CN102294029B - Preparation method and product of swine fever live vaccine - Google Patents
Preparation method and product of swine fever live vaccine Download PDFInfo
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Abstract
The invention discloses a preparation method and product of a swine fever live vaccine. The preparation method comprises the following steps: (1) culturing a swine-derived continuous cell line; (2) inoculating the swine-derived continuous cell line into a seed virus for producing the swine fever live vaccine, thereby obtaining a swine fever weak-virus vaccine strain; (3) carrying out virus multiplication on the swine fever weak-virus vaccine strain; (4) determining the virus value of the multiplied virus suspension by an immunofluorescence method; and (5) adding a freeze-drying protective agent and antibiotics into the qualified virus suspension to carry out vaccine preparation and freeze-drying. Since the swine fever live vaccine is prepared from the cell line, the difference of quality among different batches is small, and the invention has the characteristics of simple and stable technique, high yield, low cost and the like, is easy to operate, and has feasibility and amplification of industrialized mass production. In addition, the immunofluorescence method, which has the advantages of high detection sensitivity, high speed, high specificity, high accuracy, high repetitiveness and reliable result, is used for determining the virus value of the multiplied virus suspension. The swine fever live vaccine disclosed by the invention can 100% protect the attack of swine fever strong virus.
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 is obtained by this preparation method, belong to the production field of live vaccines of hog cholera.
Background technology
Swine fever (Classical swine fever, CSF) is a kind of height social disease that is caused by swine fever virus, 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 epidemic 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 to 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 the effective vaccine of research safety.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. France " 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 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 at present more satisfactory in the world swine Fever Vaccine.
Hog cholera lapinised virus is inoculated in the swine fever cell vaccine that bovine testicle cell is prepared from, has production cost low, be convenient to batch production production, the advantages such as untoward reaction is few, shortcoming is that malicious valency is unstable with bull testis cell culture hog cholera lapinised virus (HCLV), and differences between batches are very large, the product that rabbit inspection is qualified, the highest and low toxicity valency differs greatly.
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 accurate quantitative analysis, surveys malicious result and is subject to rabbit body individual variation and weather condition impact, 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; the 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 completely immanoprotection action.
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), with live vaccines of hog cholera production kind poison inoculation continuous cell line, and screening obtains the swine fever attenuated vaccine strain; (2), the swine fever attenuated vaccine strain is carried out virus multiplication; (3), adopt immunofluorescence method to measure the (TCID of virus of proliferation suspension
50) malicious valency; (4), add freeze drying protectant in the virus liquid that detects after qualified and antibiotic is joined Seedling, lyophilization, and get final product.
The present invention is directed to aborning existing series of problems of swine fever cell vaccine, from improving through following several stages: (1), the screening of continuous cell line and evaluation: hog cholera lapinised virus is inoculated different continuous cell lines, to 37 ℃ of cultivations 4～5 days, results virus, to gather in the crops virus goes down to posterity, use immunofluorescence method each generation virus of inoculating different continuous cell lines is detected, testing result shows that hog cholera lapinised virus can be in PT cell line, BT cell line, ST cell line, MPK cell line, SK6 cell line, PK 2a cell line, CPK cell line, RKC cell line, MDBK cell line, mdck cell system, CRFK cell line, Vero/slam cell line, Vero-E6 cell line, Marc-145 cell line, bhk cell system, breed on BGM cell line or the DK cell line.(2), the preparation of seed culture of viruses: hog cholera lapinised virus is gone down to posterity in 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 continuous cell line of a strain, its microbial preservation number is: CGMCC No.3891; Classification And Nomenclature 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, 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 (3)
50) during malicious valency, the monoclonal antibody that adopts is preferably anti-CSFV monoclonal antibody (Beijing Jianxiang Hemu Bio-tech Co., Ltd. is externally on sale), the label that adopts 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, access 96 porocyte culture plates with the 100ul/ hole;
(2) dilution of vaccine virus sample: live vaccines of hog cholera viral suspension that will titration is made 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: access 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 anti-CSFV monoclonal antibody 50 μ L of FITC labelling 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 anti-CSFV monoclonal antibody 50 μ L of working concentration, put in the wet box 37 ℃ and hatch 45～60min, remove primary antibodie, with PBST washing 3 times, every hole adds anti-Mus two anti-(SIGMA) 50 μ L of FITC labelling of working concentration, 37 ℃ of black outs are hatched 30～45min, remove two anti-, with PBST washing 3 times;
4. have or not the specificity fluorescent speckle to occur at the fluorescence microscopy Microscopic observation, be recorded on the test data sheet paper and use Spearman Karber method and calculate virus titer;
(5) result effectively judges: negative hole fluorescence is negative in all tests; Must be in normal scope with reference to contrast.
