CN101255189A - Animal vaccine immunopotentiator and production method thereof - Google Patents

Abstract

The invention provides a bursatin of immunopotentiator kind which is a small peptide which is a monomer with amino acid sequence COOH-Gly-His-Lys-NH2 or a multimer with end-to-end of a plurality of the sequences. The immunopotentiator of the invention has sufficient stimulating activity to various of animal lymphocyte in vitro, and has sufficient immuno-enhancing activity to newcastle disease (ND) vaccine, H5 and H9 Avian Influenza vaccine.

Description

A kind of animal vaccine immunopotentiator and production method thereof

Technical field

The present invention relates to a kind of animal vaccine immunopotentiator, specifically a kind of small-molecular peptides, it has significant immunoenhancement result to part virus and bacterial vaccine.The invention still further relates to the method for this small-molecular peptides of preparation.

Background technology

Present China Animal diseases more complicated, original disease also is not controlled effectively, and new epidemic situation comes one after another again, makes us hard to guard against, has brought enormous economic loss to aquaculture.

Immunostimulant can significantly improve the body immune system function, strengthens the immune effect of vaccine, reduces disease and takes place, thereby become the hot subject of Recent study.

Immunostimulant substantially can be divided into following a few class by its source: 1. microbiology class, as some mushroom classes in milk-acid bacteria, Corynebacterium, yeast cells wall, Ganoderma mycelium and the fungi etc., this para-immunity toughener mainly acts on body fluid and reticuloendothelial system; 2. biotic factor class, as thymic factor, vegetable polysaccharides, transfer factor, etc., this para-immunity toughener mainly stimulates T, B cytodifferentiation and growth, rapid induction antigen is in the lip-deep expression of lymphoblast; 3. synthetic class, as LEVAMISOLE HCL, liposome, adjuvant etc., they can make downtrod phagocytic cell and lymphocyte restore funcitons or strengthen the phagocytic function of scavenger cell.An important feature of LEVAMISOLE HCL is in immunity of organisms not effect just often, and when hypoimmunity, the effect that recovers and strengthen its immunologic function is arranged when especially cellular immune function is low or damaged; 4. natural class, as propolis, herbal medicine etc., this class preparation can increase huge cytophilic phagocytic function and reach the effect of raise immunity; 5. micro-factor type, as selenium, vitamin-E, vitamin A etc., they improve body's immunological function by the relative populations that antioxygenation changes immune cell.

At present known various immunostimulants (as LEVAMISOLE HCL, lipopolysaccharides, liposome, bursopoietin and some cytokines etc.) all because of cost too high or effect instability etc. former thereby be not used widely.

Summary of the invention

The objective of the invention is to provide a kind of animal vaccine immunopotentiator of Cheap highly effective safety at above-mentioned deficiency.

The present invention discovers that a kind of small-molecular peptides can strengthen the vaccine immunity of animals effect, and this small-molecular peptides sequence is COOH-Gly-His-Lys-NH ₂Or its tandem repetitive sequence, this small-molecular peptides structure is similar to the molecule result of chicken bursa element, and unit molecule puts in order just the opposite, thereby with its called after class capsule element (sBursin).

Join in the good ND of deactivation (or bird flu H5, H9) antigen with certain density class capsule element, make the plain vaccine of ND vaccine capsule (or the plain vaccine of bird flu H5 class capsule, the plain vaccine of H9 class capsule), behind the immunized chicks, different days is gathered separation of serum, measures serum titer with hemagglutination-inhibition test (HI).The negative control that does not contain class capsule element is set in test simultaneously, and the blank of not using vaccine immunity, the vaccine (no matter be ND, or AI H5 or AI H9) that the result contains class capsule element is than the vaccine that does not contain class capsule element high 1.2 titres of on average tiring, and immunoenhancement result is remarkable.

Another object of the present invention is to provide the method for the described class capsule element of preparation, comprises the structure of construction of recombinant plasmid, genetic engineering bacterium, the abduction delivering and the separation and purification of class capsule element:

With reference to codon usage frequency in some ribosomal protein of intestinal bacteria, select Gly, His and the most frequently used codon GGT, CAC and the AAA of Lys, hold the synthesising law of C end according to amino acid from N, with 5 '-GGTCACAAA-3 ' as class capsule plain gene.According to artificial base synthetic efficient, the present invention has designed the polyphone gene of 9 class capsule elements shown in sequence table SEQ ID No.1, below with (GGTCACAAA) ₉Expression.

