CN101181240A - Technique for concentrating chicken ND-IB-AI-EDS tetrad oil emulsion killed vaccine antigen - Google Patents
Technique for concentrating chicken ND-IB-AI-EDS tetrad oil emulsion killed vaccine antigen Download PDFInfo
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- CN101181240A CN101181240A CNA2006101294046A CN200610129404A CN101181240A CN 101181240 A CN101181240 A CN 101181240A CN A2006101294046 A CNA2006101294046 A CN A2006101294046A CN 200610129404 A CN200610129404 A CN 200610129404A CN 101181240 A CN101181240 A CN 101181240A
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Abstract
The invention provides a concentration technology of virus antigen during the preparation process of a chicken ND-IB-AI-EDS quadruple inactivated oil-emulsion vaccine. The main contents of the method are that: newcastle disease virus (NDV), infectious bronchitis virus (IBV) and H9 subtype avian influenza virus (AIV) are concentrated by four steps of rough filtration, fine filtration, ultrafiltration and material collection via an internal pressure hollow fiber ultrafilter. After the concentration, the three virus antigens are mixed with egg drop syndrome virus (EDSV) for the preparation of the chicken ND-IB-AI-EDS quadruple inactivated oil-emulsion vaccine.
Description
Technical field
This method is the concentration technique of virus antigen in the chicken ND-IB-AI-EDS tetrad oil emulsion inactivated vaccine preparation process.
Background technology
Development along with China's poultry husbandry, breed scale and cultivation density are increasing, because the breeding cycle of laying hen and kind chicken is longer, be subjected to the danger of disease infringement also big, force plant to inoculate different types of single Seedling for continually the chicken group, not only expend great amount of manpower and material resources, return the chicken group and cause serious stress, influence fertility performance.Bigeminy, three and multiple vaccines, inoculation once can prevent two kinds, three kinds even multiple disease, makes vaccination become easier and economical and practical, uses more and morely in production practices, is subjected to liking of numerous raisers.The production of multiple vaccines, whether abundance is the key of decision vaccine quality to unit volume endoantigen content, produces needed antigen amount and change the oil-water ratio in the vaccine emulsifying process merely and all can not reach multi-joint Seedling by change virus multiplication condition raising viral level.Therefore the antigen concentration technique of application of advanced improves the antigen concentration in the vaccine, guarantees antigenic content enough in the unit vaccine, reduces immunizing dose simultaneously as far as possible, is the key technology of being badly in need of solution during present multiple vaccines is produced.
The Hollow Fiber Ultrafiltration technology has characteristics such as unit volume intimal surface volume is big, power consumption is low, efficient is high, delay is low.Its operation principle is that stock solution is passed through pump, become under the turbulence state in hollow fiber ultrafiltration membrane or film outer surface distributes and flows at certain pressure, solvent or become ultrafiltrate through collection less than the dam material permeance film of molecular weight of film, then dammed greater than the dam material and the colloidal solid of molecular weight of film, as will turn back in the stock solution time, the concentration of its stock solution can raise gradually, is commonly called as concentrated solution, so repeatedly, thus realize the concentrated purpose of stock solution.The pressure type hollow fiber ultrafilter concentrator that present technique is used Tianjin University of Technology's membrane separation technique institute production concentrates virus antigen, is intended to solve the low problem of long-term chicken ND-IB-AI-EDS tetrad oil emulsion inactivated vaccine antigen amount.
Summary of the invention
1 goal of the invention the objective of the invention is to set up reliable, high efficiency technique for concentrating chicken ND-IB-AI-EDS tetrad oil emulsion killed vaccine antigen; concentration with four kinds of viruses in the tetrad Seedling of guaranteeing to produce all reaches the antigen amount, can protect the chicken group to avoid Avian pneumo-encephalitis virus (NDV), H9 type bird flu virus (AIV), infectious bronchitis virus (IBV), subtract the invasion and attack of egg drop syndrome virus (EDSV).
The concrete steps of 2 inventions
2.1 equipment: this method adopts the pressure type hollow fiber ultrafilter of being produced by Tianjin University of Technology's membrane separation technique institute, select the stainless steel machinery pump for use, adopt the polytetrafluoroethylene material of low noise, corrosion-resistant, pollution-free, easy sterilization, complete machine is without mechanical friction, no high vibration, easy cleaning.Interface is airtight, and it is closed circuit to circulate, and is convenient to the sterile working.This equipment is double-filtration, and one-level filter post can be held back gel-shaped materials such as particulate matter, fragment and albumen in the viral liquid, for cascade filtration is created a good filtration environment.Secondary filter post molecular cut off 40KD is the major part of this filtering device, viral liquid during by doughnut filter post moisture etc. filtered in cyclic process less than the material of molecular cut off, and virus is trapped, thereby reaches the spissated purpose of viral liquid.
