CN109453373A - A kind of ablation method of foot and mouth disease virus - Google Patents
A kind of ablation method of foot and mouth disease virus Download PDFInfo
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- CN109453373A CN109453373A CN201710796711.8A CN201710796711A CN109453373A CN 109453373 A CN109453373 A CN 109453373A CN 201710796711 A CN201710796711 A CN 201710796711A CN 109453373 A CN109453373 A CN 109453373A
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- mouth disease
- disease virus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/10—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person
- A61K41/17—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person by ultraviolet [UV] or infrared [IR] light, X-rays or gamma rays
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5252—Virus inactivated (killed)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
- C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention provides a kind of ablation methods of foot and mouth disease virus, comprising the following steps: Psoralens resistance compound is added in foot and mouth disease virus cell culture fluid, carries out lighting process then to get the foot and mouth disease virus solution of inactivation.The present invention have it is following the utility model has the advantages that 1, greatly simplify inactivation technology and shorten the inactivation technology time；2, inactivation technology stability is improved；3, antigen losses are reduced；4, nontoxic compared to other inactivator psoralens, immune side reaction will not be caused to animal；5, play the role of eliminating other bacteriums and virus, be conducive to the resistance tocrocking for improving vaccine, improve product quality, extend storage time.
Description
Technical field
The present invention relates to foot and mouth disease virus inactivation technology fields, and in particular to a kind of ablation method of foot and mouth disease virus.
Background technique
Aftosa (FMD) is acute one kind as caused by foot and mouth disease virus (FMDV), hot, high degree in contact infectiousness and can
The animal epidemic of quick long-distance communications, infecting object is that mainly poultry kind and other domestic and wild artiodactyl are dynamic for pig, ox, sheep etc.
Object, susceptible animal up to more than 70.Currently, it is also most successful arrange that vaccine immunity, which is most important in many anti-measures processed of aftosa,
One of apply, wherein conventional inactivated vaccine is the main vaccine that prevention and control are immunized in current aftosa.
Inactivated vaccine is that will have infective pathogen either physically or chemically to be inactivated with appropriate, loses it and causes a disease
Property, but still retain its immunogenicity, for manufacturing vaccine.Therefore, inactivator appropriate is selected, is gone out to foot and mouth disease virus
Work is the important link of inactivated vaccine manufacture.The inactivation technology of foot and mouth disease virus is used at present third in binary ethylenimine (BEI) and β
The inactivation mode of ester, this method is although widely used in producing but will cause higher antigen losses and inactivation time is long, work
Skill control is complicated, is the key factor for causing current aftosa inactivated virus vaccine production efficiency low.
Psoralen is also known as furocoumarin, is now widely used in Blood disinfection, can not only inactivate in blood
Known and unknown pathogenic microorganism, can also remove single blood constituent such as leucocyte in blood plasma, blood platelet and cell because
Son avoids the generation of transfusion reaction.However, tiny nucleic acid virus is resistant for the deactivation of psoralen, this be because
Very fine and close for this viral capsid, psoralen is difficult to pass through.
And foot and mouth disease virus is as a kind of tiny nucleic acid virus, under conventional reaction condition, psoralen molecules can not
Into inside virion so that photo-crosslinking RNA make it is virally inactivated；And foot and mouth disease virus is sensitive to environmental condition, it is unstable, often
The inactivation condition of rule be easy to cause antigen losses.Therefore there has been no the reports that foot and mouth disease virus inactivation is carried out using psoralen at present
Road.
Summary of the invention
For the defects in the prior art, the present invention provides a kind of ablation methods of foot and mouth disease virus.
The purpose of the present invention is what is be achieved through the following technical solutions:
The present invention provides a kind of ablation methods of foot and mouth disease virus, comprising the following steps: trains in hoof-and-mouth disease poison cell
Psoralens resistance compound is added in nutrient solution, carries out lighting process then to get the foot and mouth disease virus solution of inactivation.
Preferably, the conductance of the virocyte culture liquid system is 0.5~15mS/cm；More preferable conductance is 1~
10mS/cm.The conductance of the virocyte culture solution is too low, will lead to viral dissociation；Conductance is excessively high, will lead to psoralen point
Son cannot be introduced into.
Preferably, after Psoralens resistance compound is added in the virocyte culture solution, during carrying out ultraviolet lighting, control
Virocyte culture-liquid temp is 26~38 DEG C.Temperature is excessively high and processing overlong time will lead to proteasome degradation virus, and warm
It spends the low psoralen molecules that are unfavorable for and enters virus.
Preferably, the molar concentration of the Psoralens resistance compound of the addition is the 100- of foot and mouth disease virus molar concentration
20000 times.
