CN102018957B - Production method of hog cholera live vaccine - Google Patents
Production method of hog cholera live vaccine Download PDFInfo
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- CN102018957B CN102018957B CN 201010237909 CN201010237909A CN102018957B CN 102018957 B CN102018957 B CN 102018957B CN 201010237909 CN201010237909 CN 201010237909 CN 201010237909 A CN201010237909 A CN 201010237909A CN 102018957 B CN102018957 B CN 102018957B
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Abstract
The invention relates to a production method of hog cholera live vaccine, comprising the following technical steps: (1) cloning and identifying a hog cholera lapinized low virulent strain; (2) breeding cloned strain seed virus; (3) breeding cells for production; (4) breeding virus liquid for production; and (5) matching vaccine, carrying out split charging and freeze-drying. The method provided by the invention has the characteristics of simple and stable production technology, good virus immunogenicity and genetic stability, small risk of exogenous virus pollution, low production cost and the like, and is convenient for inspection. The hog cholera live vaccine produced by the production method has the advantages of good genetic stability, high purity and strong immunogenicity.
Description
Technical field
The present invention relates to a kind of production method of live vaccines of hog cholera, belong to the veterinary biologics technical field.
Background technology
Swine fever (Classical Swine Fever; CSF) be by swine fever virus (Classical Swine Fever Virus; CSFV) a kind of height contact that causes, the pig infectious disease of lethal; All there is generation a lot of in the world countries and regions, have brought serious economy loss to animal husbandry production.OIE (OIE) classifies swine fever as legal circular property eqpidemic disease, and China divides it into type of zoonosis.
Mainly contain 3 kinds of effects live vaccines of hog cholera preferably at present in the world; The rabbitization low virulent strain (C strain) of promptly Japanese GPE strain, the Thiverval strain of France and China, wherein the rabbitization low virulent strain is used the widest model also for significant contribution has been made in the control and the elimination of many national swine fevers in the world.At present, China also carries out the main means of compulsory immunization swine fever attenuated live vaccines as the control swine fever.
Though three kinds of vaccines all can be effective to the anti-system of swine fever, also all exist certain deficiency.Wherein GPE strain, Thiverval strain are produced with pig source continuous cell line, and its risk that exists known or unknown exogenous virus to pollute is bigger, in case control badly, are easy to cause certain existing or newfound infectious disease large tracts of land popular.Wherein the rabbitization attenuated live vaccines has two kinds of production technologies at present; First kind of technology is with the production of rabbit body, gets tissue or lymph node or the spleen seedling of inoculation rabbit, and this technology can effectively avoid exogenous virus to pollute; Guaranteed viral hereditary stability simultaneously; Virus virulence does not return by force, but needs to use a large amount of rabbit, and the quality control difficulty is higher and production cost is higher; Second kind of technology is to use cattle, sheep primary cell or the production of pig passage cell; This technology avoids using in a large number laboratory animal; But the risk (because cattle, sheep primary cell or pig passage cell are to a lot of known or unknown exogenous virus susceptibles) and the viral hereditary stability that exist exogenous virus to pollute are poor slightly, and virus has independently returns strong risk.In addition; Hog cholera lapinised virus forms through rabbit body continuous passage domestication; Kind of poison does not pass through clone purification, the kind poison of dimension different batches to the kind poison of a collection of rabbit or same batch to the rabbit of different batches infectious and immunogenicity often there is some difference.This shows, need a kind of new live vaccines of hog cholera production technology badly and overcome the deficiency that existing several kinds of vaccines exist.
Summary of the invention
The objective of the invention is to overcome the weak point of the existing production technology of live vaccines of hog cholera, provide a kind of and carry out cell culture, gather in the crops the method that viral liquid is produced live vaccines of hog cholera with cloning hog cholera lapinised virus kind poison inoculation rabbit source cell or cell commonly used.This method have production technology simple, be easy to characteristics such as quality control, the exogenous virus pollution risk is little, production cost is low.Good, the pure property of the live vaccines of hog cholera hereditary stability of utilizing the present invention to produce is high, virus titer is high, immunogenicity is strong.
Technical scheme of the present invention is: a strain that obtains through viral terminal point dilution technology with the hog cholera lapinised virus strain virus obtains highly to adapt to rabbit source cell or cell commonly used, genotype is single and the pure lines hog cholera lapinised virus clone strain CSFVCC-1 of ability genetic stability; This Strain has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 26th, 2010; Preserving number is CGMCC No.4058; CSFV CC-1 is carried out cell culture through rabbit source cell and/or cell commonly used, gather in the crops viral liquid; The viral liquid of results is added freeze drying protectant commonly used, fully mix the back packing after lyophilisation and process freeze-dried live vaccine, every part toxic amount is not less than 6000TCID
50Or 400RID.