2.2 enriched material: Avian pneumo-encephalitis virus (NDV), H9 type bird flu virus (AIV), the viral liquid of infectious bronchitis virus (IBV), after above three kinds of viral liquid are concentrated by ultrafiltration apparatus respectively, and subtract egg drop syndrome virus (EDSV) and be mixed in proportion the preparation that is used for vaccine.
2.3 viral liquid concentrates and the vaccine production flow process:
2.3.1 coarse filtration is poured viral liquid in the charging basket into, leaches tissue residual in the viral liquid or cell mass through 300 purpose copper mesh, effusive viral liquid is called coarse filtration liquid.
2.3.2 fine straining is under the pressure of 0.15MPa, coarse filtration liquid is pressed in the one-level filter post, under the pressure that continues, coarse filtration liquid flows in hollow fiber column, flow out one-level filter post (being called fine straining liquid) less than the dam viral liquid of molecular weight of film by hollow-fibre membrane, materials such as the protein of macromolecule are trapped within the post.
2.3.3 ultrafiltration is injected fine straining liquid in the secondary filter post under the pressure of 0.15MPa, under the pressure that continues, fine straining liquid flows at the hollow fiber column internal recycle, flows out by hollow-fibre membrane less than the dam solvent of molecular weight of film, and virion is trapped.Should not contain virion in the effusive solvent.Compare with the volume of virus stock solution used by the amount of calculating effusive solvent, can calculate the multiple that viral liquid is concentrated.
2.3.4 rewinding when viral liquid has reached cycles of concentration, stops ultrafiltration when by the volume calculated ratio.With viral concentrated solution remaining in the hollow fiber column, flow in the material collecting device from outlet.Whole viral concentration process finishes.
2.3.5 Avian pneumo-encephalitis virus (NDV), H9 type bird flu virus (AIV), the viral liquid of infectious bronchitis virus (IBV) after viral liquid deactivation concentrates and the formaldehyde that subtracts adding 0.2% in the egg drop syndrome virus (EDSV) carry out deactivation.
2.3.6 concentrated ND, IB, AI (H that vaccine production will be up to the standards through deactivation
9), the EDS virus antigen mixes by proper proportion, adds a small amount of tween 80, behind the preparation water, with the mixed of oil phase by 1: 3, adds final concentration and be 0.01% thimerosal, emulsifying is prepared into ND-IB-AI-EDS and concentrates the tetrad oil emulsion inactivated vaccine.
Description of drawings
What play a decisive role in Fig. 1 inner pressed hollow fiber microfiltration membrane component pattern figure ultra-filtration process is ultrafilter membrane.Hollow fiber ultrafiltration membrane be with macromolecular material adopt that the special process manufacturing forms non-to becoming semipermeable membrane, it is the doughnut capillary micropore tube wall that gathers.Stock solution flows in film or outside the film under pressure, wherein, solvent or small-molecule substance see through film becomes ultrafiltrate through collection, other macromolecular substances and colloidal solid are then stopped by face, be recycled mobile stock solution and take away and become concentrated solution, thus reach material separation, concentrate and the purpose of purification.
Fig. 2 hollow fiber membrane ultrafiltration device filter post instance graph
The specific embodiment
Example 1 fowl ND-IB-AI-EDS tetrad oil emulsion inactivated vaccine virus antigen concentrates and immune contrast test
1, materials and methods
1.1 viral liquid: NDV, IBV, AIV (H
9Hypotype), EDS live virus liquid and inactivation of viruses liquid provide by this chamber.
1.2 key instrument equipment: mesopore cellulosic ultrafiltration device, Tianjin University of Technology's membrane separation technique institute is produced; Colloid mill, the new light in Shenyang Machines Plant product; Homogenizer is available from East China, Shanghai homogenizer factory.
