CN109929776A - Bacterial strain and its application and vaccine and preparation method thereof - Google Patents

Abstract

The present invention relates to field of biotechnology, in particular to bacterial strain and its application and vaccine and preparation method thereof.The present invention provides a kind of Mink Parvovirus Enteritis, hemorrhagic pneumonia bivalent inactivated vaccine and its preparation process, (bacterium) seed culture of viruses of the vaccine is DL15 plants of MEVB plants of the Mink Parvovirus strain and mink pseudomonas aeruginosa G type of China's prevalence, JL08 plants of Type B, WD01 plants of c-type, WF05 plants of I type, DL03 plants of F type, as being mixed in proportion with after formalin-inactivated respectively after the Canine Parvovirus Enteritis of antigen and pseudomonas aeruginosa strains culture, aluminium hydroxide gel is proportionally added into be formulated, the vaccine immunity phase is 6 months, 80% or more immune protective rate, it can be used to prevent Mink Parvovirus Enteritis and mink hemorrhagic pneumonia simultaneously, reduce the immunization amount and mink stress reaction of raiser, market application prospect is preferable.

Description

Bacterial strain and its application and vaccine and preparation method thereof

The application be the applying date be 2016.02.17, entitled " bacterial strain and its application and vaccine and its preparation side Method ", application No. is the divisional applications of the invention of 201610089542.X.

Technical field

The present invention relates to field of biotechnology, in particular to bacterial strain and its application and vaccine and preparation method thereof.

Background technique

Fur-bearing animal refers mainly to the animal that product is fur coat raw material processed.With the development of economy, the improvement of people's living standards With the continuous growth of demand, China fur industry shows powerful development trend.China's fur animal farming is concentrated mainly on The ground such as Shandong, Hebei, Liaoning and Heilungkiang, the kind of cultivation mainly have mink, racoon dog, fox, a beaver rabbit, produce per year standard mink skin 6,000,000, Raw fox skin 2,000,000, wherein raw fox skin accounts for the 35% of Gross World Product, and standard mink skin accounts for the 12% of Gross World Product.

Mink hemorrhagic pneumonia is a kind of acute infectious disease for causing mink by pseudomonas aeruginosa, mostly occurs in autumn temperature Warm current is wet, is clinical special disease with cough, expiratory dyspnea, haematemesis, nosebleed, with lung large-area hemorrhage, pulmonary hepatization, pulmonary edema is Major lesions feature, disease incidence 20%-50%, the death rate 100%.Sick ermine morbidity is anxious, dead fast, often sudden and violent in region Hair property is popular.In recent years since mink farming scale and intensive degree significantly improve, the disease incidence of mink hemorrhagic pneumonia is aobvious It writes and rises, which deepens year by year, it has also become endangers one of the Infectious Diseases of China's mink farming.

Drug resistance problems are also easy to produce in view of the characteristics of incidence of the disease and the bacterium, it is often highly difficult to cure the disease with medicine, goes out Live vaccine is the most effectual way for preventing the disease, is prepared into polyvalent inactivation epidemic disease after the bacterial strain of serotype popular in mink is inactivated Seedling can effectively prevent the disease caused by similar serological type strain, and " biological characteristics of mink pseudomonas aeruginosa separation strains compare Analysis and serotype investigation " (Bai Xue, Chai Xiuli, Yan Xijun, Gao Han, Zhang Hailing, Zhao Jianjun, Zhang Lei, Shao Xiqun, Luo Guoliang, king Long phoenix), " mink hemorrhagic pneumonia bivalent inactivated vaccine and preparation method thereof " (patent No.: ZL201010203879.1, Yan Xi Army, Bai Xue, Chai Xiuli, Luo Guoliang, Zhao Jianjun, Zhang Hailing, Shao Xiqun, Zhang Lei, Gao Han) it discloses China and causes mink bleeding Property pneumonia pseudomonas aeruginosa predominant serotypes be G type and Type B, and utilize this 2 kinds of serological type strain DL15 (national strains Deposit number CGMCC NO.3811) and JL08 (national culture presevation CGMCC NO.3812) having developed can be used for prevents mink The bivalent inactivated vaccine of hemorrhagic pneumonia, the product in 2011 obtain the clinical test official written reply of the Ministry of Agriculture, and carry out simultaneously Clinical application.Obtain within 2016 national novel chiral synthon certificate (three classes).

Mink viral enteritis is that mink enteritis parvovirus (Mink enteritis parvovirus, MEV) causes Using violent diarrhea as the acute strong, highly contagious disease of Major Clinical spy's disease, the death rate is up to 80% or more, should Disease is worldwide widely current, under field conditions (factors), the equal infectious of the ermine of different cultivars and all ages and classes, but young ermine Infectious extremely strong, with the development of special livestock breeding, viral enteritis, which has become, endangers one of three big epidemic diseases of mink farming, Huge economic loss is brought to mink breeding industry, nineteen forty-seven is found by Canadian scholar Schofield and reported MEV to cause earliest Disease.Jiang Tingxiu in 1981 etc. first reported China and mink viral enteritis occur, and hereafter the disease gradually spreads the whole nation, gives Mink breeding industry brings huge economic loss.My unit Wu Wei etc. is with monoclonal antibody to the mink enteritis parvovirus of isolated in China System pattern is carried out, determines that China's mink enteritis parvovirus is mainly caused by Type B parvovirus, selects poison on this basis Power is stable, good Strain MEVB plants of immunogenicity, and develops Mink Parvovirus inactivated vaccine, obtains within 2010 National novel chiral synthon certificate (three classes), and apply on the market rapidly.Control the disease preferably.

The country does not have the bigeminy vaccine of Mink Parvovirus Enteritis, mink hemorrhagic pneumonia also at present, in the market also without This kind of Combined vaccine uses.And using single seedling need that mink is repeatedly immunized, immunizing dose is big, and immune programme is cumbersome, increases Human and material resources and financial resources cost, while also easily causing the stress reaction of mink.

This project develops that " needle is anti-on the basis of studying successfully Mink Parvovirus Enteritis and mink hemorrhagic pneumonia Two disease " Mink Parvovirus Enteritis and hemorrhagic pneumonia bivalent inactivated vaccine, technically captured connection seedling in two kinds at The problem of point parvovirus and the antigenic component of pseudomonas aeruginosa interfere with each other, vaccine contg is with when production technology.Originally it grinds Study carefully on the basis of mink hemorrhagic pneumonia bivalent inactivated vaccine is developed, newly joined the prevalence occurred in recent years in vaccine strain Strain I type, c-type and F type, are prepared as five bivalent inactivated vaccine of mink hemorrhagic pneumonia (G type, Type B, I type, c-type and F type), can be right The prevalence of the new serological type strain of mink hemorrhagic pneumonia plays good prevention effect, can solve domestic original vaccine to this Disease can only provide the problem of part protection, the more effective generation for preventing mink hemorrhagic pneumonia.By being ground to bigeminy vaccine System and key technology innovation, develop a kind of efficient, Small side effects, the feasible Combined vaccine of marketing, it is immune to solve single seedling Challenge in program meets industry and the urgent need of raiser, has a vast market application prospect.

