CN104928260A - Infectious bovine rhinotracheitis virus IBRV-JN03 isolate and application thereof - Google Patents
Infectious bovine rhinotracheitis virus IBRV-JN03 isolate and application thereof Download PDFInfo
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Abstract
The invention discloses an infectious bovine rhinotracheitis virus IBRV-JN03 isolate and application thereof. The preservation number of the infectious bovine rhinotracheitis virus is CGMCC No.10396 and the infectious bovine rhinotracheitis virus is preserved in the China General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms, and has relatively high virus titer and good immunogenicity; after calves are immunized by an aluminium hydroxide adjuvant inactivated vaccine prepared through the infectious bovine rhinotracheitis virus, relatively high antibodies are generated, so that the infectious bovine rhinotracheitis virus IBRV-JN03 isolate is a good infectious bovine rhinotracheitis vaccine candidate virus strain. Therefore, an infectious bovine rhinotracheitis diagnostic reagent, inactivated vaccine, attenuated vaccine and genetic engineering vaccine prepared on the basis of the virus can be used for diagnosis and vaccine prevention and control of the endemic infectious bovine rhinotracheitis and a wide market application prospect is realized.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of infectious bovine rhinotrachetis virus IBRV-JN03 strain isolated and application thereof.
Background technology
Infectious bovine rhinotrachetis virus (Infectious bovine rhinotracheitis virus, IBRV) infectious bovine rhinotrachetis caused, that the one of ox is acute, hot, contagious disease, clinically in multiple types of presentation, particularly this disease can cause immunosuppression, and secondary bacterial infection also causes more serious respiratory tract disease.At present, this disease is worldwide widely current, and this disease drastically influence international ox industry production trade, is classified as category-B transmissible disease by OIE (OIE).China detects IBRV in late 1970s from the milk cow of New Zealand's import, owing to there is no good immune protection measure, this virus infection is all had in the cows in many provinces of China subsequently, sickness rate is 10% ~ 90%, on the fattening of cows, give milk and breed impact greatly, causing huge financial loss to cattle farm.
The external prevention and control to this transmissible disease are based on vaccine immunization at present, wherein inactivated vaccine is because in use having good security, be used widely in the milk cows that breeding oxen, cow and economic worth are high, good control action kou is played to the popular of infectious bovine rhinotrachetis.But China there is no related vaccines product, and the Ministry of Agriculture not yet permits that external IBRV vaccine enters Chinese market, the more important thing is, along with the variation of IBRV, even if allow external vaccine import, its kind of poison is also poor to the protection of domestic epidemic strain.Therefore, in the urgent need to being separated epidemic strain, the IBRV Typical Representative strain that screening immunogenicity is strong, develops vaccine the generation preventing this disease, reduces the loss that it causes cattle-raising.For solving this difficult problem, we are separated the epidemic isolates of China's different zones, screen, obtain suitable vaccine candidate strain, make the prevention and control of infectious bovine rhinotrachetis have more specific aim and validity, in the applied research of infectious bovine rhinotrachetis inactivated vaccine, attenuated vaccine and live vector vaccine, there is important value.Meanwhile, the present invention is that the research and development of infectious bovine rhinotrachetis diagnostic reagent are laid a good foundation.
Summary of the invention
The invention provides strain infectious bovine rhinotrachetis virus (IBRV-JN03) strain isolated, this preservation is in China Committee for Culture Collection of Microorganisms's common micro-organisms center of No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is CGMCC No.10396, preservation date on February 2nd, 2015, Classification And Nomenclature: infectious bovine rhinotrachetis virus infectious bovine rhinotracheitis virus.The object of this invention is to provide a strain infectious bovine rhinotrachetis virus strain, the malicious valency of the strain provided is 8.75 × 10
8.5tCID
50/ mL, meets the requirement that production of vaccine antigen amount is high.
For achieving the above object, the present invention adopts following scheme:
One strain infectious bovine rhinotrachetis virus IBRV-JN03 strain isolated, it is characterized in that, be be separated from infectious bovine rhinotrachetis Prevalent district to obtain, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number CGMCC No.10396.
Preferably, comprise following nucleotide sequence, described nucleotide sequence is as shown in SEQ ID NO.3.