The present invention substitutes primary cell with cell line and makes live vaccines of hog cholera, can solve the problem of cattle source external source pathogen contamination, by the strict control of 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 the characteristics such as production technology simple and stable, easy to operate, output is large, cost is low, but possess feasibility and the amplification 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 through the Immunization result of the test, and 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 specific test result of live vaccines of hog cholera poison valency.
The specific embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not consisted of any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can make amendment or replace the details of technical solution of the present invention and form, but these modifications and replacing all fall within the scope of protection of the present invention.
Screening and the evaluation of embodiment 1 continuous cell line
Hog cholera lapinised virus is inoculated different continuous cell lines, to 37 ℃ of cultivations 4～5 days, results virus, to gather in the crops virus goes down to posterity, use immunofluorescence method each generation virus of inoculating different continuous cell lines is detected, testing result shows that hog cholera lapinised virus can be in PT cell line, BT cell line, ST cell line, MPK cell line, SK6 cell line, PK 2a cell line, CPK cell line, RKC cell line, MDBK cell line, mdck cell system, CRFK cell line, Vero/slam cell line, Vero-E6 cell line, Marc-145 cell line, bhk cell system, breed on BGM cell line or the DK cell line.The present invention cultivates hog cholera lapinised virus with cell line (PT, BT, ST, MPK, SK6, PK 2a, CPK, RKC, MDBK, MDCK, CRFK, Vero/slam, Vero-E6, Marc-145, BHK, BGM or DK); the 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 virus liquid after qualified to detection, 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.Add Growth of Cells liquid and continue to cultivate in 37 ℃, when forming good monolayer, be used for continuing 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 evaluation of cell seed culture of viruses meets fever virus lapinized Chinese Strain seed culture of viruses standard fully, and pig is had no side effect safely, 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% Cells for production seed culture of viruses, and (cultivate in rolling bottle, cell density reaches 5-10 * 10
7/ ml; Add 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 and water insoluble carrier (scraps of paper, nonwoven polyester fiber slide glass) combination is cultivated, and cell density reaches 5-10 * 10
7-8/ ml), put 37 ℃ and continue cultivation, connect malicious rear the 5th day and do to gather in the crops for the first time to change liquid, to gather in the crops every 4 days later on and change liquid 1 time, the venom of results is put preservation below-15 ℃; The check of seedling venom: test by " People's Republic of China's veterinary drug allusion quotation " (version in 2005) appendix 15,19 pages, should be without antibacterial, mycete, mycoplasma growth.The cell venom has no side effect safely to pig, 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 prescription of above-mentioned used maintenance medium 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, access 96 porocyte culture plates with the 100ul/ hole;
2, the dilution of vaccine virus sample: live vaccines of hog cholera viral suspension that will titration is made 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: access 100ul Virus Sample, 6～8 repetitions of each dilution factor.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 anti-CSFV monoclonal antibody 50 μ L of FITC labelling 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 anti-CSFV monoclonal antibody 50 μ L of working concentration, put in the wet box 37 ℃ and hatch 45～60min, remove primary antibodie, with PBST washing 3 times, every hole adds anti-Mus two anti-(SIGMA) 50 μ L of FITC labelling of working concentration, 37 ℃ of black outs are hatched 30～45min, remove two anti-, with PBST washing 3 times;
4. have or not the specificity fluorescent speckle to occur at the fluorescence microscopy Microscopic observation, be recorded on the test data sheet paper and use Spearman Karber method and calculate virus titer;
(5) result effectively judges: negative hole fluorescence is negative in all tests; Must be in normal scope with reference to contrast.
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 synchronously inoculation, goes down to posterity by 1: 3, wherein 1 bottle as negative control, 2 bottles go down to posterity after with MOI value 0.1 virus inoculation, put in 37 ℃ of Rotary Machines and cultivate, inoculate and gather in the crops virus liquid 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, 2 bottles after cell covers with monolayer with MOI0.1 Pigs Inoculated Pestivirus, add maintenance medium, put in 37 ℃ of Rotary Machines and cultivate, inoculate and gathers in the crops virus liquid after 5 days, freeze thawing 2 times, mensuration viral level (TCID
50).
