CN110042103A - A kind of pair of pig has CpG-ODN and its application of specific immunity stimulation - Google Patents
A kind of pair of pig has CpG-ODN and its application of specific immunity stimulation Download PDFInfo
- Publication number
- CN110042103A CN110042103A CN201910229571.5A CN201910229571A CN110042103A CN 110042103 A CN110042103 A CN 110042103A CN 201910229571 A CN201910229571 A CN 201910229571A CN 110042103 A CN110042103 A CN 110042103A
- Authority
- CN
- China
- Prior art keywords
- cpg
- pig
- preparation
- odn
- pfar4
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Virology (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Communicable Diseases (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Epidemiology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses CpG-ODN and its applications that a kind of pair of pig has specific immunity stimulation, and the nucleotide sequence of the CpG-ODN is as shown in SEQ ID NO:1.The CpG-ODN can improve congenital immunity and specificity humoral to pig and the fall out effect of cell-mediated immune response, can be transferred to plasmid vector pFAR4 construction recombination plasmid pFAR4-CpG, and prepare the preparation containing recombinant plasmid pFAR4-CpG.Said preparation can not only effectively activate the innate immune function of pig, as immunoactivator, successfully substitution or part substitute antibiotics；The acquired immunity function of pig, especially cellular immunity can also be effectively activated, immunologic adjuvant is can be used as and improves vaccine protecting effect；And it is small to body toxic side effect, there is great potential, can be widely applied to pig-breeding industry, advantageously reduce aquaculture cost, reduce live pig disease incidence, improve its survival rate and breeding efficiency.
Description
Technical field
The present invention relates to technical field of bioengineering, and in particular, to a kind of pair of pig has specific immunity stimulation
CpG-ODN and its application.
Background technique
CpG motif (CpG motifs) refers to that a kind of Cytosine-phosphate-guanine (CpG) with non-methylation is core
The specific structure of heart Sequence composition.A large number of studies show that CpG motif is a kind of strong non-specific immunostimulating DNA sequence
Column, can directly or indirectly activate T cell, B cell, Dendritic Cells, natural killer cells (Natural killer cell,
) and the panimmunities cell such as macrophage NK.DNA containing CpG motif is known as CpG-DNA；Artificial synthesized is contained
The oligodeoxynucleotide (Oligodeoxynucleotide, ODN) of non A-G hepatitis is known as CpG-ODN.Due to
CpG-ODN specific can increase antigen immunogenicity, activation panimmunity effector cell, generate certain cell factors, and make auxiliary
Helping property Th2 type (T he1per 2-type, Th2), which reacts to react to Th1 type (T he1per 1-type, Th1), to be changed, therefore
Sato etc. is called immunostimulatory sequence (Immuno-stimulatory sequences, ISS).
For a long time, it is believed that DNA is only the carrier of hereditary information, the understanding in relation to its immunological role is always
It is a blank.The nineties in 19th century, it has been found that give cancer patient's bacterial injection extract energy the substantially reduced state of an illness, at that time people
Think that the lipopolysaccharides in bacterial extract is the active constituent to work.Until later, the prapes branch bar such as Tokunaga
The mouse experiment in vivo discovery that bacterium BCG vaccine (Bacilli Calmette-Guerin, BCG) carries out, it is pretreated with DNA enzymatic
BCG does not have antitumor action, this illustrates that DNA of bacteria is only the main matter for mediating this anti-tumor effect.Then,
The researchs such as Yamamoto find that the DNA ingredient in bacterium is ox BCG main cause with anti-tumor activity, and DNA of bacteria has
Direct immunostimulation and antitumor action.Experimental study is carried out by the CpG-ODN of synthesis, finds exempting from for DNA of bacteria
Epidemic disease stimulation, it is only related with wherein having the specific non-methylated CpG nucleotide of flank.
Different from DNA of bacteria, the DNA molecular of mammal itself does not have immunostimulation.Mammal and other
The frequency that CpG motif occurs in vertebrate DNA molecular is very low, and the 5th carbon of cytimidine is former in 60%~90% CG motif
Methylation (Bird, 1986) occurs for son.This makes the DNA of bacterium become a kind of pathogen-associated molecular pattern
(Pathogenassociated molecular patterns, PAMPs) contains non-methylation CG sequence during infection
The DNA of bacteria of column discharges, and is stimulated immune system as a kind of " danger signal ", so that it is clear to promote body generation immune response
Except pathogen.