According to the restriction enzyme site of carrier, at (GGTCACAAA) ₉The upstream and downstream of sequence is introduced restriction enzyme site respectively, and respectively adds several protectiveness bases in both sides.The present invention is expression vector with pET32a, at (GGTCACAAA) ₉The upstream and downstream of sequence is introduced the restriction enzyme site of EcoR I (GAATTC) and Sal I (GTCGAC) respectively, and 4 protectiveness bases are respectively added in both sides simultaneously, and institute's synthetic gene order is as follows:

F：5’-TCAGGAATTC(GGTCACAAA) ₉GTCGACCCAC-3’

In order to make the synthetic oligonucleotide can become two strands, synthesized corresponding complementary sequence: R:5 '-GGTGGTCGAC (TTTGTGACC) simultaneously ₉GAATTCCTGA-3 '.

After above oligonucleotide balanced mix, in boiling water, hatch 10min after, with frozen water cooling, transfer to 4 ℃ of water-baths then immediately, handle the not end of coupling with the Klenow enzyme again, obtain the tandem nucleotide two strands of 9 class capsule elements.。

Behind the polyphone class capsule plain gene usefulness EcoR I and Sal I double digestion with two strandsization, be connected with the carrier pET32a that handles with enzyme.Connect product and transform BL21 (DE3) host bacterium according to a conventional method.

Picking transforms bacterium colony, extracts plasmid after the enlarged culturing, and enzyme is delivered order-checking after cutting preliminary evaluation, and the recombinant plasmid of acquisition is pET32a-sBursin.

Reorganization bacterium that will cultivation overnight in the LB nutrient solution is inoculated into by 1% volume and continues in the 2YT substratum to cultivate 4h, gathers in the crops bacterium after inducing 4h with IPTG (final concentration is 1mmol/L).With ultrasonic wave or frozen-thawed with bacteria breaking after, collect cracking, the lysate of preliminary purification is this toughener product after quantitative.

Be to be understood that, consider the degeneracy of codon, those skilled in the art can be according to the preferences that transforms host's codon, do not changing under the condition of expressing the plain aminoacid sequence of class capsule, the plain encoding gene of class capsule is made amendment, and the gained gene prepared recombinant expression vector by appropriate means, and transform the host and obtain genetic engineering bacterium.Yet these do not change essence of the present invention, all should fall into protection scope of the present invention.

The recombination classes capsule element that the present invention obtained has tangible stimulating activity to the lymphocyte of external multiple animal, and newcastle disease (ND) vaccine, bird flu H5, bird flu H9 vaccine are had tangible immune-enhancing activity.According to method provided by the present invention, can obtain the biological immunopotentiator of cheap highly effective and safe, for the industrialization of class capsule element provides possibility.

Description of drawings

Fig. 1 is the electrophoresis result of recombinant plasmid pET32a-sBursin through EcoR I and Sal I single endonuclease digestion and double digestion, and wherein 1 is segment (containing the about altogether 100bp of restriction enzyme site and protectiveness base) before sBursin (9 the repeat polyphone) clone; The 2nd, Marker DL2000; The 3rd, pET32a-sBursin is through EcoRI and SalI double digestion; The 4th, pET32a-sBursin cuts through the EcoRI enzyme; The 5th, pET32a-sBursin cuts through the SalI enzyme; The 6th, pET32a-sBursin is through EcoRI and SalI double digestion;

Fig. 2 is the stimulating activity curve of class capsule element to the rabbit splenocyte;

Fig. 3 is the stimulating activity curve of class capsule element to mouse boosting cell;

Fig. 4 is the stimulating activity curve of class capsule element to the chicken bursa cell;

Fig. 5 is common chicken 5 an ages in days immunity ND seedling immunity alive comparing result;

Fig. 6 is common chicken 12 an ages in days immunity ND seedling immunity alive comparing result;

Fig. 7 is common chicken ND deactivation vaccine immunity comparing result;

Fig. 8 is common chicken H9 deactivation vaccine immunity comparing result;

Fig. 9 is common chicken H5 deactivation vaccine immunity comparing result;

Figure 10 is a SPF chicken ND deactivation vaccine immunity comparing result;

Figure 11 is a SPF chicken H5 deactivation vaccine immunity comparing result;

Figure 12 is a SPF chicken H9 deactivation vaccine immunity comparing result;

Figure 13 is a SPF chicken ND seedling immunity alive comparing result.