1.2MOI research
Select well-grown PT cell, press MOI value 0.01,0.05,0.1,0.3 and 0.5 access virus liquid, 2 rolling bottles of each MOI value inoculation are established a negative contrast of rolling bottle simultaneously.Inoculate and gather in the crops virus liquid after 5 days, viral level (TCID is measured in freeze thawing 2 times
50).
1.3 the virus harvest time, the research of harvesting frequency
Select well-grown PT cell, press MOI value 0.1 and add the hog cholera venom, connecing poison rear 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 rear 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 2.1 connect malicious mode result of study, monolayer connects malicious mode swine fever virus titre and is higher than and connects synchronously malicious mode, sees table 1 for details.
Table 1 is synchronously inoculated comparative result with monolayer
2.2MOI the 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 2MOI result of study
2.3 virus 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 for the third time result of study behind table 5 virus inoculation
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, changes liquid every results on the 4th later on, and 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, test sample: the method according to the embodiment of the invention 2 has prepared three batches of live vaccines of hog cholera (cell line source), and 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 as can be seen from Table 8 three batches of live vaccines of hog cholera (cell source) that primary cell is made, differences between batches are large, and viral level is low, use three batches of live vaccines of hog cholera (cell line source) that 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 2 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 antibody against swine fever virus detection 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 antibody against swine fever virus detection kit in rear 21 days and 27 days in immunity and detect (blocking-up rate 〉=40% is antibody positive); Immunity was extracted immune animal (5/batches) in rear 28 days and is carried out counteracting toxic substances with control animal (3).
Three, result of the test
Rear 21 days of immunity is 85% for examination vaccine (cell line source) Antibody qualification rate, and control vaccine (cell source) Antibody qualification rate is 68%; Immunity was 91% for examination vaccine (cell line source) Antibody qualification rate in rear 27 days, control vaccine (cell source) Antibody qualification rate is 76%, shows for examination vaccine (cell line source) antibody to tell on significantly better than control vaccine (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).
Sensitivity and the specific test of the immunofluorescence method check live vaccines of hog cholera of test example 5 using monoclonal antibodies
One, the sensitivity of immunofluorescent detection method:
The sensitivity check: with cell maintenance medium CSFV being diluted is 10
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 simultaneously inoculation does not 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 can't detect 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 immunofluorescent detection method:
Specific 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 can't detect 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), extension rate is that 10,000 times of group testing results are defective, and extension rate is that to organize testing results be qualified (viral level is lower than the former) for 50,000 times and 100,000 times.Illustrate with rabbit body-shaping thermal response method and survey poison, the result is subject to rabbit body individual variation and weather condition impact, and the rabbit full-boiled process is surveyed poison and belonged to a kind of nonspecific reaction, and surveying poison with it can not accurate quantitative analysis, and measures swine fever virus TCID with immunofluorescence method
50Can accurate quantitative analysis.
Table 10
Claims (9)
1. the preparation method of a live vaccines of hog cholera may further comprise the steps: (1), subculture cell line; (2), the good 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), add freeze drying protectant in the virus liquid that detects after qualified and antibiotic is joined Seedling, lyophilization, and get final product;
Wherein the microbial preservation of swine fever attenuated vaccine strain described in the step (2) number is: CGMCC No.3891.
2. according to preparation method claimed in claim 1, it is characterized in that: the training method of the described continuous cell line of step (1) is that monolayer adherence cultivation, suspension culture or immobilization are cultivated.
3. according to preparation method claimed in claim 1, it is characterized in that: the continuous cell line described in the step (1) includes but not limited to: PT cell line, BT cell line, ST cell line, MPK cell line, SK6 cell line, PK 2a cell line, CPK cell line, RKC cell line, MDBK cell line, mdck cell system, CRFK cell line, Vero/slam cell line, Vero-E6 cell line, Marc-145 cell line, bhk cell system, BGM cell line or DK cell line.
4. according to preparation method claimed in claim 1, it is characterized in that: in the step (3) live vaccines of hog cholera production kind poison is inoculated on the cultured continuous cell line with infective dose 0.1-0.5 and cultivates.
5. according to preparation method claimed in claim 1, it is characterized in that: in the step (3) live vaccines of hog cholera production kind poison is inoculated on the cultured continuous cell line 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.