Nineteen ninety-five, the immunostimulation that Arthur M.Krieg et al. passes through the artificial synthesized different sequence C pG-ODN of analysis
Activity, obtain the prerequisite basic law of CpG-ODN structure institute with stimulation ability: (1) irritation sequence must contain
Unmethylated CpG motif.(2) end CpG motif 5' is two purine nucleotides, and the end 3' is the sequence tool of two pyrimidine nucleotides
There is stronger effect of stimulation.Its basic structure is 5'-X1X2CGY1Y2-3', and wherein X1 represents a purine, X2 represent one it is fast
Purine or a thymidine, Y1 and Y2 represent pyrimidine.(3) CpG-ODN need to have certain length, in a CpG-ODN
In can have one or more CpG motifs, be separated by base between the special purine in CpG motif two sides and pyrimidine and CpG motif
Difference can all influence the immunostimulation types and intensity of CpG motif.
Although CpG-ODN reported at present has certain immunostimulation, but can effectively promote the congenital of pig simultaneously
The CpG-ODN of property and acquired immunity has not been reported.In addition, existing reported most of CpG-ODN in order to prevent its easily
Degradation, needs thioated, the cost of synthesis is very high, is not suitable for existing pig-breeding.It is a kind of novel therefore, it is necessary to research and develop
Efficient CpG-ODN, can effectively promote the congenital and acquired immunity of pig, while reduce artificial synthesized cost.
Summary of the invention
The purpose of the invention is to overcome the above-mentioned deficiency of the prior art, a kind of pair of pig is provided and is pierced with specific immunity
Swash the CpG-ODN of effect, the CpG-ODN can be improved to the congenital immunity and specificity humoral of pig and cell-mediated immune
The fall out effect of reaction.
Another object of the present invention is to provide a kind of recombinant plasmid containing above-mentioned CpG-ODN, preparation cost is low.
Another object of the present invention is to provide above-mentioned CpG-ODN, recombinant plasmids in preparation treatment and/or prevention pig disease
Preparation in application.
Another object of the present invention is to provide a kind for the treatment of and/or the preparations of prevention pig disease.
To achieve the goals above, the present invention is achieved by following scheme:
The present invention is quick by the peripheral blood mononuclear cells IFN-r and SI of foundation by design more than 50 CpG motifs of synthesis
The directed screening mode of the high efficient and reliable of measurement filters out best pig CpG motif.Again by cytologic experiment, it is with the motif
Benchmark, using the CpG motif of multiple copies as CpG-ODN, screening obtains optimal CpG-ODN, and is transferred to plasmid vector
PFAR4 construction recombination plasmid pFAR4-CpG prepares the preparation containing recombinant plasmid pFAR4-CpG.Said preparation can not only be effectively
The innate immune function for activating pig, as immunoactivator, successfully substitution or part substitute antibiotics；Pig can also effectively be activated
Acquired immunity function, especially cellular immunity can be used as immunologic adjuvant and improves vaccine protecting effect, reduce live pig morbidity
Rate.
Therefore, the CpG-ODN that a kind of pair of pig has immunostimulation is claimed in the present invention, and nucleotide sequence is such as
Shown in SEQ ID NO:1, which can be improved to the congenital immunity and specificity humoral of pig and cell-mediated immune anti-
The fall out effect answered.
A kind of recombinant plasmid containing above-mentioned CpG-ODN is also claimed in the present invention, which is to connect CpG-ODN
It accesses obtained in plasmid vector pFAR4, as pFAR4-CpG.Used connection method is this technical field of bioengineering
Routine techniques.
Above-mentioned CpG-ODN, recombinant plasmid is also claimed in the preparation of preparation treatment and/or prevention pig disease in the present invention
Application.
Preferably, the pig disease is to be drawn by pig enterotoxigenic Escherichia coli, pig Epidemic abortion and breathing syndrome virus
The disease risen.
A kind of preparation treated and/or prevent pig disease is also claimed in the present invention, and the preparation is to contain 10~100 μ
The PBS solution of the above-mentioned recombinant plasmid of g/mL.
Preferably, the PBS solution also contains each component of following weight percent: 0.6~1.0%NaCl, 0.01~
0.04%KCl, 0.0124~0.0164%Na2HPO4, 0.014~0.034%KH2PO4, balance deionized water.
It is highly preferred that the PBS solution also contains each component of following weight percent: 0.7%NaCl, 0.02%KCl,
0.0134%Na2HPO4, 0.024%KH2PO4, balance deionized water.
Preferably, when the preparation is as pig immunoactivator, injection dosage is 0.1~0.5mL/kg.
Specifically, when the preparation uses as pig immunoactivator, for preventing pig disease when, be to be sent out in the disease of pig
Sick peak period the last week is subcutaneously injected 0.1~0.5mL preparation by per kilogram animal weight, is administered again after 3~7 days.