The prior art is that this two Pseudomonas aeruginosa strains PYG culture medium is increased bacterium training by the culture technique ventilated with rolling bottle It supports, receives bacterium after 37 DEG C of 180r/m shaking table culture 12h, 40 are respectively reached with the bacterium amount that bacterial plate counts method measures two plants of bacterium × 10⁸CFU/ml is mixed G type bacterial strain and Type B bacterial strain in the ratio of 2:1；(1) with final concentration of 0.15% formalin pair Pseudomonas aeruginosa is inactivated, and for 24 hours in 37 DEG C of inactivations, is during which shaken 2~3 times；(2) bacterium solution after inactivation is added into 20% ratio The aluminium hydroxide gel of example, can be obtained mink pseudomonas aeruginosa multivalent inactivated vaccine after mixing.Original culture medium: PYG training Support base composition: peptone 2%, glucose 1%, sodium chloride 0.5%, yeast powder 0.3% are prepared with deionized water, have matched tune pH 7.2~7.4,116 DEG C of 30min of value.But rolling bottle ventilation culture thallus is used, inoculative proportion is 3%~4%, ventilation culture 12 ~15h, since the rolling bottle production technology of vaccine falls behind, rolling bottle number is more to be easy to pollute when connecing bacterium, and incubation time is long, produces bacterium Number is few, between 20~4,000,000,000 CFU/ml, is only applicable to the small lot production of vaccine development early period.

Select 3 kinds of current Major Epidemic serotype G, Type B and Klebsiella Pneumoniae K1 type bacterial strain systems of pseudomonas aeruginosa For at inactivated vaccine.It has been immunized using 10% inactivation Klebsiella Pneumoniae as the vaccine of adjuvant, protective rate 83%；Be immunized with 10%Al (OH)₃The vaccine of adjuvant, protective rate 71%；And as blank control group, the 6th group for only having injected physiological saline is small The survival rate of mouse is 33%.Relative to traditional Al (OH)₃Adjuvant, Klebsiella Pneumoniae itself can be used as vaccine composition, protect Body is protected from the infection of the pathogen, while having good immunoregulatory activity concurrently again, strengthens body to P. aeruginosa The immune response of bacterium produces higher antibody level and provides better immunoprotection, and veterinary clinic has certain answer With value.

Vaccine strain serotype used in the mink hemorrhagic pneumonia of above several developments mainly includes 2 kinds or 3 kinds, i.e. G Type, Type B or G type, c-type, I type, according to our unit carry out mink hemorrhagic pneumonia epidemiological survey, 2010 to 2015 Newly there is F type, I type and C type in the popular serotype in year, and since the cross protection rate between various popular serotypes is low, greatly About 20%-30%.It is therefore necessary to all possible popular serotype is added.

Summary of the invention

In view of this, the present invention provides bacterial strain and its applications and vaccine and preparation method thereof.The vaccine immunity phase is 6 months, 80% or more immune protective rate can be used to prevent simultaneously Mink Parvovirus Enteritis and mink hemorrhagic pneumonia, Immunization amount and mink stress reaction, the market application prospect for reducing raiser are preferable.

In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:

The present invention also provides a kind of P. aeruginosa bacterial strain, deposit number is CGMCC No.11989.

It is CGMCC No.11988, CGMCC No.11990 and CGMCC the present invention also provides deposit number Application of the P. aeruginosa bacterial strain of No.11989 in preparation mink hemorrhagic pneumonia vaccine.

It is CGMCC No.11988, CGMCC No.11990 and CGMCC the present invention also provides deposit number The P. aeruginosa bacterial strain of No.11989 prepare Mink Parvovirus Enteritis, answering in hemorrhagic pneumonia bivalent inactivated vaccine With.

The present invention provides a kind of mink hemorrhagic pneumonia vaccines, be CGMCC No.11988 by deposit number of the present invention, The P. aeruginosa bacterial strain and DL15 plants of mink pseudomonas aeruginosa G type, B of CGMCC No.11990 and CGMCC No.11989 JL08 plants of type are made.

The present invention also provides a kind of Mink Parvovirus Enteritis, the bivalent inactivated vaccine of hemorrhagic pneumonia, You Benfa Bright deposit number be CGMCC No.11988, CGMCC No.11990 and CGMCC No.11989 P. aeruginosa bacterial strain and DL15 plants of mink pseudomonas aeruginosa G type, JL08 plants of Type B and MEVB plants of Mink Parvovirus strain are made.

In some specific embodiments of the invention, the preparation method of bivalent inactivated vaccine includes the following steps:

Step 1: taking deposit number of the present invention is CGMCC No.11988, CGMCC No.11990 and CGMCC DL15 plants of the P. aeruginosa bacterial strain and mink pseudomonas aeruginosa G type of No.11989, JL08 plants of Type B be inoculated in training respectively Pseudomonas aeruginosa pentavalent seedling semi-finished product bacterium solution after inactivation is made after mixing, inactivation after feeding base culture；

Step 2: taking MEVB plants of inoculating cells of Mink Parvovirus strain, harvest inactivates obtained parvovirus semi-finished product；

Step 3: taking thin made from pseudomonas aeruginosa pentavalent seedling semi-finished product bacterium solution and step 2 after step 1 inactivation obtained Small virus semi-finished product are mixed to prepare mixed liquor；

Step 4: mixed liquor made from step 3 being taken to mix with adjuvant.

In some specific embodiments of the invention, preservation of the present invention in the preparation method step 1 of bivalent inactivated vaccine Number is CGMCC No.11988, P. aeruginosa bacterial strain and the mink of CGMCC No.11990 and CGMCC No.11989 Respectively after culture medium culture, the mixed ratio is 1:1:1:1:1 for DL15 plants of pseudomonas aeruginosa G type, JL08 plants of Type B (v/v), the bacterium number of every plant of bacterium is at least 4,000,000,000 CFU/mL after mixing.

In some specific embodiments of the invention, preparation method step 2 parvovirus of bivalent inactivated vaccine half at The content of parvovirus is 10 in product^6.00TCID₅₀。

In some specific embodiments of the invention, step 1 described in the preparation method step 3 of bivalent inactivated vaccine is made Inactivation after the ratio of parvovirus semi-finished product mixing made from pseudomonas aeruginosa pentavalent seedling semi-finished product bacterium solution and step 2 be 2: 1。

In some specific embodiments of the invention, culture medium described in the preparation method step 1 of bivalent inactivated vaccine For PYG culture medium, MgSO preferably is added in PYG culture medium₄, urea, sodium citrate, vitamin, Na₂HPO₄Buffer salt or KH₂PO₄One of buffer salt is a variety of；

Preferably, culture medium prescription: peptone 3%, glucose 2%, yeast powder 0.3%, sodium chloride 0.5%, Na₂HPO₄2.5g/L, KH₂PO₄0.83g/L, MgSO₄0.3g/L, sodium citrate 0.3g/L, urea 2g/L, vitamin liquid 10mL/L；

Inoculation described in step 1 is according to 3% inoculating strain of PYG culture volume；Described in step 2 inoculation for according to 2% virus inoculation of CRFK cell suspension volume.

