CN106466295A - A kind of vaccine diluent and preparation method and application - Google Patents

A kind of vaccine diluent and preparation method and application Download PDF

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Publication number
CN106466295A
CN106466295A CN201610903368.8A CN201610903368A CN106466295A CN 106466295 A CN106466295 A CN 106466295A CN 201610903368 A CN201610903368 A CN 201610903368A CN 106466295 A CN106466295 A CN 106466295A
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vaccine
live
diluent
levamisole
carbomer
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周远成
王敖
蔡雨函
李碧
牛婷
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Sichuan Huashen Animal Biolog Products Co Ltd
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Sichuan Huashen Animal Biolog Products Co Ltd
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    • C12N2770/00011—Details
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    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

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Abstract

The present invention relates to animal biological product field, in particular to a kind of vaccine diluent and preparation method and application, described vaccine diluent includes following component：Carbomer 0.1～100g/L and levamisole 0.1～200g/L；Solvent for use is any one in water, normal saline or phosphate buffer.This vaccine diluent can solve the problems, such as vaccine immunity after that NAT is low, generation time length, antibody are held time is short, poultry attenuated live vaccines can be coordinated to use, such as single Seedling of virus type live vaccine such as live vaccines of hog cholera, pseudorabies disease live-vaccine, Porcine reproductive and respiratory syndrome live vaccine, porcine epizootic diarrhea live vaccine, transmissible gastroenteritis of swine live vaccine, porcine rotaviruses disease live-vaccine, fowlpox live vaccine, chicken new city live vaccine and connection Seedling.

