CN101879311A - Method for preparing live vaccines of hog cholera and product thereof - Google Patents
Method for preparing live vaccines of hog cholera and product thereof Download PDFInfo
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Abstract
The invention discloses a method for preparing live vaccines of hog cholera and a product thereof. The preparation method comprises the following steps of: (1) culturing porcine passage cell lines; (2) inoculating the porcine passage cell lines with live vaccine production seed viruses of the hog cholera to obtain attenuated vaccine strains of the hog cholera; (3) performing virus multiplication on the attenuated vaccine strains of the hog cholera; (4) measuring the virus titer of multiplication virus suspension by adopting an immunofluorescence method; and (5) adding a freeze-drying protective agent and antibiotics into the virus suspension which is detected to be qualified for vaccine matching and freeze-drying. The preparation method has the advantages of producing the live vaccines of the hog cholera by using the cell lines so as to achieve small quality differences among batches and the characteristics of simple and stable process, easy operation, high yield, low cost, the feasibility and extendibility of industrial production and the like, and measuring the virus titer of the multiplication virus suspension by adopting the immunofluorescence method so as to achieve sensitive, fast, specific and accurate detection, high repeatability and reliable results. The live vaccines of the hog cholera prepared by the method can completely protect pigs from the attacks of violent hog cholera viruses.
Description
Technical field
The present invention relates to a kind of production method of live vaccine for animals, relate in particular to a kind of preparation method of live vaccines of hog cholera and the product that obtains by this preparation method, belong to the production field of live vaccines of hog cholera.
Background technology
(Hog cholera is a kind of height social disease that is caused by swine fever virus HC) to swine fever, and in OIE disease register is listed it by OIE (OIE), is the infectious disease of legal essential report.In China, swine fever is one of great eqpidemic disease, lists swine fever one of in 17 kinds of animal epidemics of a class in " the sick register of planting of one, two, three class animal epidemics ", and the eruption and prevalence of swine fever causes serious economic loss for the pig industry of the world and China.
Vaccine is the important means of control swine fever, comprises inactivated vaccine and attenuated vaccine.Each state is all making great efforts to study vaccine safely and effectively.Through using for many years, generally recognized as safe is effective, does not have the attenuated vaccine strain of remaining pathogenicity to have three kinds: 1. Chinese rabbitization attenuated vaccine; 2. Japanese GPE cell weak-toxic vaccine; 3. French " Thiveosal " cold variation low virulent strain.China system (C system) hog cholera lapinised virus vaccine of succeeding in developing of Chinese scholar wherein from nineteen fifty-seven, except that China's extensive use, and has been generalized to Eurasian a lot of nationally, makes these state controls or has eliminated swine fever.This vaccine is acknowledged as more satisfactory in the world swine Fever Vaccine at present.
Hog cholera lapinised virus is inoculated in the swine fever cell vaccine that bovine testicle cell is prepared from, advantages such as it is low to have production cost, is convenient to batch production production, and untoward reaction is few, shortcoming is with bull testis cell culture hog cholera lapinised virus (HCLV), poison valency instability, differences between batches are very big, the product that the rabbit inspection is qualified, the highest and low toxicity valency differs greatly, and use bull testis, Ox blood serum in the production process, the probability of polluting bovine viral diarrhea virus is arranged, cause immuning failure.
The height of vaccine valence is the whether qualified key index of vaccine, the proliferative ability of HCLV in cell a little less than, do not produce cytopathogenic effect, its mensuration of tiring is continued to use rabbit body-shaping thermal response method (rabbit full-boiled process) always.But the outstanding deficiency that this method exists is: the rabbit full-boiled process is surveyed poison and is belonged to a kind of nonspecific reaction, and surveying poison with it can not be accurately quantitative, surveys malicious result and is subject to rabbit body individual variation and weather condition influence, and the test period is long, waste time and energy.
Summary of the invention
Technical problem to be solved by this invention is the existing deficiency of production that overcomes the swine fever cell vaccine; a kind of preparation method of new live vaccines of hog cholera is provided; this method can be measured vaccine valence fast and accurately; prepared vaccine safety is good, immune efficacy is high, and the swine fever strong virus attack is had immanoprotection action completely.