In some specific embodiments of the invention, go out described in the preparation method step 1 or step 2 of bivalent inactivated vaccine It is living to use formalin-inactivated；The dosage of the formaldehyde is the 0.2% of bacterium solution total amount.

Preferably, inactivation described in step 1 or step 2 is to be slowly added to formalin, Bian Jia by the 0.2% of bacterium solution total amount Side stirring, 36~37 DEG C of stirrings inactivate 24 hours.

In some specific embodiments of the invention, adjuvant described in the preparation method step 4 of bivalent inactivated vaccine is The mixed proportion of 40% (w/v, in terms of g/mL) aluminium hydroxide gel, mixed liquor made from step 3 and the adjuvant is 6:1.

Specific packet is cultivated in some specific embodiments of the invention, in the preparation method step 1 of bivalent inactivated vaccine Include following steps:

Step 1: first order seed breeding: taking DL15 plants, JL08 plants, WD01 plants, WF05 plants, DL03 plants of bacterial strain freeze-drying lactobacillus Each 1 unpacking, is diluted with sterile saline, is inoculated with the PYG agar medium (culture medium prescription: peptone respectively 3%, glucose 2%, yeast powder 0.3%, sodium chloride 0.5%, Na₂HPO₄2.5g/L, KH₂PO₄0.83g/L, MgSO₄ 0.3g/ L, sodium citrate 0.3g/L, urea 2g/L, vitamin liquid 10mL/L, agar powder 2g/L), 36~37 DEG C are cultivated 18~24 hours, 5 or more colonies typicals are selected respectively to be mixed in a small amount of PYG fluid nutrient medium, then are inoculated with PYG agar slant culture-medium respectively (the culture medium prescription: peptone 3%, glucose 2%, yeast powder 0.3%, sodium chloride 0.5%, Na₂HPO₄2.5g/L KH₂PO₄0.83g/L, MgSO₄0.3g/L, sodium citrate 0.3g/L, urea 2g/L, vitamin liquid 10mL/L, agar powder 2g/L) Several, 36~37 DEG C are cultivated 18~24 hours, as first order seed.2~8 DEG C of preservations, validity period are no more than 15, and passage is not More than 5 generations；

Step 2: secondary seed breeding: first order seed being taken to be inoculated with several 5mL PYG fluid nutrient medium (trainings respectively Support based formulas: peptone 3%, glucose 2%, yeast powder 0.3%, sodium chloride 0.5%, Na₂HPO₄2.5g/L, KH₂PO₄ 0.83g/L, MgSO₄0.3g/L, sodium citrate 0.3g/L, urea 2g/L, vitamin liquid 10mL/L) in, 36~37 DEG C of shakings 12 Hour, then be inoculated in larger amount of PYG fluid nutrient medium respectively in 3% ratio, 36~37 DEG C shake 12 hours, are purely examined After qualification, as secondary seed, 2~8 DEG C of preservations are set, should be no more than 4；

Step 3: fermentation seed liquid preparation: the secondary seed being inoculated in fermentation medium, in temperature 37~37.5 DEG C, fermented and cultured 7h under the conditions of 100~120r/min of mixing speed, 70~90L/min of charge flow rate, 30~70Kpa of pressure, Obtain fermentation seed liquid；

Step 4: one grade fermemtation: fermentation seed liquid being inoculated in 3% ratio equipped with 250~300L PYG culture medium (institute The culture medium prescription stated: peptone 3%, glucose 2%, yeast powder 0.3%, sodium chloride 0.5%, Na₂HPO₄2.5g/L KH₂PO₄0.83g/L, MgSO₄0.3g/L, sodium citrate 0.3g/L, urea 2g/L, vitamin liquid 10mL/L) 500L fermentation Tank, be added defoaming agent GPE, cultivate 6~7h after be separately added into glucose to final concentration of 1% and urea to final concentration 2g/L make For supplementary carbon source, main control parameters are as follows during fermentation:: 37~37.5 DEG C of tank temperature, 80~110r/min of mixing speed, into 80~120L/min of throughput, tank press 30~70Kpa, 12h harvest, through purely after the assay was approved, as semi-finished product bacterium solution, and bacterium number Stablize in 25,000,000,000 CFU/mL；

Step 5: second order fermentation: fermentation seed liquid being inoculated in 4% ratio equipped with 500L~600L PYG culture medium (the culture medium prescription: peptone 3%, glucose 2%, yeast powder 0.3%, sodium chloride 0.5%, Na₂HPO₄2.5g/L KH₂PO₄0.83g/L, MgSO₄0.3g/L, sodium citrate 0.3g/L, urea 2g/L, vitamin liquid 10mL/L) 1000L hair Fermentation tank is separately added into glucose to final concentration of 1% and urea to final concentration 2g/L as supplementary carbon source after cultivating 6~7h, adds Enter defoaming agent GPE, main control parameters are as follows during fermentation:: 37~37.5 DEG C of tank temperature, 80~100r/min of mixing speed, 90~136L/min of charge flow rate, tank press 30~70Kpa, 12h harvest, through purely after the assay was approved, as semi-finished product bacterium solution, and bacterium Number can be stablized in 25,000,000,000 CFU/ml.

In some specific embodiments of the invention, cultivated in the preparation method step 2 of bivalent inactivated vaccine specifically: Basic seed culture of viruses is diluted with cell maintenance medium, synchronizes and is inoculated in CRFK cell suspension in 2% ratio, sets 37 DEG C of trainings It supports 3~4, when typical case CPE occurs in 80% or more cell, can harvest, -20 DEG C of preservations.

In some specific embodiments of the invention, the culture medium and fermentor of pseudomonas aeruginosa provided by the invention Culture medium is improved on the basis of the original PYG culture medium of culture process (glucose-peptone-yeast powder), passes through addition MgSO required for bacterial metabolism₄, sodium citrate, vitamin hydroful foot high-speed fermentation nutritional need, and pass through addition Na₂HPO₄, KH₂PO₄The stabilization that salt maintains PH value and thallus osmotic pressure is buffered, in addition, supplementing grape in 6~7h of fermented and cultured Sugar and urea pass through the foundation of 100L, 500L, 1000L fermentor fermentation process step by step, it is determined that the bacterium number of seed liquor, inoculation Ratio, seed liquor holding time, passage number, fermentation harvest time make bacterium number that can stably reach 25,000,000,000 CFU, than original side Method bacterium number improves 5~10 times.

