CN104353072B - Application of the bacterium ghost in viral vaccine adjuvant is prepared - Google Patents

Abstract

The invention discloses application of the bacterium ghost in viral vaccine adjuvant is prepared, and the invention also discloses a kind of scale fermentation processes of bacterium ghost.The beneficial effects are mainly as follows：The present invention provides the large-scale producing method of bacterium ghost, and solid foundation has been established for the merchandized handling of ghost vaccine；In addition, the adjuvant function of have rated ghost of present system, the result shows that ghost can greatly improve the immune efficacy of viral vaccine, and ghost is simple as adjuvant application method, can a degree of production cost for reducing vaccine, the prevention and control to human and animal's viral infectious are of great significance.

Description

Application of the bacterium ghost in viral vaccine adjuvant is prepared

Technical field

The present invention relates to bacterium ghost scale fermentation preparation and ghost as adjuvant in viral vaccine Using.The invention belongs to genetic engineering and immunological technique field.

Background technology

Bacterium ghost (bacterial ghost, BG) is by controlling PhiX174 bacteriophages in Gram-negative bacteria The ghost that the expression of crack protein E makes host strain that cracking occur and produces.The bacteriolyze channel sized of E protein mediation is by cell Individual difference influences, and aperture is between 40nm to 200nm.Once being formed, bacterial cytoplasm content is in interior exosmosis pressure for passage Under the action of flowed out by passage it is extracellular, formed one completion empty bacterial.The bacteriolysis of E protein mediation has been applied successfully In various Escherichia coli, salmonella, Actinobacillus pleuropneumoniae, Friedlander's bacillus, helicobacter pylori, cholera arc Bacterium, haemophilus influenzae, Pasteurella etc..The bacteriolyze of the so big explanation E protein mediation of scope may be in all grams Occur in negative bacterium.

Ghost can be used as a kind of good vaccine in itself.Its shell mechanism is not destroyed, retains as viable bacteria After birth structure and associated antigen protein, and outer membrane contain natural immunocyte identified by pattern recognition receptors it is highly conserved Structure PAMP (pathogen-associated molecular patterns), for example, lipopolysaccharides, peptide glycan, CpG, OmpA, Pili etc., can be effectively by antigen presenting cell (APCs) phagocytosis, processing and submission.This is also ghost and conventional inactivated vaccine phase One outstanding advantage of ratio.At present, ghost is immunized dynamic in mouse, rabbit, pig etc. by approach such as vein, subcutaneous, gases Good immune protective effect is achieved in thing model.With Actinobacillus pleuropneumoniae ghost through gas immunity inoculation pig, induction For the complete protection of pleuropneumonia caused by A.pleuropneumoniae.Comma bacillus ghost subcutaneous injection mouse lures Serum is led to produce high-caliber anticholera antibody and be enough the infection for protecting newborn mice to exempt from comma bacillus.In addition, bacterium Slough off and be perfect carrier, substantial amounts of allogenic material can be received.Recombinant protein can be inserted into thin by specific film anchoring structure After birth, or be filled in born of the same parents week gap and cell cavity, to obtain restructuring ghost multivalence seedling and multi-joint seedling.

Ghost production is simple, can be collected after fermentation with the method that centrifugation is resuspended, and the form that can be freezed is stored in Room temperature, without complicated purification work as protein vaccine.Preferable ghost is free of the contents such as endochylema, nucleic acid, therefore not It is safe to use there are the problem of virulence enhancing, it is environmentally safe.