Present invention also offers a kind of separation method of infectious bovine rhinotrachetis virus IBRV-JN03 strain isolated, comprise the steps:
A. gather morbidity ox sample, after process, inoculation MDBK cell, is separated and obtains cytopathogenic effect strain;
B. with IBR-F and IBR-R for primer, with PCR method carry out detection amplification, amplify infectious bovine rhinotracheitis virus specific fragment in isolated strain.
C. isolated strain is added MDBK cell suspension, adopt Reed-Muench method to calculate the titre of isolated strain;
D. each isolated strain immunogenicity comparative analysis: prepare inactivation antigen, immune mouse and rabbit, produce higher antibody horizontal, filter out immunogenicity best, to obtain final product.
Above-mentioned infectious bovine rhinotrachetis virus IBRV-JN03 strain isolated is preparing the application in infectious bovine rhinotracheitis vaccine, and described infectious bovine rhinotracheitis vaccine is inactivated virus vaccine, subunit vaccine, attenuated live vaccine, recombinant vaccine or DNA vaccination.
The application of above-mentioned infectious bovine rhinotrachetis virus IBRV-JN03 strain isolated in preparation infectious bovine rhinotrachetis diagnostic reagent, is characterized in that: described infectious bovine rhinotrachetis virus detects relevant reagent to pathogenic pathogen antigen, antibody and nucleic acid etc.
In a second aspect of the present invention, the present invention proposes and a kind ofly identify and screen the method for IBRV, comprise the following steps: the separation andpreconcentration of (1) virus: adopt morbidity ox nasal secretion, ox diarrheic stools sample, morbidity cattle tissue (lung, liver, spleen, lymphoglandula) sample, after treatment, treatment solution is inoculated MDBK cell, be separated acquisition 6 strain cytopathogenic effect strain, called after IBRV-SD1, IBRV-JN03, IBRV-LY10707, IBRV-LY9038, IBRV-JNDN and IBRV-3029., detect by PCR method for primer with IBR-F and IBR-R (SEQ ID NO.1 and SEQ ID NO.2), institute's isolated strain all amplifies IBRV specific fragment.With IBR-gCF and IBR-gCR for primer, amplification gC gene (SEQ ID NO.4) also carries out phylogenetic trees phenotypic analysis, and result shows, above-mentioned institute isolated strain all belongs to BHV
1.1type.Isolated viral strain is respectively done 10 times of serial dilutions, 6 porocyte culture plates of inoculation MDBK cell monolayer, form plaque, carry out plaque purification in low melting-point agarose.Extract the RNA of cell culture and virus respectively, after reverse transcription becomes cDNA, apply the RT-PCR detection method that this laboratory is set up, exogenous virus detection is carried out to bovine viral diarrhea virus (BVDV), bovine enteroviruses (BEV), bovine rota (BRV), bovine coronavirus (BcoV), is feminine gender.After institute's isolated viral strain negative staining, electron microscopic observation, in typical simplexvirus particle shape, has cyst membrane.Isolated strain is added MDBK cell suspension, adopt Reed-Muench method to calculate the TCID of isolated strain
50.Result shows, and the titre of 6 strain isolated strains is 7.35 × 10
5.5~ 8.75 × 10
8.5tCID
50/ mL.
(2) isolated strain immunogenicity comparative analysis: to institute's separation and purification and virus titer is 10
7.0~ 10
8.5tCID
50the 2 strain IBRV of/mL carry out immunogenicity comparative studies, at 37 DEG C, isolated strain through 0.2 ‰ formalin-inactivated process 36h, prepare aluminum hydroxide adjuvant vaccine, the inactivation of virus liquid of same dose respectively inoculates 10 mouse and 5 rabbits, taking periodic blood, application ELISA method measures the antibody horizontal that virus produces, and filters out the IBRV-JN03 strain that immunogenicity is best.
In third aspect present invention, the present invention proposes a kind of method of production of infectious bovine rhinotrachetis inactivated vaccine.The IBRV-JN03 virus of separation and purification is carried out mass propgation, and supernatant liquor adds 0.2 ‰ formaldehyde in 37 DEG C of deactivation 24h, carries out steriling test, then fully mixed with aluminum hydroxide adjuvant 4:1 by virus liquid, make inactivated vaccine after deactivation completes.