Figure 14 is the SDS-PAGE gel electrophoresis figure of expressing protein, and wherein swimming lane 1 is the molecular weight of albumen standard, swimming lane the 2, the 3rd, empty carrier expression product, swimming lane the 4, the 5th, recombinant vectors expression product (being class capsule element).

Embodiment

Further set forth the present invention below in conjunction with specific embodiment.Should be appreciated that these embodiment only are used to illustrate the present invention, and can not limit protection scope of the present invention.

Embodiment 1

According to the preferences of intestinal bacteria to different Gly, His, Lys, design can be expressed the gene 5 '-GGTCACAAA-3 ' of this tripeptides, and with 9 these same gene polyphones.Gene behind the polyphone advances in the pET32a vector plasmid by restriction enzyme (EcoR I and Sal I) clone, transformed into escherichia coli BL21 (DE3) host bacterium, and by the sequencing screening positive clone.The reorganization bacterium called after E.coli BL21/pET32a-sBursin that obtains.(this bacterial strain is delivered Chinese microbial preservation council common micro-organisms preservation center on September 26th, 2007, and preserving number is CGMCC No.2188).This bacterial strain is after the suitable culture base uploaded for 100 generations, and inherited character is still very stable.Animal experiment shows that the extract of reorganization bacterium lysate has tangible immuno-potentiation to animal vaccine.

Concrete grammar is as follows:

The structure of 1 reorganization bacterium

1.1 class capsule element (COOH-Gly-His-Lys-NH ₂) the determining of gene order

According to the inclined to one side preferendum of intestinal bacteria to codon, design 5 '-GGTCACAAA-3 ' is as the encoding gene of class capsule element, and intestinal bacteria have good expression efficiency to this gene.

1.2 class capsule plain gene serial sequence is synthetic

1.2.1 design polyphone gene order is according to artificial base synthetic efficient, the present invention has designed the polyphone gene of 9 class capsule elements, shown in sequence table SEQ ID No.1.

Have the restriction enzyme site of the polyphone gene order of restriction enzyme site 1.2.2 design is synthetic, at (GGTCACAAA) with reference to the pET32a carrier ₉The upstream and downstream of gene order add the restriction enzyme site of EcoR I (GAATTC) and Sal I (GTCGAC) respectively, and 3 protectiveness bases are respectively added in both sides simultaneously, and institute's synthetic gene order is as follows

F：5’-TCAGGAATTC(GGTCACAAA) ₉GTCGACCCAC-3’。

In order to make the synthetic oligonucleotide can become two strands, synthesized corresponding complementary strand simultaneously, sequence is as follows: R:5 '-GGTGGTCGAC (TTTGTGACC) ₉GAATTCCTGA-3 '.

1.2.3 the two strandsization of single stranded oligonucleotide is because the clone and the expression of gene needs two strands, in order to obtain double-stranded polyphone class capsule plain gene, under the effect of Klenow enzyme, connection is spent the night with F and R strand.

1.3 the clone of polyphone class capsule plain gene

Behind the polyphone class capsule plain gene usefulness EcoR I and Sal I double digestion with two strandsization, be connected with the carrier pET32a that handles with enzyme.Connect product and transform BL21 host bacterium according to a conventional method.

1.4 screening

The bacterium colony that grows of picking 4 at random, extract plasmid after the enlarged culturing, cut the positive plasmid of preliminary evaluation with enzyme and serve the sea and give birth to worker's biotechnology Services Co., Ltd and carry out sequencing, the result screens 2 strain positive strains, called after E.coli BL21/pET32a-sBursin.

The characteristic check of 2 reorganization bacterium

2.1 form and biochemical characteristic check that gramstaining is negative, are typical intestinal bacteria form.

2.2 the cultural characters inspection is well-grown on the LB of pH 6.4～7.4 agar or other appropriate medias.

2.3 genetic characteristics inspection

The Molecular Detection of goal gene or identify and can cut or sequencing is finished that concrete scheme is as follows by PCR, enzyme:

2.3.1PCR identify

2.3.1.1 primer

Because clone's segment is less, therefore when identifying with PCR, this Project design primer at the part nucleic acid of pET32a carrier multiple clone site upstream and downstream carry out, concrete primer sequence design is as follows:

Upstream primer F:5 '-TTCGAACGCCAGCACATG-3 '

Downstream primer R:5 '-TTGTTAGCAGCCGGATCTC-3 '

Be mixed with 40 μ mol/L with the TE damping fluid and store concentration.