Preferably, when the preparation is as pig immunologic adjuvant, injection dosage is 0.2~1mL/kg.
Specifically, when the preparation is as pig immunologic adjuvant, 0.2~1mL CpG/ epidemic disease is subcutaneously injected by per kilogram pig weight
Seedling (mixes corresponding preparation and part vaccine), is administered again after 3~7 days.
Compared with prior art, the invention has the following advantages:
(1) CpG-ODN provided by the invention can be improved to the congenital immunity and specificity humoral of pig and cell-mediated
The fall out effect of immune response can be transferred to plasmid vector pFAR4 construction recombination plasmid pFAR4-CpG, and be prepared containing weight
The preparation of group plasmid pFAR4-CpG, said preparation production cost are low.
(2) preparation provided by the invention containing CpG-ODN can not only effectively activate the innate immune function of pig, as
Immunoactivator, successfully substitution or part substitute antibiotics；The acquired immunity function of pig can also be effectively activated, especially carefully
Born of the same parents are immune, can be used as immunologic adjuvant and improve vaccine protecting effect；And it is small to body toxic side effect, there is great potential, it can be extensive
Applied to pig-breeding industry, production cost is advantageously reduced, live pig disease incidence is reduced, improves its survival rate and breeding efficiency.
Detailed description of the invention
Fig. 1 is that CpG-poly series DNA stimulates pig lymphocyte IP-10 expression quantity situation.Wherein, 1 to the 10 of abscissa
It is relative expression's content of plasmid CpG-poly-1 to CpG-poly-10 stimulation pig lymphocytes in vitro IP-10 respectively, 11 are
Relative expression's content of CpG-ODN stimulation IP-10；Numerical value is the average value of 3 individuals, 9 RT-qRCR in figure；Same time
Interior difference CpG-poly stimulation generates the relative amount of IP-10 compared with PBS group, and * indicates significant difference p < 0.05.
Fig. 2 is the double digestion qualification result of recombinant plasmid.Wherein, swimming lane M is Marker DL2000, and swimming lane 1 is pFAR4-
CpG digestion products.
Fig. 3 is that recombination bacteria plasmid extracts testing result.Wherein, swimming lane M is Marker DL2000, and swimming lane 1 is pFAR4-
CpG plasmid band (about 2400bp).
Fig. 4 is the building flow chart for recombinating bacteria plasmid.
Specific embodiment
With reference to the accompanying drawings of the specification and specific embodiment is made the present invention and is further elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
The screening of 1 specific C pG-ODN of embodiment
1, the present embodiment uses existing genetic engineering or chemical method, and design has synthesized more than 50 CpG motifs (partially such as table
Shown in 1), pass through the directed screening mode of the peripheral blood mononuclear cells IFN-r and SI of the foundation high efficient and reliable quickly measured, screening
Best pig CpG motif out (the results are shown in Table 2).
The structure feature of 1 in-vitro screening pig specific C pG ODN of table
Note: CpG motif is marked with underscore, and wherein lowercase indicates to use thio-modification, and capitalization is indicated without sulphur
Generation modification.
The different pig specific C pG ODN of table 2 is in vitro to the effect of stimulation of Swine PBMC
Note: above data is 5 individual average values；Same letter indicates not significant p > 0.05 of difference, different alphabets
Show significant difference p < 0.05.
As shown in Table 2, CpG10 can induce stronger lymphopoiesis and IFN-γ induces ability.Therefore, it selects
CpG10 is used for the motif determination of amount research of next step as motif.
2, on the basis of CpG10 motif, using the CpG motif of multiple copies as CpG-ODN, and it is transferred to plasmid vector
In pFAR4, construction recombination plasmid pFAR4-CpG, and number (as shown in table 3).Again by cytologic experiment, screening obtains best
CpG-ODN sequence.
CpG-poly sequence used in 3 the present embodiment of table
| Plasmid number | The number of motif containing CpG |
| CpG-poly-1 | 1 |
| CpG-poly-2 | 3 |
| CpG-poly-3 | 4 |
| CpG-poly-4 | 7 |
| CpG-poly--5 | 10 |
| CpG-poly-6 | 13 |
| CpG-poly-7 | 19 |
| CpG-poly-8 | 25 |
| CpG-poly-9 | 49 |
| CpG-poly-10 | 73 |
Recombinant plasmid containing CpG-poly series progression is subjected to stimulated in vitro pig lymphocyte respectively, culture 6h is laggard
The extraction of row cell total rna detects relative expression's content of IP-10.RT-qPCR testing result such as Fig. 1 of IP-10 expression contents
It is shown.
