CN101050448A - Oral recombined DNA vaccine for accelerating growth of animal, and application - Google Patents

Abstract

This invention relates to preparation and application of orally administered recombinant DNA vaccine for accelerating animal growth, which Salmonella typhimurium CSO22/pGMCSF-SS (CCTCC M206141) is containing fusion expression plasmid pGMCSF-SS. Salmonella typhimurium CSO22/pGMCSF-SS is prepared by: cloning GMCSF, fusing with plasmid pcS/2SS containing somatostatin gene to obtain fusion expression plasmid pGMCSF-SS, and transforming Salmonella typhimurium. This invention also discloses the application of Salmonella typhimurium CSO22/pGMCSF-SS in orally administered recombinant DNA vaccine for accelerating animal growth.

Description

A kind of oral recombined DNA vaccine and application that promotes growth of animal

Technical field

The invention belongs to animal gene engineering technology field, be specifically related to a kind of oral recombined DNA vaccine and application that promotes growth of animal.

Background technology

Growth of animal is the result of combined factors influences such as heredity, nutrition, environment and hormone regulation, wherein strengthen breed improvement, full nutrition is provided and provides adapt circumstance to become the effective way that improves breeding performonce fo animals, but its effect finally is to be achieved by neuroendocrine integration, regulation and control.At present, be one of focus of current research by changing hormone secretion with the regulation and control growth of animal.Rise and the development learned along with hormone immunity, in the hormone immunity and technology (Hormone Immunoneutralization HIN) arises at the historic moment, promptly initiatively induce or passive input hormone antagonist antibody with in the biological activity of endogenous hormone.The growth of animal is a complex physical process, formed with tethelin is the growth axis of core, tethelin (Growth Hormone, GH) can regulate the nutrient distribution of body, promote bone growth, promote protein synthesis and reduce fatty deposits, the generation of GH, release is subjected to hypothalamus somatotropin releasing factor (Growth Hormone Release Factor, GHRF) and Somatostatin (Somatostatin, SS) dual regulation, GHRF promotes the secretion of GH, exists remarkable negative correlation between the concentration of SS and the speed of growth in the body, thereby has pointed out the release that can promote GH by the concentration that reduces SS in the body.Somatostatin (SS) is 14 peptide hormones that hypothalamus discharges, has restraining effect widely in animal body, particularly growth and immune response are had tangible influence, impel many scholars to be devoted to promote growth of animal by levels such as exhaustion or the corresponding raising of the immunity intravital SS level of neutralization GH.SS is with after vector construction becomes antigen, and the antibody that is produced combines with SS, can block SS and receptor acting; Simultaneously under the effect of liver, the removing of SS in the acceleration cycle, thus promote the release of multiple hormones such as GH.To synthesize peptide SS as far back as Spencer in 1981 etc. and be connected in people's alpha-globulin, make complex antigen (synthetic peptide vaccine), immunity sheep in 3 age in week, the test group speed of growth is 17.6% of a control group, has confirmed that SS active immunity can promote growth of animal.The food consumption of SS immunity lamb reduces by 10%, but feed conversion rate improves 14%, 16 weeks of lamb promptly reach the 30kg slaughter weight, the contrast lamb then needs 20 weeks (Spencer GSG etc., Increased growth in lambs following auto-immunization against somatostatin, Anim.Prod, 1981,32:376-382).Chemosynthesis such as Ceng Yixiang the SS gene, and in intestinal bacteria, successfully expressed the beta-galactosidase enzymes (fusion rotein of β-gal) and SS, in order to preparation SS gene engineering vaccine (Ceng Yixiang etc., the expression of inhibiting hormone of growth gene in intestinal bacteria, Acta Genetica Sinica, 1989 (3): 282-288).By immune rat test, obtained certain gaining effect, but need use affinity chromatography, thereby production cost is higher owing to from thalline, extract SS.This recombinant vaccine promptly has only a haptenic group on a carrier molecule because a molecule only links to each other with a β-gal molecule in addition, and immunogenicity is relatively poor.Liu Yongqing etc. utilize the pET32 of escherichia coli high-level expression system, efficiently, stably expressed SS, immunogenicity SS subunit vaccine (Liu Yongqing etc. have preferably been obtained, Somatostatin (SS) gene efficiently expressing in the pET32 expression system, Agricultural University Of Nanjing's journal, 2003,26 (1): 61-65).This fusion rotein has better immunogenicity and gaining effect, but will apply as vaccine, must be through purge process, and this expression product is to be carrier with sulphur hydrogen reduction albumen, the difficulty of purifying.In addition, sulphur hydrogen reduction protein carrier effect during animal experiment is too strong, (the Liu Yongqing etc. that apply of this vaccine have also been influenced, Somatostatin (SS) and sulphur hydrogen reduction albumen (Thioredoxin) merge 2 kinds of genetically engineered fusion rotein SS-N/X generating and the expression characterization of SS-N/S mutually, China animal doctor journal, 2003,23 (2): 172-176).Xu Wenzhong etc. are cloned into the fusion gene (HBsAg/SS) of SS and HBsAg among the vaccinia virus expression plasmid pGJP-5, by with the vaccinia virus Tiantan strain cells infected in reorganization and plaque selection techniques acquisition contain recombinant virus (the VV-HBsAg/SS) (Xu Wenzhong etc. of fusion gene, the structure of Somatostatin and hepatitis B surface antigen blending gene vaccinia virus recombinant and expression thereof, Agricultural University Of Nanjing's journal, 1992 (3): 74-80), it has kept the original infectivity of vaccinia virus, and the HBsAg/SS heterozygosis particle of expression has the immunogenicity of SS.VV-HBsAg/SS is inoculated in the chicken embryo cultivates, can produce can HBsAg expression/SS vaccinia virus live vector seedling, make lyophilized powder again, be named as to swash and give birth to No. 1 bovine vaccine.The weak point of this seedling is vaccinia virus recombinant as live vector seedling direct immunization animal, behind the initial immunity, produces antiviral body in the animal body, and virus can not massive duplication during immunity once more, influences immune effect; Secondly the risk that also has the rise of attenuated virus virulence.For remedying this shortcoming, Du Nianxing etc. in succession development and design the growth chalone as one genetic engineering subunit seedling, successively made up many strain gene engineerings body, made vaccine is called for short respectively to swash gives birth to No. 2 seedlings and swashs and give birth to No. 3 seedlings.Rise along with gene vaccine, Du Nianxing etc. have made up somatostatin gene vaccine gene engineering body according to the advantage of gene vaccine again, it is reported behind its dna gene vaccine, expression plasmid can be expressed the 2-3 month in zooblast, constantly secretory immune is former, induce to produce SS antibody, thereby promote growth of animal.But do not see about the application of this dna vaccination on piglet, and produce this vaccine and need large quantity extracting plasmid, might exist intracellular toxin to remove clean in the leaching process and cause animal endotoxicosis equivalent risk, in addition intramuscular injection easily make animal produce bigger stress, be unfavorable for the growth of large animal.