Preferable adjuvant is can to cause appropriate immunologic enhancement with minimum immunostimulation, and is had no adverse reaction.Aluminium Salt adjuvant is most widely used adjuvant on current live vaccine and vaccine for man, has been widely used in the cause of diseases such as bacterium, virus Microorganism vaccine, confirms although the adjuvant has obtained long-term practice in raising antibody level and secure context, exists many Shortcoming：It cannot freeze；Difference is big between batch；With failing during many polypeptide vaccine antigen co-immunizations recombinantly or synthetically Effective immune response is excited, makes it difficult to the needs for meeting new generation vaccine development.Typically, oil/water emulsion-type adjuvant with Inject based on delivery approach, including the oil-in-water such as M F59, QS21, A S02 and Montanide or water-in-oil type adjuvant, it is this kind of Adjuvant strengthens the immunogenicity of vaccine antigen by muscle or inoculated with subcutaneous injections.In addition, body endogenous cytokine is such as IL-2, IL-12, GM-CSF etc., can also make new vaccine adjuvant, be mainly inoculated with by injecting pathway, and that is triggered is immune Acknowledgement type is similar to oil/water emulsion-type adjuvant.But such adjuvant mostly cost is higher.At present, simple mucosal route type assistant Agent is less, mainly based on E.coli LT toxin and its mutant, induces the responsing reaction based on mucosal immunity, secretion is special Different in nature sIgA, also generation system immune response, but intensity is weak compared with injecting pathway.This kind of adjuvant is delivered opposite compared with mucosal route Safety, even if adjuvant there are certain toxicity, but in effective dose carry out mucous membrane deliver it is still safer, but also with deliver The remarkable advantages such as simplicity, hurtless measure.The new vaccine adjuvant for possessing two kinds of immunization routes of injection and mucous membrane is favored.In difference Immunization route under, this kind of adjuvant both can induce out systemic immunity response, can also produce mucosal immunity effect, existing humoral immunity Also there is cellular immunity.Since ghost possesses intact natural bacterium shell mechanism, it can excite humoral and cellular immune response should at the same time Answer.The surface adhesion structure such as its pili, cilium makes it targeting and is attached on specific tissue, glutinous such as intestines and stomach and respiratory tract Film surface, is easily captured by body phagocyte, such as the M cell recognitions of PP knots (Peyer ' s Patches), thus can effectively pass Vaccine antigen is sent to mucous membrane surface and the related mucosal immune response of induction.

At present, domestic and international researcher focus of attention is that ghost is used to prevent bacterium infection as a kind of new generation vaccine.And The adjuvant function of systematic evaluation ghost is simultaneously applied to the immune there is not yet relevant report of viral vaccine as adjuvant. In addition, the present invention overcomes the small bottleneck problem of conventional ghost preparative-scale, salmonella is prepared in large quantities using fermentation tank Ghost, realizes the large-scale production of ghost.

The content of the invention

An object of the present invention is to provide application of the bacterium ghost in viral vaccine adjuvant is prepared.

Wherein, the adjuvant function of the bacterium ghost is mainly reflected in the immune effect that its energy specificity improves viral vaccine The protective rate of power, the antibody duration for improving viral vaccine and viral vaccine.

In the present invention, the ghost comes from various bacteria, preferably from Salmonella strains.The bacterium bacterium Sloughing off can apply through subcutaneous, mouth, part, mucous membrane, lung and/or nose.

The second object of the present invention is to provide a kind of large-scale preparation method of bacterium ghost, comprises the following steps：

(1) bacterium of the structure containing punching plasmid pBV-E, wherein the punching plasmid pBV-E is by by PhiX174 What the crack protein E gene clonings of bacteriophage were obtained into pBV-220 plasmids；

(2) bacterial colony containing punching plasmid pBV-E on the LB agar plates containing carbenicillin is transferred to and contained In the LB fluid nutrient mediums of carbenicillin, when 37 DEG C of 200rpm shaken cultivations 10-14 are small, as primary seed solution；By 5% (v/v) inoculum concentration, primary seed solution is transferred in the LB fluid nutrient mediums containing carbenicillin, and 37 DEG C of 200rpm cultures 6 are small When, as secondary seed solution；LB fluid nutrient mediums are added into fermentation tank and carry out in-situ sterilization；By 5% (v/v) inoculum concentration, take Secondary seed solution, which is transferred in fermentation tank while adds carbenicillin, ferments；Primary fermentation bar is induced by Programmed control Part：37 DEG C, 200rpm, 10L air/minute, by Bacteria Culture to OD600=0.75~1.0；

(3) 42 DEG C of induction E protein expression are warming up to, at the time point when induction 2.5 is small, add final concentration 150-200 μ g/ The chloramphenicol of ml；When induction all times are 4 small；

(4) thalline is collected, 70-90 DEG C of processing, freezes.

Wherein, it is preferred that the nucleotide sequence of the crack protein E genes of PhiX174 bacteriophages such as SEQ ID NO.1 institutes Show.Wherein, it is preferred that the concentration of carbenicillin is 100 μ g/ml in each step.

Wherein, it is preferred that the bacterium is Salmonella strains, in one particular embodiment of the present invention, described Salmonella strains be salmonella Sm6.