Technical problem to be solved by this invention is achieved through the following technical solutions:
A kind of preparation method of live vaccines of hog cholera may further comprise the steps: (1), cultivation pig source continuous cell line; (2), with live vaccines of hog cholera production kind poison inoculation pig source continuous cell line, screening obtains the swine fever attenuated vaccine strain; (3), the swine fever attenuated vaccine strain is carried out virus multiplication; (4), adopt immunofluorescence method to measure the (TCID of virus of proliferation suspension
50) malicious valency; (5), in the viral liquid that detects after qualified, add freeze drying protectant and antibiotic is joined Seedling, lyophilization, promptly.
The present invention is directed to the existing aborning a series of problems of swine fever cell vaccine, from improving: (1) through following several stages, the screening and evaluation of pig source continuous cell line: hog cholera lapinised virus is inoculated different pigs source continuous cell line, to 37 ℃ of cultivations 4～5 days, results virus, to gather in the crops virus goes down to posterity, using immunofluorescence method detects each generation virus of inoculating different pigs source continuous cell line, testing result shows that hog cholera lapinised virus can be in PT cell line, MPK cell line, SK6 cell line, PK 2a cell line is bred on CPK cell line or the RKC cell line.(2), the preparation of seed culture of viruses: hog cholera lapinised virus is gone down to posterity on PT cell line, adapt to PT cell line.Use limiting dilution assay and carry out the two-wheeled clone purification, screen the swine fever attenuated vaccine strain of the suitable pig source continuous cell line of a strain, its microbial preservation number is: CGMCC No.3891; The classification name is: swine fever virus; The preservation time is: on May 27th, 2010; Depositary institution is: China Committee for Culture Collection of Microorganisms common micro-organisms center; The preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.(3) foundation of immunofluorescence method: use anti-CSFV monoclonal antibody and set up immunofluorescence method, adopting said method carries out viral TCID
50Mensuration.
Preferably, the training method of the described pig of step (1) source continuous cell line is that monolayer adherence cultivation, suspension culture or immobilization are cultivated.
In the step (2) live vaccines of hog cholera production kind poison is inoculated into the PT cell monolayer with infective dose (MOI) 0.1-0.5 and fastens and cultivate, inoculate 5 and do for the first time results and change liquid, change liquid every results on the 4th later on, results are no more than 5 times.
Adopt immunofluorescence method to measure the (TCID of virus of proliferation in the step (4)
50) during malicious valency, the monoclonal antibody that is adopted is preferably anti-CSFV monoclonal antibody (Beijing Jianxiang Hemu Bio-tech Co., Ltd. is externally on sale), the label that is adopted is preferably FITC or IPMA; Used fixative is preferably 80% acetone, 10% formaldehyde or 75% ethanol of pre-cooling.Preferred, described immunofluorescence method may further comprise the steps:
(1) passage, prepare Tissue Culture Plate: choose well-grown PT cell, after pancreas enzyme-EDTA digestion, add cell growth medium and prepare cell suspension, application station is expected blue dilution counting, and adjusting cell density is 2.5～3.5 * 10
5Individual/ml, insert 96 porocyte culture plates with the 100ul/ hole.
(2) vaccine virus dilution of sample: will be titrating the live vaccines of hog cholera viral suspension make 10 times of doubling dilutions, note from a dilution factor to change suction nozzle when moving to next dilution factor, the whirlpool mixing is all wanted in each dilution.
(3) virus inoculation (connecing poison synchronously): insert 100 μ l samples, 6～8 repetitions of each dilution factor.Add 100 μ l culture medium as negative control, every block of plate is established 2 row negative controls at least.Cover lid is at 37 ℃ ± 1 ℃, 4～6%CO
2Cultivated in the incubator 3～5 days.
(4) immunofluorescence detects
1. incline liquid in the plank in strong alkali solution, with PBST washing 2 times, pat dry residual liquid, the fixative (80% acetone or 10% formaldehyde or 75% ethanol) that every hole adds 50～100 μ L pre-coolings is 20min fixedly.