Summary of the invention

Task of the present invention is to overcome the defective of existing somatostatin gene immunological technique, develops a kind of simple, effective, safe oral reorganization promotes growth dna vaccination, to reach the purpose that promotes growth of animal.

Purpose of the present invention specifically comprises:

First purpose of the present invention is to obtain GM-CSF and SS gene fusion expression plasmid.

Second purpose of the present invention is the Salmonella typhimurium strain that obtains to contain above-mentioned fusion expression plasmid.

The 3rd purpose of the present invention is the oral recombined DNA vaccine that obtains to promote growth of animal.

The 4th purpose of the present invention is that above-mentioned fusion plasmid is applied to prepare on the oral recombined DNA vaccine that promotes growth of animal.

The present invention implements by following technical solution:

A kind ofly promote that the preparation of the gene vaccine of growth of animal is at first to clone to obtain granulocyte-macrophage colony stimutaing factor (GM-CSF) gene, the plasmid of this gene of encoding can strengthen the immune effect of dna vaccination, then its fusion is inserted among the existing Somatostatin plasmid pcS/2SS, obtain fusion expression plasmid pGMCSF-SS, this fusion expression plasmid is transformed in the attenuated salmonella typhimurium, obtains having the oral recombined DNA vaccine that promotes growth of animal.Successfully be applied in the present invention promote in the growth of animal, as in weanling pig, promoting the application of growth.

The concrete technical scheme of the present invention is as follows:

One, the structure of pGM-CSF plasmid

1, granulocyte-macrophage colony stimutaing factor (GM-CSF) gene clone and sequential analysis

(kind is a DLY to take tri-crossbreeding, derive from Jiangsu Province Agriculture Science Institute herding institute) peripheral blood, extract the total RNA of monocyte, adopt conventional RT-PCR method clone pig granulocyte-macrophage colony stimutaing factor (GM-CSF) (in the GenBank login, accession number is DQ108393 to this gene order of the applicant clone).The cDNA open reading frame of GM-CSF is 435bp, and 144 amino acid of encoding compare with the reference sequences that is used as pcr template, and the homology of its Nucleotide is 97.5%, and amino acid whose homology is 95.1%.The GM-CSF sequence of same sheep, people, ox, mouse and dog relatively, the homology of its Nucleotide is respectively 86.2%, 80.5%, 81.7%, 69.7% and 81.8%, and amino acid whose homology is respectively 78.5%, 71.3%, 70.3%, 55.9% and 71.5%.

2, the structure of pGM-CSF plasmid

With pcDNA3.1 (-) (available from Invitrogen company) is plasmid vector, makes up pig GM-CSF gene eucaryon expression plasmid pGM-CSF (preparation as embodiment 11.1 as described in) in detail and order-checking.

Two, the structure of GM-CSF and SS fusion expression plasmid

1, the amplification of GM-CSF encoding gene

Adopt the Primer5.0 primer-design software, according to the pig GM-CSF sequence of having logined at GenBank, design upstream and downstream primer (primer structure as embodiment 22.2 as described in), with eukaryon expression plasmid pGM-CSF (as embodiment 1) is that template is carried out PCR acquisition GM-CSF gene, use QIAquick PCR Purification Kit purifying then, quantitatively standby.

3, the structure of GM-CSF and SS fusion expression plasmid

The GM-CSF gene fragment and the pcS/2SS of purifying, recovery are carried out EcoR I and XhoI double digestion respectively, with low melting point agarose gel electrophoresis Separation and Recovery GM-CSF purpose fragment and the big fragment of pcS/2SS.After 4 ℃ of connections of spending the night, use CaCl ₂Legal system is equipped with fresh competence bacterium DH5 α and is used for recombinant plasmid transformed, screens afterwards and identifies positive fusion expression plasmid pGMCSF-SS (seeing embodiment 2), and qualification result is referring to accompanying drawing 5.

Three, the preparation of vaccine

1, the conversion of plasmid

Adopt the heat-shocked method that positive colony plasmid pGMCSF-SS, original plasmid pcS/2SS and the eukaryon expression plasmid pGM-CSF that the previous step screening obtains transformed attenuated salmonella typhimurium (seeing embodiment 3 and 4) respectively.

2, vaccine production

Get the single bacterium colony of original plasmid pcS/2SS, eukaryon expression plasmid pGM-CSF and fusion expression plasmid pGMCSF-SS and be seeded to respectively on the LB liquid nutrient medium, be cultured to OD in 37 ℃, 150rpm jolting ₆₀₀0.3-0.4 4 ℃, the centrifugal 10min of 1500g abandon supernatant, adjust bacterial concentration to 10 with the PBS solution of sterilization ¹⁰CFU/mL obtains testing the standby vaccine of usefulness.