The ewcastle disease inactivated vaccine semi-finished product of adjuvant will not added to be mixed with the ghost, SPF chickens are immunized.Assistant will not be added The inactivated avian influenza vaccine semi-finished product of agent are mixed with the ghost, and SPF chickens are immunized.2 type pig annulus of adjuvant will not be added Viral vaccine semi-finished product are mixed with the ghost, and pig is immunized.The vaccine for not adding ghost is set at the same time as compareing, as a result It was found that compared to the vaccine for being not added with ghost, addition ghost specific can improve the immune of viral vaccine as the vaccine of adjuvant Effect, the antibody duration for improving viral vaccine, and finally improve the protective rate of viral vaccine.

The beneficial effects are mainly as follows：

1st, the present invention provides the large-scale producing method of bacterium ghost, heavily fortified point has been established for the merchandized handling of ghost vaccine Real basis；

2nd, the systematic adjuvant function of have rated ghost, ghost can greatly improve the immune efficacy of viral vaccine, And ghost is simple as adjuvant application method, can it is a degree of reduce vaccine production cost, to human and animal virus The prevention and control of sexually transmitted disease are of great significance.

Brief description of the drawings

Fig. 1 is the clastogram figure of pBV-E/Sm6；

Fig. 2 is Sm6 ghost scanning electron microscope (SEM) photographs；

Fig. 3 is ND_HI antibody bioactivity result；

Fig. 4 is ewcastle disease HI antibody duration testing results；

Fig. 5 is bird flu HI antibody duration testing results.

Embodiment

Below by experiment and the present invention will be further described in conjunction with the embodiments, it should be understood that these embodiments The purpose of illustration is only used for, is never limited in protection scope of the present invention.

Embodiment 1：The prepare with scale of Bacterium enteritidis ghost and identification

(1) structure of the Bacterium enteritidis Sm6 containing punching plasmid pBV-E

Primer is designed according to GenBank pnagus medius PhiX174 Lysis gene Es：

Lysise-U(5'-AGGGAATTCATGGTACGCTGGACTTTGTGG-3')

Lysise-L(5'-AGGGGATCCGAGCTCTCACTCCTTCCG-3')

Upstream and downstream primer 5' ends introduce EcoR I and BamH I restriction enzyme sites respectively, by Invitrogen (on Sea) company's synthesis.Using Phage PhiX174 double-stranded DNA as template amplification Lysis gene E, its nucleotide sequence such as SEQ ID Shown in NO.1.Pcr amplification reaction system is 50 μ L, wherein：22 μ L, the PrimeSTAR MAX Premix (2 of deionized water of sterilizing ×)25μL；10 μM of each 1 μ L of upstream and downstream primer；1 μ L (DNA containing 12.5ng) of PhiX174 double-stranded DNAs.The loop parameter of amplification For：1) 94 DEG C of 20s, 55 DEG C of 10s, 72 DEG C of 20s, 30 circulations；2)72℃ 5min.PCR product is coagulated with 0.9% agarose Gel electrophoresis detect, and E genetic fragments are recycled with plastic recovery kit.Using BamH I and EcoR I to E genetic fragments and pBV-220 Plasmid carries out double digestion respectively, and endonuclease reaction system is as follows：15 μ L of E genes or pBV-220 plasmids；BamH I 2μL；EcoR I 2μL；10×Thermo Scientific FastDigest buffer 5μL；26 μ L of sterile deionized water, 50 μ L of cumulative volume, 37 DEG C of water-bath 1h, glue reclaim E genetic fragments and linear pBV-220 carriers.

