CN104975038A - Riemerella anatipestifer pre-bacterial ghost vaccine preparation method - Google Patents
Riemerella anatipestifer pre-bacterial ghost vaccine preparation method Download PDFInfo
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Abstract
The present invention relates to a method for conversion preparation of a riemerella anatipestifer pre-bacterial ghost vaccine by using phage PhiX174 E gene and staphylococcal nuclease A gene, and an application method. According to the technical scheme, the phage PhiX174 E gene and the staphylococcal nuclease A gene are coupled to form the series coupling gene through the flexible coupling arm (Gly4Ser), a temperature control expression vector pBV-E-SN is further constructed, the pBV-E-SN is transformed into riemerella anatipestifer protoplast through electric shock conversion, the positive clones are screened and are subjected to bacterial amplification culture at a temperature of 28 DEG C, the bacteria are separated, a protection agent is added, and sub-packaging and freeze-drying are performed. According to the present invention, the prepared live bacterial vaccine is immunized through drinking water, such that the local immunity on the respiratory tract mucous membrane and the digestive tract mucous membrane can be irritated, and the humoral immunity can be stimulated.
Description
Technical field
The invention belongs to poultry farming control and prevention of disease field, relate to a kind of preparation method of bacterial vaccine, especially relate to a kind of utilize the E gene of Phage PhiX174 and Staphylococcus nuclease A gene transformation to prepare bacterium shadow vaccine before Riemerellosis Anatipestifer method and application method.
Background technology
Infectious serositis of duck, be by Riemerellosis Anatipestifer (
riemerella Anatipestifer, RA) cause duck, goose, turkey and other poultry and wild fowl the acute or Chronic exposure sexually transmitted disease of one, main infringement 2-7 duckling in age in week, with nervous symptoms and fibrinous pericarditis, serohepatitis and airsacculitis for feature.
Current, Riemerellosis Anatipestifer infects harm to aquaculture, and except the morbidity that directly causes animal and death, the feed conversion rate that inapparent infection causes reduces, growth retardation and the indirect economic loss that brings is also very serious.Although Riemerellosis Anatipestifer is to some antibiotic sensitive, be difficult in body remove completely; Simultaneously due to antibiotic unreasonable use, its resistance strengthens day by day, often causes the drug residue problem of poultry product to become increasingly conspicuous.The scientific approach of this disease of prevention and control, is on the basis of strengthening feeding and management, inoculates suitable Riemerellosis Anatipestifer vaccine.
Although there have been many research reports at present in the vaccine of Riemerellosis Anatipestifer, such as, Riemerellosis Anatipestifer deactivation vaccine, multivalence composite adjuvant inactivated vaccine and Novel bacteria shadow vaccine etc.But still there is the problem of following several respects: 1. traditional vaccine preparation technology, by changing antigenic structure in the chemicals inactivation process such as formaldehyde, and reduces immunogenicity, causes immune protective rate to reduce.2. current reported vaccine is all by injecting immune, and immunological stress is large, and under large-scale cultivation condition, immune operation is wasted time and energy.3. deactivation vaccine can only cause humoral immunization, does not produce or only produces slight cellular immunization and local mucosa-immune, can not provide and support anti-infectious mucosa-immune barrier reliably.
Summary of the invention
In view of the deficiencies in the prior art, the object of the present invention is to provide a kind of E gene and Staphylococcus nuclease A gene transformation Riemerellosis Anatipestifer clinical separation strain utilizing Phage PhiX174, prepare the technical scheme of bacterium shadow vaccine and application thereof before novel Riemerellosis Anatipestifer.Technical scheme provided by the present invention is to effectively control Riemerellosis Anatipestifer infection, and the use, minimizing drug residue etc. that reduce antibacterials in poultry farming process are significant.
In order to solve the problem existing for background technology, the present invention is by the following technical solutions:
Bacterial strain based on the 2 type Riemerellosis Anatipestifer clinical separation strains (being numbered LY1) selecting this laboratory qualification to preserve.This bacterial strain still no pathogenicity, thus use safety after 3 pickup kind duckling rejuvenation.After the bacterial strain recovery of freezen protective, to be prepared into protoplastis for subsequent use for test method routinely.
Utilize conventional molecular biological technology, the E gene (being called for short E) of Phage PhiX174 is flexibly connected arm (Gly with Staphylococcus nuclease A gene (being called for short SN) by coding
4ser)
3linker connect, obtain the tandem gene E-SN of E, SN, further it be connected with Human liver glutathione carrier pBV220, structure Human liver glutathione carrier pBV-E-SN.By the pBV-E-SN electric Riemerellosis Anatipestifer protoplastis transforming preparation of test method routinely, screening positive clone obtains Riemerellosis Anatipestifer recombinant bacterial strain.