Four, the immune efficacy of vaccine check

Select the two-way cross piglet of the kind of 40 wean for " growing up ", be divided into 4 groups at random, every group 10, male and female half and half, wherein being numbered 1 group is control group, being numbered the group of 2-4 oral respectively is gene vaccine pcS/2SS, pGMCSF-SS and the pGM-CSF+pcS/2SS of carrier with the attenuated salmonella typhimurium, analyzes the influence of these gene vaccines to production performance, antibody horizontal, hormone secretion and the metabolite concentration of weanling pig.The result shows, compares with control group, and the pGMCSF-SS immune group has improved day weight gain and the efficiency of feed utilization of piglet, is respectively 6.6% and 15.7%.Three test group have all produced than strong immune response the immunity of SS simultaneously.Compare with control group, Somatostatin antibody P/N value all improves.The content of GH has trend of rising in 2 groups, 3 groups and the 4 groups of blood plasma, and the content of control group GH all compares steadily in whole experiment periods, and the 3rd group GH content is higher than 2 groups and 4 groups (P＞0.05) generally.The content of the 2nd group of IGF-I is in higher level always, and the 3rd group and 4 groups is in lower secretion level after the 2nd week, and the 3rd group of IGF-I excretory peak value is higher than the peak value of other group.The 1st group, the content of 2 groups and 4 groups T3 are downward trend, and the 3rd group of gesture that is rising, and in immunity the 8th week of back, the 3rd group T3 secretion level is significantly higher than 2 groups and 4 groups (P＜0.05), with the control group significant difference.The content of the 1st group of T4 constantly reduces, and the 2nd group, 3 groups and 4 groups have continuous trend of rising, and the secretion of 2 groups and 3 groups T4 remains on higher level always; Immunity does not have obvious influence to piglet glucose and non-esterified fatty acid (NEFA) and urea nitrogen concentration.

The present invention clone's GM-CSF gene can improve the immune effect of SS gene vaccine, and the attenuated salmonella typhimurium that contains the pGMCSF-SS plasmid is better than the attenuated salmonella typhimurium immunity jointly that contains the pGM-CSF+pcS/2SS plasmid.Can produce stronger immunne response with the attenuated salmonella typhimurium oral immunity weanling pig that contains the pGMCSF-SS plasmid, improved the secretion level of GH, IGF-I, T3, T4 keep higher concentration always, have improved day weight gain and the efficiency of feed utilization of weanling pig.

Description of drawings

Fig. 1 is the electrophorogram of the present invention's GM-CSF segment of cloning by the RT-PCR amplification.

Fig. 2 is the pGM-CSF plasmid enzyme restriction qualification result that the present invention prepares.

Fig. 3 is the fusion expression plasmid pGMCSF-SS constructing technology route map that the present invention prepares.

Fig. 4 is the dna fragmentation GM-CSF pcr amplification product electrophorogram that the present invention prepares.

Fig. 5 is that the fusion expression plasmid pGMCSF-SS enzyme that the present invention prepares is cut qualification result: each swimming lane is expressed as follows among the figure: M.-DL2000; (1.pGMCSF-SS/ HindIII+EcoR I); 2.pGMCSF-SS/ (HindIII+XhoI); (3.pGMCSF-SS/ EcoR I+XhoI).

Fig. 6 is that fusion expression plasmid pGMCSF-SS enzyme of the present invention is cut qualification result.Each swimming lane is expressed as follows among the figure: M.-DL2000; (1.pGMCSF-SS/ HindIII+EcoR I); 2.pGMCSF-SS/ (HindII+XhoI); (3.pGMCSF-SS/ EcoR I+XhoI).

Fig. 7 is the effect of oral recombined DNA vaccine of the present invention to each stage piglet day weight gain.

Fig. 8 is an oral recombined DNA vaccine of the present invention to the effect of feed conversion rate of each piglet in stage.

Embodiment

The structure of embodiment 1 eukaryon expression plasmid pGM-CSF

1.1 reverse transcription and pcr amplification

Adopt RT-PCR test kit (with reference to RNA PCR Kit (AMV) the Ver.3.0 test kit working instructions of precious biotechnology (Dalian) company limited (TaKaRa)), the cell total rna 1-2 μ g that gets extraction carries out reverse transcription, and reaction cumulative volume 10 μ L wherein contain:

MgCl ₂ 2μL

OligodT primer (2.5pmol/ μ L) 0.5 μ L

DNTP Mixture (each 10mM) 1 μ L

RNA enzyme inhibitors (40U/ μ L) 0.25 μ L

ThermoScript II (5U/ μ L) 0.5 μ L

10×RT Buffer 1μL

RNase Free dH ₂O makes reaction cumulative volume 10 μ L.

Carry out reverse transcription reaction by following condition: 30 ℃ of 10min

42℃30min

99℃5min

5℃5min

React 1 circulation.RT product-20 ℃ preservation, standby.

10 μ L reverse transcription products are joined 40 μ L (wherein contain 5 * PCR Buffer, 10 μ L, each 0.5 μ L of upstream and downstream primer, Taq ^TMEnzyme 0.25 μ L adds sterile purified water to 40 μ L at last) system in, cumulative volume is 50 μ L.

Carry out PCR reaction by following condition: 94 ℃ of sex change 2min, then 94 ℃ of sex change 30sec, 59 ℃ of annealing 30sec, 72 ℃ extend 1min, 35 circulations are extended 7min for back 72 ℃ again.

At last, with PCR product 1.2% agarose gel electrophoresis, as molecular weight standard, observe electrophoresis result with DL2000.(seeing accompanying drawing 1) 1.2 enzymes are cut with being connected with QIAquick PCR Purification Kit and pcr amplification product are reclaimed and purifying (with reference to the PCR product purification test kit working instructions of the Shanghai China biological company limited of Shun production).PCR product behind the purifying and expression vector pcDNA3.1 (-) use EcoRI and XhoI double digestion respectively.

Enzyme is cut system: 10 * H buffer, 6 μ L

GM-CSF (or pcDNA3.1) 20 μ L

EcoR I and XhoI are respectively 1.5 μ L

DdH ₂O is to cumulative volume 60 μ L,

37 ℃, reaction 2-3h, enzyme cut the back with hanging down the big fragment of melting point agarose gel electrophoresis Separation and Recovery GM-CSF purpose fragment and pcDNA3.1 (-).