2. direct immunofluorescence: PBST washing is 2 times, pats dry residual liquid, and every hole adds the FITC labelling CSFV monoclonal antibody 50 μ L of working concentration, puts in the wet box 37 ℃ and hatches 30～45min, removes antibody, with PBST washing 3 times;
3. indirect immunofluorescence: PBST washing is 2 times, pat dry residual liquid, every hole adds the CSFV monoclonal antibody 50 μ L of working concentration, put in the wet box 37 ℃ and hatch 45～60min, remove one anti-, with PBST washing 3 times, every hole adds worker-do anti-Mus two anti-(SIGMA) 50 μ L of FITC labelling of concentration, black out is hatched 30～45min for 37 ℃, removes two and resists, with PBST washing 3 times;
4. under fluorescence microscope, observe and have or not the specificity fluorescent speckle to occur, be recorded on the test data sheet paper and use Spearman Karber method and calculate virus titer.
(5) result effectively judges: negative hole fluorescence feminine gender in all tests; Must be in normal scope with reference to contrast; If sample is lower than 50% or be higher than 50% in the positive hole of high dilution in the positive hole of minimum dilution factor, can report the result is below or above this titre 50%.
The present invention substitutes the bull testis primary cell with cell line and makes live vaccines of hog cholera, can solve cattle source external source cause of disease pollution problems, and the strictness control by raw material and condition of culture guarantees that the vaccine of producing is pure, guarantees the safety of vaccine.Little with mass discrepancy between each batch of producing swine fever live vaccine with cell line, have production technology simple and stable, easy to operate, characteristics such as output is big, cost is low, possess the feasibility and the scalable property of industrialized great production.In addition, the present invention adopts the immunization method of monoclonal antibody to detect swine fever cell venom viral level, detects sensitivity, and special fast, accurately, repeatability is high, reliable results.Vaccine can be 4 ℃ of preservations after utilizing production of the present invention and using the live vaccines of hog cholera lyophilizing of novel freeze drying protectant preparation.Through producing, check, a series of improvement of preservation can improve the immune efficacy of live vaccines of hog cholera greatly, show that through immune challenge test result the prepared live vaccines of hog cholera of the present invention can 100% protection to the swine fever strong virus attack.
Description of drawings
Fig. 1 live vaccines of hog cholera immunofluorescence testing result; A: negative control, B: vaccine test sample.
Fig. 2 immunofluorescence detects the sensitivity tests result of live vaccines of hog cholera poison valency.
Fig. 3 immunofluorescence detects the specificity result of the test of live vaccines of hog cholera poison valency.
The specific embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
The screening and the evaluation of embodiment 1 pig source continuous cell line
Hog cholera lapinised virus is inoculated different pigs source continuous cell line, to 37 ℃ of cultivations 4～5 days, results virus will be gathered in the crops virus and go down to posterity, and use immunofluorescence method each generation virus of inoculating different pigs source continuous cell line is detected, testing result shows that hog cholera lapinised virus can be in PT cell line, MPK cell line, SK6 cell line, PK 2a cell line, CPK cell line is bred on the RKC cell line.The present invention cultivates hog cholera lapinised virus with cell line (PT, MPK, SK6, PK 2a, CPK, RKC); harvesting virus liquid; adopt the malicious valency of determination of immunofluorescence method virus of proliferation suspension; adding freeze drying protectant and antibiotic are joined Seedling, lyophilization in the viral liquid after detection is qualified, make swine fever passage cell Seedling.