The present invention newly joined close several on the basis of mink hemorrhagic pneumonia bivalent inactivated vaccine is developed in vaccine strain Epidemic strain I type, c-type and the F type that year occurs, are prepared as five bivalent inactivated vaccine of mink hemorrhagic pneumonia, can be to mink hemorrhagic The prevalence of the new serological type strain of pneumonia plays good prevention effect, can solve can only providing the disease for domestic original vaccine The problem of part is protected, so as to effectively prevent the generation of mink hemorrhagic pneumonia.The vaccine immunity phase is 6 months, is immunized and protects 80% or more shield rate can be used to prevent simultaneously Mink Parvovirus Enteritis and mink hemorrhagic pneumonia, reduce raiser Immunization amount and mink stress reaction, market application prospect it is preferable.

Biological deposits explanation

Bacterial strain WD01, classification naming: pseudomonas aeruginosa Pseudomonas aeruginosa. was in 01 month 2016 11 Day is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and address is BeiChen West Road, Chaoyang District, BeiJing City No. 1 institute 3, Institute of Microorganism, Academia Sinica, deposit number are CGMCC No.11988；

Bacterial strain WF05, classification naming: pseudomonas aeruginosa Pseudomonas aeruginosa. was in 01 month 2016 11 Day is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and address is BeiChen West Road, Chaoyang District, BeiJing City No. 1 institute 3, Institute of Microorganism, Academia Sinica, deposit number are CGMCC No.11990；

Bacterial strain DL03, classification naming: pseudomonas aeruginosa Pseudomonas aeruginosa. was in 01 month 2016 11 Day is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and address is BeiChen West Road, Chaoyang District, BeiJing City No. 1 institute 3, Institute of Microorganism, Academia Sinica, deposit number are CGMCC No.11989.

Detailed description of the invention

In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technical description to be briefly described.

Fig. 1 shows that pseudomonas aeruginosa antibody ELISA immunosorbent adsorption test measurement result after Combined vaccine is immunized in mink；

Fig. 2 shows that parvovirus hemagglutination inhibition antibody measurement result after Combined vaccine is immunized in mink；

Fig. 3 shows that parvovirus neutralizing antibody measurement result after Combined vaccine is immunized in mink.

Specific embodiment

The invention discloses bacterial strain and its application and vaccine and preparation method thereof, those skilled in the art can be used for reference Present disclosure is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to this field skill It is it will be apparent that they are considered as being included in the present invention for art personnel.Method of the invention and application by compared with Good embodiment is described, related personnel obviously can not depart from the content of present invention, in spirit and scope to as described herein Methods and applications are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.

Bacterial strain uses therefor, raw material and reagent are equal in bacterial strain provided by the invention and its application and vaccine and preparation method thereof It is available on the market.Wherein, the deposit number of bacterial strain DL15 is CGMCC No.3811；The deposit number of bacterial strain JL08 is CGMCC No.3812；MEVB plants of screening techniques of Strain are see " research of China's mink enteritis parvovirus Major Epidemic type " Wu Wei；Nie Jin Zhen；Wang Kejian；Cheng Shipeng；Chinese animal doctor's journal, 04 phase in 1996.

Culture medium prescription in specific embodiments of the present invention are as follows: peptone 3%, glucose 2%, yeast powder 0.3%, chlorine Change sodium 0.5%, Na₂HPO₄2.5g/L, KH₂PO₄0.83g/L, MgSO₄0.3g/L, sodium citrate 0.3g/L, urea 2g/L, dimension Raw element liquid 10mL/L.

Below with reference to embodiment, the present invention is further explained:

The preparation of embodiment 1 Mink Parvovirus Enteritis, hemorrhagic pneumonia bivalent inactivated vaccine

1. vaccine bacterium kind standard:

1.1 pseudomonas aeruginosa bacterial standards

1.1.1 form and this bacterium of biochemical characteristic are gram-Negative bacillus.Biochemical characteristic should meet this in systematic bacteriology The characteristic of bacterium.

1.1.2 cultural character is seeded on PYG agar plate, is cultivated 18~24 hours, is visually observed at 36~37 DEG C, Bacterium colony is flat wet, and edge is irregular, and generation diffuses marennin or brown pigment in culture medium；It is inoculated with PYG Liquid Culture In base, 36~37 DEG C are cultivated 18~24 hours, and liquid is uniformly muddy, generate green or brown pigment.

1.1.3 serological characteristic is formed with pseudomonas aeruginosa blood grouping serum, and DL15 plants should be G type, and JL08 plants should be B Type, WD01 plants should be c-type, WF05 plant and should be I type, DL03 plant and should be F type.

1.1.4 virulence strain DL15 plants, JL08 plants, WD01 plants, WF05 plants, DL03 plants is inoculated with PYG fluid nutrient medium respectively In 37 DEG C shaken cultivation 12 hours, bacterium solution culture virulence should meet claimed below.

1.1.4.1 DL15 plants, WD01 plants and WF05 plants are with 16~18g mouse 10, every mouse hypodermic inoculation viable bacteria 0.2ml (70,000,000 CFU/0.2ml containing viable bacteria), mouse should be all dead in 48 hours；With 2~10 monthly ages susceptible mink 5 of health Only, every intratracheal instillation viable bacteria 1ml (200,000,000 CFU/ml containing viable bacteria).Mink should be all dead in 72 hours.

1.1.4.2 JL08 plants and DL03 plant are with 16~18g mouse 10, and every subcutaneous vaccination viable bacteria 0.2ml is (containing viable bacteria 1.2 hundred million CFU/0.2ml), mouse should be all dead in 48 hours；With 2~10 monthly ages health susceptible mink 5, every intratracheal It instills viable bacteria 1ml (400,000,000 CFU/ml containing viable bacteria).Mink should be all dead in 72 hours.

1.1.5 immunogenicity

1.1.5.1 mouse is divided into 5 groups, every group 10, single bacterium type is subcutaneously injected respectively with 16~18g of weight mouse 50 Vaccine 0.2ml divides 5 groups, every group 10, is respectively subcutaneously injected 70,000,000 CFU/0.2ml's together with control mice 50 after 21 days The JL08 strain of DL15 plants, WD01 plants or WF05 plants of virulent liquid or 1.2 hundred million CFU/0.2ml or DL03 plants of virulent liquid, it is continuous to see It examines 7.Control mice should be all dead, and immune mouse should at least protect 8.

1.1.5.2 susceptible mink 25 of 2~10 monthly ages health of mink divides 5 groups, every group 5, distinguishes intramuscular injection list Bacterial type vaccine 1ml divides 5 groups, every group 5, intratracheally instills 200,000,000 CFU/ml's respectively together with control mink 25 after 21 days DL15 plants, WD01 plants or WF05 plants or 400,000,000 CFU/ml of JL08 strain or DL03 plants of virulent liquid, are observed continuously 7.Compare mink Should be all dead, immune mink should at least protect 4.

1.1.6 it is purely checked with PYG culture medium (note 2), it should be pure.

1.1.7 5~10 generation of algebra basic seed.