Carry out ligation by following system then, connect purpose fragment and carrier, linked system is:

10×ligase buffer 2uL

GM-CSF 6μL

pcDNA3.1(-)5μL

T ₄DNA Ligase 1μL

DdH ₂O is to cumulative volume 20 μ L, and 4 ℃ are spent the night.Connect product-20 ℃ preservation, standby.

1.3 preparation of competence bacterium and recombinant plasmid transformed competence colibacillus bacterium

Preparing fresh competence bacterium DH5 α, to be used for the recombinant plasmid transformed method as follows:

(1) (1L LB solid medium is formed: 10g sodium-chlor, 5g yeast extract, 10g Tryptones from the LB flat board, the 15g agar powder, pH 7.0) the single bacterium colony of the new activatory E.coli of picking DH5 α, be inoculated in (1L LB liquid nutrient medium composition: 10g sodium-chlor, 5g yeast extract, 10g Tryptones in the 10mL LB liquid nutrient medium, pH 7.0), about 37 ℃ of following shaking culture 12h, until the logarithmic growth later stage.This bacteria suspension is inoculated in the 100mL LB liquid nutrient medium 37 ℃ of shaking culture 2.5h to OD with 1: 100 ratio ₆₀₀About=0.5;

(2) nutrient solution with step (1) changes in the centrifuge tube, places 10min on ice, then in 4 ℃ of centrifugal 10min of following 3000g;

(3) remove the supernatant of step (2) nutrient solution, with the CaCl of the 0.05mol/L of precooling ₂Solution 10mL is suspension cell gently, place 30min on ice after, 4 ℃ of centrifugal 10min of following 3000g;

(4) discard the supernatant of step (3) nutrient solution, add the CaCl that the 4mL precooling contains the 0.05mol/L of 15% glycerine ₂Solution, suspension cell is placed 10min on ice gently, the competent cell suspension.

(5) competent cell is distributed into the aliquot of 100 μ L, can be used at once transforming, and perhaps is stored in-70 ℃ of refrigerators.

To connect product transformed competence colibacillus bacterium DH5 α, method is as follows:

(6) from-70 ℃ of refrigerators, get 100 μ L competent cell suspensions, place on ice and melt;

(7) add connection product D NA solution, shake up gently, place 30min on ice;

Thermal shock 90s in (8) 42 ℃ of water-baths places cooled on ice 3min rapidly behind the thermal shock;

(9) Xiang Guanzhong adds 400 μ L LB liquid nutrient mediums (not containing penbritin), and 37 ℃ of shaking culture 1h behind the mixing make the bacterium state that restore normal growth, and the ammonia benzyl antibiotics resistance gene of expressible dna coding.

(10) get 100 μ l after above-mentioned bacterium liquid is shaken up and coat on the screening flat board that contains penbritin (the Amp final concentration is 50mg/mL), face up and place half an hour, treat that bacterium liquid is absorbed the back by substratum fully and is inverted culture dish, cultivate 16-24h for 37 ℃.

1.4 the screening of positive colony, evaluation and order-checking

Picking positive colony from the flat board in last step; being inoculated in the LB liquid nutrient medium that contains Amp (final concentration is 50mg/mL) cultivates; with plasmid small volume of reagent box extracting plasmid (with reference to the plasmid extraction kit working instructions of the Shanghai China biological company limited of Shun); EcoRI and XhoI double digestion; the enzyme system of cutting is: 10 * H buffer, 1.6 μ L; recombinant plasmid 9 μ L, EcoR I and XhoI are respectively 0.8 μ L, add ddH ₂O is to cumulative volume 16 μ L, and 37 ℃, reaction 2-3h observes enzyme and cuts the result, and electrophoretic band conforms to the purpose stripe size, serves extra large Ying Jun Bioisystech Co., Ltd and checks order.The positive colony called after pig pGM-CSF that obtains.(seeing accompanying drawing 2)

The structure of embodiment 2 fusion expression plasmid pGMCSF-SS

2.1 plasmid pGM-CSF extraction and purification

The single bacterium colony of the new activatory pGM-CSF of picking from the LB flat board; be seeded in the 10mL LB liquid nutrient medium; 37 ℃, 240rpm overnight incubation; 1: 250 by volume dilution proportion is in proper volume LB; continue to cultivate 12h; centrifugal collection thalline, extracting and purifying pGM-CSF plasmid (with reference to the plasmid extraction kit working instructions of the Shanghai China biological company limited of Shun).

2.2GM-CSF the amplification of encoding gene

Referring to accompanying drawing 4, adopt the Primer5.0 primer-design software, be that template is carried out PCR and obtained the GMCSF gene with the eukaryon expression plasmid pGM-CSF (embodiment 1) of pig.Upstream primer (P1) is: 5 '-GTAT CTCAGAAGGATGTGGC-3 ' has introduced restriction enzyme XhoI site (being its sequence in the square frame), and downstream primer (P2) is: 5 '-GGCG

TAACTTTTTGACTGGC-3 ' introduces restriction enzyme EcoRI site (in the square frame is its sequence).Primer is synthetic to be finished by Shanghai Ying Jun Bioisystech Co., Ltd.

With the pGM-CSF plasmid is template, carries out the amplification of GM-CSF gene according to following PCR reaction system.

5×PCR buffer 10μL

MgCl ₂(25mmol/L)4μL

dNTP(10mmol/L)1μL

PGMCSF plasmid 0.5 μ L

Each 0.5 μ L (P1, P2 primer concentration are 20pmol/ μ L) of upstream and downstream primer

Taq archaeal dna polymerase (5U/ μ L) 0.5 μ L

DdH ₂O is to cumulative volume 50 μ L

Reaction conditions: 94 ℃ of pre-sex change 2min, 94 ℃ of sex change 30sec then, 58.5 ℃ of annealing 30sec, 72 ℃ are extended 1min, 35 circulations, last 72 ℃ are extended 7min; 1.2% agarose gel electrophoresis detects, and glue reclaims, and uses QIAquick PCR Purification Kit (with reference to the PCR product purification test kit working instructions of the Shanghai China biological company limited of Shun) purifying then, and is quantitatively standby.