2.3.1.2PCR reaction system

dNTP(25mmol/L each) 4μL

10 * Buffer (contains Mg ²⁺) 5 μ L

F primer 1 μ L

R primer 1 μ L

Template 1 μ L

Taq enzyme (5U/ μ L) 0.5 μ L

H ₂O 38.5μL

The preparation of template: from picking list bacterium colony on the solid medium flat board, add 100mL sterilization distilled water, heating was got 1 μ L as template after 5 minutes in boiling water bath.

2.3.1.3PCR response procedures

95℃5min

72℃10min

After the PCR reaction finishes, get 10 μ LPCR products, 1.5% agarose gel electrophoresis, observations.

2.3.1.4 the result judges

The PCR product of expection is 223bp, if electrophoretic band coincide with expection, can tentatively be judged to the positive, otherwise negative.

2.3.2. enzyme is cut evaluation

2.3.2.1 test materials

Plasmid extraction kit (vast Tyke biological gene technology company limited), EcoRI and SalI restriction enzyme, DNA MarkerDL2000 (precious biotechnology company limited), agarose (worker's biotechnology company limited is given birth in Shanghai), water-bath, nucleic acid electrophoresis apparatus etc.

2.3.2.2 test method

Culture propagation and plasmid extract

Inoculate single reorganization bacterium and contain in the LB substratum of 100 μ g/ml acillins (Amp) in 5ml, 37 ℃ of shaking culture are spent the night, and extract plasmid according to the plasmid extraction kit specification sheets next day, and simple step is as follows:

Collect in the precipitation and 1.5ml Eppendorf centrifuge tube of 1.5ml bacterium liquid, add 100 μ l solution I, vibration adds 150 μ l solution II to thoroughly suspending, and softly puts upside down centrifuge tube immediately for several times, makes the abundant cracking of thalline, and the thalline after the cracking becomes limpid.Subsequently centrifuge tube was placed 2 minutes on ice, added 150 μ l solution III, gentleness is put upside down centrifuge tube for several times immediately, puts room temperature and places 5 minutes.Centrifugal 12 minutes of 12000rpm shifts supernatant behind the centrifugal adsorption column that shifts, and washs centrifugal adsorption column with 420 μ l binding buffer liquid boxes, 750 μ l rinsing liquids respectively, washes out plasmid with 50 μ l elutriants.

The enzyme of plasmid is cut with electrophoresis and is identified

Plasmid carries out enzyme by following embodiment respectively and cuts digestion after extracting and finishing:

System I:

Recombinant plasmid 6 μ l

10×Buffer H 2μl

EcoRI enzyme 0.5 μ l

SalI enzyme 0.5 μ l

H ₂O 11μl

System II:

Recombinant plasmid 6 μ l

10×Buffer H 2μl

EcoRI enzyme 0.5 μ l

H ₂O 11.5μl

System III:

Recombinant plasmid 6 μ l

10×Buffer H 2μl

SalI enzyme 0.5 μ l

H ₂O 11.5μl

Get the pET32a carrier of not reorganization simultaneously and make negative control with EcoRI and SalI double digestion, reaction system is as follows:

PET32a plasmid 6 μ l

10×Buffer H 2μl

EcoRI enzyme 0.5 μ l

SalI enzyme 0.5 μ l

H ₂O 11μl

Behind above each system difference mixing, after 1 hour, carry out electrophoresis in 37 ℃ of water-baths digestion with 1.2% sepharose 100V, take out observations after 1 hour.

2.3.2.3 the result judges

The result of restriction enzyme digestion and electrophoresis as shown in Figure 1.For carrier, with the purpose segment of EcoRI and SalI double digestion under UV-light band very a little less than, so enzyme be essential will be with the above plasmid of 0.5 μ g, need careful resolution simultaneously during observations.Electrophoresis time surpasses 1 hour, helps distinguishing recombinant plasmid and nonrecombinant carrier size.

The restriction enzyme digestion and electrophoresis result meets Fig. 1 result's, is judged to the positive, otherwise negative.