1.1.8 virulent standard

1.1.8.1 form and biochemical characteristic, cultural character, serological characteristic and the same 1.1.1,1.1.2 of virulence, 1.1.3, 1.1.4。

1.1.8.1 the inspection of PYG culture medium is purely used, it should be pure.

1.1.8.1 5~10 generation of subalgebra is planted.

1.1.9-20 DEG C of freeze-drying lactobacillus of fungi preservation, it is 36 months that storage life, which is fixed tentatively,.

1.2 MEVB plants of Mink Parvovirus should meet following standard

1.2.1 seed culture of viruses cell maintenance medium work is serially diluted for 10 times by viral level, takes 10^-4、10^-5、10^-6、 10^-7Four A dilution, each dilution are inoculated with 4 holes, and every hole 0.1ml supplements CRFK cell suspension 0.9ml, set 37 DEG C and cultivate and observe 3 ~4, calculate TCID₅₀, every ml viral level answers >=10^6.50 TCID₅₀。。

1.2.2 erythrocyte agglutination valence is carried out according to note 3.1, and seed culture of viruses answers >=1:64 to swine erythrocyte agglutination titer.

1.2.3 virulence seed culture of viruses takes orally 2~10 monthly age health mink 5, every 15ml, observes 10,4 or more minks Occur classical symptom (see note 1).

1.2.4 vaccine (inactivation provirus content >=10 are made according to this regulation in immunogenicity^6.00TCID₅₀) after, muscle connects Kind 2~10 monthly age health mink 5, every 1ml, together with the identical control group mink of condition 5, oral challenge mink after 21 days SMPV-11 plants of parvovirus (enteron aisle poison) 10ml (contains 100 ID₅₀), it observes 10, immune mink should all be protected, right It all falls ill according to mink, classical symptom occurs.

1.2.5 specific 1: 100 diluted seed culture of viruses and 1: 10 times of diluted Mink Parvovirus specific serum equivalent are mixed It closes, after 37 DEG C neutralize 1 hour, is inoculated with 4 hole of CRFK cell, while setting 4 hole of virus control group, culture observation 4 days, test group is answered There is not CPE, CPE should all occur in control group.

1.2.6 pure to test by existing " Chinese veterinary pharmacopoeia " annex, seed culture of viruses should be dirty without bacterium, mould, mycoplasma Dye；Seed culture of viruses should be polluted without exogenous virus.

1.2.7 6~14 generation of algebra basic seed.

1.2.8 basic virus seed conservation cell culture wet poison -70 DEG C of storage lives of kind are 3 years；Vacuum freeze drying is placed on- In 70 DEG C, storage life is 5 years.

1.2.9 virulent standard is examined

1.2.9.1 to the median infective dose (ID of mink₅₀) SMPV-11 plants of internal organs poison of Mink Parvovirus are taken, use physiology salt Water is made 10% emulsion, and multigelation twice and filters, every milliliter of 2000 unit of addition penicillin of filtrate, streptomycin sulphate 2000 μ g are placed in 4 DEG C of effects overnight, as the virulent stoste of inspection；Stoste carries out 10 times with physiological saline and is serially diluted, and takes original Liquid, 10^-1、10^-2、10^-3Four dilutions, each dilution gavage 3~10 monthly age health mink 4, every 10ml, observation 10 Day, calculate ID₅₀, SMPV-11 strain virus is that every 10ml stoste answers >=100 ID to mink median infective dose standard₅₀。

1.2.9.2 it examines with 100 ID of virulent virulence standard₅₀SMPV-11 plants of internal organs poison of Mink Parvovirus gavage 3~10 monthly age health minks 4,3~5d mink all falls ill after attacking poison, classical symptom occurs, virus is to pig in mink excrement Red blood cell HA potency answers >=1: 256.

1.2.10 exogenous virus is examined is carried out by note 2.2, and seed culture of viruses should be polluted without exogenous virus.

1.2.11 it examines and uses 4~6 generation of generation with virulent.

1.2.12 examining with virulent virus seed conservation internal organs seed culture of viruses is 4 years in -70 DEG C of storage lives.

2 vaccine preparations and the inspection of semifinished product

2.1 mink hemorrhagic pneumonia inactivated vaccines

2.1.1 production is prepared with seed

2.1.1.1 first order seed breeding takes DL15 plants, JL08 plants, WD01 plants, WF05 plants, DL03 plants of bacterial strains jellies with identification Each 1 unpacking of dry strain, is diluted with sterile saline, is inoculated with PYG agar medium, 36~37 DEG C of cultures 18~24 respectively Hour, 5 or more colonies typicals are selected respectively and are mixed in a small amount of PYG fluid nutrient medium, then inoculation PYG agar slant training respectively It is several to support base, 36~37 DEG C are cultivated 18~24 hours, as first order seed.2~8 DEG C of preservations, validity period are no more than 15, passage No more than 5 generations.

2.1.1.2 secondary seed breeding takes first order seed to be inoculated in several 5ml PYG fluid nutrient mediums respectively, and 36~37 It DEG C shaking 12 hours, then is inoculated in larger amount of PYG fluid nutrient medium respectively in 3% ratio, 36~37 DEG C shake 12 hours, warp Purely after the assay was approved, as secondary seed, 2~8 DEG C of preservations are set, should be no more than 4.

2.1.2 seedling culture medium PYG improved culture medium.

2.1.2.1 seedling is prepared with bacterium solution

Fermentation seed liquid preparation: the 2.1.1.2 secondary seed is inoculated in fermentation medium, in temperature 37~37.5 DEG C, fermented and cultured 7h under the conditions of 100~120r/min of mixing speed, 70~90L/min of charge flow rate, 30~70Kpa of pressure, Obtain fermentation seed liquid；

One grade fermemtation: being inoculated in the 500L fermentor equipped with 250~300L culture medium in 3% ratio for fermentation seed liquid, Defoaming agent GPE is added, glucose is added to final concentration of 1% and urea to final concentration of 2g/L as supplement after cultivating 6~7h Carbon source, main control parameters are as follows during fermentation:: 37~37.5 DEG C of tank temperature, 80~110r/min of mixing speed, charge flow rate 80~120L/min, tank press 30~70Kpa, 12h harvest, through purely after the assay was approved, as semi-finished product bacterium solution, bacterium number are stablized In 25,000,000,000 CFU/ml.

Second order fermentation: fermentation seed liquid is inoculated in the fermentation of the 1000L equipped with 500L~600L culture medium in 4% ratio Tank, 6~7h of culture are added glucose to final concentration of 1% and urea to final concentration of 2g/L as supplementary carbon source, defoaming are added Agent GPE, main control parameters are as follows during fermentation:: 37~37.5 DEG C of tank temperature, 80~100r/min of mixing speed, inlet air flow 90~136L/min is measured, tank presses 30~70Kpa, 12h harvest, through purely after the assay was approved, as semi-finished product bacterium solution, bacterium number can Stablize in 25,000,000,000 CFU/ml.