In addition, the present invention proposes the application of a strain IBRV in diagnosis and prevention infectious bovine rhinotrachetis.According to the embodiment of the present invention, the IBRV clinical separation strain filtered out has higher titre, and has good immunogenicity.Based on this virus, prepare diagnostic antigen, infectious bovine rhinotrachetis inactivated vaccine, live vector vaccine and attenuated vaccine, have very strong specific aim to ox diagnosis and immunity, there is wide market application foreground.
Accompanying drawing explanation
Fig. 1 .IBRV-JN03 virus strain causes MDBK cytopathy.Wherein, A: produce cytopathic MDBK cell; B: normal MDBK cell.
Fig. 2 isolated viral strain PCR detects.Wherein, 1:IBRV-SD1; 2:IBRV-JN03; 3:IBRV-LY10707; 4:IBRV-LY9038; 5:IBRV-JNDN; 6:IBRV-3029; 6: the virus of separation; 7: negative control; 8: positive control; M:DL2000DNA Marker, is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom.
Fig. 3 IBRV-JN03 virus strain specificity, pure property detect.1:BVDV positive control; 2:BVDV negative control; 3:IBRV-JN03 BVDV detects; 4:BcoV positive control; 5:BcoV negative control; 6:IBRV-JN03 BcoV detects; 7:BRV positive control; 8:BRV negative control; 9:IBRV-JN03 BRV detects; 10:BEV positive control; 11:BEV negative control; 12:IBRV-JN03 BEV detects; 13:IBRV-JN03 IBRV detects; 14:IBRV negative control.M:DL1000DNA Marker, is followed successively by 1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, 100bp from top to bottom.
Fig. 4 IBRV particle Electronic Speculum detects.Visible have cyst membrane, in typical simplexvirus particle shape.
Fig. 5 utilizes IBRV totivirus indirect ELISA method to the Analysis of Immunogenicity of virus.A: the immune antibody analysis of mouse; B: the immune antibody analysis of rabbit.
Fig. 6 utilizes IBRV gB competition stop band restrain method to carry out the detection of calf immune antibody.%IN (inhibiting rate) value is equal to or greater than 35 for positive.
The level determination of neutralizing antibody after the immunity of Fig. 7 calf.
Concrete embodiment
IBRV of the present invention obtains from screening and separating the various clinical sample infecting IBRV, and having higher virus titer and good immunogenicity, is the good infectious bovine rhinotracheitis vaccine candidate strain of a strain.Below in conjunction with specific embodiment, the present invention is described in detail.The method that the present invention applies can adopt method conventional in virus research field, and is not limited only to the concrete record of the embodiment of the present invention.Experimental technique used in following embodiment if no special instructions, is ordinary method.Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
In following embodiment materials more used and reagent as follows: 100mm culture dish, 96 well culture plates are Costar product; DMEM cell culture medium, foetal calf serum, trypsinase are that HyClone company produces; DNA Marker DL2000, MiniBEST Viral RNA/DNA Extraction Kit, LA Taq enzyme, dNTP, RNA enzyme inhibitors, is all purchased from precious biotechnology (Dalian) company limited; Toluylene red, aluminum hydroxide adjuvant available from Sigma.
The isolation identification of embodiment 1 infectious bovine rhinotrachetis virus
The separation of 1.1 infectious bovine rhinotrachetis viruses
Observe cows incidence, contriver gathers morbidity ox nasal secretion, ox diarrheic stools sample, morbidity cattle tissue (liver, spleen, the lymphoglandula) sample that clinical principium is diagnosed as infectious bovine rhinotrachetis.For ight soil and nose of an ox swab sample, dilute with PBS (100U/mL penicillin, 100g/mL Streptomycin sulphate) 1:5.The centrifugal 10min of 3000r/min, the supernatant liquor obtained obtains pathological material of disease treatment solution after 0.22 μm of membrane filtration is degerming.After the fragmentation of morbidity cattle tissue (liver, spleen, lymphoglandula) sample application tissue grinder instrument, multigelation 3 times, dilutes with PBS (100U/mL penicillin, 100g/mL Streptomycin sulphate) 1:3.The centrifugal 10min of 8000r/min, the supernatant liquor obtained obtains pathological material of disease treatment solution after 0.22 μm of membrane filtration is degerming.