The Riemerellosis Anatipestifer engineering bacteria ordinary method 28 DEG C that obtains is expanded bacterium cultivate, separating thallus, mixes with lyophilized vaccine equal-volume, is sub-packed in 10mL cillin bottle, lyophilize under clean environment, namely obtains bacterium shadow vaccine before Riemerellosis Anatipestifer.
The formula of lyophilized vaccine is made up of the component of following weight part: skimming milk 8-12 part, sucrose 2-6 part, trehalose 3-10 part, xitix 2-6 part, glycerol 12-20 part, purified water 50-70 part.
Preferred further, lyophilized vaccine is made up of the component of following weight part: skimming milk 8-10 part, sucrose 2-4 part, trehalose 3-8 part, xitix 2-4 part, glycerol 12-16 part, purified water 60-70 part.
By immunity of drinking water, this vaccine is normally bred at the upper respiratory tract that temperature is lower, stimulates respiratory tract local mucosal immune response (SIgA), and is eliminated gradually; The bacterium that the upper respiratory tract is bred and enter gastral bacterial part and break through Mucosa Barrier and enter blood, induces entrained Human liver glutathione carrier to produce lysis genes product immediately.Wherein, E gene product forms cross-film duct at bacterial cell membrane, and bacterium content flows out and forms bacterium shadow, and continuing stimulates body to produce cellular immunization and humoral immunization; DNA of bacteria is degraded into the fragment being less than 100bp by SN gene product, by thorough for bacterium deactivation.
Compared with prior art, the present invention relates to utilize the two lysis genes of temperature control to prepare Riemerellosis Anatipestifer before bacterium shadow vaccine tool have the following advantages and marked improvement:
(1) compared with traditional inactivated vaccine, before forming bacterium shadow (at respiratory tract reproductive stage) and enter body in after formation bacterium shadow, bacterial surface structures is substantially complete, and antigenicity is substantially unaffected, and immune protective rate is high.
(2) bacterium shadow vaccine before Riemerellosis Anatipestifer provided by the present invention, by immunity of drinking water, avoid injecting immune stress, application is convenient, adapts to large-scale cultivation.
(3) traditional inactivated vaccine and current reported relevant Riemerellosis Anatipestifer bacterium shadow vaccine, by injecting immune, can only cause humoral immune reaction, protected effect has certain limitation.Technical scheme provided by the present invention, utilize the temperature contrast of duck body different sites, before the Riemerellosis Anatipestifer of preparation, bacterium shadow can not the expression of inducing lysis gene at the lower respiratory tract of temperature, thus normally can breed and stimulate the mucosa-immune reaction of respiratory tract local; Enter the rising (41 DEG C-42 DEG C) of the vaccine in body due to temperature, lysis genes starts to express immediately, thus forms bacterium shadow stimulation body generation humoral immunization and cellular immunization.Mucosa-immune due to respiratory tract reacts (SIgA) Temporal variation, before being increased to the vaccine strains being enough to remove inoculation, having bacterium and continues ingress engine body and form bacterium shadow and excite humoral immune reaction, play the slow releasing function of similar oil adjuvant killed vaccine at SIgA.Thus, scheme provided by the present invention both can cause respiratory tract and gastral mucosa-immune react and provide the first line of defence of opposing bacteriological infection, also can cause humoral immune reaction and provide lasting protection.
(4) the two lysis genes of the series connection of temperature control abduction delivering is utilized, make the Most bacterial entering internal body form bacterium shadow lose activity and retain immunogenicity, minority fails to be formed in time the bacterium in cross-film duct, and its DNA is degraded rapidly to small segment and inactivation by SN.In addition, the bacterial strain of selection is low pathogenicity clinical separation strain, and the security that vaccine uses is guaranteed in triple guarantee.
Embodiment
Be below specific embodiments of the invention, technical scheme of the present invention is described further, but protection scope of the present invention is not limited to these embodiments.Every do not deviate from the present invention's design change or equivalent substituting include within protection scope of the present invention.
