017-07-10 | 2017-10-27 | 中国农业科学院北京畜牧兽医研究所 | B plants of infectious bovine rhinotrachetis virus IBRV and its application |
| CN107299088B (en) * | 2017-07-10 | 2020-11-03 | 中国农业科学院北京畜牧兽医研究所 | Infectious bovine rhinotracheitis virus IBRV-B strain and application thereof |
| CN108467902A (en) * | 2018-04-25 | 2018-08-31 | 江南大学 | A kind of method of quick detection Bovine Rhinotracheitis Virus |
| CN117070476A (en) * | 2023-10-13 | 2023-11-17 | 金宇保灵生物药品有限公司 | Bovine herpesvirus 4 strain and application thereof in preparation of inactivated vaccine |
| CN117070476B (en) * | 2023-10-13 | 2023-12-19 | 金宇保灵生物药品有限公司 | Bovine herpesvirus 4 strain and application thereof in preparation of inactivated vaccine |
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