Salmonella strain Sm6 is isolated by the spontaneous diseased chicken group in this research department.Taken with aseptic inoculation ring freeze in Common LB plate streakings, while compareed in the LB plate streakings containing ampicillin (Amp), 37 DEG C of incubator overnight incubations.It is secondary Day well-grown single bacterium colony of picking is inoculated in 37 DEG C of shaking overnight incubations in 5mL LB fluid nutrient mediums.After taking 1mL to activate Nutrient solution be added in 100mL LB fluid nutrient mediums, 37 DEG C shaking culture 2.5-3h make OD_600nmReach 0.4 or so.Sterile Under the conditions of bacterial cultures is poured into after sterilizing in the centrifuge tube of ice precooling, ice bath 10min, 4 DEG C of 1600rpm centrifuge 10min, Supernatant is abandoned to the greatest extent.With the 0.1M CaCl of 10mL ice precoolings₂Gentle to have hanged bacterial precipitation, ice bath places 30min.4 DEG C of 1100rpm from Supernatant is abandoned to the greatest extent after heart 10min.With the 0.1M CaCl of 4mL ice precoolings₂Precipitation is resuspended in solution again, adds final concentration of 15% Sterile glycerol is packed as 200 μ l/ pipes after mixing, be placed in ice cube, add the 10 μ L of connection product of precooling in competent cell. The ice bath 30min in ice cube, then put 42 DEG C of heat shock 90s, ice bath 2min immediately after heat shock, adds and is preheating to 37 DEG C in advance 100 μ L LB nutrient solutions of antibiotic-free, 37 DEG C are shaken culture 1h, and 3500rpm centrifuges 3min afterwards, discards unnecessary supernatant, remaining 100 μ L, mix thalline, take 100 μ L to be spread evenly across on the LB tablets containing 50 μ g/mL ampicillins, and 37 DEG C of cultures 12~ 16h.Random picking colony, is inoculated in LB culture mediums of the 5mL containing 50 μ g/mL ampicillins and is incubated overnight, using alkaline lysis Method extracts Plasmid DNA in a small amount, is identified respectively with PCR and double digestion, gained positive colony, is pBV-E/Sm6.

(2) fermentation of ghost

By the Bacterium enteritidis containing punching plasmid pBV-E on LB agar plates (containing 100 μ g/ml carbenicillins) Sm6 monoclonals are transferred in the 100ml triangular flasks of the fluid nutrient mediums of LB containing 10ml (containing 100 μ g/ml carbenicillins), 37 DEG C 200rpm shaken cultivations are stayed overnight (when 10-14 is small), as primary seed solution.By 5% (v/v) inoculum concentration, primary seed solution is turned The 500ml conical flasks of the fluid nutrient mediums of LB containing 150ml (containing 100 μ g/ml carbenicillins) are connected to, 37 DEG C of 200rpm cultures 6 are small When, as secondary seed solution.10L pH7.2 are added into 15-L fermentation tanks (BIOSTAT C-15, Sartorius, Germany) LB fluid nutrient mediums carry out in-situ sterilization.By 5% (v/v) inoculum concentration, secondary seed solution is taken to be transferred in fermentation tank while add Enter carbenicillin and ferment to 100 μ g/ml of final concentration.Achieve following parameter：Temperature, flowing, stirrer, pH, pO₂.It is logical Programmed control induction primary fermentation condition (37 DEG C, 200rpm, 10L air/minute) is crossed by Bacteria Culture to exponential phase (OD600=0.75~1.0).42 DEG C of induction E protein expression are warming up to, at the time point when induction 2.5 is small, add final concentration The chloramphenicol of 170 μ g/ml；When induction all times are 4 small.With taking 10ml fermentation-likes when 0.5 is small during induction before induction Product, detect OD value, until optical density no longer reduces, by sample coating LB tablets when 37 DEG C of cultures 12 are small, bacterium colony counts Method calculates ghost concentration.4 DEG C of 80000 × g are centrifuged 10 minutes and are collected thalline, are washed precipitation twice with the PBS of pH7.0, will be precipitated Handled 3 minutes in 70-90 DEG C.By required ghost concentration is finally freezed, PBS or the freeze drying protectant for adding respective volume are (bright Glue-sucrose or skimmed milk), 5ml vaccine bottles are sub-packed in, every bottle of 1ml (contains ghost 5 × 10¹⁰Cfu), using VirTis GENESIS Freeze dryer by sample freeze 48 it is small when.It is stored in -20 DEG C.

(3) clastogram is drawn measures with lysis efficiency

With taking 10ml fermented samples during induction when 0.5 is small before fermentation inducement, OD value is detected, until optical density No longer reduce, by sample coating LB tablets when 37 DEG C of cultures 12 are small, colony counting method calculates lysis efficiency.

Experimental result：Bacterium colony count shows, containing punching plasmid pBV-E Bacterium enteritidis Sm6 after induction 1 it is small when Through starting bacteriolyze, 4 can fully crack (Fig. 1) when small, and lysis efficiency reaches 99.999%

(4) living stems of the scanning electron microscopic observation of ghost and ghost

The ghost before freezing is taken, 3% glutaraldehyde is resuspended, and overnight, ethanol is dehydrated step by step, penta 2 ester interchange of acetic acid for 4 DEG C of fixations, Metal spraying after drying, scanning electron microscopic observation.Empty balloon-shaped structure is presented in experimental result such as Fig. 2, most microorganisms, and form is complete It is whole, and the bacteriolyze duct of visible 200nm or so.