Above-mentioned pathological material of disease treatment solution is inoculated in eugonic individual layer MDBK cell on 24 well culture plates, every hole inoculation 0.2mL, sets up Positive control wells and negative control hole, in 37 DEG C of incubators, 1h is made in sense, discard sense and make liquid, add maintenance medium (DMEM of 2% foetal calf serum), in 5%CO
2, 37 DEG C of cultivations, 24h observation of cell pathology, receives poison after cultivating 36h.Again inoculate MDBK cell after cell toxicant multigelation 3 times, so go down to posterity, until cell produces typical cytopathy.
After pathological material of disease treatment solution inoculation MDBK monolayer cell, after 36h there is not cytopathy in cell, virus blind passage is to 2nd generation, there is local cells pathology in nose of an ox swab sample (IBRV-JN03) and fecal sample (IBRV-SD1 and IBRV-JNDN), harvested cell cultivates poison, typical CP is produced after again inoculating MDBK cell 18h, main manifestations is cells infected generation graininess pathology, the contracting of cell circle, be gathered into grape cluster sample group, monolayer cell forms cavity, find that there is several nuclear giant cell sometimes, last major part or all cell come off (see Fig. 1) from bottle wall.Other does not occur that sample (IBRV-LY10707, IBRV-LY9038 and IBRV-3029) blind passage of pathology has all occurred cytopathy to the 3rd generation, and then growth conditions is good for the MDBK cell not connecing poison contrasted with it.
The qualification of 1.2 infectious bovine rhinotrachetis viruses
1.2.1 virus PCR detects
According to the IBRV (accession number AJ004801.1) delivered in GenBank gD gene conserved sequence and utilize Primer5.0 primer-design software to design 1 pair of primers designed, primer sequence is as follows:
Table 1 IBRV PCR detects primer
Amplified fragments size is 422bp, is synthesized, be diluted to 20 μm of ol/L ,-20 DEG C of preservations by Shanghai biotechnology company limited.
The MDBK cell virus nutrient solution getting the above-mentioned isolated strain of 200 μ L extracts sample as viral DNA, operates by MiniBEST Viral DNA Extraction Kit specification sheets.With the viral DNA extracted for template, with the detection primer pair IBR-F of design, IBR-R carries out pcr amplification respectively, and reaction system is as follows: DNA profiling 5 μ L, 10 × PCR buffer (Mg2+plus) 2.5 μ L, dNTP (2.5mM) 4 μ L, upstream primer (20 μm of ol/L) 0.75 μ L, downstream primer (20 μm of ol/L) 0.75 μ L, Taq enzyme 0.5 μ L, mend to cumulative volume 25 μ L with sterilizing deionized water, increase on control thermal cycler.PCR circulating reaction condition is: 94 DEG C of sex change 30s, 58 DEG C of annealing 45s, and 72 DEG C extend 30s, 35 circulations, and last 72 DEG C extend 10min.Amplified production detects through agarose gel electrophoresis, result shows all isolated strains and all increases and obtain the specific band (Fig. 2) that a size is 422bp, and serve marine life engineering company limited and carry out sequencing analysis, measurement result (SEQ ID NO.3) carries out homology analysis with the domestic and international IBRV gene delivered on GenBank, and sequencing result is 98% ~ 100% with the sequence homology of (the Bovine herpesvirus type 1.1 isolate NVSL challenge 97-11) that announced.
1.2.2 viral major function gB gene, gC gene, gD gene, gE gene sequencing
According to the gB gene of the IBRV (accession number AJ004801.1) delivered in GenBank, gC gene, gD gene and gE gene, devise 4 pairs of Auele Specific Primers (see table 2).Extract the genome of all separation IBRV strain cell cultures poison, carry out pcr amplification to the gB gene of its major function, gC gene, gD gene and gE gene, reaction system is as follows: cDNA template 5 μ L, 2 × GC buffer (Mg
2+plus) 25 μ L, dNTP (2.5mM) 8 μ L, upstream primer (20 μm of ol/L) 1 μ L, downstream primer (20 μm of ol/L) 1 μ L, LA Taq enzyme 0.5 μ L, mends to cumulative volume 50 μ L with sterilizing deionized water, increases on control thermal cycler.PCR circulating reaction condition is: 94 DEG C of sex change 30s, and respective annealing temperature is in table 2, and annealing 45s, 72 DEG C extend 45 ~ 90s, 35 circulations, and last 72 DEG C extend 10min.PEASY-Simple-T1 carrier cloning, sequencing analysis is inserted after the DNA fragmentation purifying obtained increasing.Sequential analysis shows that the nucleotide homology of other strain of IBRV that the gB gene of each isolated strain of IBRV, gC gene, gD gene, gE gene and GenBank log in is more than 95%, shows that IBRV strain there are differences, but can protect the infection of epidemic isolates.Carry out phylogenetic trees phenotypic analysis with the gC gene of the IBRV-JN03 strain that increases (SEQ ID NO.4), result shows, isolated strain belongs to BHV
1.1type.