The method of embodiment 2 usefulness producing swine fever live vaccine with cell line
(1) seedling going down to posterity and cultivating with cell: PT cell line is through pancreas enzyme-EDTA cell dispersion liquid had digestive transfer culture, and one to five goes down to posterity.Continue to cultivate in 37 ℃ with cell growth medium, when forming good monolayer, be used to continue to go down to posterity or virus inoculation;
(2) breeding of cell seed culture of viruses: use cell maintenance medium, fresh spleen poison is made 0.3% viral suspension, inoculate well-grown PT cell line monolayer, put 37 ℃ and continue to cultivate.Cultivate venom every harvesting on the 5th and use seed culture of viruses as producing; The cell seed culture of viruses is identified: the cell seed culture of viruses identifies and meets hog cholera lapinised virus strain seed culture of viruses standard fully that pig safety is had no side effect, and the every 1ml of cell seed culture of viruses contains virus＞100,000 a rabbit infective dose;
(3) breeding of seedling venom: get the PT cell line culture bottle that forms good monolayer, discard nutritional solution, inoculation contains 3% production, and (cultivate in rolling bottle, cell density reaches 5-10 * 10 with the cell seed culture of viruses
7/ ml; Add the suspension culture or the DNAcarrier free suspension culture of adhering to carrier in bioreactor, cell density reaches 5-10 * 10
7-8/ ml; Immobilization is cultivated above-mentioned cell line is combined and cultivates with water insoluble carrier (scraps of paper, nonwoven polyester fiber slide glass), and cell density reaches 5-10 * 10
7-8/ ml), put 37 ℃ of continuation and cultivate, connect poison back work on the 5th results first time and change liquid, change liquid 1 time every results on the 4th later on, the venom of results is put below-15 ℃ and is preserved; The check of seedling venom: test for 15,19 pages by " People's Republic of China's veterinary drug allusion quotation " (version in 2005) appendix, should not have antibacterial, mycete, mycoplasma growth.The cell venom has no side effect to pig safety, and each is received time virus-culturing fluid and measures with rabbit respectively, and every 1ml contains virus＞100,000 a rabbit infective dose;
Above-mentioned nutrient solution used prescription is: 92%DMEM liquid, 5-8% calf serum, add an amount of antibiotics, pH value is adjusted into 7.0.The above-mentioned used prescription of keeping liquid is: 95%DMEM liquid, 3.5-5% calf serum, add an amount of antibiotics, pH value is adjusted into 7.2.
The virus titer of the immunofluorescence method check live vaccines of hog cholera of test example 1 using monoclonal antibody
1, passage, prepare Tissue Culture Plate: choose well-grown PT cell, after pancreas enzyme-EDTA digestion, add cell growth medium and prepare cell suspension, application station is expected blue dilution counting, and adjusting cell density is 2.5～3.5 * 10
5Individual/ml, insert 96 porocyte culture plates with the 100ul/ hole.
2, vaccine virus dilution of sample: will be titrating the live vaccines of hog cholera viral suspension make 10 times of doubling dilutions, note from a dilution factor to change suction nozzle when moving to next dilution factor, the whirlpool mixing is all wanted in each dilution.
3, virus inoculation (connecing poison synchronously): insert the 100ul Virus Sample, each dilution factor 6～-8 repetition.Add the 100ul culture medium as negative control, every block of plate is established 2 row negative controls at least.Cover lid is at 37 ℃ ± 1 ℃, 4～6%CO
2Cultivated in the incubator 3～5 days;
4, immunofluorescence detects
1. incline liquid in the plank in strong alkali solution, with PBST washing 2 times, pat dry residual liquid, the fixative (80% acetone or 10% formaldehyde or 75% ethanol) that every hole adds 50～100 μ L pre-coolings is 20min fixedly.
2. direct immunofluorescence: PBST washing is 2 times, pats dry residual liquid, and every hole adds the FITC labelling CSFV monoclonal antibody 50 μ L of working concentration, puts in the wet box 37 ℃ and hatches 30～45min, removes antibody, with PBST washing 3 times;
3. indirect immunofluorescence: PBST washing is 2 times, pat dry residual liquid, every hole adds the CSFV monoclonal antibody 50 μ L of working concentration, put in the wet box 37 ℃ and hatch 45～60min, remove one anti-, with PBST washing 3 times, every hole adds anti-Mus two anti-(SIGMA) 50 μ L of FITC labelling of working concentration, black out is hatched 30～45min for 37 ℃, removes two and resists, with PBST washing 3 times;
4. under fluorescence microscope, observe and have or not the specificity fluorescent speckle to occur, be recorded on the test data sheet paper and use Spearman Karber method and calculate virus titer.