2.1.3 it purely examines and tests by existing " Chinese veterinary pharmacopoeia " annex, it should be pure.

2.1.4 count plate carries out count plate by existing " Chinese veterinary pharmacopoeia " annex with PYG agar plate；Every milliliter Bacterium number is not less than 2 × 10¹⁰CFU。

2.1.5 it inactivates and is slowly added to formalin by the 0.2% of bacterium solution total amount, stirring while adding, 36~37 DEG C of stirrings are gone out It is 24 hours living.

2.1.3 the inspection of semifinished product

2.1.3.1 steriling test is tested by existing " Chinese veterinary pharmacopoeia " annex, answers asepsis growth.

2.1.3.2 inactivation is examined and is tested by existing " Chinese veterinary pharmacopoeia " annex, answers asepsis growth.

2.1.4 with seedling by steriling test and inactivation G type after the assay was approved, Type B, c-type, I type, F type bacterium solution according to 1: 1:1:1:1 (V/V) mixing is mink hemorrhagic pneumonia vaccine pentavalent seedling semi-finished product bacterium solution after mixing well, every plant after mixing The bacterium number of bacterium is at least 4,000,000,000 CFU/mL.

Mink Parvovirus Enteritis inactivated vaccine:

2.2 Mink Parvovirus Enteritis inactivated vaccines

2.2.1 the production preparation of seed culture of viruses

2.2.1.1 seed culture of viruses breeding is suitably diluted basic seed culture of viruses with cell maintenance medium, is synchronized and is connect in 2% ratio Kind is set 37 DEG C and is cultivated 3~4, when typical case CPE occurs in 80% or more cell, can harvest, -20 DEG C in CRFK cell suspension It saves, indicates harvest date, seed culture of viruses algebra, loading amount etc..

2.2.1.2 virus seed identification should meet regulation by 1.2.1~1.2.6 progress.

2.2.1.3 it is no more than 12 months for -20 DEG C of virus seed conservation.

2.2.1.4 virus seed subculture was no more than for 4 generations.

2.2.2 the preparation of seedling virus liquid

2.2.2.1 the preparation of cat kidney passage cell (CRFK plants) prepares CRFK cell according to a conventional method.CRFK cell should accord with Close following standard: cell should be without bacterium, mould and mycoplasma contamination；Cell rotates culture in MEM nutrient solution under the conditions of 37 DEG C, Fine and close single layer should be formed in 36 hours；Cell base algebra was 43~48 generations, was no more than for 10 generations using generation.For production of vaccine With.

2.2.2.2 seed culture of viruses will be produced by, which being inoculated with, is inoculated in the cat kidney passage cell suspension turn prepared in 1~2% ratio In bottle.

2.2.2.3 rotating and culturing under the conditions of cell suspension is uniformly mixed 37 DEG C of postposition with seed culture of viruses with observation by culture, continuously Observation 4 days, the cell of waste pollution or growth failure.

2.2.2.4 harvesting can harvest when CPE occurs in 70% or more cell, set -20 DEG C and freeze, then receive after melting Cell culture is obtained in sterilization container, several bottles are 1 group, are frozen at -20 DEG C or less spare.

2.2.2.5 the inspection of seedling virus liquid carries out virus liquid viral level survey by 1.2.1.1,1.2.1.6 methods Fixed and steriling test, every milliliter of viral level answer >=10^6.00TCID₅₀, virus liquid should be without bacterium, mould contamination；If virus liquid is sick Malicious content is 10^5.50~10^6.00TCID₅₀Between, 5 times of concentrations are carried out by ultrafiltration, until meeting regulation.

It is molten by 0.2% addition formaldehyde of virus liquid total amount after filtering after 2.3 inactivate the virus liquid thawing that will examine qualification Liquid, it is stirring while adding, it mixes well, sets and inactivated 24 hours at 37 DEG C, should be stirred continuously during inactivation.

2.4 the inspection of semifinished product

2.4.1 steriling test is separately sampled as unit of group by the virus liquid of harvest, by existing " Chinese veterinary pharmacopoeia " annex It carries out, it should be without bacterium, fungus growth.

2.4.2 inactivation is examined to take to inactivate after liquid makees 1: 5 dilution is inoculated with 4 bottles, every bottle of 5ml CRFK cell suspension respectively 0.4ml sets and cultivates at 37 DEG C, changes liquid within 24 hours, passes for 1 generation after continuing culture 3 again, and lesion, harvest culture should not occur in cell Negative, judgement inactivation qualification is presented in liquid hemagglutination test.

The preparation and packing of 3 Combined vaccines

Pseudomonas aeruginosa pentavalent seedling semi-finished product bacterium solution after qualified inactivation will be examined to press with parvovirus semi-finished product bacterium solution The ratio of 2:1 (V/V) mixes, then mixed liquor and 40% (w/v, in terms of g/mL) aluminium hydroxide gel are mixed by 6:1 ratio uniform, After mixing well, quantitative separating seals bottleneck.

1 Combined vaccine of table configuration component ratio is groped

It chooses PA vaccine G type progress vaccine formulation ratio to grope, according to production of vaccine content standard, sets containing for Combined vaccine Amount: PA G type is 4,000,000,000 CFU/ml, and the content of parvovirus is 10^6.00TCID₅₀, 4 groups are carried out to vaccine on the basis of this concentration The mixing of different proportion (V/V), later intramuscular injection are immunized mink, 5/group, measure different groups of immune PAs of the mink after 28 days ELISA value and MEV HI value, the results showed that, antibody is relatively high when C group PA:MEV=2:1, is optimum proportioning, therefore really The PA:MEV configuration proportion for determining Combined vaccine is 2:1.

4 product inspections

After 4.1 characters are stood, upper layer is khaki clear liquid, and lower layer is pale precipitation, in uniformly mixed after shaking Suspension.

4.2 steriling tests are tested by existing " Chinese veterinary pharmacopoeia " annex, equal asepsis growth.

Susceptible mink 5 of 2~10 monthly ages health of 4.3 safety verifications, each hind leg inside muscle vaccine inoculation 3ml (divide 3 Point injection, every 1.0ml), it is observed continuously 15.

4.4 efficacy test

4.4.1 mink hemorrhagic pneumonia efficacy test

4.4.1.1 mouse is divided into 5 groups, every group 10, bigeminy epidemic disease is subcutaneously injected respectively with 16~18g of weight mouse 50 Seedling 0.2ml divides 5 groups, every group 10, every group is subcutaneously injected 70,000,000 CFU/ respectively together with control mice 50 after 21 days The DL15 strain of 0.2ml, WD01 plants or WF05 plants virulent liquid or 1.2 hundred million CFU/0.2ml JL08 strain or DL03 plants virulent Liquid is observed continuously 7.Control mice should be all dead, and immune mouse should at least protect 8.