2.3GM-CSF structure with the SS fusion expression plasmid

Technological line as shown in Figure 3, GM-CSF gene fragment and pcS/2SS (original plasmid) that purifying, recovery are obtained carry out EcoR I and XhoI double digestion by following system respectively, and reaction system is: 10 * H buffer, 6 μ L, GM-CSF15 μ L (or pcS/2SS 20 μ L), EcoR I and XhoI are respectively 1.5 μ L, add ddH ₂O is to cumulative volume 60 μ L, and 37 ℃, reaction 2-3h is after enzyme is cut, with low melting point agarose gel electrophoresis Separation and Recovery GM-CSF purpose fragment and the big fragment of pcS/2SS.

Carry out ligation by following system then: 10 * ligase buffer, 2 μ L, GM-CSF 6 μ L, pcS/2SS 5 μ L, T4DNA ligase1 μ L, add ddH ₂O is to cumulative volume 20 μ L, and 4 ℃ are spent the night CaCl ₂Legal system is equipped with fresh competence bacterium DH5 α and is used for recombinant plasmid transformed (concrete steps are with embodiment 1.3).

2.4 the screening and the evaluation of positive recombinant plasmid (pGMCSF-SS)

The picking positive colony, be inoculated in the LB liquid culture that contains Amp, extract plasmid DNA, carry out enzyme and cut evaluation, with EcoR I and XhoI double digestion, EcoR I and HindIII, HindIII and XhoI double digestion evaluation recombinant plasmid, the enzyme system of cutting is: 10 * H buffer, 1.6 μ L, recombinant plasmid 9 μ L, EcoR I and XhoI (or HindIII) are respectively 0.8 μ L, add ddH respectively ₂O is to cumulative volume 16 μ L, and 37 ℃, reaction 2-3h, 1.2% agarose electrophoresis detects, and electrophoretic band conforms to the purpose stripe size (seeing accompanying drawing 5), serves extra large Ying Jun Bioisystech Co., Ltd and carries out dna sequencing.Fusion expression plasmid is named as pGMCSF-SS.

Embodiment 3 is the preparing carriers oral recombined DNA vaccine with the attenuated salmonella typhimurium

3.1pGMCSF-SS plasmid transforms attenuated salmonella typhimurium

Employing prepares fresh competence attenuated salmonella typhimurium with above-mentioned method, with fusion expression plasmid pGMCSF-SS transformed competence colibacillus attenuated salmonella typhimurium (the concrete operations step is with embodiment 1.3).

Attenuation mouse typhus Salmonella typhimurium (Salmonella typhimurium) CSO22/pGMCSF-SS that contains fusion expression plasmid is deposited in Chinese typical culture collection center (CCTCC) on December 19th, 2006, and preserving number is CCTCC M206141.

3.2 the evaluation of plasmid in the reorganization Salmonella typhimurium

The positive colony list colony inoculation that picking step 3.1 obtains 37 ℃ of overnight incubation in the LB liquid nutrient medium that contains Amp; extracting plasmid from Salmonella typhimurium (with reference to the plasmid extraction kit working instructions of the Shanghai China biological company limited of Shun) is identified with 1.2% agarose gel electrophoresis.To from Salmonella typhimurium, return back to E.coli DH5 α (method is with embodiment 1.3) with the heat-shocked method by extractive plasmid, extractive plasmid is used HindIII+EcoR I, HindIII+XhoI, EcoR I+XhoI double digestion respectively, enzyme is cut system: 10 * H buffer, 1.6 μ L, recombinant plasmid 9 μ L, EcoR I and XhoI are respectively 0.8 μ L, add ddH ₂O is to cumulative volume 16 μ L, and 37 ℃, reaction 2-3h, 1.2% agarose gel electrophoresis identifies that qualification result is as shown in Figure 6.

3.3 the preparation of recombination attenuated salmonella typhimurium vaccine

The attenuated salmonella typhimurium list colony inoculation of picking pGMCSF-SS is in the LB liquid nutrient medium that contains Amp, and 37 ℃, 150rpm jolting are cultured to OD ₆₀₀0.3-0.4 4 ℃, the centrifugal 10min of 1500g abandon supernatant, adjust bacterial concentration to 10 with the PBS solution (the PBS lysate is the phosphate buffered saline buffer of pH 7.4,10mmol/L) of sterilization ¹⁰CFU/mL, standby.

The application in the promotes growth on piglet of embodiment 4 oral recombined DNA vaccines of the present invention

4.1 vaccine production:

It is described respectively with pcS/2SS, pGM-CSF transformed competence colibacillus Salmonella typhimurium to press embodiment 1.3 methods, the attenuated salmonella typhimurium vaccine that contains pcS/2SS, pGM-CSF and three kinds of plasmids of pGMCSF-SS afterwards according to the described preparation of 3.3 methods, and adjust bacterial concentration to 10 ¹⁰CFU/mL, standby.

4.2 experimental animal:

Experimental animal is planted the pig farm from Agricultural University Of Jiangxi, and the selection source is identical, body weight is close (about 8.8kg), and the wean of 35 ages in days " greatly enhances " 40 of binary piglets, be divided into 4 groups at random, 10 every group, male and female half and half, wherein the 1st group is control group, oral PBS damping fluid; The 2nd group of oral immunity contains the attenuated salmonella typhimurium of original plasmid pcS/2SS; The 3rd group of oral immunity contains the attenuated salmonella typhimurium bacterium liquid of fusion expression plasmid pGMCSF-SS; The 4th group of while oral immunity contains the attenuated salmonella typhimurium bacterium liquid and the attenuated salmonella typhimurium bacterium liquid that contains the pcS/2SS plasmid of pGM-CSF plasmid.