Description

A kind of vaccine diluent and preparation method and application
Technical field
The present invention relates to animal biological product field, in particular to a kind of vaccine diluent and preparation method thereof with Application.
Background technology
Currently for all no specific medicine of common animal viral disease, therefore vaccine immunity is optimal Preventions.Clinically common livestock and poultry virus class live vaccine mainly has live vaccines of hog cholera, pseudorabies disease live-vaccine, pig numerous Grow and respiration syndrome live vaccine, porcine epizootic diarrhea live vaccine, transmissible gastroenteritis of swine live vaccine, porcine rotavirus disease work epidemic disease Seedling, fowlpox live vaccine, chicken Newcastle disease live vaccine etc..All produced using lyophilized technique in addition to indivedual special kinds in live vaccine, because This is used after being required to when using be dissolved using vaccine diluent.The diluent of all kinds of live vaccine mainly has normal saline and phosphorus Phthalate buffer.Now widely used vaccine diluent is poor with reference to antibody generation time length, entirety degree after corresponding vaccine immunity, After inoculation, swinery herd immunity antibody horizontal dispersion is big, have impact on the immune effect of vaccine.
Taking Pseudorabies viruses as a example, research show using current popular strain preparation gene-deleted vaccine immunity latter 28 days To China, new epidemic isolates and popular conventional strain all can be protected completely.But PRV needs just to be provided that within latter 28 days about in immunity 100% immune protective efficiency, and after immunity, 1,2,3 weeks immune protective rates are respectively 5%, 67% and 92%.Clinically generally adopt Carry out initial immunity with 1～3 age in days collunarium or carry out intramuscular injection immunity, therefore according to both immunity sides in 6～10 week old Formula, piglet just avoids the infection of PRV completely in 29～31 ages in days or 10～14 week old.Current immunization protocol exists for a long time The Blank immunization phase, and this Blank immunization phase be PRV infection and morbidity peak period.In addition, clinically selected swine farms are pressed Significantly raise according to marketing period growing and fattening pigs gE antibody positive rate after the immunity of existing immune programme for children, be indicated above existing part vaccine immunity Duration shorter it is impossible to provide immunoprotection over a long time for growing and fattening pigs.In view of outstanding vaccine diluent is in dilution vaccine Also there are protection and maintenance effect to vaccine valence simultaneously, thereby may be ensured that the immune effect of vaccine.Therefore, for solving existing epidemic disease Variety of problems existing for Seedling, provides a kind of new generation vaccine diluent to be highly desirable to.
In view of this, the special proposition present invention.
Content of the invention
The first object of the present invention is to provide a kind of vaccine diluent, and described vaccine diluent can solve vaccine and exempt from Short problem that after epidemic disease NAT is low, generation time length, antibody are held time, can coordinate swine Fever Vaccine, porcine pseudorabies Vaccine, porcine reproductive and respiratory syndrome vaccine, Porcine Epidemic Diarrhea vaccine, transmissible gastroenteritis of swine vaccine or porcine rotaviruses Disease vaccine etc. is used.
The second object of the present invention is to provide a kind of preparation method of described vaccine diluent, and the method is simply easy to Operation.
In order to realize the above-mentioned purpose of the present invention, spy employs the following technical solutions：
A kind of vaccine diluent, including following component：
Carbomer 0.1～100g/L and levamisole 0.1～200g/L；
Solvent for use is any one in water, normal saline or phosphate buffer.
It is further preferred that vaccine diluent as above, including following component：
Carbomer 0.1～50g/L and levamisole 0.1～100g/L.
It is further preferred that vaccine diluent as above, including following component：
Carbomer 0.1～10g/L and levamisole 0.1～20g/L.
In the present invention, main component is Carbomer and levamisole, and wherein levamisole has the effect strengthening immunity, card Ripple nurse is a kind of polymer, forms gel in aqueous, is likely to be of the function of slow release, this material can also be formed with vaccine Effect with vaccine in antibody in complex prevention animal body fluid.
Preferably, vaccine diluent as above, described vaccine diluent solvent for use is the phosphorus containing following component Phthalate buffer：
100～150mM sodium chloride, 0.5～4mM potassium chloride, 1～100mM disodium hydrogen phosphate and 0.1～10mM di(2-ethylhexyl)phosphate Hydrogen potassium.
Preferably, vaccine diluent as above, described vaccine diluent solvent for use is the phosphorus containing following component Phthalate buffer：
130～140mM sodium chloride, 2～3mM potassium chloride, 1～30mM disodium hydrogen phosphate and 1～5mM potassium dihydrogen phosphate.
Due to vaccine, contain substantial amounts of bioactive substance particularly in live vaccine, thus carry out dilute under buffer system Release effect best.