Table 2 is for the primer pair of IBRV gB, gC, gD and gE gene that increases
1.2.3 pure property detects
Get 200 μ L MDBK cell virus nutrient solutions and extract sample as viral RNA, operate by MiniBEST Viral RNA Extraction Kit specification sheets.The RNA sample extracted is according to TaKaRa pimeScript
tMrT-PCR Kit specification sheets carries out RT-PCR Reactive Synthesis cDNA.With the cDNA of synthesis for template, apply the RT-PCR detection method that this laboratory is set up, exogenous virus detection is carried out to bovine viral diarrhea virus (BVDV), bovine enteroviruses (BEV), bovine rota (BRV), bovine coronavirus (BcoV).
Primer and reaction conditions needed for table 3 RT-PCR
Reaction system is as follows: cDNA template 5 μ L, 10 × PCR buffer (Mg
2+plus) 2.5 μ L, dNTP (2.5mM) 4 μ L, upstream primer (20 μm of ol/L) 0.75 μ L, downstream primer (20 μm of ol/L) 0.75 μ L, Taq enzyme 0.5 μ L, mend to cumulative volume with sterilizing deionized water to be 25 μ L, to increase on control thermal cycler.PCR circulating reaction condition is: 94 DEG C of sex change 30s, and each viral annealing temperature is in table 3, and annealing 45s, 72 DEG C extend 45s, 35 circulations, and last 72 DEG C extend 10min.Detect through RT-PCR, each isolated strain of IBRV detects BVDV, BcoV, BRV and BEV for negative through exogenous virus.
1.3 virus plaques purifying
Virus-culturing fluid is done 10 times of serial dilutions to 10
-6, inoculation grows up to 6 porocyte culture plates of good MDBK cell monolayer, every hole virus inoculation diluent 100 μ L, and each extent of dilution virus liquid inoculates 2 holes, puts in cell culture incubator and adsorbs 1h, and every 15min shake once makes viral distribu-tion even.Discard 6 orifice plate inner virus liquid after absorption, the 2 × DMEM cell culture fluid then got containing 4%FBS adds short mix in 2% low melting-point agarose of equivalent, and every hole adds 2mL, and room temperature is solidified rearmounted 37 DEG C and contained 5%CO
2cell culture incubator in continue cultivate.Occur after single plaque until suitable extent of dilution, every hole adds 500 μ L0.1% toluylene reds, selects typical plaque repeated cloning with aseptic rifle choicest, and inoculation individual layer MDBK cell continues to cultivate propagation.After all isolated viral strains are taken turns choose spot purifying through 6, on MDBK cell, all expanding pure culture for the last time receiving poison as planting poison.
The negative staining electron microscopic observation of 1.4 viruses
Draw respectively cytopathy venom 50 μ L be added drop-wise to be coated with Formvar film copper mesh on, absorption 3 ~ 5min, unnecessary viral suspension is sucked with filter paper, after the drying of Formvar film, drip 2% phosphoric acid tungsten solution carry out negative staining 30min, under room temperature after seasoning, observe under transmission electron microscope (PHILIPS).By showing single virus particle after all isolated viral strain negative staining of electron microscopic observation enlarged culturing, in typical simplexvirus particle shape (see Fig. 4), there is cyst membrane.The virus particle of other form is showed no in all isolated viral strain cell culture and virus liquid.