4.3 immunization method:

Be subjected to the preceding 12h fasting of animal (35 age in days weanling pig) immunity of immunity to prohibit water, 30min before the immunity, oral in advance 7.5% NaHCO ₃(5mL/ head) with in and hydrochloric acid in gastric juice, immunizing dose is 4 * 10 ¹⁰The CFU/ head.2 weeks with same dose, same procedure booster immunization once behind the initial immunity.

4.4 feeding and management:

Test is adopted once and is weaned with 35 age in days weanling pigs, from mother not from the son.Weanling pig changes little swinery over to after 5-7d is raised on former hurdle.Little pig house is a single-column type, and the south is a playground, for the weanling pig free movement.Carry out raising the phase in advance of 7d before on-test earlier, piggy is carried out expelling parasite and castration raising in advance in the phase.After the phase of raising in advance finished, the starting weight just of weighing was analyzed by statistics, and difference is not remarkable, enters trial period, and trial period is 56d.Test pig day feeds 3 times, and automatic drinking bowl is freely drunk water, and keeps cleaning, health in the house, in time removes fecaluria in the house, keeps the house inner drying.

Prepare with blood plasma 4.5 weigh:

With initial immunity was 0 week, took by weighing morning the weight of test pig respectively on an empty stomach in the 0th, 2,4,6,8 weeks, and adopted the method at the bottom of the knock-out, the feed intake when record is weighed at every turn; Precaval vein is taked blood sample when weighing, heparin sodium anti-freezing, preparation blood plasma,-20 ℃ of preservations, in order to the detection of antibody, hormone and metabolite concentration (with reference to chief editors such as Yang Liguo. enzyme immunoassay technique, [M]. Nanjing: press of Agricultural University Of Nanjing, 1998:138-139).

4.6 testing index

4.6.1 day weight gain and feed conversion rate

Write down weighing and feed intake of each weanling pig, calculate day weight gain and the feed conversion rate of each weanling pig in stage.

Reorganization oral DNA vaccine of the present invention is to the influence of weanling pig growth performance:

As described in Table 1, the 3rd group of full phase average daily gain of test pig of the present invention is the highest, reaches 427.3 ± 40.70g, has improved 6.6%, 8.4% and 17.2% than 1 group, 2 groups and 4 groups respectively.The 3rd group of feed conversion rate is the highest, is 1.67: 1; The 1st group the poorest, up to 1.98: 1.2 groups, 3 groups and 4 groups have been improved 1.0%, 15.7% and 9.6% than control group respectively, and the day weight gain in each stage of weanling pig and feed conversion rate are respectively shown in accompanying drawing 7 and accompanying drawing 8:

The different Somatostatin dna vaccinations of table 1 are to the influence of weanling pig growth performance

Project	Group

	1	2	3	4
Starting weight (kg)					8.68 ±0.39a	8.88 ±0.46a	8.93 ±0.31a	8.78 ±0.43a

(%) feed conversion rate feed conversion rate relatively (%) is compared in end heavy (kg) weightening finish (kg) experimental period (d) average daily gain g/d weightening finish

31.12 ±1.66a 22.44 ±1.45 56 400.7 ±25.98ab 100 1.98∶1 100

30.96 ±1.05a 22.08 ±0.97 56 394.3 ±17.31ab 98.4 1.96∶1 99.0

32.86 ±2.31a 23.93 ±2.28 56 427.3 ±40.70a 106.60 1.67∶1 84.3

29.2 ±1.41a 20.42 ±1.09 56 364.6 ±19.50b 90.99 1.79∶1 90.4

Annotate: person inequality represents significant difference with the line data lowercase.

4.6.2 Somatostatin antibody test

Adopt indirect ELISA method to detect the long chalone antibody of antibiosis in the blood plasma.Step is as follows:

Bag is by the every hole of standard growth chalone antigen 100ng/100 μ L, and 4 ℃ of reaction 12h abandon reaction solution, and washing is 6 times on automatic washer, each 30 seconds; Add confining liquid (5% skimmed milk solution) 200 μ L/ holes, 37 ℃ of reaction 1.5h abandon reaction solution, wash each 30 seconds 6 times; 50 times of piglet diluted plasmas add blood plasma to be measured 100 μ L/ holes, establish control wells (zeroing hole: do not add any reaction solution simultaneously; Non-specific adsorption hole: replace blood plasma) with the PBS damping fluid, three repetitions of each sample, 37 ℃ of reaction 1h abandon reaction solution, wash each 30 seconds 6 times; Add the anti-pig IgG-HRP of rabbit dilution in 1: 5000, every hole 100 μ L, 37 ℃ of reaction 1h abandon reaction solution, wash each 30 seconds 6 times; Add tmb substrate liquid, every hole 150 μ L, 37 ℃ of reaction 25min; Add 2mol/L sulfuric acid termination reaction, every hole 50 μ L; The OD450 reading.Be judged to the positive with P/N value 〉=2, wherein P is the light absorption value of blood sample to be measured, the negative contrast blood sample of N light absorption value, and its measurement result sees Table 2.

Oral recombinant vaccines of the present invention the 2nd all P/N values behind the oral immunity for the first time just reaches the peak, and other 3 group P/N value peak periods all in the 4th week after exempting from, organize all decline to some extent of P/N values for 4 subsequently, but test group all are higher than control group (P＞0.05).The pGMCSF-SS group is up to still being higher than other each group the 6th week.

The ratio of total antibody positive pig is significantly higher than control group (P＜0.05) in the whole antibody monitoring phase (full phase), and the antibody positive pig does not appear in control group.Being maximum with the positive ratio of pGM-CSF+pcS/2SS immune group in three test group, reaching 30%, secondly is pcS/2SS group and pGMCSF-SS, and positive ratio is 20%, and difference is remarkable (P＞0.05) not.