Preferably, vaccine diluent as above, including following component：
Carbomer 1g/L and levamisole 5g/L；
Solvent is the phosphate buffer containing following component：
137mM sodium chloride, 2.7mM potassium chloride, 10mM disodium hydrogen phosphate and 2mM potassium dihydrogen phosphate；PH=6.8～7.4.
The preparation method of vaccine diluent as above, including：
Carbomer and levamisole are dissolved in sterilizing, thus obtaining the product after solvent.
Preferably, the preparation method of vaccine diluent as above, the condition of described sterilizing is：
106 DEG C～121 DEG C autoclaving 15min～30min.
Application in dilution animal viral live vaccine for the vaccine diluent as above；
Described animal viral live vaccine includes one or more of following vaccine：
Live vaccines of hog cholera, pseudorabies disease live-vaccine, Porcine reproductive and respiratory syndrome live vaccine, Porcine Epidemic Diarrhea are lived Vaccine, transmissible gastroenteritis of swine live vaccine, porcine rotaviruses disease live-vaccine, fowlpox live vaccine and chicken Newcastle disease live vaccine.
Preferably, application as above, is diluted according to head part that vaccine is indicated, 1～2ml/ head part.
Specific embodiment
Below in conjunction with embodiment, embodiment of the present invention is described in detail, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and be not construed as limiting the scope of the present invention.Unreceipted concrete in embodiment Condition person, the condition according to normal condition or manufacturer's suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, are Can be by the commercially available conventional products bought and obtain.
Embodiment 1
Present embodiments provide a kind of vaccine diluent, including：
Carbomer and levamisole are dissolved in normal saline and obtain final concentration of Carbomer 10g/L, levamisole 10g/L Vaccine diluent, using sodium hydroxid adjust pH to 6.8～7.4.
Vaccine diluent is pressed certain volume subpackage standby after 106～121 DEG C of autoclavings 15 minutes.
Embodiment 2
Present embodiments provide a kind of vaccine diluent, including：
Carbomer and levamisole are dissolved in the vaccine that water obtains final concentration of Carbomer 1g/L, levamisole 10g/L Diluent, adjusts pH to 6.8～7.4 using sodium hydroxid.
Vaccine diluent is pressed certain volume subpackage standby after 106～121 DEG C of autoclavings 30 minutes.
Embodiment 3
Present embodiments provide a kind of vaccine diluent, including：
Every 1L diluent contains：100mM sodium chloride, 0.5mM potassium chloride, 100mM disodium hydrogen phosphate, 0.1mM biphosphate Potassium, 1g Carbomer, 20g levamisole.
The preparation method of this vaccine diluent is：
Above material is dissolved in and is injected in water, add sodium hydroxid and adjust pH to 6.8～7.4, divide by certain volume Dress is standby after 106～121 DEG C of autoclavings 25 minutes.
Embodiment 4
Present embodiments provide a kind of vaccine diluent, including：
Every 1L diluent contains：150mM sodium chloride, 0.5mM potassium chloride, 1mM disodium hydrogen phosphate, 10M potassium dihydrogen phosphate, 10g Carbomer, 1g levamisole.
The preparation method of this vaccine diluent is：
Above material is dissolved in and is injected in water, add sodium hydroxid and adjust pH to 6.8～7.4, divide by certain volume Dress is standby after 106～121 DEG C of autoclavings 25 minutes.
Embodiment 5
Present embodiments provide a kind of vaccine diluent, including：
Every 1L diluent contains：137mM sodium chloride, 2.7mM potassium chloride, 10mM disodium hydrogen phosphate, 2mM potassium dihydrogen phosphate, 1g Carbomer, 5g levamisole.
The preparation method of this vaccine diluent is：
Above material is dissolved in and is injected in water, add sodium hydroxid and adjust pH to 6.8～7.4, divide by certain volume Dress is standby after 106～121 DEG C of autoclavings 25 minutes.
Embodiment 6
The using method of the vaccine diluent described in embodiment 1～5：
Take the diluted freeze-dried live vaccine of preparation, show that head part is diluted according to vaccine, 1～2ml/ head part, dilute Release rear Nasal immunization or intramuscular injection immunity.
Experimental example 1 pseudorabies live vaccine immunity control experiment
1st, material：
1), vaccine：Pseudorabies disease live-vaccine SA215 strain, lot number 201510001, by Sichuan China god's veterinary biologicses Company limited provides.
2), laboratory animal：21～28 age in days ablactational baby pig.
3), cell culture associated materials and reagent：PK15 cell is by Sichuan Huashen Animal Biolog Products Co., Ltd. poultry Biological product Key Laboratory of Sichuan Province provides, and hyclone and DMEM culture medium are purchased from Thermo Fisher Scientific.
2nd, animal immune experiment
21 age in days ablactational baby pig 15, are randomly divided into 3 groups, respectively 1,2,3 groups of labelling；
Immunity metering uses according to vaccine description, the wherein 1 group vaccine diluent using embodiment 5 preparation, 2 groups of uses Vaccine carries diluent, and 3 groups of immune vaccine diluents (i.e. without vaccine), are negative control.
Immunity before and immunity after 7,14,21,28 days collection pig vena cava anterior blood, separate serum after carry out neutralizing antibody survey Fixed.