Plasmid CpG-poly-1, CpG-poly-2, CpG-poly-3, CpG-poly-4 and CpG- as seen from Figure 1
Poly-6, CpG-poly-7, CpG-poly-8 stimulation pig lymphocytes in vitro IP-10 relative expression quantity with compare it is not significant
Difference, it is extremely significant (p < 0.01) with positive control CpG-ODN difference.The relative expression quantity of IP-10 is under CpG-poly-5 is stimulated
3.21 times of control, but variance analysis is not significantly different with compareing, and with positive control CpG-ODN difference it is extremely significant (p <
0.01).The relative expression quantity of IP-10 is 4.5 times of control when CpG-poly-10 stimulates lymphocyte, not with control group difference
Significantly, with positive control CpG-ODN difference extremely significant (p < 0.01) with.The phase of IP-10 when CpG-poly-9 stimulates lymphocyte
It is not significant with control group difference extremely significant (p < 0.01) and positive control CpG-ODN difference to 12 times that expression quantity is control.Sun
Property control group CpG-ODN stimulation lymphocyte when IP-10 relative expression quantity be compare 9 times, it is extremely significant with control group difference
(p < 0.01), it is not significant with CpG-poly-10 difference.
Comprehensive analysis is it is found that CpG-poly-9 has optimal stimulus effect to the secretion of pig lymphocytes in vitro IP-10.Cause
This, using CpG-poly-9 as optimal CpG-ODN sequence, nucleotide sequence is as shown in SEQ ID:1.
Embodiment 2
Existing genetic engineering can be used or be chemically synthesized the nucleotide sequence (SEQ ID NO:1) of CpG-ODN.Synthesis
Afterwards, construct pFAR4-CpG recombinant plasmid, by the nucleotide sequence (SEQ ID NO:1) of target gene CpG and pFAR4 segment into
Row connection (as shown in figs. 2 to 4), obtain recombinant plasmid, be stored in -15 DEG C it is spare.
A kind of preparation treated and/or prevent pig disease, the preparation are to contain the sterile of the 30 above-mentioned recombinant plasmids of μ g/mL
PBS solution (i.e. pFAR4-CpG preparation).
The sterile PBS solution (1L) also contains each component of following weight percent: 0.6%NaCl, 0.01%KCl,
0.0124%Na2HPO4, 0.014%KH2PO4, balance deionized water.
The preparation method of the sterile PBS solution: after each component is mixed in proportion, using damp and hot autoclaving
At 115 DEG C, sterilize 20min.Damp and hot autoclaving is sterilizing methods commonly used in the art, and operation is referred to existing skill
Art carries out.
Prevention: being disease incidence peak period the last week in pig, by per kilogram animal weight when as pig immunoactivator
0.1mL pFAR4-CpG preparation is subcutaneously injected, is administered again after 3 days.
Treatment:, (will be corresponding by per kilogram pig weight subcutaneous injection 0.2mL CpG/ vaccine when as pig vaccine adjuvant
PFAR4-CpG preparation and part vaccine mix), it is administered again after 3 days.
Embodiment 3
Existing genetic engineering can be used or be chemically synthesized the nucleotide sequence (SEQ ID NO:1) of CpG-ODN.Synthesis
Afterwards, construct pFAR4-CpG recombinant plasmid, by the nucleotide sequence (SEQ ID NO:1) of target gene CpG and pFAR4 segment into
Row connection (as shown in figs. 2 to 4), obtain recombinant plasmid, be stored in -15 DEG C it is spare.
A kind of preparation treated and/or prevent pig disease, the preparation are to contain the sterile of the 30 above-mentioned recombinant plasmids of μ g/mL
PBS solution (i.e. pFAR4-CpG preparation).
The sterile PBS solution (1L) also contains each component of following weight percent: 0.7%NaCl, 0.02%KCl,
0.0134%Na2HPO4, 0.024%KH2PO4, balance deionized water.
The preparation method of the sterile PBS solution: after each component is mixed in proportion, using damp and hot autoclaving
At 117 DEG C, sterilize 21min.Damp and hot autoclaving is sterilizing methods commonly used in the art, and operation is referred to existing skill
Art carries out.
Prevention: being disease incidence peak period the last week in pig, by per kilogram animal weight when as pig immunoactivator
0.2mL pFAR4-CpG preparation is subcutaneously injected, is administered again after 4 days.
Treatment:, (will be corresponding by per kilogram pig weight subcutaneous injection 0.3mL CpG/ vaccine when as pig vaccine adjuvant
PFAR4-CpG preparation and part vaccine mix), it is administered again after 4 days.