The titer determination of 1.5 viruses
MDBK cell suspension good for growth conditions is paved with 96 orifice plates, and after then cell virus nutrient solution being done to the dilution of continuous 10 times, add in 96 porocyte plates, each extent of dilution adds 8 holes, every hole 100 μ L, 37 DEG C of 5%CO
2cultivate sense in incubator and make 1h, discard virus liquid, every hole adds the DMEM nutrient solution of 2% bovine serum, 37 DEG C of 5%CO
2cultivate, 4 ~ 6d is observation of cell pathology situation day by day, carries out TCID by Reed-Muench computing method to all isolated strains
50measure, result is as shown in the table:
The isolation identification of table 4 virus
The immunogenic comparative analysis of embodiment 2 IBRV strain isolated
The breeding of 2.1 viruses
Get the MDBK cell that growth conditions is good, wash 2 times with PBS, virus titer is greater than 10
7.0tCID
50/ mL, and take turns IBRV strain isolated IBRV-JN03 and IBRV-LY9038 after plaque purification, by 100TCID through 6
50amount carry out virus inoculation respectively, add containing 2% (v/v) horse serum, the DMEM cell maintenance medium of pH value 7.0,37 DEG C of 5%CO
2cultivate, when cytopathy reaches 70% ~ 80%, results virus-culturing fluid, multigelation 2 ~ 3 times, 4 DEG C of high speed centrifugations, obtain supernatant liquor to obtain described IBRV antigen.
The comparative analysis of 2.2 viral immunogenic
Viral IBRV-JN03 and the IBRV-LY9038 supernatant liquor of above-mentioned amplification is respectively added 0.2 ‰ formaldehyde in 37 DEG C of deactivation 24h, carries out steriling test after deactivation completes, then virus liquid is fully mixed with aluminum hydroxide adjuvant 4:1, make inactivation antigen.By (IBRV-JN03 and IBRV-LY9038) aluminum hydroxide adjuvant inactivation of viruses liquid back intradermal injection immunity BALB/c mouse (every 0.3mL) of preparation, new zealand rabbit (every 1mL), the each immune BALB/c mouse 10 of every strain virus, new zealand rabbit 5, simultaneously immune in contrast with the DMEM aluminum hydroxide adjuvant of 5 BALB/c mouse and 3 same volumes of new zealand rabbit intradermal injection, booster immunization after 2 weeks, weekly through mouse tail and the blood sampling of new zealand rabbit ear, totally 5 times, the centrifugal 20min of 2000r/min, separation of serum, IBRV totivirus indirect ELISA method is utilized to detect mouse (Fig. 5 A respectively, OD value), rabbit (Fig. 5 B, OD value) immune IgG antibody level in serum.Preliminary immunity test result shows, IBRV-JN03 strain inactivated vaccine can produce antibody sooner than IBRV-LY9038 inactivated vaccine, and the antibody horizontal produced after booster immunization is higher.
The development of embodiment 3 IBRV inactivated vaccine
The breeding of 3.1 viruses
Get the MDBK monolayer cell that growth conditions is good, wash 2 times with PBS, by vaccine candidate strain (IBRV-JN03) cell culture fluid good for above-mentioned immunogenicity, by 100TCID
50virus quantity inoculate, add the DMEM cell maintenance medium 37 DEG C of 5%CO containing 2% (v/v) horse serum
2cultivate, when cytopathy is 70% ~ 80%, results virus-culturing fluid, multigelation 2 ~ 3 times, 4 DEG C of high speed centrifugations, obtain vial supernatant, and viral level mensuration is carried out in sampling, and titre is 6.75 × 10
8.5tCID
50/ mL, in the IBRV isolated strain reported, this virus strain has higher titre, more meets the requirement of production of vaccine to high resistance commercial weight.
The preparation of 3.2 viral inactivation vaccines
By vaccine candidate strain IBRV-JN03 cell culture supernatant good for above-mentioned immunogenicity, at 37 DEG C, utilize final concentration be 0.2 ‰ formaldehyde shake well is carried out to IBRV nutrient solution, inactivation treatment 36h.Respectively get 2 parts of appropriate inactivation of virus liquid to inoculate MDBK cell respectively and carry out deactivation inspection and through MDBK cell blind passage 2 generation, per in generation, all cultivates 36 ~ 72h, observation of cell pathology situation, establishes positive-virus to contrast and cell controls simultaneously.The test set of inoculation inactivation of viruses liquid and negative control group are without CPE, and typical IBRV pathology appears in positive controls, to test set carry out 2 generation blind passage, have not yet to see CPE, show that the deactivation of detected inactivation of viruses liquid is complete.Finally mixed with the volume ratio of aluminum hydroxide adjuvant according to 4:1 by the IBRV of deactivation, after stirring, fully mixing, makes deactivation vaccine, is sub-packed in clear vial, and observing vaccine is beige homogenous suspension, has a small amount of precipitation after leaving standstill at the bottom of bottle.