Detailed description of the present invention:
The clone of 1 hog cholera lapinised virus and evaluation
1.1 the clone of hog cholera lapinised virus
Carry out with the terminal point dilution method.
(1) in 24 porocyte plates, inserts the cell culture wave carrier piece, and inoculate an amount of rabbit source cell or cell commonly used, at CO
237 ℃ of incubators with cell culture to monolayer;
(2) swine fever virus rabbitization low virulent strain kind poison (China Veterinery Drug Inspection Office's preservation and supply, CVCC AV1412) is diluted to 0.1TCID
50/ 0.1ml is with 0.1ml/ hole dose inoculation (1) cell plates;
(3) put CO
2Incubator is cultivated 3d for 37 ℃, takes out each hole culture fluid and cell wave carrier piece;
(4) the cell wave carrier piece is fixed, carried out the swine fever fluorescent antibody staining;
(5) culture fluid with the positive hole of fluorescence staining dilutes 24 orifice plates that the cell culture wave carrier piece is inserted in inoculation again, carries out time cloning again;
(6) carry out repeatedly 3～5 times, promptly obtain cloning hog cholera lapinised virus kind poison, called after CSFV CC-1 (this strain has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 26th, 2010, protecting minus sign is CGMCC No.4058);
1.2 the evaluation of clone strain
Through identifying that clone strain CSFV CC-1 has following characteristic:
(1) adaptability of pair cell: CSFV CC-1 inoculation rabbit source cell is carried out cell culture and sampling and measuring poison valency, cultivate the highest malicious valency>=10 through rolling bottle
6.0TCID
50/ 0.1ml is through microcarrier or the highest malicious valency of suspension culture>=10
6.8TCID
50/ 0.1ml; CSFV CC-1 inoculation cell commonly used is carried out cell culture and sampling and measuring poison valency, cultivate the highest malicious valency>=10 through rolling bottle
5.0TCID
50/ 0.1ml is through microcarrier or the highest malicious valency of suspension culture>=10
6.0TCID
50/ 0.1ml;
(2) hereditary stability: through the continuous passage of rabbit source cell and carry out gene sequencing, the 30th generation and the 1st generation cloning kind poison nucleotide homology are 99.9% with CSFV CC-1, and amino acid identity is 100%; Through using the cell continuous passage always and carrying out gene sequencing, the 30th generation and the 1st generation cloning kind poison nucleotide homology are 99.5% with CSFV CC-1, and amino acid identity is 99.9%;
(3) biological characteristics: CSFV CC-1 is carried out the hog cholera antibody inhibition test, and the fluorescence that hog cholera antibody ability specificity suppresses this kind poison produces; With CSFV CC-1 with 15TCID
50(or 1RID)/dosage intravenous inoculation healthy rabbits only 95% produces typing heat or light-duty heat as a result;
(4) immunogenicity: with the 150 RID/ non-immune health pig of immunizing dose intramuscular inoculation only, antibody on average the highest titre reaches 1: 128 with CSFV CC-1, and average duration is 400d; 2 weeks are attacked 1ml swine fever crossdrift blood poison (10 in the immunity back
5MLD/1ml), 100% protection, high fever≤41% and continue to be no more than 3d;
(5) safety: with CSFV CC-1 (>=10
5.0TCID
50/ 0.1ml) 10ml injection rabbit, 100ml injection pig, 5ml injection Cavia porcellus, 1ml injection mice all do not cause death;
The preparation of 2 vaccines
2.1 plant the breeding of poison
With the seed lot cell of CSFV CC-1 seed lot kind poison well-grown rabbit source cell of inoculation or cell commonly used, cultivate and receive poison after 3～4 days, as producing with kind of a poison;
Kind of poison check: by " People's Republic of China's veterinary drug allusion quotation " (Chinese veterinary drug allusion quotation committee. three ones of in 2005 versions of People's Republic of China's veterinary drug allusion quotation. Chinese agriculture publishing house; 2006; The present invention is hereinafter to be referred as " Chinese veterinary drug allusion quotation ") method of regulation tests, should meet steriling test and exogenous virus touchstone and stipulate;
2.2 produce going down to posterity and cultivating with cell
The seed lot cell carries out cell culture after digestion, disperseing, when forming monolayer or growing to proper density, be used to continue to go down to posterity or virus inoculation;
Cellular assay: the method by " Chinese veterinary drug allusion quotation " regulation is tested, and answers accords with production, check to use the cell standard code;
2.3 produce breeding with viral liquid
To produce kind of the well-grown production of poison inoculation and use cell, and carry out cell culture, the viral liquid of results is preserved below-15 ℃ after 3～4 days;
Produce with viral liquid check: the method by " Chinese veterinary drug allusion quotation " regulation is tested, and should meet steriling test and exogenous virus touchstone regulation; And every ml toxic amount is no less than 60000 (or 4000RID);
2.4 join Seedling, packing and lyophilizing
Viral liquid with being up to the standards adds suitable stabilizing agent and antibiotics, mixing, and quantitatively packing, every part toxic amount is not less than 6000TCID
50(or 400RID), lyophilisation;
Product inspection: the method by " Chinese veterinary drug allusion quotation " regulation is tested, and should meet steriling test and exogenous virus touchstone regulation; Inject responsive pigling with 10 parts, should not cause manifest symptom; Every part toxic amount is no less than 6000TCID
50(or 400RID).