Embodiment 4
Existing genetic engineering can be used or be chemically synthesized the nucleotide sequence (SEQ ID NO:1) of CpG-ODN.Synthesis
Afterwards, construct pFAR4-CpG recombinant plasmid, by the nucleotide sequence (SEQ ID NO:1) of target gene CpG and pFAR4 segment into
Row connection (as shown in figs. 2 to 4), obtain recombinant plasmid, be stored in -15 DEG C it is spare.
A kind of preparation treated and/or prevent pig disease, the preparation are to contain the sterile of the 50 above-mentioned recombinant plasmids of μ g/mL
PBS solution (i.e. pFAR4-CpG preparation).
The sterile PBS solution (1L) also contains each component of following weight percent: 0.8%NaCl, 0.03%KCl,
0.0144%Na2HPO4, 0.0245%KH2PO4, balance deionized water.
The preparation method of the sterile PBS solution: after each component is mixed in proportion, using damp and hot autoclaving
At 118 DEG C, sterilize 22min.Damp and hot autoclaving is sterilizing methods commonly used in the art, and operation is referred to existing skill
Art carries out.
Prevention: being disease incidence peak period the last week in pig, by per kilogram animal weight when as pig immunoactivator
0.3mL pFAR4-CpG preparation is subcutaneously injected, is administered again after 5 days.
Treatment:, (will be corresponding by per kilogram pig weight subcutaneous injection 0.4mL CpG/ vaccine when as pig vaccine adjuvant
PFAR4-CpG preparation and part vaccine mix), it is administered again after 5 days.
Embodiment 5
Existing genetic engineering can be used or be chemically synthesized the nucleotide sequence (SEQ ID NO:1) of CpG-ODN.Synthesis
Afterwards, construct pFAR4-CpG recombinant plasmid, by the nucleotide sequence (SEQ ID NO:1) of target gene CpG and pFAR4 segment into
Row connection (as shown in figs. 2 to 4), obtain recombinant plasmid, be stored in -15 DEG C it is spare.
A kind of preparation treated and/or prevent pig disease, the preparation are to contain the sterile of the 40 above-mentioned recombinant plasmids of μ g/mL
PBS solution (i.e. pFAR4-CpG preparation).
The sterile PBS solution (1L) also contains each component of following weight percent: 1.0%NaCl, 0.04%KCl,
0.0164%Na2HPO4, 0.034%KH2PO4, balance deionized water.
The preparation method of the sterile PBS solution: after each component is mixed in proportion, using damp and hot autoclaving
At 121 DEG C, sterilize 25min.Damp and hot autoclaving is sterilizing methods commonly used in the art, and operation is referred to existing skill
Art carries out.
Prevention: being disease incidence peak period the last week in pig, by per kilogram animal weight when as pig immunoactivator
0.1mL pFAR4-CpG preparation is subcutaneously injected, is administered again after 3 days.
Treatment:, (will be corresponding by per kilogram pig weight subcutaneous injection 0.2mL CpG/ vaccine when as pig vaccine adjuvant
PFAR4-CpG preparation and part vaccine mix), it is administered again after 3 days.
Comparative example 1
Experimental method uniquely the difference is that, is substituted the partial sequence of CpG-ODN used, compares ODN1 with embodiment 4
Sequence is as follows: 5 '-AAAAAACGATCGTCAAAAAA-3 '.
Comparative example 2
Experimental method uniquely the difference is that, is substituted the partial sequence of CpG-ODN used, compares ODN2 with embodiment 4
Sequence is as follows: 5 '-CCCCCACGATCGTCCCCCCC-3 '.
Comparative example 3
Experimental method uniquely the difference is that, is substituted the partial sequence of CpG-ODN used, compares ODN3 with embodiment 4
Sequence is as follows: 5 '-TTTTTACGATCGTCTTTTTT-3 '.
6 cytologic experiment of embodiment
With the peripheral blood lymphocytes (PBMC) of 30~40 age in days pigs (great Bai × Yorkshire × Du Luoke tri-crossbreeding)
For experimental cell, be respectively adopted recombinant plasmid pFAR4-CpG preparation that the embodiment of the present invention 2~5 is prepared and comparative example 1~
3 are used as immunostimulant, and enterotoxigenic Escherichia coli (Enterotoxigenic E.coli) is used as pathogen antigen,
Using compare ODN1,2,3 groups as ODN effect comparison, analyze the expression of Immunity active factor IP-10 and IFN-γ, as a result
It is shown in Table 4.Recombinant plasmid pFAR4-CpG and E.coli co-cultures Swine PBMC s, and discovery can significantly raise Immunity active factor IFN-
The expression of γ, IP-10, the effect of comparison ODN1,2,3 groups and vaccine to Immunity active factor, as a result illustrate that pFAR4-CpG has
The effect of good anti-escherichia coli infection, also illustrates that pFAR4-CpG has good congenital immunity hoisting power.