(5) result effectively judges: negative hole fluorescence feminine gender in all tests; Must be in normal scope with reference to contrast; If sample is lower than 50% or be higher than 50% in the positive hole of high dilution in the positive hole of minimum dilution factor, can report the result is below or above this titre 50%.
The optimization Test of test example 2 swine fever virus condition of culture
1 method
1.1 connecing malicious mode studies
1.1.1 well-grown PT cell 2 rolling bottles digestion is selected in inoculation synchronously for use, goes down to posterity by 1: 3, wherein 1 bottle as negative control, 2 bottles of backs of going down to posterity are put in 37 ℃ of Rotary Machines and are cultivated with MOI value 0.1 virus inoculation, inoculate the viral liquid of results after 5 days, viral level (TCID is measured in freeze thawing 2 times
50).
1.1.2 in the cell of monolayer inoculation 1.1.1 had digestive transfer culture, treat for 2 bottles to inoculate swine fever virus with M0I 0.1 after cell covers with monolayer, add and keep liquid, put in 37 ℃ of Rotary Machines and cultivate, inoculate the viral liquid of results after 5 days, viral level (TCID is measured in freeze thawing 2 times
50).
1.2 MOI research
Select well-grown PT cell for use, press MOI value 0.01,0.05,0.1,0.3 and 0.5 and insert viral liquid, 2 rolling bottles of each MOI value inoculation are established a negative contrast of rolling bottle simultaneously.Inoculate the viral liquid of results after 5 days, viral level (TCID is measured in freeze thawing 2 times
50).
1.3 viral harvest time, the research of harvesting frequency
Select well-grown PT cell, press MOI value 0.1 and add swine fever virus liquid, connecing poison back the 1st day respectively, 2,3,4, got the supernatant sample, measure viral level (TCID in 5th
50), made for the first time results in back 5 days and change liquid connecing poison, change liquid once every results on the 4th later on, receive in each and get the supernatant sample time every day, measure viral level (TCID
50), results are no more than 5 and receive inferior.
2 result of the tests
Show that 2.1 connect malicious mode result of study monolayer connects malicious mode swine fever virus titre and is higher than and connects malicious mode synchronously, sees table 1 for details.
Synchronous and the monolayer inoculation comparative result of table 1
2.2 MOI development test result shows that MOI is that 0.1～0.5 o'clock virus titer is the highest, sees table 2 for details.
Table 2 MOI result of study
2.3 viral harvest time and harvesting frequency development test result show, do to gather in the crops for the first time to change liquid in 5th behind the virus inoculation, later on every 4 days, it is higher that results are no more than 5 virus titers, sees table 3,4,5,6,7 for details.
Gather in the crops for the first time result of study behind table 3 virus inoculation
After-crop result of study behind table 4 virus inoculation
Gather in the crops result of study behind table 5 virus inoculation for the third time
The 4th results result of study behind table 6 virus inoculation
The 5th results result of study behind table 7 virus inoculation
3 brief summaries
According to above experimental result, the present invention determines that the optimal culture condition of swine fever purified virus on the PT cell is: connect poison during cell monolayer, the MOI value is 0.1～0.5, inoculates 5 and does to gather in the crops for the first time to change liquid, and later on every 4 days, results are no more than 5 times.
The malicious valency determination test of test example 3 live vaccines of hog cholera
One, test material
1, for test agent: according to the method for the embodiment of the invention 1 prepared three batches of live vaccines of hog cholera (the cell line source, lot number is examination PT200801, examination PT200802, examination PT200803;
2, live vaccines of hog cholera (cell source) is available from biotech firm, and lot number is 20080415,20080621,20081005;
Two, test method
Rabbit is examined qualified three batches of live vaccines of hog cholera (cell line source) carry out viral TCID with three batches of live vaccines of hog cholera (cell source) application direct immunofluorescence method
50Measure, the results are shown in Table 8.