Defining of some special terms that the present invention relates to:
1. rabbit source cell: be meant rabbit primary cell or passage cell;
2. cell commonly used: be meant bull testis primary cell, Testis Caprae seu Ovis primary cell, Ren caprae seu ovis primary cell, cattle BT cell line, pig SK-6 cell line, pig ST cell line, pig PK-15 cell line, pig IBRS-2 cell line;
3. cell culture: be phalangeal cell under suitable temperature and density, cultivate or cell microcarrier cultivation or cell suspension cultures through rolling bottle.
Positive effect of the present invention
The present invention obtains highly to adapt to the pure lines kind poison of cell and ability genetic stability through with the hog cholera lapinised virus clone, inoculates cell and carries out cell culture, produces viral liquid, joins Seedling, packing, lyophilizing at last.Compared with prior art, the present invention has the following advantages:
(1) with respect to prior art, clone of the present invention, purification plant poison, guaranteed to plant the heredity and the biological phenotypic unicity of poison, improved the hereditary stability and the biological safety of vaccine;
(2) with respect to prior art, clone of the present invention, purification plant poison, the viral liquid poison valency of production is higher, the vaccine active constituent content in the unit volume is higher;
(3) with respect to prior art, clone of the present invention, purification plant poison, vaccine immunogenicity is more stable, reliable;
(4) with respect to existing rabbit system Seedling technology, production technology of the present invention is simple, has avoided long-time, highly intensive labours such as letting animals feed, observation animal, has reduced production cost, has reduced the risk of antibacterial, mycete and mycoplasma contamination simultaneously.
Specific embodiment
Embodiments of the invention are for further describing technical scheme of the present invention, but are not construed as limiting the invention.
Embodiment 1
The clone of 1 hog cholera lapinised virus and evaluation
1.1 the clone of hog cholera lapinised virus
Carry out with the terminal point dilution method.
(1) in 24 porocyte plates, inserts the cell culture wave carrier piece, press 0.4ml/ hole inoculation RK-13 cell line, at CO
237 ℃ of incubators with cell culture to monolayer;
(2) swine fever virus rabbitization low virulent strain kind poison (CVCC AV1412) is diluted to 0.1TCID
50/ 0.1ml is with 0.1ml/ hole dose inoculation (1) cell plates;
(3) put CO
2Incubator is cultivated 3d for 37 ℃, takes out each hole culture fluid and cell wave carrier piece;
(4) the cell wave carrier piece is fixed, carried out the swine fever fluorescent antibody staining;
(5) culture fluid with the positive hole of fluorescence staining dilutes 24 orifice plates that the cell culture wave carrier piece is inserted in inoculation again, carries out time cloning again;
(6) carry out repeatedly 5 times, promptly obtain cloning hog cholera lapinised virus kind poison, called after CSFV CC-1;
1.2 the evaluation of clone strain
Through identifying that clone strain CSFV CC-1 has following characteristic:
(1) adaptability of pair cell: CSFV CC-1 inoculation RK-13 cell is carried out cell culture and sampling and measuring poison valency, through the highest malicious valency of suspension culture>=10
6.8TCID
50/ 0.1ml;
(2) hereditary stability: through the continuous passage of RK-13 cell and carry out gene sequencing, the 30th generation and the 1st generation cloning kind poison nucleotide homology are 99.9% with CSFV CC-1, and amino acid identity is 100%;
(3) biological characteristics: CSFV CC-1 is carried out the hog cholera antibody inhibition test, and the fluorescence that hog cholera antibody ability specificity suppresses this kind poison produces; With CSFV CC-1 with 15TCID
50(or 1RID)/dosage intravenous inoculation healthy rabbits only 95% produces typing heat or light-duty heat as a result;
(4) immunogenicity: with the 150RID/ non-immune health pig of immunizing dose intramuscular inoculation only, antibody on average the highest titre reaches 1: 128 with CSFV CC-1, and average duration is 400d; 2 weeks are attacked 1ml swine fever crossdrift blood poison (10 in the immunity back
5MLD/1ml), 100% protection, high fever≤41% and continue to be no more than 3d;
(5) safety: with CSFV CC-1 (>=10
5.0TCID
50/ 0.1ml) 10ml injection rabbit, 100ml injection pig, 5ml injection Cavia porcellus, 1ml injection mice all do not cause death;