Observe daily after immunity all pigs body temperature (METHOD FOR CONTINUOUS DETERMINATION 7 days), search for food, mental status, record is with respect to comparison Organize normal and abnormal pig.
3rd, the mensure of neutralizing antibody
Conventionally pass on PK15 cell, referring next to《Republic of China Veterinary Pharmacopoeia》2005 editions three annex 《Neutralization laboratory method》The fixed virus diluted blood therapy for clearing away heat of middle record is neutralized the mensure of antibody titer to all samples.With group it is Unit carries out data statisticss.
4th, experimental result
1), each group clinical manifestation situation after immunity
After the immunity of different diluent, daily measure piglet body temperature, METHOD FOR CONTINUOUS DETERMINATION during 7 days all piglet body temperature all low In 40.0 DEG C, the person that has no abnormal body temperature.All experiment pig are searched for food, drink water, the mental status is all normal.Shown in statistical result table 1.
Clinicing symptom observation result statistics after table 1 immunity
2), neutralizing antibody Changing Pattern after immunity
Serum is all gathered before immunity and after immunity, according to《Republic of China Veterinary Pharmacopoeia》2005 editions requirements are neutralized TPPA, result show experiment pig neutralizing antibody meansigma methodss using diluent described in embodiment 17 after immunity, 14, 21st, 28 days all different degrees of higher than common diluent group, described in immune latter 28 days embodiments 1, diluent neutralizing antibody is than common Diluent group is high 1 times.Neutralizing antibody measurement result is as shown in table 2.
Neutralizing antibody measurement result after table 2 immunity
Group Before immunity Latter 7 days of immunity Latter 14 days of immunity Latter 21 days of immunity Latter 28 days of immunity
1 <2 6.6±0.89 36.6±7.56 62±2.0 122.33±8.89
2 <2 5±1.33 21.67±4.44 33±5.33 66±2.67
3 <2 <2 <2 <2 <2
Experimental example 2 live vaccines of hog cholera immunity control experiment
1st, material
1), vaccine：Swine fever heat resisting protective live vaccine (cell source), lot number 201506002, biological by Sichuan China mythical animals Products Co., Ltd provides.
2), laboratory animal：21～28 age in days ablactational baby pig.
3), hog cholera antibody blocks ELISA detection kit and is purchased from IDEXXX company.
4), vaccine diluent：The vaccine diluent of embodiment 5 preparation；The physiology salt that conventional vaccine diluent vaccine carries Water is as diluent.
2nd, animal immune experiment
21 age in days ablactational baby pig 15, are randomly divided into 3 groups, respectively 1,2,3 groups of labelling；
Immunity metering uses according to vaccine description, the wherein 1 group diluent using preparation in embodiment 5, and 2 groups use epidemic disease Seedling carries diluent, and 3 groups of immune vaccine diluents are to for negative control.
Immunity before and immunity after 7,14,21,28 days collection pig vena cava anterior blood, separate serum after carry out neutralizing antibody survey Fixed.
Observe daily after immunity all pigs body temperature (METHOD FOR CONTINUOUS DETERMINATION 7 days), search for food, mental status, record is with respect to comparison Organize normal and abnormal pig.
3rd, the mensure of antibody
Block the mensure that ELISA antibody kit description carries out hog cholera antibody according to swine fever.Line number is entered in units of group According to statistics.
4th, result statistics
1), each group clinical manifestation situation after immunity
After the immunity of different diluent, daily measure piglet body temperature, METHOD FOR CONTINUOUS DETERMINATION during 7 days all piglet body temperature all low In 40.0 DEG C, the person that has no abnormal body temperature.All experiment pig are searched for food, drink water, the mental status is all normal.Shown in statistical result table 3.
Clinicing symptom observation result statistics after table 3 immunity
2), antibody change after immunity
All gathering serum before immunity and after immunity, carrying out antibody titer mensure according to kit specification, it was found that exempting from 7 after epidemic disease, 14 all pig antibody be feminine gender, be within latter 21 days, 28 days the positive using the immunity of embodiment 1 diluent group, antibody hinders Disconnected rate be higher than conventional dilution liquid group, and matched group before immunity, immunity after be feminine gender within 7,14,21,28 days.Measurement result counts Data is as shown in table 4.
Neutralizing antibody measurement result (blocking-up rate) after table 4 immunity
Finally it should be noted that：Various embodiments above only in order to technical scheme to be described, is not intended to limit；To the greatest extent Pipe has been described in detail to the present invention with reference to foregoing embodiments, but it will be understood by those within the art that：Its Still the technical scheme described in foregoing embodiments can be modified, or to wherein some or all of technical characteristic Carry out equivalent；And these modifications or replacement, do not make the essence of appropriate technical solution depart from various embodiments of the present invention skill The scope of art scheme.