2.3.3. order-checking is identified

A kind of simply and easily authentication method identifies by sequencing that exactly method is as follows:

2.3.3.1 microbial culture

The single recombinant clone of picking is inoculated into 5ml and contains in the LB substratum of 100 μ g/ml acillins (Amp), and 37 ℃ of shaking culture are spent the night.

2.3.3.2 order-checking is measured

It is aseptic subpackaged in the Eppendorf pipe of 1.5ml to get 0.5ml bacterium liquid, delivers order-checking (worker is given birth in Shanghai).

2.3.3.3 sequential analysis

Sequencing result and expected sequence are compared, and the sequencing result is consistent with expection, shows the positive clone of genetic engineering bacterium that screening obtains.

The separation and purification of 3 expression products

3.1 the preparation of bacterium liquid

With the engineering strain streak inoculation in the SOB plate culture medium that contains penbritin, 37 ℃ are spent the night, choose smooth mellow and full single bacterium colony, be inoculated in 4 25 milliliters of SOB liquid nutrient mediums that contain penbritin, under 30 ℃ of conditions in the shaking table shaken over night, then it is transferred in 4 250 milliliters of SOB liquid nutrient mediums that contain penbritin, shook 2.5 hours at shaking table under 37 ℃ of conditions, then above-mentioned 1 liter of culture is inoculated in 9 liters of SOB liquid nutrient mediums that contain penbritin, 37 ℃ are cultured to bacterium and enter plateau in 15 liters of fermentor tanks.

3.2 the extraction of inclusion body protein, purifying and evaluation

(1) the reorganization bacterium after 100ml is induced is sub-packed in the centrifuge tube of 50ml, and the centrifugal 30min of 4000rpm/min discards supernatant.

(2) every pipe adds cell pyrolysis liquid 10ml, fully behind the mixing, the bacterium liquid of two pipes is merged to a 100ml.

Small beaker in.The PMSF of N,O-Diacetylmuramidase 20 μ l, the 0.1mmol/Lol/L of adding 0.1g/ml and the Aprotinin (trypsin inhibitor) of 5 μ l, room temperature is put and is stirred 30min on the magnetic stirring apparatus.

(3) small beaker is placed on carries out ultrasonic degradation in the mixture of ice and water, ultrasonic power is 200W, work 10s, and intermittently 10s carries out 20 circulations altogether; If bacterium liquid thickness, continue so several times ultrasonic, up to bacterium liquid become limpid till.

(4) the bacterium liquid after ultrasonic is sub-packed in the centrifuge tube, 4 ℃, the centrifugal 10min of 8000rpm, cleer and peaceful precipitation in the reservation is got the supernatant of 200 μ l, adds the albumen sample-loading buffer of 50 μ l, is the sample of ultrasonic degradation supernatant.

(5) with the ice-cold solution 2 (washings) of 20ml that the precipitation after ultrasonic is resuspended, mixing slowly shakes under the room temperature, behind the effect 10min, and 4 ℃, the centrifugal 10min of 8000rpm.Carry out altogether three times, wash for the last time centrifugal after, outwell supernatant, keep precipitation.

(6) will precipitate with the solution 3 (washings) of 20ml resuspended, mixing.Carry out and (5) identical operations, precipitation is the inclusion body that SDS-PAGE extracts.

(7) with 20ml solution 4 (solubilization of inclusion bodies liquid) dissolution precipitation, get the lysate of 200 μ l, add the sample-loading buffer of 50 μ l, be the sample of inclusion body.Respectively get above-mentioned bacterium liquid supernatant sample, the supernatant sample of ultrasonic treatment and inclusion body sample are respectively got 10 μ l, and the SDS-PAGE electrophoresis detection result with 12% after electrophoresis is finished, with the dyeing of Coomassie brilliant blue staining fluid, after the decolouring, takes pictures with gel imaging system.The result as shown in figure 14, expression product size with expect consistent.