The preparation of 2 vaccines
2.1 plant the breeding of poison
With the well-grown RK-13 seed lot cell of CSFV CC-1 seed lot kind poison inoculation, cultivate and receive poison after 3～4 days, as producing with kind of a poison;
Plant the poison check: the method by " Chinese veterinary drug allusion quotation " regulation is tested, and the result meets steriling test and the exogenous virus touchstone is stipulated;
2.2 produce going down to posterity and cultivating with cell
Seed lot cell RK-13 carries out cell suspension cultures after digestion, disperseing, when growing to proper density, be used to continue to go down to posterity or virus inoculation;
Cellular assay: the method by " Chinese veterinary drug allusion quotation " regulation is tested, and the cell standard code is used in accords with production, check as a result;
2.3 produce breeding with viral liquid
To produce kind of the well-grown production of poison inoculation and use cell, and carry out cell suspension cultures, the viral liquid of results is preserved below-15 ℃ after 3～4 days;
Produce with viral liquid check: the method by " Chinese veterinary drug allusion quotation " regulation is tested, and the result meets steriling test and exogenous virus touchstone regulation; And every ml toxic amount＞60000TCID
50(or 4000RID);
2.4 join Seedling, packing and lyophilizing
Viral liquid with being up to the standards adds suitable stabilizing agent and antibiotics, mixing, and quantitatively packing, every part toxic amount is not less than 6000TCID
50(or 400RID), lyophilisation;
Product inspection: the method by " Chinese veterinary drug allusion quotation " regulation is tested, and the result meets steriling test and exogenous virus touchstone regulation; Inject responsive pigling with 10 parts, the result does not cause manifest symptom; Every part toxic amount＞6000TCID
50(or 400RID).
Embodiment 1
The clone of 1 hog cholera lapinised virus and evaluation
1.1 the clone of hog cholera lapinised virus
Carry out with the terminal point dilution method.
(1) in 24 porocyte plates, inserts the cell culture wave carrier piece, press 0.4ml/ hole inoculation SK-6 cell line, at CO
237 ℃ of incubators with cell culture to monolayer;
(2) swine fever virus rabbitization low virulent strain kind poison (CVCC AV1412) is diluted to 0.1TCID
50/ 0.1ml is with 0.1ml/ hole dose inoculation (1) cell plates;
(3) put CO
2Incubator is cultivated 3d for 37 ℃, takes out each hole culture fluid and cell wave carrier piece;
(4) the cell wave carrier piece is fixed, carried out the swine fever fluorescent antibody staining;
(5) culture fluid with the positive hole of fluorescence staining dilutes 24 orifice plates that the cell culture wave carrier piece is inserted in inoculation again, carries out time cloning again;
(6) carry out repeatedly 5 times, promptly obtain cloning hog cholera lapinised virus kind poison, called after CSFV CC-1;
1.2 the evaluation of clone strain
Through identifying that clone strain CSFV CC-1 has following characteristic:
(1) adaptability of pair cell: CSFV CC-1 inoculation SK-6 cell is carried out cell culture and sampling and measuring poison valency, through the highest malicious valency of suspension culture>=10
6.0TCID
50/0.1ml;
(2) hereditary stability: through the continuous passage of SK-6 cell and carry out gene sequencing, the 30th generation and the 1st generation cloning kind poison nucleotide homology are 99.5% with CSFV CC-1, and amino acid identity is 99.9%;
(3) biological characteristics: CSFV CC-1 is carried out the hog cholera antibody inhibition test, and the fluorescence that hog cholera antibody ability specificity suppresses this kind poison produces; With CSFV CC-1 with 15TCID
50(or 1RID)/dosage intravenous inoculation healthy rabbits only 95% produces typing heat or light-duty heat as a result;
(4) immunogenicity: with the 150RID/ non-immune health pig of immunizing dose intramuscular inoculation only, antibody on average the highest titre reaches 1: 128 with CSFV CC-1, and average duration is 400d; 2 weeks are attacked 1ml swine fever crossdrift blood poison (10 in the immunity back
5MLD/1ml), 100% protection, high fever≤41% and continue to be no more than 3d;
(5) safety: with CSFV CC-1 (>=10
5.0TCID
50/ 0.1ml) 10ml injection rabbit, 100ml injection pig, 5ml injection Cavia porcellus, 1ml injection mice all do not cause death;
The preparation of 2 vaccines