PFAR4-CpG preparation induces effect to IFN-γ, IP-10 after 4 E.coli of table infection Swine PBMC
7 zoopery of embodiment
1, congenital immunity protection (prevention disease) zoopery
Take health 3 age in days piggys (great Bai × Yorkshire × Du Luoke tri-crossbreeding) about 3kg, every group of 8 piglets, with
Machine is divided into 9 groups, and the raising situation of each group is identical, as experimental subjects, the embodiment of the present invention 2~5 is respectively adopted and is prepared into
The recombinant plasmid pFAR4-CpG preparation arrived as immunostimulant, using compare ODN1,2,3 groups as ODN effect comparison, produce intestines
Toxin escherichia coli (Enterotoxigenic E.coli) is used as pathogen, when being used to prevent, by per kilogram animal
Intramuscular injection 0.3mL pFAR4-CpG preparation under heavy weight hide.After three days, piglet is injected again with identical preparation.Exempt from for the second time
Three days after epidemic disease, all piglets are all to every piglet 1010The dosage oral challenge of CFU.Preparation is protected as congenital immunity
(prevention disease) research preventive effect.Experimental result is as shown in Table 5,6.
The preventive effect of 5 pFAR4-CpG preparation of table attacks the disease incidence after poison
| Experimental group | 0a D/Tb | 1D/T | 3D/T | 7D/T | 14D/T |
| PBS | 27.5% ± 0.3% | 75% ± 9.6% | 75% ± 4.3% | 87.5% ± 9.2% | 100% |
| K88/K99 | 27.5% ± 0.2% | 50% ± 4.7% | 62.5% ± 5.4% | 75% ± 6.3% | 50% ± 0.3% |
| Embodiment 2 | 32.5% ± 1.6% | 32.5% ± 2.2% | 31% ± 4.6% | 17.5% ± 0.4% | 13.4% ± 0.6% |
| Embodiment 3 | 0 | 10% ± 0.7% | 25% ± 0.2% | 12.5% ± 1.4% | 0 |
| Embodiment 4 | 25% ± 2.3% | 22.5% ± 0.5% | 25% ± 0.3% | 12.5% ± 0.3% | 6.2% ± 0.3% |
| Embodiment 5 | 15% ± 0.5% | 12% ± 0.2% | 14.5% ± 1.4% | 5.5% ± 0.2% | 0 |
| Comparative example 1 | 37.5% ± 6.4% | 62.5% ± 10.3% | 87.5% ± 12.6% | 87.5% ± 13.3% | 100% |
| Comparative example 2 | 34% ± 6.6% | 62.5% ± 10.5% | 75% ± 17.2% | 87.5% ± 14.5% | 100% |
| Comparative example 3 | 41% ± 10.1% | 62.5% ± 10.7% | 87.5% ± 23.3% | 87.5% ± 6.6% | 100% |
Note: it a: respectively indicates the 0th, 1,3,7,14 day after attacking poison；B: diarrhea piglet number/total piglet number.
The preventive effect of 6 pFAR4-CpG preparation of table attacks 7 days after poison specific antibody titers value log2(x) value
2, acquired immunity protects (vaccine adjuvant) zoopery
Take health 3 age in days piggys (great Bai × Yorkshire × Du Luoke tri-crossbreeding) about 3kg, every group of 3 piglets, with
Machine is divided into 9 groups, and the raising situation of each group is identical, as experimental subjects, the embodiment of the present invention 2~5 is respectively adopted and is prepared into
The recombinant plasmid pFAR4-CpG preparation arrived as vaccine adjuvant, using compare ODN1,2,3 groups as ODN effect comparison, it is existing
The colibacillosis of piglet K88/K99 bivalent gene engineering inactivated vaccine conduct of commercially available Shanghai Hai Li biologics Co., Ltd
Experiment vaccine, injects pFAR4-CpG+ vaccine preparation, the 7th day extraction blood sample after injection.It takes a blood sample about in foreneck venae subcutaneae
3mL injects sterile Eppendorf (Ep) pipe.Multiple proportions gradient dilution, ELISA reaction are carried out to positive serum.Exempt from as acquired
Epidemic disease protects preparation (vaccine adjuvant) to study therapeutic effect.Experimental result is as shown in table 7.