4.4.1.2 susceptible mink 25 of 2~10 monthly ages health of mink divides 5 groups, every group 5, distinguishes intramuscular injection two Vaccines 1ml divides 5 groups, every group 5, every group intratracheally instills 200,000,000 CFU/ml's respectively together with control mink 25 after 21 days The JL08 strain of DL15 plants, WD01 plants or WF05 plants virulent liquid or 400,000,000 CFU/ml or DL03 plants of virulent liquid, are observed continuously 7.It is right Should be all dead according to mink, immune mink should at least protect 4.

4.4.1.3ELISA

Establish the ELISA method for mink hemorrhagic pneumonia, antibody yin and yang attribute critical value OD₄₅₀It indicates, G type DL15 plants, JL08 plants of Type B, WD01 plants of c-type, WF05 plants of I type, that the critical value of DL03 plants of F type is set as 0.3100, OD value is big The positive is then judged in critical value；Conversely, being then determined as feminine gender.With the immune rear pseudomonas aeruginosa of ELISA method monitoring Combined vaccine G, the variation of B, C, I, F type antibody, 0,7,14,21,28,60,90 after each group is inoculated with ELISA method measurement respectively after being immunized, The green pseudomonad antibody level of 120,180 Copper in Serum.

4.4.2 Canine Parvovirus Enteritis efficacy test

4.4.2.1 serum antibody measurement with 2~10 monthly ages healthy susceptible mink (Mink Parvovirus HI antibody titer≤ 1: 4) 5, each intramuscular inoculation vaccine 1ml after 21 days, together with control mink 5, take a blood sample respectively, measure HI and SN antibody titer, Immune group mink antibody titer should all >=1: 32, control group mink antibody titer should all≤1: 4.

4.4.2.2 Immunization susceptible mink (antibody titer≤1 Mink Parvovirus HI: 4) 5 of 2~10 monthly ages health Only, each intramuscular inoculation vaccine 1ml, it is each to take orally 100 ID together with control mink 5 after 21 days₅₀Mink Parvovirus SMPV- 11 plants of internal organs poison are observed 9, and control group mink should all fall ill, and immune mink should all be good for work.

4.4.3 residual formaldehyde measurement is measured by existing " Chinese veterinary pharmacopoeia " annex respectively, should meet regulation.

5 effects are with purposes for preventing Mink Parvovirus Enteritis and by G type, Type B, c-type, F type, the verdigris vacation of I type Mink hemorrhagic pneumonia caused by monad.Duration of immunity is 5 months.

Intramuscular injection on the inside of 6 usage and dosage hind legs.Every inoculation 1ml of mink.Intramuscular injection, 50~70 days after birth The young ermine in age is inoculated with simultaneously with female ermine, and kind ermine breeds first 30~60 days and is inoculated with, every 1ml.

2 safety testing of embodiment

The 2 safety testing scope of examination of table

1.1 safety testing

201501, the single dose inoculation of 201502,201,503 3 batches of vaccines, the inoculation of single dose repeated inoculation, overdose Different days mink carries out safety evaluatio, and a single dose is inoculated with 60~70 age in days children ermines, and 1ml/ is only；Single dose repeats to connect 60~70 age in days children ermines of kind, 2 inoculations midfeather 15 days are inoculated with 1ml/ only every time；Overdose is inoculated with 60~70 age in days children ermines It is bred as ermine with the 3-6 monthly age, 3ml/ only, divides 3 points of injections.Every group of inoculation mink 5, hind leg inside intramuscular inoculation, and set up not Immunized controls group.It is observed continuously after inoculation 15, observation whole body clinical symptom and inoculation localized variation, and after inoculation preceding 10 Day, the daily morning records Temperature changing.

1.2 safety testing results:

201501, mink does not occur locally and systemically adverse reaction after 201502,201,503 3 batches of vaccine immunities, essence Mind, appetite, body temperature and excrement variation without exception.Mink body temperature is identical as basal body temperature after inoculation, is kept at 38.6~41.5 Between DEG C, injection site is without enlargement after all inoculation mink inoculations, and without festering, young ermine upgrowth situation is good.Illustrate the vaccine It is safer to mink.

3 Mink Parvovirus Enteritis of table, hemorrhagic pneumonia bivalent inactivated vaccine mink safety testing result

The effect evaluation of embodiment 3MEV-PA bivalent inactivated vaccine

One, the potency test of MEV-PA bivalent inactivated vaccine

1 immune grouping

1.1 mink

Take the 3 monthly ages susceptible mink 120 of health (the susceptible mink standard of health: Mink Parvovirus serum HI antibody titer ≤ 1: 4, pseudomonas aeruginosa G, B, C, I, F mink serum macroscopic agglutination is feminine gender) 4 groups are randomly divided into, every group 40, divide For MEV-PA group, MEV group, PA group and control group, muscle vaccinates 1ml/ only on the inside of hind leg respectively, control group the same manner note Penetrate isometric physiological saline.

1.2 mouse

With 16~18g of weight mouse 50, it is divided into 5 groups, every group 10, vaccine 0.2ml is subcutaneously injected respectively.2 minks go out Hemorrhagic pneumonia Efficacy evaluation

2.1 mink

2.1.1 attacking malicious protection

Take 30 it is immune after 21 days, together with control mink 30, points 6 groups, every group 5 are immune and control group mink exists respectively Malicious PA DL15 plants, JL08 plants, WD01 plants, WF05 plants, DL03 plants of bacterial strains and MEV SMPV-11 are attacked within 21st, 90,180 after immune Strain, each strain attack 5 minks, observe 7, record death toll, and malicious protecting effect is attacked in measurement.

2.1.2 antibody determination

The 5 mink blood sample detection PA's of every group of acquisition on the 0th, 7,14,21,28,60,90,120,180 is anti-after immune G, B, C, I, F type ELISA antibody.

2.2 mouse

21 days after 50 mouse immunes, together with control mice 50, divide 5 groups, every group 10, every group is subcutaneously injected respectively The DL15 strain of 70000000 CFU/0.2ml, WD01 plants or WF05 plants virulent liquid or 1.2 hundred million CFU/0.2ml JL08 strain or DL03 The virulent liquid of strain, is observed continuously 7, records death toll.

3 Mink Parvovirus Enteritis Efficacy evaluations

3,1 antibody determination

5 mink blood sample detections of every group of acquisition on the 0th, 7,14,21,28,60,90,120,180 after vaccine immunity SN the and HI antibody of MEV.

3.2 attack malicious protection

Above-mentioned immune and control group mink attacks poison 1 PADL15 plants of MLD, JL08 on the 21st, 90,180 after immune respectively Strain, WD01 plants, WF05 plants, DL03 plants of bacterial strains and 100 ID₅₀SMPV-11 plants of internal organs poison of Mink Parvovirus, each strain attack 5 Mink is observed 7, and death toll is recorded, and malicious protecting effect is attacked in measurement.