6. according to preparation method claimed in claim 1, it is characterized in that: when adopting immunofluorescence method to measure the malicious valency of virus of proliferation in the step (4), the monoclonal antibody that adopts is the swine fever virus resistant monoclonal antibody; The fluorescent marker that adopts is FITC or IPMA; Used fixative is 80% acetone, 10% formaldehyde or 75% ethanol of pre-cooling.
7. according to preparation method claimed in claim 1, it is characterized in that: when adopting immunofluorescence method to measure the malicious valency of virus of proliferation in the step (4), its step comprises: (a) passage, prepare Tissue Culture Plate: choose well-grown 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, access 96 porocyte culture plates with the 100ul/ hole; (b) dilution of vaccine virus sample: live vaccines of hog cholera viral suspension that will titration is made 10 times of doubling dilutions; (c) virus inoculation: access 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 ° of C ± 1 ° C, 4～6%CO
2Cultivated in the incubator 3～5 days; (d) 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 ° of C 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 ° of C and hatch 45～60min, remove primary antibodie, with PBST washing 3 times, every hole adds the anti-Mus two anti-50 μ L of FITC labelling of working concentration, 37 ° of C of black out are hatched 30～45min, remove two and resist, with PBST washing 3 times; 4. have or not the specificity fluorescent speckle to occur at the fluorescence microscopy Microscopic observation, be recorded on the test data sheet paper and use Spearman Karber method and calculate virus titer; (e) result effectively judges: negative hole fluorescence is negative in all tests; Must be in normal scope with reference to contrast.
8. the live vaccines of hog cholera that is obtained by any one preparation method of claim 1-7.
9. the purposes of live vaccines of hog cholera claimed in claim 8 in preparation prevention or treatment swine fever biological product.
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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 |
| CN102727882B (en) * | 2011-10-27 | 2014-05-14 | 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 |
| KR20140036262A (en) * | 2011-05-27 | 2014-03-25 | 시노벳 (베이징) 바이오테크놀로지 컴퍼니 리미티드 | Combined vaccines for prevention of porcine virus infections |
| CN102727883B (en) * | 2011-05-27 | 2014-05-14 | 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 |
| CN103083653B (en) * | 2011-10-28 | 2015-07-22 | 辽宁成大动物药业有限公司 | Method for producing classical swine fever live vaccine by using microcarrier high-density cell culture technology |
| CN103777009B (en) * | 2012-10-18 | 2016-03-02 | 辽宁成大动物药业有限公司 | A kind of method using horseradish peroxidase labeling antibody to detect the weak malicious virus titer of swine fever |
| CN102994445B (en) * | 2012-12-14 | 2015-10-07 | 山东滨州沃华生物工程有限公司 | A kind of indirect immunofluorescene assay technology of using carries out the method for screening in viral sensitive cells clone |
| CN103105493A (en) * | 2013-02-07 | 2013-05-15 | 新疆天康畜牧生物技术股份有限公司 | Novel method for replacing detection of rabbit thermal response of hog cholera lapinized virus strain |
| CN103698518A (en) * | 2013-12-30 | 2014-04-02 | 山东滨州博莱威生物技术有限公司 | Method for detecting virus content of swine fever live vaccine through indirect immunofluorescence |
| CN103954760A (en) * | 2014-04-11 | 2014-07-30 | 中国兽医药品监察所 | Method used for evaluating immune efficacy of cell line sourced hog cholera live vaccine via detecting virus cell infective dose |
| CN105734007B (en) * | 2016-03-10 | 2019-06-07 | 金宇保灵生物药品有限公司 | A method of the screening MDBK cell line sensitive to bovine viral diarrhoea/mucosal virus |
| CN107201334B (en) * | 2016-03-16 | 2021-02-19 | 金宇保灵生物药品有限公司 | Bovine kidney cell capable of suspension culture and suspension culture method and application thereof |
| CN106344917A (en) * | 2016-10-17 | 2017-01-25 | 四川华神兽用生物制品有限公司 | Swine fever mucosal immunity live vaccine composition and preparation method of vaccine |
| CN108020664A (en) * | 2017-12-06 | 2018-05-11 | 中国水产科学研究院珠江水产研究所 | Detect the kit and method of viral level in II type grass carp reovirus vaccines |
| CN115877015B (en) * | 2023-01-09 | 2023-08-15 | 华南农业大学 | Method for monitoring antigen expression quantity of baculovirus expression system in suspension culture |
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