Take it is lyophilized before with it is lyophilized after ghost stoste, coating LB tablets are as a result sterile on tablet when 37 DEG C of cultures 12 are small Drop out and now, show that prepared ghost exists without viable bacteria.

Effect of 2 ghost of embodiment as newcastle disease vaccine immunologic adjuvant

Newcastle disease oil-emulsion inactivated vaccine is Harbin dimension section biology skill with not adding the ewcastle disease inactivated vaccine semi-finished product of adjuvant Art development company produces.Bacterium enteritidis ghost is prepared by 1 method of embodiment.

(1) adjuvant effect of subcutaneous inoculation approach detection ghost

1) packet transaction

7 age in days SPF chickens totally 60, are randomly divided into 4 groups, every group 15, that is, test I group of (newcastle disease oil-emulsion inactivated vaccine Group, neck subcutaneous inoculation, 0.3ml/ only), experiment II group (ghost vaccine adjuvant group, ewcastle disease inactivated vaccine semi-finished product are mixed with ghost Composition and division in a proportion example is 1:2 (volume ratios), ghost are 5 × 10⁹Cfu, neck subcutaneous inoculation, 0.3ml/ only), experiment III group of (LPS adjuvant Group, ewcastle disease inactivated vaccine and 100 μ g/ml LPS 1:1 mixing, neck subcutaneous inoculation, 0.3ml/ is only) and control group (PBS, neck Subcutaneous inoculation, 0.3ml/ is only).It is denoted as first immunisation day the 0th day, is immunized 2 times altogether, each immunization interval two weeks.Two exempt from one week ( 21 days) after collunarium is carried out to experiment chicken, eye droppings attacks Virulent Newcastle Disease Virus strain, and every chicken attacks toxic agent amount as 10⁵EID₅₀.Attack poison Observe its clinical symptoms daily afterwards, observe 15 days.Calculate the protective rate of vaccine.

2) viral hemoagglutination suppresses antibody titer and duration detection

Exempt from two weeks (the 14th days) and two in one and exempt from (the 21st day) wing venous blood sampling in one week, carry out blood clotting (HA) and suppress with blood clotting (HI) test.

HA is tested：96 hole " V " shape micro-reaction plates are taken, each hole adds 50 μ L PBS from left to right, adds 50 μ L in the 1st hole of left side NDV chick embryo allantoic liquids, after mixing, inhale 50 μ L to the 2nd hole, after blowing and beating for several times repeatedly, are diluted to the 11st hole successively, suction discards 50 μ L, through doubling dilution, the extension rate of chick embryo allantoic liquid is 21-211, and the 12nd hole is not added with chick embryo allantoic liquid, as control, then Add 1% chicken erythrocyte suspension, 50 μ L to each hole from left to right, vibration mixes, and 37 DEG C of standing 25min, are observed as a result, with experimental The middle HA-HI test (HAU) that can make the highest extension rate that complete aggegation occurs for equivalent red blood cell as NDV in chick embryo allantoic liquid, is used Log2 is represented.

HI is tested：The NDV HA-HI tests divided by 4 measured by HA experiments are 4 unit HAU, and 4 units are prepared according to extension rate NDV liquid.And blood coagulation tests are carried out, verify 4 unit antigens, take 96 hole " V " shape micro-reaction plates, 1-10 holes respectively add 50 from left to right μ LPBS, 11 and 12 holes add 4 unit NDV liquid and serum control respectively；1st hole adds 50 μ L serum to be checked, and pressure-vaccum mixes repeatedly Afterwards, take 50 μ L to the 2nd hole to mix, be diluted to the 10th hole successively, 50 μ L are abandoned in suction, and through doubling dilution, the extension rate of serum is 2¹- 2¹⁰；Respectively add 50 μ L, 4 unit NDV liquid from the 1st to the 10th hole, the 12nd hole adds 50 μ L serum, and concussion mixes, and puts 37 DEG C of incubator effects 20min；Respectively add 1% chicken red blood cell, 50 μ L from the 1st to the 12nd hole, concussion mixes, 37 DEG C of standing 20min, to completely inhibit aggegation Serum highest extension rate as HI titres, represented with log2.