It is highly preferred that the molar concentration of the Psoralens resistance compound of the addition is foot and mouth disease virus molar concentration
200-10000 times.The concentration of the psoralen is 1~100mg/L.Psoralen molecules when this concentration is than in range,
It can guarantee that opposite virus concentration is absolutely excessive, conducive to the progress of inactivation reaction, prevented also from other impurities molecule to psoralen
The absorption of molecule and non-specific crosslinking cause relative concentration to reduce, and are unfavorable for the inactivation of virus.
Preferably, the Psoralens resistance compound be psoralen and its derivative, specifically include AMT, S-59, TMP,
8-MOP。
It is highly preferred that the Psoralens resistance compound is AMT, S-59.
Preferably, the parameter that the lighting process specifically uses includes: a length of 300-400nm of light wave, more preferable 340-
380nm, Values In Ultraviolet Irradiance exposure dose 3-8mW/cm2, the processing time is 10-100 minutes.
Preferably, the method that the lighting process specifically uses is to lead to the virocyte culture solution added with psoralen
Cross the long and narrow pipeline penetrated with ultraviolet light.
Ablation method of the present invention is applicable not only to foot and mouth disease virus, applies also for the inactivation of other viruses, equally
It can reach effect of the invention.
The prior art is compared, the present invention have it is following the utility model has the advantages that
1, inactivation technology is greatly simplified, the inactivation technology time is shortened；
2, the stability of inactivation technology is improved；
3, antigen losses are reduced；
4, other inactivators are compared, psoralen is nontoxic, and immune side reaction will not be caused to animal；
5, play the role of eliminating other bacteriums and virus, be conducive to the resistance tocrocking for improving vaccine, improve product quality, prolong
Long storage time.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection scope.
The embodiment provides a kind of ablation methods of foot and mouth disease virus, comprising the following steps: in hoof-and-mouth disease
Psoralens resistance compound is added in poison cell culture solution, carries out lighting process then to get the foot and mouth disease virus solution of inactivation.
The conductance of the virocyte culture liquid system is 0.5~15mS/cm；More preferable conductance is 1~10mS/cm.
After Psoralens resistance compound is added in the virocyte culture solution, during carrying out ultraviolet lighting, it is thin to control virus
Born of the same parents' culture-liquid temp is 26~38 DEG C.Temperature is excessively high and processing overlong time will lead to proteasome degradation virus, and temperature is too low
It is unfavorable for psoralen molecules to enter inside viruses molecule.
The molar concentration of the Psoralens resistance compound of the addition is the 100-20000 of foot and mouth disease virus molar concentration
Times.More preferably 200-10000 times.
The Psoralens resistance compound is psoralen and its derivative, specifically includes AMT, S-59, TMP, 8-MOP.
The Psoralens resistance compound is AMT, S-59.
The parameter that the lighting process specifically uses includes: a length of 340-380nm of light wave, Values In Ultraviolet Irradiance 3-8mW/cm2, place
Managing the time is 10-100 minutes.
The method that the lighting process specifically uses is will to pass through to have added with the virocyte culture solution of psoralen
The long and narrow pipeline that ultraviolet light penetrates.
Embodiment 1
The ablation method for present embodiments providing a kind of aftosa vaccine carries out scale virus using psoralen photochemistry
Antigens inactive, the specific steps are as follows:
(1) the A type foot and mouth disease virus cell culture fluid of BHK21 cell suspension cultures obtains after 0.22 μm of micro-filtrate membrane filtration
Clarify virocyte culture solution.
(2) virocyte culture solution is concentrated 10 times through 300kD hollow-fibre membrane, and with PB solution filter wash virocyte solution
Its conductance is reduced to 1mS/cm.
(3) psoralen S-59 extremely clarification virocyte culture solution is added to final concentration 0.625mL/L.The S-59's rubs
Your concentration is 200 times of foot and mouth disease virus molar concentration.
(4) the virocyte culture solution added with psoralen is passed through into transparent pipeline, and applying Values In Ultraviolet Irradiance is 8mW/
cm2Ultraviolet light flows through the residence time holding of the virocyte culture solution of transparent pipe under ultraviolet light 10 minutes, maintains simultaneously
Temperature is at 38 DEG C.
(5) the virocyte culture solution after the present embodiment method carries out ultraviolet irradiation is according to plaque ethods measurement TCID50
0；The virocyte culture solution 146S content of sucrose density gradient method measurement inactivation front and back is respectively 51.31 μ g/mL and 50.29 μ
g/mL。
Embodiment 2
The ablation method for present embodiments providing a kind of aftosa vaccine carries out scale virus using psoralen photochemistry
Antigens inactive, the specific steps are as follows:
(1) the O-shaped foot and mouth disease virus cell culture fluid of BHK21 attached cell shaking flask culture is at 4 DEG C, through 6000g centrifugation 15
Clarification virocyte culture solution is obtained after minute.