Somatostatin antibody P/N value and positive pig ratio in the oral different dna vaccination weanling pig blood plasma of table 2

Group	The immunity back time (week)				The P/N maximum value	Positive pig ratio (%) SS Ab
							2	4	6	8
	1 2	1.06 ±0.13Aa 1.34 ±0.25Aa	1.31 ±0.09Aa 1.41 ±0.21Aa	1.17 ±0.13Aa 1.30 ±0.21Aa							1.28 ±0.15Aa 1.38 ±0.14Aa	1.95 3.05	0 A 20 B

3 4

1.50 ±0.13Aa 1.29 ±0.16Aa

1.44 ±0.15Aa 1.47 ±0.20Aa

1.43 ±0.16Aa 1.27 ±0.20Aa

1.37 ±0.20Aa 1.28 ±0.17Aa

2.39 2.05

20 B 30 B

Annotate: the antibody positive rate of full phase is meant the percentage ratio that detects the piglet of antibody positive number of times 〉=1 in the whole antibody monitoring phase.

4.6.3 the detection of GH, IGF-I, T3 and T4 in the blood plasma

The test kit that adopts Beijing Fu Rui bio-engineering corporation to produce detects GH, T3 and T4, and the test kit that adopts Tianjin Jiuding Medical Biological Engineering Co., Ltd to produce detects IGF-I, and concrete operations are undertaken by the working instructions of the test kit that above-mentioned unit provides.Control group GH concentration is a little more than test group during on-test, prolongation along with the immunity time, the content of testing GH in 2,3,4 groups of blood plasma has trend of rising, pcS/2SS, pGM-CSF+pcS/2SS immune group peaked when the 6th week, the maximum value of fusion expression plasmid pGMCSF-SS group GH content appeared at for the 8th week, and the content of control group GH all compares steadily in whole experiment periods.The GH content of pGMCSF-SS immune group is higher than 2,4 groups (P＞0.05) generally.This may be the Somatostatin antibody intravital SS that neutralized, and has promoted the synthetic and secretion of GH, and prompting SS gene vaccine has obvious effect to keeping body inner growth hormone (GH) higher level, and GM-CSF has certain immuno-potentiation (seeing Table 3).

The change in concentration (ng/mL) of GH in the table 3 weanling pig different times blood plasma

Group	The immunity back time (week)
						0	2	4	6	8
	1 2 3 4	0.73±0.14Aa 0.65±0.15Aa 0.57±0.15Aa 0.51±0.11Aa	0.76±0.20Aa 0.54±0.12Aa 0.69±0.14Aa 0.61±0.03Aa	0.68±0.10Aa 0.73±0.20Aa 0.65±0.07Aa 0.61±0.08Aa	0.76±0.37Aa 0.95±0.17Aa 0.90±0.17Ab 0.74±0.08Aa						0.73±0.45Aa 0.93±0.12Aa 0.98±0.30Ab 0.63±0.14Aa

Annotate: same column (OK) data capitalization (small letter) alphabetical person inequality, expression significant difference (P＜0.05), down together.

Control group IGF-I concentration is a little more than test group during on-test, and along with the prolongation of immunity time, the variation of its concentration is similar to the dynamic change of GH.1 group, 2 groups and 4 groups of excretory climaxs are in the 2nd week, and for the highest, but difference is not remarkable with the pGM-CSF+pcS/2SS group; The secretion of 2 groups of IGF-I is higher than other group since the 2nd week always, and 3 groups and 4 groups are in lower secretion level after the 2nd week.The 3rd group secretion peak appeared at for the 4th week, was higher than the peak secretion level of other group.This may be because the decline of SS level has promoted the synthetic and secretion of GH, and the increase of GH excretory has improved the secretion level of IGF-I in the blood, but group difference not obvious (P＞0.05) (seeing Table 4).

The change in concentration (ng/mL) of IGF-I in the table 4 weanling pig different times blood plasma

Group	The immunity back time (week)
						0	2	4	6	8
	1 2 3	325.67 ±15.22Aab 318.39 ±4.06Aa 293.30	367.51 ±9.85Aa 350.44 ±14.53Aa 344.63	318.89 ±26.94Aab 307.48 ±17.51Aa 350.46	283.46 ±22.82Ab 321.74 ±15.42Aa 286.66						304.42 ±22.05Aab 329.51 ±20.97Aa 297.41

4

±17.09Aa 283.80 ±17.90Aac

±23.15Ab 386.66 ±8.28Ab

±8.02Ab 306.31 ±6.44Aac

±17.72Aa 280.01 ±8.05Aa

±13.02Aa 310.85 ±12.05Ac

Along with the prolongation of immunity time, the secretion of 1 group, 2 groups and 4 groups T3 is downward trend, and their climax appeared at for 0 and 2 weeks.And the secretion of the 3rd group of T3 is the gesture of rising, and the climax was compared with 0 all secretion levels in the 8th week, significant difference (P＜0.05).Although in the later stage, each secretion level of organizing T3 all descends to some extent, and the T3 secretion level of pGMCSF-SS immune group (3 groups) is significantly higher than 2 groups and 4 groups, with 1 group of significant difference.This may be the pGMCSF-SS vaccine immunodepletion SS in the body, remove SS to T3 excretory restraining effect, promoted T3 secretion (seeing Table 5).