Table 8
Use three batches of live vaccines of hog cholera (cell source) that the bull testis primary cell is made as can be seen from Table 8, differences between batches are big, and viral level is low, use three batches of live vaccines of hog cholera (cell line source) that pig source continuous cell line is made, differences between batches are little, and viral level is higher than live vaccines of hog cholera (cell source).
The immune effect test of test example 4 live vaccines of hog cholera of the present invention
One, test material
1, for the examination vaccine: the method according to embodiment 1 is produced three batches of live vaccines of hog cholera (cell line source), and lot number is examination PT200801, examination PT200802, examination PT200803;
2, control vaccine: three batches of live vaccines of hog cholera (cell source) are available from biotech firm, and lot number is 20080415,20080621,20081005;
3, IDEXX swine fever virus antibody assay kit is available from the prosperous bio tech ltd of Beijing Ai Deshi unit
Two, test method
Getting three batches and supply examination vaccine (cell line source) and three batches of control vaccines (cell source) to do immune contrast test on two pig farms, six batches of vaccines are diluted to 1 part by operation instruction with it respectively, is 10 for examination vaccine (cell line source) viral level
4.0TCID
50/ head part, control vaccine (cell source) viral level＞3000RID/ head part, intramuscular inoculation 21-30 age in days piglet.Carried out antibody titer with IDEXX swine fever virus antibody assay kit in back 21 days and 27 days in immunity and detect (blocking-up rate 〉=40% is an antibody positive); Immunity was extracted immune animal (5/batches) in back 28 days and is carried out counteracting toxic substances with control animal (3).
Three, result of the test
Back 21 days of immunity, control vaccine (cell line source) antibody qualification rate is 85%, control vaccine (cell source) antibody qualification rate is 68%; Immunity was 91% for examination vaccine (cell line source) antibody qualification rate in back 27 days, and control vaccine (cell source) antibody qualification rate is 76%, shows that control vaccine (cell line source) antibody tells on significantly better than live vaccines of hog cholera (cell line source)
After the immunity 28 days, it is blood poison (10 that extraction immune group pig (5/batches) is together injected the swine fever crossdrift with control animal (3)
4.0TCID
50/ ml), and 1ml/ pig, challenge test is end in 16 days behind counteracting toxic substances, the results are shown in Table 9.
Table 9
Conclusion: this test shows for examination vaccine (cell line source) immune effect significantly better than control vaccine (cell source).
The sensitivity and the specificity test of the immunofluorescence method check live vaccines of hog cholera of test example 5 using monoclonal antibodies
One, the sensitivity of immunofluorescence detection method:
The sensitivity check: is 10 with cell maintenance medium with the CSFV dilution
3, 10
2, 10
1, 1TCID
50Four dilution factors, 96 porocyte culture plates, 10 holes that 90% monolayer PT cell is covered with in each dilution factor virus liquid inoculation, the cell maintenance medium that inoculation does not simultaneously add virus contrasts 10 holes, cultivated 3～4 days for 37 ℃, carry out direct fluorescent antibody staining after fixing, washing, microscopy.10 viral negative control holes all detect less than specificity fluorescent, and each dilution poison cell hole that connects all can detect specificity fluorescent.The immunofluorescence method of setting up with anti-swine fever monoclonal antibody can detect 1 TCID
50The swine fever virus particle of unit, sensitivity is good, the results are shown in Figure 2:
Two, the specificity of immunofluorescence detection method:
Specificity test: pseudorabies virus (PRV), rabies virus (RV), pig parvoviral (PPV), pig circular ring virus (PCV) dilution are 1000TCID with cell maintenance medium
50, every kind of 96 porocyte culture plates, 10 holes that 90% monolayer PT cell is covered with in the inoculation of virus dilution liquid; Not virus inoculation negative control hole of 10 swine fever virus (CSFV) positive control hole and 10 is set simultaneously, cultivated 3～4 days for 37 ℃, carry out fluorescent antibody staining, washing, microscopy after fixing.PRV, RV, PPV, PCV connect the poison cell hole and negative control hole all detects less than specificity fluorescent, and CSFV inoculation hole all can detect specificity fluorescent.The immunofluorescence method specificity of setting up with the anti-swine fever monoclonal antibody of our company's preparation is good, the results are shown in Figure 3.