Two, the effect evaluation result of MEV-PA bigeminy vaccine:

1 mink hemorrhagic pneumonia efficacy test results

1.1 measurement results show that each serotype antibody turns sun from 7 days antibody, and highest on the 21st continues to 180d.With list Seedling control is compared, and the antibody level difference for joining each serotype in seedling is not significant (P > 0.005).

1.1 attack malicious protection

1.1.1 mink MEV-PA Combined vaccine and the mono- seedling of PA are immunized after mink 30,90,180 respectively, together with control group, divide Do not attack DL15 plants of malicious PA G type, JL08 plants of Type B, WD01 plants of c-type, WF05 plants of I type, DL03 plants of F type, every kind of serotype and control Attack poison group 5/group of random selection.Poison is attacked the result shows that each group protected number 4/5 or more, and control group equal 100% is dead to 180 days It dies, vaccine protecting effect is preferable, and duration of immunity was up to 180 days.Compared with the mono- seedling of PA, the protecting effect of vaccine without significant difference (P > 0.005)。

1.1.2 mouse MEV-PA Combined vaccine and the mono- seedling of PA attack poison on the 21st the result shows that PA MEV- after mouse is immunized respectively Mouse is immunized to DL15 plants of G type, JL08 plants of Type B in PA Combined vaccine and the mono- seedling of PA, WD01 plants of C type, WF05 plants of I type, DL03 plants of F type Malicious protective rate of attacking be 100% (10/10).2 Mink Parvovirus Enteritis efficacy tests

The measurement of 2.1 serum antibodies

Hemagglutination inhibition antibody (HI) uses HI method detection Combined vaccine and MEV seedling to be immunized the 0th, 7,14,21 after mink respectively, Anti- MEV antibody after MEV HI antibody level in mink serum on the 28th, 60,90,120,180, Combined vaccine and MEV seedling are immune the Antibody average value >=2 on the 7th^5.9, continue within 28th~60 compared with High antibody level, be 2^9.5~2^10.4, until antibody on the 180th is higher, It is 2⁸~2^8.6.From graphical trend, compared with the immune rear level of the mono- seedling of MEV, MEV antibody is integrally relatively low after Combined vaccine is immune, But otherness is not significant (P > 0.05).

2.2 neutralizing antibodies (SN) detect Combined vaccine with endpoint titration respectively and MEV seedling is immunized the 0th, 7,14 after mink, Anti- MEV after anti-MEV SN antibody level in mink serum on the 21st, 28,60,90,120,180, Combined vaccine and MEV seedling are immune is anti- Body antibody average value >=10 on the 7th^0.8, continue within 28th~60 compared with High antibody level, be 10¹~10^1.29, until antibody on the 180th is equal It is higher, it is 10^0.9.Compared with the immune rear level of the mono- seedling of MEV, MEV antibody is integrally relatively low after Combined vaccine is immune, but otherness is not shown It writes (P > 0.05).

2.3 attack malicious MEV-PA Combined vaccine and the mono- seedling of MEV is immunized after mink 30,90,180 respectively, every group of random selection 5 Only, each to take orally 100 ID together with control 5₅₀SMPV -11 plants of internal organs poison of Mink Parvovirus.Poison is attacked the result shows that 180 Day, it is 5/5 that each group, which protects number, equal 5/5 morbidity of control group, it was demonstrated that vaccine protecting effect is preferable, and duration of immunity was up to 180 days.With The mono- seedling of MEV is compared, and the protecting effect difference of Combined vaccine is not significant (P > 0.05).

4 Combined vaccine of table is immunized mink and attacks poison protection result

5 Combined vaccine of table is immunized mouse and attacks within 21st poison protection result

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Claims (10)

1. a kind of P. aeruginosa bacterial strain, which is characterized in that its deposit number is CGMCC No.11989.

2. application of the P. aeruginosa bacterial strain according to claim 1 in preparation mink hemorrhagic pneumonia vaccine.

3. P. aeruginosa bacterial strain according to claim 1 is preparing Mink Parvovirus Enteritis, hemorrhagic pneumonia two Join the application in inactivated vaccine.

4. a kind of mink hemorrhagic pneumonia vaccine, which is characterized in that by P. aeruginosa bacterial strain described in claim 1 and water DL15 plants of ermine pseudomonas aeruginosa G type, JL08 plants of Type B be made.

5. the bivalent inactivated vaccine of a kind of Mink Parvovirus Enteritis, hemorrhagic pneumonia, which is characterized in that by claim 1 The P. aeruginosa bacterial strain and DL15 plants of mink pseudomonas aeruginosa G type, JL08 plants of Type B and Mink Parvovirus strain MEVB plants are made.

6. the preparation method of bivalent inactivated vaccine according to claim 5, which comprises the steps of:

Step 1: taking P. aeruginosa bacterial strain and DL15 plants of mink pseudomonas aeruginosa G type, Type B described in claim 1 JL08 plants are inoculated in mixing after culture medium culture respectively, pseudomonas aeruginosa pentavalent seedling semi-finished product bacterium after inactivation are made after inactivation Liquid；

Step 2: taking MEVB plants of inoculating cells of Mink Parvovirus strain, harvest inactivates obtained parvovirus semi-finished product；

Step 3: taking pseudomonas aeruginosa pentavalent seedling semi-finished product bacterium solution and tiny disease made from step 2 after step 1 inactivation obtained Malicious semi-finished product are mixed to prepare mixed liquor；

Step 4: mixed liquor made from step 3 being taken to mix with adjuvant.

7. preparation method according to claim 6, which is characterized in that P. aeruginosa described in claim 1 in step 1 Bacterial strain and DL15 plants of mink pseudomonas aeruginosa G type, JL08 plants of Type B are respectively after culture medium culture, the mixed ratio For 1:1:1:1:1, the bacterium number of every plant of bacterium is at least 4,000,000,000 CFU/mL after mixing.

8. preparation method according to claim 6 or 7, which is characterized in that parvovirus in step 2 parvovirus semi-finished product Content be 10^6.00TCID₅₀。

9. according to the described in any item preparation methods of claim 6 to 8, which is characterized in that go out made from step 1 described in step 3 The ratio of the mixing of parvovirus semi-finished product made from pseudomonas aeruginosa pentavalent seedling semi-finished product bacterium solution and step 2 is 2:1 after work.

10. according to the described in any item preparation methods of claim 6 to 9, which is characterized in that culture medium described in step 1 is in PYG MgSO is added in culture medium₄, sodium citrate, vitamin, urea, Na₂HPO₄Buffer salt or KH₂PO₄One of buffer salt is more Kind；

Inoculation described in step 1 is according to 3% inoculating strain of PYG culture volume；Inoculation described in step 2 is thin according to CRFK Inactivation described in the 2% virus inoculation step 1 or step 2 of born of the same parents' suspension volume uses formalin-inactivated；The dosage of the formaldehyde is bacterium solution The 0.2% of total amount；

Adjuvant described in step 4 is 40% (w/v, in terms of g/mL) aluminium hydroxide gel, mixed liquor made from step 3 and the adjuvant Mixed proportion is 6:1.