1.3 deactivation provirus antigen concentration test: use NDV, IBV, the AIV (H of mesopore cellulosic ultrafiltration device with results
9) concentrate 4 times, calculate the volume of concentration time and filtered water, and gather respectively concentrate before, concentrate back virus sample and isobaric flushing water, backwash water sample, use the micromethod working sample HA of 1% chicken red blood cell tired, calculate viral rejection.Earlier carry out pretreatment before the IBV sample determination with 1% trypsin.
1.4 virus antigen concentration test after the deactivation: carry out concentration test with the ND after the deactivation, IB, AI virus liquid, the concentrated forward and backward virus sample of mensuration is tired to the HA of 1% chicken red blood cell, calculates viral rejection.Earlier carry out pretreatment before the IBV sample determination with 1% trypsin.
1.5 vaccine production: concentrated ND, IB, the AI (H that will be up to the standards through deactivation
9) virus antigen mixes by proper proportion, adds a small amount of tween 80, behind the preparation water, with the mixed of oil phase by 1: 3, adds final concentration and be 0.01% thimerosal, emulsifying is prepared into ND-IB-AI and concentrates triple oil emulsion inactivated vaccine.Use unconcentrated ND, IB, AI (H9) virus antigen to prepare non-concentrated triple vaccine simultaneously by identical antigen volume ratio.
1.6 immune contrast test: the non-immunized chicks of 20 ages in days is divided into 2 groups, 10 every group, use the concentrated vaccine immunity for the 1st group, the 2nd group with non-concentrated vaccine immunity, every chicken inoculation 0.25ml.Preceding and immune back 1,4,8,12 week detection ND, IB, AI (H9) serum antibody HI tire with immunity respectively.
2, experimental result
2.1 thickening efficiency is measured: the application filter carries out 4 times to NDV, IBV, AIV respectively by design in advance and concentrates, and before gather concentrating respectively, concentrate back virus sample and filtered solution, isobaric flushing water, backwash water sample, working sample is tired to the HA of 1% chicken red blood cell, calculates viral rejection.The results are shown in Table 2-1, table 2-2, table 2-3, table 2-4.
Table 2-1: the HA before and after inactivation of viruses liquid concentrates tire (Log2)
Table 2-1 result shows that 3 kinds of viral liquid concentration tests all get a desired effect, and after viral liquid concentrated 4 times, its HA had improved two titres before tiring and concentrating.It is zero after testing that the HA of while filtered solution tires, and illustrates that this device has good preciseness, and the rejection of virus is 100%.
Table 2-2: the HA that concentrates back four flushing liquors tires
By table 2-2 as can be known, all can detect HA in the sample of twice isobaric flushing and tire, particularly the HA of isobaric for the first time flushing sample tires and approaches to concentrate provirus liquid.Therefore can be with the isobaric flushing twice of an amount of sterilized water after concentrating, flushing liquor is recycled, and improves the viral response rate; Backwash and anti-top are washed and are failed to detect HA in the sample and tire, and proves virus-free or few virus existence in backwash and the anti-top washing liquid, can discard after washing.
Table 2-3: the relation of viral cycles of concentration and its change in volume
Table 2-3 result show, the cycles of concentration of virus with concentrate before and after the change in volume of viral liquid and HA tire and change linearly linear relationship, can in actual production, determine cycles of concentration according to the proportionate relationship of relief liquor and stock solution volume or the variation that HA tires.
Table 2-4: concentrated effect relatively before and after the viral liquid deactivation
Table 2-4 result show, concentrates before inactivation of virus He after the deactivation, for not influence of concentrated effect, therefore, can select the opportunity that concentrates according to the characteristic of different virus and the production technology of preservation condition and vaccine in the vaccine development process.
2.2 different time serum HI antibody horizontal testing result sees Table 2-5 after the immune contrast test immunity.
Table 2-5 result show, after the concentrated vaccine immunity, it is faster than non-concentrated vaccine group that antibody produces speed; And the contemporaneity after immunity, concentrated vaccine immune group antibody horizontal shows that apparently higher than non-concentrated vaccine group the immune effect of concentrated vaccine is better than non-concentrated vaccine.Analyze according to its antibody downward trend, be not difficult to find out that this vaccine has longer immune duration.
Table 2-5: concentrated vaccine and non-concentrated vaccine immune effect are relatively
3, brief summary and discussion
3.1 by table 2-1, table 2-2 result as can be known, use mesopore cellulosic ultrafiltration device and can carry out effectively concentratedly to NDV, IBV and AIV, its viral rejection is 100%, by isobaric flushing liquor is recycled, in theory can 100% reclaim virus, therefore, this system has the very high viral response rate.In addition, this system concentrates 10000ml virus liquid required time and is no more than 20min, shows that this equipment has higher thickening efficiency, can be used for the concentration technology of extensive virus antigen.