Embodiment one: the preparation of bacterium shadow before Riemerellosis Anatipestifer
the toxicity checking of 1.1 clinical separation strainsthe bacterial strain that the Riemerellosis Anatipestifer (being numbered LY1) one strain be separated from clinical egg duck is accredited as 2 types through serotype has carried out virulence checking.This bacterial strain does not produce gelatinase; in inoculation duckling continuous 3 generations, do not show pathogenic; but the protection of more than 90% can be produced with homotype strong virus attack, illustrate that this bacterial strain is that a strain had no pathogenicity or virulence are lower and has again good immunogenic bacterial strain, can be used as vaccine candidate strain.
the preparation of protoplastisafter being activated by LY1 bacterial strain, be cultured to OD
600when being 0.7, bacterium liquid is placed in ice-water bath 30min, gets 10mL bacterium liquid, under 4 DEG C of conditions, the centrifugal 10min of 5000r/min, abandons supernatant, with the washing of 10mL ice-water bath aqua sterilisa, and recentrifuge, repeated washing like this three times.Suspend with ice-water bath high osmotic buffer, add the lysozyme soln of preheating to 5mg/mL, after 37 DEG C of water-bath 30min, then the EDTA solution adding preheating is to 0.01mol/L, 37 DEG C of water-bath 20min.In mechanism, get the bacterium liquid of process on slide glass every 5min, azaleine dyes, the generation situation of oily Microscopic observation protoplastis.When the rate of formation of protoplastis reaches more than 95%, the centrifugal 20min of 5000r/min, abandons supernatant, then adds TSB regeneration culture medium centrifuge washing 3 times, abandons supernatant.Finally protoplastis TSB regeneration culture medium is suspended.
the structure of Human liver glutathione carrierby Phage PhiX174 crack protein E gene order (E, GI:9626372) and Staphylococcal Nuclease A gene order (SN, GI:21281729) by 15 flexible amino acid linker (Gly
4ser)
3series connection is that E-SN is dual-gene, proceeds in cloning vector pMD19T-simple, builds recombinant cloning vector pMD-E-SN.Then respectively with EcoRI, PstI Human liver glutathione carrier pBV220 is carried out double digestion with recombinant cloning vector pMD-E-SN and is connected, build restructuring Human liver glutathione carrier pBV-E-SN.
the screening of electricity conversion and positive colonyget 10 μ L bacteriolyze plasmids to add in 40 μ L LY1 protoplasm somatocytes, carefully mix, move in the electric shock cup of 0.1cm after placing 5min on ice.Electricity ginseng is set to: 15Kv/cm, 50 μ Fd, 150 Ω, 5ms, carry out electroporated.After electric shock, in electric shock cup, add 960 μ LTSB regeneration culture mediums immediately, be transferred to by bacterium liquid in sterilizing 1.5 mL centrifuge tube, 28 DEG C, 150 r/min shake bacterium 2h.Centrifugal bacterium liquid, discard 800 μ L supernatants, with the resuspended thalline of remaining liq, regenerate soft agar medium (35ug/mL Amp+) with the TSB being chilled to 45 DEG C and mix, be poured into above TSB regenerated solids substratum (35 μ g/mL Amp+), 28 DEG C of cultivations.Single bacterium colony that picking flat board grows, be inoculated in the test tube containing the TSB substratum of Amp+ (100 μ L/mL), 28 DEG C of jolting overnight incubation, are accredited as the positive by bacterium liquid PCR.
, front bacterium shadow vaccine preparation:with 1:100 ratio, the little bacterium liquid that shakes is seeded to 200 mL containing in the fresh TSB substratum of Amp+ resistance, mixing, gets appropriate bacterium liquid and survey the OD before shaking bacterium cultivation
600, then 28 DEG C of joltings are cultivated, and survey OD every 1h
600.Treat OD
600when being about 0.6, the centrifugal 20min separating thallus of 5000r/min, mixes with lyophilized vaccine equal-volume, is sub-packed in the lyophilize of 10mL cillin bottle under clean environment.Lyophilized vaccine is made up of the component of following weight part: skimming milk 10 parts, sucrose 3 parts, trehalose 6 parts, 3 parts, xitix, glycerol 15 parts, purified water 70 parts.
Cryodesiccated vaccine be stored in 0 DEG C-4 DEG C for subsequent use.
Embodiment two: vaccine immunity protection test
In order to verify that bacterium shadow vaccine that the present invention utilizes genetic engineering means to prepare is to the clinical protection effect of duck; select Growth of Cherry Valley Commercial meat-type duck as laboratory animal, the homology strain oil-adjuvant vaccine prepared with formalin-inactivated method carries out duckling Immunoprotection test under the same conditions.
1. materials and methods
1.1 test materials
Front bacterium shadow vaccine: by method preparation described in embodiment 1.5.
Oil adjuvant inactivated vaccine: the LY1 bacterial strain of formalin-inactivated mixes according to 1:1 ratio with mineral oil adjuvant, makes bacteria content reach 10
10cfu/mL.
Laboratory animal: 1 age in days Growth of Cherry Valley Commercial meat-type duck.