Claims (10)

1. a kind of vaccine diluent is it is characterised in that include following component：
Carbomer 0.1～100g/L and levamisole 0.1～200g/L；
Solvent for use is any one in water, normal saline or phosphate buffer.
2. vaccine diluent according to claim 1 is it is characterised in that include following component：
Carbomer 0.1～50g/L and levamisole 0.1～100g/L.
3. vaccine diluent according to claim 2 is it is characterised in that include following component：
Carbomer 1～10g/L and levamisole 1～20g/L.
4. the vaccine diluent according to any one of claims 1 to 3 is it is characterised in that molten used by described vaccine diluent Agent is the phosphate buffer containing following component：
100～150mM sodium chloride, 0.5～4mM potassium chloride, 1～100mM disodium hydrogen phosphate and 0.1～10mM biphosphate Potassium.
5. vaccine diluent according to claim 4 is it is characterised in that described vaccine diluent solvent for use is containing such as The phosphate buffer of lower component：
130～140mM sodium chloride, 2～3mM potassium chloride, 1～30mM disodium hydrogen phosphate and 1～5mM potassium dihydrogen phosphate.
6. vaccine diluent according to claim 5 is it is characterised in that include following component：
Carbomer 1g/L and levamisole 5g/L；
Solvent is the phosphate buffer containing following component：
137mM sodium chloride, 2.7mM potassium chloride, 10mM disodium hydrogen phosphate and 2mM potassium dihydrogen phosphate；PH=6.8～7.4.
7. the preparation method of the vaccine diluent described in any one of claim 1～6 is it is characterised in that include：
Carbomer and levamisole are dissolved in sterilizing, thus obtaining the product after solvent.
8. the preparation method of vaccine diluent according to claim 7 is it is characterised in that the condition of described sterilizing is：
106 DEG C～121 DEG C autoclaving 15min～30min.
9. application in dilution animal viral live vaccine for the vaccine diluent described in any one of claim 1～6；
Described animal viral live vaccine includes one or more of following vaccine：
Live vaccines of hog cholera, pseudorabies disease live-vaccine, Porcine reproductive and respiratory syndrome live vaccine, Porcine Epidemic Diarrhea work epidemic disease Seedling, transmissible gastroenteritis of swine live vaccine, porcine rotaviruses disease live-vaccine, fowlpox live vaccine and chicken Newcastle disease live vaccine.
10. application according to claim 9 is it is characterised in that be diluted according to head part that vaccine is indicated, 1～2ml/ Head part.
CN201610903368.8A 2016-10-17 2016-10-17 A kind of vaccine diluent and preparation method and application Pending CN106466295A (en)

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CN108245675A (en) * 2018-02-02 2018-07-06 四川华神兽用生物制品有限公司 A kind of live vaccine dilution and preparation method thereof, application and vaccine product
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CN111467488A (en) * 2020-04-17 2020-07-31 内蒙古必威安泰生物科技有限公司 Water-soluble composite immunologic adjuvant and application thereof
CN111467488B (en) * 2020-04-17 2023-09-01 内蒙古必威安泰生物科技有限公司 Water-soluble composite immunoadjuvant and application thereof

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Application publication date: 20170301