The change in concentration (nmol/L) of T3 in the table 5 weanling pig different times blood plasma

Group	The immunity back time (week)
						0	2	4	6	8
	1 2 3 4	1.26±0.08Aa 0.74±0.11Ba 0.73±0.09Ba 0.75±0.11Ba	1.04±0.15Aa 1.17±0.19Ab 1.0±0.22Aab 1.28±0.14Ab	1.05±0.07Aa 1.04±0.13Aab 1.15±0.07Ab 0.91±0.15Aa	1.13±0.08ABa 0.92±0.08Bab 1.02±0.09ABab 1.21±0.09Ab						1.11±0.05ADa 0.77±0.10Ba 1.32±0.15CDb 0.99±0.11ABab

Prolongation along with the immunity time, the secretion of T4 constantly reduces for the 1st group, and 2 groups, 3 groups and 4 groups have continuous trend of rising, the maximum value of 2 groups and 4 groups appeared at for the 6th week, and the 3rd group just reaches 78.73 ± 4.64nmol/L in the 4th week, and the secretion of T4 after this remains on higher level always, this is consistent with the secretion of T3, illustrate the pGMCSF-SS vaccine immunodepletion SS in the body, remove SS to T4 excretory restraining effect, promoted the T4 secretion.(seeing Table 6)

The change in concentration (nmol/L) of T4 in the table 6 piglet different times blood plasma

Group	The immunity back time (week)
						0	2	4	6	8
	1 2 3 4	79.86±6.22Aa 53.92±9.56Aa 58.81±6.82Aa 68.63±2.34Aa	71.16±7.26Aa 64.85±2.37Aa 59.76±16.96Aa 58.93±15.69Aa	73.12±2.43Aa 64.49±6.31Aa 78.73±4.64Aa 79.63±6.17Aa	74.45±5.10Aa 67.53±7.45Aa 69.69±9.79Aa 79.87±9.10Aa						64.44±2.40Aa 55.81±2.26Aa 68.63±0.20Aa 67.17±4.47Aa

4.6.4 the mensuration of plasma glucose, blood urea nitrogen and free fatty acids

The test kit that adopts Shanghai Rongsheng Bioisystech Co., Ltd to produce detects glucose (Glu), adopt Nanjing to build up test kit detection blood urea nitrogen (BUN), free fatty acids (NEFA) that bio-engineering research is produced, concrete operations are undertaken by the test kit working instructions.

The concentration of 1 group, 3 groups and 4 groups glucose is higher than 3 groups during on-test.Behind dna gene vaccine, from Dynamic Variation Analysis, the change in concentration of pcS/2SS immune group glucose is little, the pGM-CSF+pcS/2SS immune group presents downward trend, the situation that rises then appears in fusion expression plasmid pGMCSF-SS immune group, compare significant difference (P＜0.05) with former weeks to the dying weeks.And that the concentration of control group glucose changes always is not obvious.Prompting SS genetic immunization is not obvious to the concentration affects of plasma glucose, and fusion expression plasmid pGMCSF-SS then can improve the concentration of plasma glucose.(seeing Table 7)

The change in concentration (mmol/L) of glucose in the table 7 weanling pig different times blood plasma

Group	The immunity back time (week)
						0	2	4	6	8
	1 2 3 4	5.01±0.40Aa 5.07±0.30Aa 3.99±0.39Aa 4.71±0.76Aa	5.74±0.99Aa 4.75±0.51Aa 4.57±0.47Aa 4.09±0.29Aa	5.10±0.23Aa 5.20±0.45Aa 4.31±0.46Aa 4.40±0.48Aa	5.07±0.17Aa 4.58±0.27Aa 5.21±0.31Ab 5.10±0.34Aa						5.49±0.33Aa 5.14±0.41Aa 5.45±0.77Ab 4.31±0.37Aa

From Dynamic Variation Analysis, the 4th week of concentration pcS/2SS immune group of blood plasma free fatty acid peaks, the secretion peak of pGM-CSF+pcS/2SS immune group is in 0 week, and control group and pGMCSF-SS immune group peaked in the 6th week, thereafter control group sharply descends, the immunity back is during the 8th week, and the concentration of 2 groups, 3 groups and 4 groups blood plasma free fatty acids is significantly higher than control group (P＜0.05).Prompting SS genetic immunization does not have obvious influence to the secretion level of blood plasma free fatty acid.(seeing Table 8)

The change in concentration (mmol/L) of free lipid acid in the table 8 weanling pig different times blood plasma

Group	The immunity back time (week)
						0	2	4	6	8
	1 2 3 4	264.25 ±49.81Ab 373.06 ±26.70Aa 246.98 ±46.73Aa 489.64 ±18.32Aa	333.75 ±19.43Aab 392.06 ±23.93Aa 221.51 ±13.15Aa 347.153 ±13.20Aa	312.61 ±18.19Aab 444.54 ±57.19Aa 297.93 ±16.15Aa 467.62 ±47.42Aa	414.51 ±52.74Aa 428.32 ±27.43Aa 341.97 ±26.71Aa 330.61 ±28.50Aa						301.04 ±42.41Aab 369.60 ±49.84ACa 277.20 ±40.11Aa 428.77 ±34.86Ba

The change in concentration of plasma urea nitrogen is not obvious in different immune period, from Dynamic Variation Analysis, the pGMCSF-SS immune group has the trend of rising, concentration at the 2nd week, 4 weeks and 6 all plasma urea nitrogens is higher than other three groups (P＞0.05), and the pcS/2SS immune group then descends gradually with the prolongation of immunity time.Control group and pGM-CSF+pcS/2SS immune group keep situation more stably always.(seeing Table 9)

The change in concentration (mmol/L) of table 9 weanling pig different times plasma urea nitrogen

Group	The immunity back time (week)
						0	2	4	6	8
	1 2 3 4	3.11±0.32Aa 3.41±0.14Aa 3.37±0.40Aa 3.02±0.28Aa	2.85±0.70Aa 3.21±0.30Aa 3.99±0.24Aa 3.08±0.43Aa	3.04±0.54Aa 2.90±0.40Aa 3.65±0.23Aa 3.09±0.14Aa	2.61±0.25Aa 3.17±0.32Aa 3.73±0.23Aa 2.86±0.42Aa						3.55±0.36Aa 3.12±0.18Aa 3.36±0.28Aa 3.17±0.14Aa

Claims (4)

1, contain the Salmonella typhimurium Salmonella typhimuriumCSO22/pGMCSF-SS of fusion expression plasmid pGMCSF-SS, its preserving number is CCTCC NO:M206141.

2, the oral recombined DNA vaccine that comprises the promotion growth of animal of claim 1.

3, a kind of fusion expression plasmid pGMCSF-SS.

4, the application of the described Salmonella typhimurium of claim 1 in the oral recombined DNA vaccine of preparation promotion growth of animal.

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