1.2 test method
1.2.1 testing program
Be health 1 age in days Growth of Cherry Valley Commercial meat-type duck 40 plumage of RA feminine gender after testing, raise to 5 ages in days, be divided into 4 groups to carry out immunity respectively at random.Front bacterium shadow vaccine immunity group carries out drinking-water immunity, guarantees that every duck soakage reaches 5 × 10
10cfu.Oil adjuvant inactivated vaccine group and vehicle control group every leg muscle inject 5 × 10
10cfu/0.5mL.Within 2 weeks, carry out the strong poison of RA2 type after immunity attack poison and observe one week.Grouping is immune and attack malicious situation in table 1.
Table 1 vaccine immunity protected effect
| Group | Quantity | Immunizing dose | Attack toxic agent amount | Survive number |
| Front bacterium shadow vaccine | 10 | 5×10 10Cfu/0.5mL/ only | 10 6Cfu/0.5mL/ only | 10/10 |
| Oil adjuvant inactivated vaccine | 10 | 5×10 10Cfu/0.5mL/ only | 10 6Cfu/0.5mL/ only | 10/10 |
| Vehicle control group | 10 | 0.5mL/ only | 10 6Cfu/0.5mL/ only | 2/10 |
| Blank group | 10 | - | - | 10/10 |
1.2.2 antibody horizontal detects
Using 2 type RA ultrasonic degradation supernatants as envelope antigen coated elisa plate, detect the Fluctuation of RA antibody in the duck serum of taking a blood sample weekly and being separated.
2. test-results
2.1 challenge test results
From the test-results of table 1; under test conditions; front bacterium shadow vaccine and oil adjuvant inactivated vaccine all can reach the protection of 100% to the protection ratio that 19 ages in days attack malicious duck; and the mortality ratio of vehicle control group reaches 80%; equal survive organized by not immune poison of not attacking, and shows that the bacterium shadow vaccine prepared has good protected effect to duck in vulnerable period.
2.2 antibody dynamic regularity
After vaccine immunity in 4 weeks, antibody horizontal continues to rise, and within 4 weeks, peaks, after immunity, within 5 weeks, still maintain higher level.Although bacterium shadow vaccine immunity group at the antibody horizontal in each stage all lower than the antibody horizontal (see Figure of description 1) of deactivation vaccine immune group, after immunity 2 weeks attack malicious result, both have equal immune protective effect.
Accompanying drawing explanation
Figure of description 1 shows Riemerellosis Anatipestifer bacterium shadow vaccine and the Antibody dynamics change of deactivation vaccine after immune Growth of Cherry Valley Commercial meat-type duck in 5 weeks.
Claims (5)
1. prepare the method for bacterium shadow vaccine before Riemerellosis Anatipestifer for one kind, it is characterized in that: utilize the E gene of Phage PhiX174 (being called for short E) and Staphylococcus nuclease A gene (being called for short SN) to build the two lysis genes pBV-E-SN of temperature control, its electricity is transformed Riemerellosis Anatipestifer protoplastis, and screening obtains positive colony.
2. the method preparing bacterium shadow vaccine before Riemerellosis Anatipestifer according to claim 1; it is characterized in that: the thalline that the positive bacterium colony that screening obtains obtains through 28 DEG C of Zengjing Granule mixes with protective material; this protectant formula is made up of the component of following weight part: skimming milk 8-12 part; sucrose 2-6 part; trehalose 3-10 part; xitix 2-6 part, glycerol 12-20 part, purified water 50-70 part.
3. the method preparing bacterium shadow vaccine before Riemerellosis Anatipestifer according to claim 2; it is characterized in that: described protectant formula composition; preferably; be made up of the component of following weight part: skimming milk 8-10 part; sucrose 2-4 part, trehalose 3-8 part, xitix 2-4 part; glycerol 12-16 part, purified water 60-70 part.
4. the method preparing bacterium shadow vaccine before Riemerellosis Anatipestifer according to any one of claim 1-3, is characterized in that: this vaccine by drinking-water via digestive tube and respiratory immunity animal.
5. the method preparing bacterium shadow vaccine before Riemerellosis Anatipestifer according to claim 4, is characterized in that: described animal is commodity egg duck, Commercial meat-type duck, kind duck, goose and turkey.
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| CN112410360A (en) * | 2021-01-18 | 2021-02-26 | 西南大学 | Chicken pathogenic bacterium ghost and preparation method and application thereof |
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| CN102260636A (en) * | 2010-05-28 | 2011-11-30 | 内蒙古蒙牛乳业（集团）股份有限公司 | Freeze-drying protective agent, and preparation method and application thereof |
| CN103146732A (en) * | 2013-01-28 | 2013-06-12 | 山东省农业科学院畜牧兽医研究所 | Efficient splitting tandem gene, efficient splitting plasmid and construction method and appliance |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112410360A (en) * | 2021-01-18 | 2021-02-26 | 西南大学 | Chicken pathogenic bacterium ghost and preparation method and application thereof |
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