(2) solution conductivity is adjusted to 10mS/cm using PB solution.
(3) psoralen AMT extremely clarification virocyte culture solution is added to final concentration 5mg/L.The molar concentration of the AMT
It is 20000 times of foot and mouth disease virus molar concentration.
It (4) is 3mW/cm with Values In Ultraviolet Irradiance by the virocyte culture solution added with psoralen2Ultraviolet light irradiates simultaneously
Maintain temperature 100 minutes at 26 DEG C.
(5) the virocyte culture solution after ultraviolet irradiation is carried out through the present embodiment method measure TCID50 according to plaque ethods
It is 0；The virocyte culture solution 146S content of sucrose density gradient method measurement inactivation front and back is respectively 4.33 μ g/mL and 4.31 μ
g/mL。
Embodiment 3
A kind of ablation method of aftosa vaccine is present embodiments provided, specific steps are substantially the same manner as Example 2, different
Place is only that: the Psoralens resistance compound that the present embodiment uses is 8-MOP.
Carrying out the virocyte culture solution after ultraviolet irradiation according to plaque ethods measurement TCID50 through the present embodiment method is
10-1.5；The virocyte culture solution 146S content of sucrose density gradient method measurement inactivation front and back is respectively 3.35 μ g/mL and 2.1
μg/mL。
Embodiment 4
A kind of ablation method of aftosa vaccine is present embodiments provided, specific steps are substantially the same manner as Example 1, different
Place is only that: the step 2 that the present embodiment uses is utilizes PB solution to adjust solution conductivity to 0.5mS/cm.
The virocyte culture solution after ultraviolet irradiation, which is carried out, through the present embodiment method measures TCID50 not according to plaque ethods
It is 0；The virocyte culture solution 146S content of sucrose density gradient method measurement inactivation front and back is respectively 22.30 μ g/mL and 20.04
μg/mL。
There are many invention concrete application approach, the above is only a preferred embodiment of the present invention.It should be pointed out that above real
It applies example and is merely to illustrate the present invention, and the protection scope being not intended to restrict the invention.For the ordinary skill of the art
For personnel, without departing from the principle of the present invention, several improvement can also be made, these improvement also should be regarded as the present invention
Protection scope.
Claims (9)
1. a kind of ablation method of foot and mouth disease virus, which comprises the following steps: in foot and mouth disease virus cell culture fluid
Then middle addition Psoralens resistance compound carries out lighting process to get the foot and mouth disease virus solution of inactivation.
2. the ablation method of foot and mouth disease virus according to claim 1, which is characterized in that the virocyte culture solution
Conductance is 0.5~15mS/cm.
3. the ablation method of foot and mouth disease virus according to claim 1, which is characterized in that the virocyte culture solution adds
After entering Psoralens resistance compound, control culture-liquid temp is 26~38 DEG C.
4. the ablation method of foot and mouth disease virus according to claim 1, which is characterized in that the Psoralens resistance of the addition
The molar concentration of compound is 100-20000 times of foot and mouth disease virus molar concentration.
5. the ablation method of foot and mouth disease virus according to claim 4, which is characterized in that the Psoralens resistance of the addition
The molar concentration of compound is 200-10000 times of foot and mouth disease virus molar concentration.
6. the ablation method of foot and mouth disease virus according to claim 1, which is characterized in that the Psoralens resistance compound
For psoralen and its derivative, AMT, S-59, TMP, 8-MOP are specifically included.
7. the ablation method of foot and mouth disease virus according to claim 6, which is characterized in that the Psoralens resistance compound
For AMT, S-59.
8. the ablation method of foot and mouth disease virus according to claim 1, which is characterized in that the lighting process specifically uses
Parameter include: a length of 300-400nm of light wave, Values In Ultraviolet Irradiance 3-8mW/cm2, the processing time is 10-100 minutes.
9. the ablation method of foot and mouth disease virus according to claim 1, which is characterized in that the lighting process specifically uses
Method be that the virocyte culture solution added with psoralen is passed through into the long and narrow pipeline that penetrates with ultraviolet light.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710796711.8A CN109453373B (en) | 2017-09-06 | 2017-09-06 | Method for inactivating foot-and-mouth disease virus |
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| CN201710796711.8A CN109453373B (en) | 2017-09-06 | 2017-09-06 | Method for inactivating foot-and-mouth disease virus |
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| CN109453373A true CN109453373A (en) | 2019-03-12 |
| CN109453373B CN109453373B (en) | 2021-08-17 |
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| US6004742A (en) * | 1993-06-28 | 1999-12-21 | Cerus Corporation | Method for inactivation of pathogens in platelets using 4' and 5' primary amino-substituted psoralens |
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