Blood clotting histamine result shows：Test I group and II group of effect that can strengthen special viral hemoagglutination and suppress antibody of experiment Valency, and higher than experiment III group (Fig. 3).

Two exempt to carry out wing venous blood sampling weekly after a week, carry out hemagglutination-inhibition test.The result shows that：Exempt from 12 weeks until two (the 112nd day), the blood clotting that two experimental groups can keep higher suppress valency potency (Fig. 4).

3) ewcastle disease attacks the protection situation and toxin expelling situation of each group after poison

After attacking poison, the chicken of control group occurs observing symptom：Neck is backward or side is reversed, and eye is half-open or fully closed, is preced with gradual change Kermesinus, hydraulically full content in crop, occurs the smelly liquid outflow of a large amount of acid when holding；It is dead since the 5th day, the 10th It is all dead, the visible larynx of dissect, tracheae, tunica serosa intestini tenuis mucosal bleeding, the swelling of glandular stomach nipple, bleeding.III group is tested in There are within 10 days two chickens to walk lamely, by 15 days without death.Test I group, test II group of chicken without observing symptom, the attack to strong poison can To reach 100% protection.

Attack the health status of observation SPF chickens, and 3d, 5d, 7d gather the pharyngeal of SPF chickens after poison is attacked daily after poison Swab and cloacal swab, are placed in the PBS containing 50% glycerine (containing penicillin, streptomysin), 4 DEG C of 2000 × g centrifugations of sample liquid 10 minutes, take supernatant to be inoculated in 9 age in days SPF chicken embryos with the dosage of inoculation of 200 μ L/ piece, discard 24 it is small when interior dead chicken embryo, 72 Collect chick embryo allantoic liquid after hour, by HA testing inspections judge SPF chickens whether toxin expelling.Test result indicates that：After poison is attacked 3d, the brush,throat and cloacal swab of III group of I group of experiment and experiment detect toxin expelling, and test II group in cloacal swab In be not detected by toxin expelling.To 10d, I group of experiment, II group of experiment can't detect toxin expelling in throat and cloaca, as a result as follows Shown in table 1.

1 ewcastle disease of table attacks the virus purification result and case fatality rate after poison

Note："-" represents that control group chicken is all dead

(2) subcutaneous inoculation and the contrast of Nasal immunization effect

21 age in days SPF chickens totally 75, are randomly divided into 5 groups, every group 15, that is, test I group of (newcastle disease oil-emulsion inactivated vaccine Group, neck subcutaneous inoculation, 20 μ l/ are only), II group of experiment (ghost vaccine adjuvant group, ewcastle disease inactivated vaccine semi-finished product and ghost vaccine Mixed proportion is 1:2, ghost is 1 × 10⁹Cfu, neck subcutaneous inoculation, 20 μ l/ are only), (newcastle disease low toxicity power is lived for III group of experiment Vaccine group, every 1/100 dosage of Nasal immunization), experiment IV group of (ghost vaccine adjuvant group, ewcastle disease inactivated vaccine semi-finished product It is 1 with ghost vaccine mixed proportion:2, ghost is 1 × 10⁹Cfu, every 1/100 dosage of Nasal immunization) and control group, exempt from Epidemic disease after three weeks carries out experiment chicken collunarium, and eye droppings, attacks Virulent Newcastle Disease Virus strain, the toxic agent amount of attacking of every chicken is 10⁵EID50.Attack Its clinical symptoms is observed after poison daily, is observed 15 days.The protective rate of vaccine is calculated, it is as a result as shown in table 2 below.

The HI antibody levels and protective rate of 2 subcutaneous inoculation of table and Nasal immunization

Group	One exempts from three weeks HI antibody averagely (log2)	Protective rate
			Test I group	7	93.3% (14/15)
Test II group	8	100% (15/15)
			Test III group	4.4	93.3% (14/15)
Test IV group	5.3	100% (15/15)
			Control group	1	0% (0/15)

Immune effect of 3 ghost of embodiment as inactivated avian influenza vaccine adjuvant

7 age in days SPF chickens totally 24, are randomly divided into 3 groups, every group 8, that is, test I group (bird flu oil-emulsion inactivated vaccine group, Chest muscle is immunized, 0.3ml/ only), experiment II group (ghost vaccine adjuvant group, AI inactivated vaccine semi-finished product are with ghost mixed proportion 1:2 (volume ratios), ghost are 5 × 10⁹Cfu, chest muscle are immunized, and 0.3ml/ is only) and control group.It is denoted as first immunisation day the 0th day, altogether It is 2 times immune, each immunization interval two weeks.Two exempt to carry out wing venous blood sampling weekly after a week, carry out blood clotting (HA) and suppress with blood clotting (HI) test.