4 reorganization bacterium lysates (class capsule element) are to lymphocytic effect

4.1 experimental technique

4.1.1 lymphocytic preparation

Aseptic technique, get the spleen of rabbit, mouse respectively, get the fabricius bursa of SPF chicken, squeeze out histocyte and dilute several times with blunt-ended forceps with PH 7.2-7.6Hanks liquid, filter above-mentioned splenocyte suspension with 200 order copper mesh, filtered liquid is divided into two parts, and a copy of it is handled a moment with the erythrocyte cracked liquid of 4～5 times of volumes, and adding 5 times of volumes does not then have Ca ⁺⁺, Mg ⁺⁺Hanks liquid, mixing 1, the centrifugal 10min of 500r/min inhales and abandons supernatant, repeated washing 2 times, trypan blue exclusion method counting is adjusted cell concn with 10% calf serum RPMI RPMI-1640 (containing two anti-) and is respectively 2 * 10 ⁶Cell/mL and 1 * 10 ⁷Cell/mL, the same packing 96 porocyte culture plates.Another part is used the lymphocyte separation medium isolated lymphocytes, the same cell numeration and packing Tissue Culture Plate.Add 10 μ g class capsule elements in above-mentioned Tissue Culture Plate hole, each concentration is established three multiple holes, and carries out repeated experiments with three Different Individual, and 37 ℃ or 41 ℃, 5%CO ₂Middle 64～the 66h that cultivates.

4.1.2 ³H-TdR mixes and the stimulation index statistical study

Cell cultures stops preceding 16 hours, and every hole adds 0.5 μ Ci ³H-TdR.After cultivating end, usefulness bull cell harvestor on glass fiber filter paper, is used PBS, 5% Tricholroacetic Acid and absolute ethanol washing cell each three times with cell harvesting successively.β-liquid scintillation instrument is measured the per minute flicker number of times (cpm value) of every porocyte, and calculates stimulation index (SI).SI=(measuring the cpm value-machine background cpm value in hole)/(negative control hole cpm value-machine background cpm value).Owing to the repeated experiments that three Different Individual are carried out is identical substantially, the SI end value is chosen the SI mean value of three repeated experiments of the body one by one in three Different Individual and is represented.

4.2 experimental result

4.2.1 stimulation to the rabbit splenocyte

Measure the immunostimulatory activity of class capsule element to the rabbit splenocyte with mtt assay, the test group that the result adds class capsule element is significantly higher than the control group (Fig. 2) that does not add class capsule element.

4.2.2 stimulation to mouse boosting cell

Measure the immunostimulatory activity of class capsule element to the kunming mouse splenocyte with mtt assay, the test group that the result adds class capsule element is significantly higher than the control group (Fig. 3) that does not add class capsule element.

4.2.3 stimulating activity to the SPF chicken bursa

Measure the immunostimulatory activity of class capsule element to SPF chicken bursa cell with mtt assay, the test group that the result adds class capsule element is significantly higher than the control group (Fig. 4) that does not add class capsule element.

5 reorganization bacterium lysates (class capsule element) are to the immuno-potentiation of vaccine

One, material:

1, the yellow chicken of 1 age in days is 350

2, extra large blue embryo is 80 pieces, 150 of SPF chicken embryo 70 embryos, 1 age in days SPF chickens

3, immunostimulant: _ pET32a-sBursin_, for simplicity, with its called after X10.

4, other medical disposable material: syringe, centrifuge tube, antigen, disposable blood clotting plate.

Two, method:

1, the mensuration of ND (La Sota strain) antigenic preparation and EID50.

2, immunostimulant is diluted to 2ml with physiological saline, class capsule fibroin concentration is about 500ug/ml, about 1500 plumage parts.Class capsule element is added in the diluent of ND living vaccine, perhaps it is added to the aqueous phase of ND inactivated vaccine, carry out eye droppings collunarium or intramuscular injection according to the routine immunization method of ND living vaccine and inactivated vaccine.

3, grouping situation:

3.1 common chicken grouping situation: (annotate: every group immune 30, other vaccine is not immune)

The immunity of the common chicken of table 1

Group

The immunity kind

Immune day age (my god)

Immunizing dose (ml)

The 1st group	ND+X10 seedling alive	5	0.2
				The 2nd group	ND seedling alive	5	0.2
The 3rd group	The blank group
				The 4th group	The ND deactivation vaccine	12	0.2
The 5th group	The ND+X10 deactivation vaccine	12	0.2
				The 6th group	The H9 deactivation vaccine	12	0.3
The 7th group	The H9+X10 deactivation vaccine	12	0.3
				The 8th group	The H5 deactivation vaccine	12	0.3
The 9th group	The H5+X10 deactivation vaccine	12	0.3
				The 10th group	ND+X10 seedling alive	12	0.2
The 11st group	ND seedling alive	12	0.2