3.2 inactivated vaccine immunity calves
3.2.1 grouping is tested
3.2.2 vaccine immunity
Choose 3 ~ 6 monthly age IBRV antigen negatives, negative antibody healthy calf 128 (IBRV gB competes stop band restrain and detects %IN value not higher than 35).Before each inoculation 3 ~ 5 days, every day observed with or without clinical exception, and all animal blood takings, for antiviral antibody level detection.First the 1st group of experimental animal respectively musculi colli inoculation lot number is 0511IBRV inactivated vaccine (IBRV-JN03), 2mL/ head ox (5.4 × 10
8.5tCID
50/ mL); 2nd group is control group, and musculi colli inoculation is not containing the enchylema of virus.Within after vaccine inoculation 21 days, carry out two to exempt from, second batch inoculation lot number be 0928IBRV inactivated vaccine (IBRV-JN03), vaccination and dosage ditto described in.Different time blood sampling after vaccine inoculation, the immune antibody utilizing Spain Hai Bolai IBRV gB to compete stop band restrain method detection calf (Fig. 6, %IN value) is tired, and measures the neutralizing antibody level (Fig. 7) after calf immunity.The immunization experiment result of different animals shows, IBRV-JN03 virus strain can induce produce high-level antibody (immunogenicity is good) and the extended period long, can as the viral candidates strain of vaccine development.
By reference to the accompanying drawings the specific embodiment of the present invention is described although above-mentioned; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various amendment or distortion that creative work can make still within protection scope of the present invention.
Claims (5)
1. a strain infectious bovine rhinotrachetis virus IBRV-JN03 strain isolated, it is characterized in that, be be separated from infectious bovine rhinotrachetis Prevalent district to obtain, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number CGMCC No.10396.
2. infectious bovine rhinotrachetis virus IBRV-JN03 strain isolated according to claim 1, is characterized in that, comprise following nucleotide sequence, and described nucleotide sequence is as shown in SEQ ID NO.3.
3. the separation method of infectious bovine rhinotrachetis virus IBRV-JN03 strain isolated according to claim 1, is characterized in that, comprise the steps:
A. gather morbidity ox sample, after process, inoculation MDBK cell, is separated and obtains cytopathogenic effect strain;
B. with IBR-F and IBR-R for primer, with PCR method carry out detection amplification, amplify infectious bovine rhinotracheitis virus specific fragment in isolated strain;
C. isolated strain is added MDBK cell suspension, adopt Reed-Muench method to calculate the titre of isolated strain;
D. each isolated strain immunogenicity comparative analysis: prepare inactivation antigen, immune mouse and rabbit, produce higher antibody horizontal, filter out immunogenicity best, to obtain final product.
4. the infectious bovine rhinotrachetis virus IBRV-JN03 strain isolated described in claim 1 and 2 is preparing the application in infectious bovine rhinotracheitis vaccine, it is characterized in that, described infectious bovine rhinotracheitis vaccine is inactivated virus vaccine, subunit vaccine, attenuated live vaccine, recombinant vaccine or DNA vaccination.
5. the application of infectious bovine rhinotrachetis virus IBRV-JN03 strain isolated according to claim 1 and 2 in preparation infectious bovine rhinotrachetis diagnostic reagent, is characterized in that: described infectious bovine rhinotrachetis virus detects relevant reagent to pathogenic pathogen antigen, antibody and nucleic acid etc.
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| 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 |
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| CN113061581A (en) * | 2021-03-22 | 2021-07-02 | 华中农业大学 | Goat-derived bovine herpes virus type 1 strain and application thereof |
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| 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 |
| CN113061581A (en) * | 2021-03-22 | 2021-07-02 | 华中农业大学 | Goat-derived bovine herpes virus type 1 strain and application thereof |
| CN113278736A (en) * | 2021-05-28 | 2021-08-20 | 深圳市易瑞生物技术股份有限公司 | Reagent and method for qualitative detection of bovine herpes virus type I |
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