The specific antibody titers value log of the therapeutic effect of 7 pFAR4-CpG+ vaccine preparation of table2(x) value
| Experimental group | IgG | IgA |
| PBS | 2.2±0.23 | 1.33±0.12 |
| K88/K99 | 4.4±0.2 | 2.5±0.23 |
| Embodiment 2 | 13.9±0.2 | 11.83±0.1 |
| Embodiment 3 | 11.6±0.13 | 14.33±0.82 |
| Embodiment 4 | 9.3±0.12 | 12.83±0.73 |
| Embodiment 5 | 7.8±0.44 | 10.23±0.54 |
| Comparative example 1 | 2.3±0.18 | 1.2±0.24 |
| Comparative example 2 | 1.2±0.12 | 0.4±0.34 |
| Comparative example 3 | 1.3±0.2 | 0.8±0.17 |
From table 5~7 it is found that pFAR4-CpG preparation prepared by the embodiment of the present invention 2~5, effect is than single in terms of prevention
The effect of vaccine will be got well.Effect is more preferable than the effect of single vaccine in terms for the treatment of.Therefore, pFAR4-CpG+ vaccine preparation is complete
It is hopeful to replace or part replaces conventional antibiotic, as a kind of novel, efficient, safe formulation application to cultivated animals--
In the diseases prevention of pig.
8 toxicity research of embodiment
The present embodiment selects 7 age in days balb/c mouse, is randomly divided into blank control group and makes the embodiment of the present invention 2~5
Standby obtained pFAR4-CpG preparation group, each animal injecting immune 0.5mL, every group of 3 repetitions.Control group fed is using raising
The methods of indices such as test, animal tissue's dissection and appearance property.Test period is 2 months, and when off-test is detected small
The death rate and lesion tissue situation of mouse.As the result is shown (table 8): the mortality of animals of pFAR4-CpG processing group and control group is equal
It is zero, anatomic tissue does not find obvious lesion, and processing group mouse is vivaciously active, illustrates pFAR4-CpG to BALB/C mice safety
Nonhazardous.
The research of 8 pFAR4-CpG formulations toxic of table
| Experimental group | Lesion tissue | The death rate (%) |
| PBS control | - | 0 |
| Embodiment 2 | - | 0 |
| Embodiment 3 | - | 0 |
| Embodiment 4 | - | 0 |
| Embodiment 5 | - | 0 |
Note: "-" indicates inorganization lesion.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of shield range can also be made on the basis of above description and thinking for those of ordinary skill in the art
Other various forms of variations or variation, there is no necessity and possibility to exhaust all the enbodiments.It is all of the invention
Made any modifications, equivalent replacements, and improvements etc., should be included in the protection of the claims in the present invention within spirit and principle
Within the scope of.
Sequence table
<110>Agricultural University Of South China
<120>a kind of pair of pig has CpG-ODN and its application of specific immunity stimulation
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1088
<212> DNA
<213>artificial synthesized sequence (Synthetic sequence)
<400> 1
gaattcgggg gacgatcgtc ggggggaagc ttgtcgacgg gggacgatcg tcgggggggg 60
gggacgatcg tcgggggggg gggacgatcg tcggggggct cgacggggga cgatcgtcgg 120
ggggggggga cgatcgtcgg ggggggggga cgatcgtcgg ggggctcgac gggggacgat 180
cgtcgggggg gggggacgat cgtcgggggg gggggacgat cgtcgggggc tcgacggggg 240
acgatcgtcg gggggggggg acgatcgtcg ggggggggga cgatcgtcgg ggggctcgac 300
gggggacgat cgtcgggggg gggggacgat cgtcgggggg ggggacgatc gtcgggggct 360
cgacggggga cgatcgtcgg ggggggggga cgatcgtcgg ggggggggga cgatcgtcgg 420
gggctcgacg ggggacgatc gtcggggggg ggggacgatc gtcggggggg gggacgatcg 480
tcggggggct cgacggggga cgatcgtcgg ggggggggga cgatcgtcgg ggggggggga 540
cgatcgtcgg ggggctcgac gggggacgat cgtcgggggg gggggacgat cgtcgggggg 600
gggggacgat cgtcgggggg ctcgacgggg gacgatcgtc gggggggggg gacgatcgtc 660
gggggggggg gacgatcgtc ggggggctcg acgggggacg atcgtcgggg gggggggacg 720
atcgtcgggg gggggggacg atcgtcgggg ggctcgacgg gggacgatcg tcgggggggg 780
gggacgatcg tcgggggggg gggacgatcg tcggggggct cgacggggga cgatcgtcgg 840
ggggggggga cgatcgtcgg ggggggggga cgatcgtcgg ggggctcgac gggggacgat 900
cgtcgggggg gggggacgat cgtcgggggg gggggacgat cgtcgggggg ctcgacgggg 960
gacgatcgtc gggggggggg gacgatcgtc gggggggggg gacgatcgtc ggggggctcg 1020
acgggggacg atcgtcgggg gggggggacg atcgtcgggg gggggggacg atcgtcgggg 1080
ggctcgag 1088
Claims (10)
1. the CpG-ODN that a kind of pair of pig has specific immunity stimulation, which is characterized in that the nucleotide of the CpG-ODN
Sequence is as shown in SEQ ID NO:1.