HA is tested：96 hole " V " shape micro-reaction plates are taken, each hole adds 50 μ L PBS from left to right, adds 50 μ L in the 1st hole of left side AIV chick embryo allantoic liquids, after mixing, inhale 50 μ L to the 2nd hole, after blowing and beating for several times repeatedly, are diluted to the 11st hole successively, suction discards 50 μ L, through doubling dilution, the extension rate of chick embryo allantoic liquid is 21-211, and the 12nd hole is not added with chick embryo allantoic liquid, as control, then Add 1% chicken erythrocyte suspension, 50 μ L to each hole from left to right, vibration mixes, and 37 DEG C of standing 25min, are observed as a result, with experimental The middle HA-HI test (HAU) that can make the highest extension rate that complete aggegation occurs for equivalent red blood cell as AIV in chick embryo allantoic liquid, is used Log2 is represented.

HI is tested：The AIV HA-HI tests divided by 4 measured by HA experiments are 4 unit HAU, and 4 units are prepared according to extension rate AIV liquid.And blood coagulation tests are carried out, verify 4 unit antigens, take 96 hole " V " shape micro-reaction plates, 1-10 holes respectively add 50 from left to right μ L PBS, 11 and 12 holes add 4 unit AIV liquid and serum control respectively；1st hole adds 50 μ L serum to be checked, and pressure-vaccum mixes repeatedly Afterwards, take 50 μ L to the 2nd hole to mix, be diluted to the 10th hole successively, 50 μ L are abandoned in suction, and through doubling dilution, the extension rate of serum is 2¹- 2¹⁰；Respectively add 50 μ L, 4 unit AIV liquid from the 1st to the 10th hole, the 12nd hole adds 50 μ L serum, and concussion mixes, and puts 37 DEG C of incubator effects 20min；Respectively add 1% chicken red blood cell, 50 μ L from the 1st to the 12nd hole, concussion mixes, 37 DEG C of standing 20min, to completely inhibit aggegation Serum highest extension rate as HI titres, represented with log2.

The result shows that：Until two exempt from 12 weeks (the 112nd day), the blood clotting that two experimental groups can keep higher suppresses valency Potency (Fig. 5).

Effect of 4 ghost of embodiment as 2 type pig circular ring virus inactivated vaccine immunologic adjuvants

7 week old SPF mouse totally 15, are randomly divided into 3 groups, every group 5, i.e., ghost adjuvant group, France's ISA15 adjuvants group and Control group.Ghost adjuvant group：By PCV2 inactivated vaccines semi-finished product and ghost according to 1:1 (volume ratio) mixes, ghost for 5 × 10⁹Cfu, dorsal sc are immunized, and 0.3ml/ is only；French ISA15 adjuvants group：PCV2 inactivated vaccines semi-finished product and France ISA15 are helped Agent is according to 15：85 (mass ratioes) mix, and dorsal sc is immunized, and 0.3ml/ is only；Control group：Every mouse injection 0.3ml PBS.It is first It is denoted as secondary immune day the 0th day, is immunized 2 times altogether, each immunization interval three weeks.It is on the day of first immunisation and quiet into end of line weekly afterwards Arteries and veins is taken a blood sample, and utilizes the antibody level of immunopcroxidase monolayer assay (IPMA) method measure serum moderate resistance PCV2.