Test example 6 immunofluorescence methods and rabbit body temperature method detect the comparative test of hog cholera lapinised virus
As can be seen from Table 10, detect hog cholera lapinised virus (bull testis cell toxicant) with rabbit body temperature method, extension rate is that 50,000 times of group testing results are defective, and extension rate is that 100,000 times of groups (viral level is lower than the former) testing result is for qualified; Same rabbit body temperature method detects hog cholera lapinised virus (embodiment 1 is prepared) (PT cell toxicant), and extension rate is that 10,000 times of group testing results are defective, and extension rate is 50,000 times and 100,000 times, and to organize testing results be qualified (viral level is lower than the former).Illustrate with rabbit body-shaping thermal response method and survey poison, the result is subject to rabbit body individual variation and weather condition influence, and the rabbit full-boiled process is surveyed poison and belonged to a kind of nonspecific reaction, and surveying poison with it can not be accurately quantitative, and measures swine fever virus TCID with immunofluorescence method
50Can be accurately quantitative.
Table 10
Claims (10)
1. the preparation method of a live vaccines of hog cholera may further comprise the steps: (1), cultivation pig source continuous cell line; (2), the good pig source continuous cell line of malicious inoculated and cultured is planted in live vaccines of hog cholera production, screening obtains the swine fever attenuated vaccine strain; (3), the swine fever attenuated vaccine strain is carried out virus multiplication; (4), adopt immunofluorescence method to measure the malicious valency of virus of proliferation suspension; (5), in the viral liquid that detects after qualified, add freeze drying protectant and antibiotic is joined Seedling, lyophilization, promptly.
2. according to according to the described preparation method of claim 1, it is characterized in that: the training method of the described pig of step (1) source continuous cell line is that monolayer adherence cultivation, suspension culture or immobilization are cultivated.
3. according to the described preparation method of claim 1, it is characterized in that: the pig source continuous cell line described in the step (1) is PT cell line, MPK cell line, SK6 cell line, PK 2a cell line, CPK cell line or RKC cell line.
4. according to the described preparation method of claim 1, it is characterized in that: the microbial preservation of swine fever attenuated vaccine strain described in the step (2) number is: CGMCC No.3891.
5. according to the described preparation method of claim 1, it is characterized in that: in the step (2) live vaccines of hog cholera production kind poison is inoculated on the continuous cell line of cultured pig source with infective dose 0.1-0.5 and cultivates.
6. according to the described preparation method of claim 1, it is characterized in that: in the step (2) live vaccines of hog cholera production kind poison is inoculated on the continuous cell line of cultured pig source with infective dose 0.1-0.5 and cultivates, inoculate 5 and do to gather in the crops for the first time to change liquid, change liquid every results on the 4th later on, results are no more than 5 times.
7. according to the described preparation method of claim 1, it is characterized in that: when adopting in the step (4) immunofluorescence method to measure the malicious valency of virus of proliferation, the monoclonal antibody that is adopted is the swine fever virus resistant monoclonal antibody; The fluorescent marker that is adopted is FITC or IPMA; Used fixative is 80% acetone, 10% formaldehyde or 75% ethanol of pre-cooling.