3.2SPF chicken grouping situation: (15 every group)

The immunity of table 2SPF chicken

Group	The immunity kind	Immune day age (my god)	Immunizing dose (ml)
				The 1st group	The blank group
The 2nd group	The ND+X10 deactivation vaccine	12	0.2
				The 3rd group	The ND deactivation vaccine	12	0.2
The 4th group	The H5 deactivation vaccine	12	0.3
				The 5th group	The H5+X10 deactivation vaccine	12	0.3
The 6th group	The H9+X10 deactivation vaccine	12	0.3
				The 7th group	The H9 deactivation vaccine	12	0.3
The 8th group	ND+X10 seedling alive	12	0.2
				The 9th group	ND seedling alive	12	0.2

Each organize in immunity back 7 days, 14,21,28,35,42,49,56 respectively blood sampling survey the antibody titer of H5, H9, ND.Blood sampling in per according to circumstances later on 15～30 days once.

Three, result and analysis:

1, common chicken result:

The common chicken immune result of table 3

Annotate: live and be expressed as the living vaccine immunity, going out is expressed as the deactivation vaccine immunity,--expression is not surveyed.

Carrying out analytical results (referring to Fig. 5～9) with SPSS software shows:

(1) with antibody titers indifference between ND seedling immunity alive and ND+X10 seedling immune group alive, there were significant differences with the blank group.

(2) between H5 deactivation vaccine and H5+X10 deactivation vaccine immune group antibody titers there were significant differences, H5+X10 group is significantly higher than H5 deactivation vaccine group.

(3) between H9 deactivation vaccine and H9+X10 deactivation vaccine immune group antibody titers there were significant differences, H9+X10 group is significantly higher than H9 deactivation vaccine group.

(4) between ND deactivation vaccine and ND+X10 deactivation vaccine immune group antibody titers there were significant differences, ND+X10 group is significantly higher than ND deactivation vaccine group.

2, SPF chicken result

The immune result of table 4SPF chicken

Carrying out analytical results (referring to Figure 10～13) with SPSS software shows:

(1) with antibody titers indifference between ND seedling immunity alive and ND+X10 seedling immune group alive, there were significant differences with the blank group.

(2) antibody titers indifference between H5 deactivation vaccine and H5+X10 deactivation vaccine immune group.

(3) the basic indifference of antibody titers between H9 deactivation vaccine and H9+X10 deactivation vaccine immune group, only in immunity in the time of back 21 days, the H9 deactivation vaccine is than H9+X10 deactivation height of seedling, significant difference.

(4) the basic indifference of antibody titers between ND deactivation vaccine and ND+X10 deactivation vaccine immune group, only in immunity in the time of back 21 days, the ND deactivation vaccine is than ND+X10 deactivation height of seedling, significant difference.

The above results shows that class capsule element of the present invention has significant immuno-potentiation.

Sequence table

＜110〉Guangdong Dahuanong Animal Healthcare Product Ltd

＜120〉a kind of animal vaccine immunopotentiator and production method thereof

<130>

<160>1

<170>PatentIn version 3.3

<210>1

<211>81

<212>DNA

＜213〉artificial sequence

<400>1

ggtcacaaag gtcacaaagg tcacaaaggt cacaaaggtc acaaaggtca caaaggtcac 60

aaaggtcaca aaggtcacaa a 81

Claims (10)

1. small-molecular peptides, it is for having aminoacid sequence COOH-Gly-His-Lys-NH ₂Monomer or the end to end concatermer of a plurality of these sequences.

2. small-molecular peptides as claimed in claim 1, it is 9 COOH-Gly-His-Lys-NH ₂End to end concatermer.

3. the gene of coding claim 1 or 2 described small-molecular peptides.

4. gene as claimed in claim 3, it has the nucleotide sequence shown in the sequence table SEQ ID No.1.

5. contain claim 3 or 4 described expression carrier.

6. expression vector as claimed in claim 5, it is derived from pET32a.

7. the host of containing claim 5 or 6 described expression vectors.

8. colon bacillus (Escherichia coli) BL21 (pET-sBur) CGMCCNo.2188, it contains the described expression vector of claim 5.

9. a method for preparing claim 1 or 2 described small-molecular peptides is characterized in that cultivating the described host of claim 7, and abduction delivering.

10. the immunostimulant that contains claim 1 or 2 described small-molecular peptides.

Images