IPMA operation sequences：By the use of 60 generation of PCV2 molecular labeling strain PCV2/PE strains as seed culture of viruses, new digestion is synchronously inoculated in PK-15 cell suspensions (2 × 10⁵/ mL), viral maintaining liquid is RPMI1640,2% calf serum of addition (FCS) and blue or green, strepto- Element, 37 DEG C of culture 6d receive poison, multigelation 3 times, and 4 DEG C of 3000 × g centrifuge 10min, and viral cultures supernatant is synchronously inoculated in newly In the PK-15 cell suspensions of digestion, dispensed with 100 μ L/ holes in the odd column of 96 orifice plates, even column sets non-virus inoculation cell pair According to putting 37 DEG C, 5%CO₂Under the conditions of culture form individual layer, fixed with acetone-PBS liquid, be IPMA reaction plates after dry, put- 20 DEG C spare.IPMA reaction plates are taken to put room temperature preheating, PBS embathes once.Swine serum to be checked is pressed 1 with PBS liquid:25、1:50、1: 100 equal proportions are serially diluted, and are compared with PCV2 positive and negative serum, and every part of diluted serum is separately added into 1 antigen hole and l A cell control well (100 μ L/ holes), puts 37 DEG C of wet box and is incubated 1h, PBS is washed 3 times, adds diluted HRP-SPA liquid (100 μ L/ Hole, Zymed Products), put 37 DEG C of wet box and be incubated 1h, PBS is washed 3 times, adds 3- amino -9- ethyl imidazol(e)s (AEC) reaction solution Colour developing, is observed with light microscope and judges result.

The result shows that：Ghost adjuvant can stimulate mouse to produce the specific antibody with ISA15 adjuvant phase same levels, more It is important that the excitation rate of ghost adjuvant is faster, shift to an earlier date week age compared with ISA15 adjuvants, it is as a result as shown in table 3 below.

3 ghost adjuvant of table is contrasted with ISA15 adjuvant immunity PCV2 vaccine effects

The foregoing is merely the preferred embodiment of the present invention, is merely illustrative for the purpose of the present invention, and nonrestrictive； Those of ordinary skill in the art understand that can carry out many to it in the spirit and scope that the claims in the present invention are limited changes Become, modification, or even equivalent change, but fall within protection scope of the present invention.

Claims (8)

1. application of the salmonella ghost in viral vaccine adjuvant is prepared, it is characterised in that the viral vaccine is selected from new City epidemic disease inactivated vaccine, inactivated avian influenza vaccine or 2 type pig circular ring virus inactivated vaccines, and the salmonella ghost is directly and institute Viral vaccine is stated to be used in mixed way.

2. application as claimed in claim 1, it is characterised in that the salmonella ghost can specificity raising viral vaccine Immune efficacy.

3. application as claimed in claim 1, it is characterised in that the salmonella ghost can improve the antibody of viral vaccine Duration.

4. application as claimed in claim 1, it is characterised in that the salmonella ghost can improve the protection of viral vaccine Rate.

5. application as claimed in claim 1, it is characterised in that the salmonella ghost through subcutaneous, mouth, mucous membrane, lung and/ Or nose is applied.

6. application as claimed in claim 1, it is characterised in that the salmonella ghost is prepared according to following steps：

(1) salmonella of the structure containing punching plasmid pBV-E, wherein the punching plasmid pBV-E is by by PhiX174 What the crack protein E gene clonings of bacteriophage were obtained into pBV-220 plasmids；

(2) the salmonella monoclonal containing punching plasmid pBV-E on the LB agar plates containing carbenicillin is transferred to and contained In the LB fluid nutrient mediums of carbenicillin, when 37 DEG C of 200rpm shaken cultivations 10-14 are small, as primary seed solution；By 5% (v/v) inoculum concentration, primary seed solution is transferred in the LB fluid nutrient mediums containing carbenicillin, and 37 DEG C of 200rpm cultures 6 are small When, as secondary seed solution；LB fluid nutrient mediums are added into fermentation tank and carry out in-situ sterilization；By 5% (v/v) inoculum concentration, take Secondary seed solution, which is transferred in fermentation tank while adds carbenicillin, ferments；Primary fermentation bar is induced by Programmed control Part：37 DEG C, 200rpm, 10L air/minute, by Bacteria Culture to OD600=0.75~1.0；

(3) 42 DEG C of induction E protein expression are warming up to, at the time point when induction 2.5 is small, add final concentration 150-200 μ g/ml's Chloramphenicol；When induction all times are 4 small；

(4) thalline is collected, 70-90 DEG C of processing, freezes.

7. application as claimed in claim 6, it is characterised in that the nucleotides sequence of the crack protein E genes of PhiX174 bacteriophages Row are as shown in SEQ ID NO.1.

8. application as claimed in claim 6, it is characterised in that the concentration of carbenicillin is 100 μ g/ml in each step.