3.2 immune comparative test result shows, compare with non-concentrated vaccine group, after the concentrated vaccine immunity, it is faster that body produces antibody speed, 4 weeks of immunity back are detected antibody, ND HI antibody horizontal rises to 10.log2 by the 8.0log2 before concentrating, and IB HI antibody horizontal rises to 8.5log2 by 6.5log2, all reaches more than the 2log2; AI HI antibody horizontal rises to 8.9log2 by 7.5log2, apparently higher than non-concentrated vaccine group, shows that the immune effect of concentrated vaccine is better than non-concentrated vaccine.Analyze according to its antibody downward trend, be not difficult to find out that this vaccine has longer immune duration.Therefore, the virus antigen concentration technique of research can be used for fully multiple vaccines produce in the concentrating of virus antigen, have good using value.
Claims (7)
1. the concentration technique of a virus antigen is used in chicken ND-IB-AI-EDS tetrad oil emulsion inactivated vaccine preparation process.
2. according to the described technique for concentrating chicken ND-IB-AI-EDS tetrad oil emulsion killed vaccine antigen of claim 1, it is characterized in that: the antigen that this method is used for Avian pneumo-encephalitis virus (NDV), infectious bronchitis virus (IBV), H9 subtype avian influenza virus (AIV) concentrates.
3. technique for concentrating chicken ND-IB-AI-EDS tetrad oil emulsion killed vaccine antigen according to claim 1 is characterized in that: comprised coarse filtration, fine straining, ultrafiltration, four steps of rewinding.
4. according to claims 3 described virus antigen concentration techniques, it is characterized in that: before virus antigen concentrated, having added the pre-filtering process was the coarse filtration program, selects 300 purpose copper mesh for use, filters out tissue residual in the viral liquid or cell mass.
5. according to claims 3 described virus antigen concentration techniques, it is characterized in that: in the fine straining process filtrate is pressed in the one-level filter post, filters out the materials such as protein of macromolecule residual in the viral liquid.
6. according to claims 3 described virus antigen concentration techniques, it is characterized in that: ultra-filtration process selects for use the dam hollow-fibre ultrafiltration device of molecular weight of 40KD to be used for concentrating of three kinds of virus compositions.Select for use the hollow-fibre membrane of this molecular cut off both can stop that the virion in the viral liquid saw through hollow-fibre membrane, again can be because of the viral liquid concentrating efficiency of the too small influence of molecular cut off.
7. virus antigen concentration technique according to claim 1, it is characterized in that: this technology also can be used for the preparation of Avian pneumo-encephalitis virus (NDV), infectious bronchitis virus (IBV), any two or more virus connection Seedlings of H9 subtype avian influenza virus (AIV) except that being used for the preparation of chicken ND-IB-AI-EDS tetrad oil emulsion inactivated vaccine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102302775A (en) * | 2011-07-06 | 2012-01-04 | 普莱柯生物工程股份有限公司 | Combined inactivated vaccine of Newcastle disease and H9 subtype avian influenza and preparation method thereof |
| CN103937754A (en) * | 2014-04-11 | 2014-07-23 | 天津瑞普生物技术股份有限公司 | Porcine reproductive and respiratory syndrome virus (PPRSV) purification method |
-
2006
- 2006-11-14 CN CNA2006101294046A patent/CN101181240A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102302775A (en) * | 2011-07-06 | 2012-01-04 | 普莱柯生物工程股份有限公司 | Combined inactivated vaccine of Newcastle disease and H9 subtype avian influenza and preparation method thereof |
| CN102302775B (en) * | 2011-07-06 | 2013-06-05 | 普莱柯生物工程股份有限公司 | Combined inactivated vaccine of Newcastle disease and H9 subtype avian influenza and preparation method thereof |
| CN103937754A (en) * | 2014-04-11 | 2014-07-23 | 天津瑞普生物技术股份有限公司 | Porcine reproductive and respiratory syndrome virus (PPRSV) purification method |
| CN103937754B (en) * | 2014-04-11 | 2017-01-11 | 天津瑞普生物技术股份有限公司 | Porcine reproductive and respiratory syndrome virus (PPRSV) purification method |
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