2.1 plant the breeding of poison
With the well-grown SK-6 seed lot cell of CSFV CC-1 seed lot kind poison inoculation, cultivate and receive poison after 3～4 days, as producing with kind of a poison;
Plant the poison check: the method by " Chinese veterinary drug allusion quotation " regulation is tested, and the result meets steriling test and the exogenous virus touchstone is stipulated;
2.2 produce going down to posterity and cultivating with cell
Seed lot cell SK-6 carries out cell suspension cultures after digestion, disperseing, when growing to proper density, be used to continue to go down to posterity or virus inoculation;
Cellular assay: the method by " Chinese veterinary drug allusion quotation " regulation is tested, and the cell standard code is used in accords with production, check as a result;
2.3 produce breeding with viral liquid
To produce kind of the well-grown production of poison inoculation and use cell, and carry out cell suspension cultures, the viral liquid of results is preserved below-15 ℃ after 3～4 days;
Produce with viral liquid check: the method by " Chinese veterinary drug allusion quotation " regulation is tested, and the result meets steriling test and exogenous virus touchstone regulation; And every ml toxic amount＞60000TCID
50(or 4000RID);
2.4 join Seedling, packing and lyophilizing
Viral liquid with being up to the standards adds suitable stabilizing agent and antibiotics, mixing, and quantitatively packing, every part toxic amount is not less than 6000TCID
50(or 400RID), lyophilisation;
Product inspection: test by " Chinese veterinary drug allusion quotation ", the result meets steriling test and exogenous virus touchstone regulation; Inject responsive pigling with 10 parts, the result does not cause manifest symptom; Every part toxic amount＞6000TCID
50(or 400RID).
Claims (2)
1. live vaccines of hog cholera; It is characterized in that this vaccine contains hog cholera lapinised virus clone strain CSFV CC-1 virus; This Strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 26th, 2010, and preserving number is CGMCC No.4058.
2. method for preparing of making the described a kind of live vaccines of hog cholera of claim 1; It is characterized in that preparing the used strain CGMCC No.4058 hog cholera lapinised virus clone strain virus of live vaccines of hog cholera and carry out cell culture, gather in the crops viral liquid through rabbit source cell and/or cell commonly used; The viral liquid of results is added freeze drying protectant commonly used, fully mix the back packing after lyophilisation and process freeze-dried live vaccine, every part toxic amount is not less than 6000TCID
50Or 400RID.
Priority Applications (1)
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|---|---|---|---|
| CN 201010237909 CN102018957B (en) | 2010-07-28 | 2010-07-28 | Production method of hog cholera live vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010237909 CN102018957B (en) | 2010-07-28 | 2010-07-28 | Production method of hog cholera live vaccine |
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| CN102018957A CN102018957A (en) | 2011-04-20 |
| CN102018957B true CN102018957B (en) | 2012-09-05 |
Family
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| CN 201010237909 Expired - Fee Related CN102018957B (en) | 2010-07-28 | 2010-07-28 | Production method of hog cholera live vaccine |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102221618B (en) * | 2011-06-23 | 2013-07-31 | 中国兽医药品监察所 | Method for establishing hog cholera lapinized virus labeled vaccine strain and preparing vaccine |
| CN102327610A (en) * | 2011-09-27 | 2012-01-25 | 南京创启生物科技有限公司 | Method by utilizing rabbit-origin cells to produce swine fever live vaccine |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1405328A (en) * | 2002-11-08 | 2003-03-26 | 武汉大学 | Fluorescent quantitative PCR kit for rapid and quantitative detecting hog choleravirus and hog cholera lapinized vaccine and its use |
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2010
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1405328A (en) * | 2002-11-08 | 2003-03-26 | 武汉大学 | Fluorescent quantitative PCR kit for rapid and quantitative detecting hog choleravirus and hog cholera lapinized vaccine and its use |
Non-Patent Citations (2)
| Title |
|---|
| 戴志红 等.猪瘟病毒FJFQ-39株的毒力鉴定.《中国预防兽医学报》.2008,第30卷(第9期),694-726. * |
| 赵耘 等.猪瘟病毒猪细小病毒猪繁殖与呼吸综合征病毒猪_省略_狂犬病病毒多重PCR方法的建立.《中国兽医杂志》.2007,第43卷(第2期),3-6. * |
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