8. according to the described preparation method of claim 1, it is characterized in that: when adopting in the step (4) immunofluorescence method to measure the malicious valency of virus of proliferation, its step comprises: (1) passage, prepare Tissue Culture Plate: choose well-grown pig source continuous cell line, after pancreas enzyme-EDTA digestion, add cell growth medium and prepare cell suspension, application station is expected blue dilution counting, and adjusting cell density is 2.5～3.5 * 10
5Individual/ml, insert 96 porocyte culture plates with the 100ul/ hole.(2) vaccine virus dilution of sample: will make 10 times of doubling dilutions by titrating live vaccines of hog cholera viral suspension; (3) virus inoculation: insert 100 μ l samples, 6～8 repetitions of each dilution factor; Add 100 μ l culture medium as negative control, every block of plate is established 2 row negative controls at least; Cover lid is at 37 ℃ ± 1 ℃, 4～6%CO
2Cultivated in the incubator 3～5 days; (4) immunofluorescence detects: the liquid in the plank that 1. inclines with PBST washing 2 times, pats dry residual liquid in strong alkali solution, and the fixative that every hole adds 50～100 μ L pre-coolings is 20min fixedly; 2. direct immunofluorescence: PBST washing is 2 times, pats dry residual liquid, and every hole adds the FITC labelling CSFV monoclonal antibody 50 μ L of working concentration, puts in the wet box 37 ℃ and hatches 30～45min, removes antibody, with PBST washing 3 times; 3. indirect immunofluorescence: PBST washing is 2 times, pat dry residual liquid, every hole adds the CSFV monoclonal antibody 50 μ L of working concentration, put in the wet box 37 ℃ and hatch 45～60min, remove one anti-, with PBST washing 3 times, every hole adds the anti-Mus two anti-50 μ L of FITC labelling of working concentration, black out is hatched 30～45min for 37 ℃, removes two and resists, with PBST washing 3 times; 4. under fluorescence microscope, observe and have or not the specificity fluorescent speckle to occur, be recorded on the test data sheet paper and use Spearman Karber method and calculate virus titer; (5) result effectively judges: negative hole fluorescence feminine gender in all tests; Must be in normal scope with reference to contrast; If sample is lower than 50% or be higher than 50% in the positive hole of high dilution in the positive hole of minimum dilution factor, reporting the result is below or above this titre 50%.
9. the live vaccines of hog cholera that obtains by any one preparation method of claim 1-8.
10. the purposes of the described live vaccines of hog cholera of claim 9 in preparation prevention or treatment swine fever biological product.
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| CN2010102197726A CN101879311A (en) | 2010-06-28 | 2010-06-28 | Method for preparing live vaccines of hog cholera and product thereof |
| CN 201010536418 CN102294029B (en) | 2010-06-28 | 2010-11-09 | Preparation method and product of swine fever live vaccine |
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| CN2010102197726A Withdrawn CN101879311A (en) | 2010-06-28 | 2010-06-28 | Method for preparing live vaccines of hog cholera and product thereof |
| CN 201010536418 Active CN102294029B (en) | 2010-06-28 | 2010-11-09 | Preparation method and product of swine fever live vaccine |
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| CN 201010536418 Active CN102294029B (en) | 2010-06-28 | 2010-11-09 | Preparation method and product of swine fever live vaccine |
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| CN102600465A (en) * | 2010-12-28 | 2012-07-25 | 华威特（北京）生物科技有限公司 | Newcastle disease (ND) vaccine, and its production method |
| CN102727882A (en) * | 2011-10-27 | 2012-10-17 | 华威特（北京）生物科技有限公司 | Combined live vaccine against porcine reproductive and respiratory syndrome, swine fever and pseudorabies, and preparation method thereof |
| CN102727883A (en) * | 2011-05-27 | 2012-10-17 | 华威特（北京）生物科技有限公司 | Combined live vaccine against porcine reproductive and respiratory syndrome and swine fever, and application thereof |
| WO2012163258A1 (en) * | 2011-05-27 | 2012-12-06 | Sinovet (Beijing) Biotechnology Co.,Ltd | Combined vaccines for prevention of porcine virus infections |
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| CN100577204C (en) * | 2007-11-30 | 2010-01-06 | 中国兽医药品监察所 | Method for producing swine fever live vaccine with cell line |
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2010
- 2010-06-28 CN CN2010102197726A patent/CN101879311A/en not_active Withdrawn
- 2010-11-09 CN CN 201010536418 patent/CN102294029B/en active Active
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Also Published As
| Publication number | Publication date |
|---|---|
| CN102294029B (en) | 2013-04-03 |
| CN102294029A (en) | 2011-12-28 |
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