2. a kind of recombinant plasmid, which is characterized in that contain CpG-ODN described in claim 1.
3. recombinant plasmid according to claim 2, which is characterized in that the recombinant plasmid is pFAR4-CpG.
4. recombinant plasmid described in CpG-ODN, Claims 2 or 3 described in claim 1 is in preparation treatment and/or prevention pig disease
Preparation in application.
5. applying according to claim 4, which is characterized in that the pig disease is popular by pig enterotoxigenic Escherichia coli, pig
Property miscarriage and breathing syndrome virus caused by disease.
6. the preparation of a kind for the treatment of and/or prevention pig disease, which is characterized in that the preparation is to contain 10~100 μ g/mL rights
It is required that the PBS solution of 2 or 3 recombinant plasmids.
7. preparation according to claim 6, which is characterized in that the PBS solution also contains each group of following weight percent
Point: 0.6~1.0%NaCl, 0.01~0.04%KCl, 0.0124~0.0164%Na2HPO4, 0.014~0.034%
KH2PO4, balance deionized water.
8. preparation according to claim 7, which is characterized in that the PBS solution also contains each group of following weight percent
Point: 0.7%NaCl, 0.02%KCl, 0.0134%Na2HPO4, 0.024%KH2PO4, balance deionized water.
9. according to any one of claim 6 to 8 preparation, which is characterized in that when the preparation is as pig immunoactivator,
Its injection dosage is 0.1~0.5mL/kg.
10. according to any one of claim 6 to 8 preparation, which is characterized in that when the preparation is as pig immunologic adjuvant,
Its injection dosage is 0.2~1mL/kg.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910229571.5A CN110042103B (en) | 2019-03-25 | 2019-03-25 | CpG-ODN with specific immunostimulation effect on pigs and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910229571.5A CN110042103B (en) | 2019-03-25 | 2019-03-25 | CpG-ODN with specific immunostimulation effect on pigs and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110042103A true CN110042103A (en) | 2019-07-23 |
| CN110042103B CN110042103B (en) | 2023-01-17 |
Family
ID=67275171
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910229571.5A Active CN110042103B (en) | 2019-03-25 | 2019-03-25 | CpG-ODN with specific immunostimulation effect on pigs and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110042103B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112322626A (en) * | 2020-10-26 | 2021-02-05 | 中国科学院昆明动物研究所 | CpG-ODN with specific immunostimulation effect on PRRSV and application thereof |
| CN112410344A (en) * | 2020-10-26 | 2021-02-26 | 中国科学院昆明动物研究所 | CpG-ODN with specific immunostimulation effect on PRRSV and application thereof |
| CN112626073A (en) * | 2020-10-26 | 2021-04-09 | 中国科学院昆明动物研究所 | CpG-ODN with specific immunostimulation effect on PRRSV and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1468957A (en) * | 2002-07-19 | 2004-01-21 | 中国人民解放军第二军医大学 | Plasmid used as human therapeutic vaccine adjuvant |
| US20070184068A1 (en) * | 2005-12-14 | 2007-08-09 | Cytos Biotechnology Ag | Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity |
| CN106490361A (en) * | 2016-10-31 | 2017-03-15 | 南宁学院 | A kind of preparation method of the CpG ODN for having immune-enhancing activity to pig |
-
2019
- 2019-03-25 CN CN201910229571.5A patent/CN110042103B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1468957A (en) * | 2002-07-19 | 2004-01-21 | 中国人民解放军第二军医大学 | Plasmid used as human therapeutic vaccine adjuvant |
| US20070184068A1 (en) * | 2005-12-14 | 2007-08-09 | Cytos Biotechnology Ag | Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity |
| CN106490361A (en) * | 2016-10-31 | 2017-03-15 | 南宁学院 | A kind of preparation method of the CpG ODN for having immune-enhancing activity to pig |
Non-Patent Citations (5)
| Title |
|---|
| "A Novel Chitosan CpG Nanoparticle Regulates Cellular and Humoral Immunity of Mice", 《生物医学与环境科学(英文版)》 * |
| KRISTINA DOMEIKA ET AL.: "Characteristics of oligodeoxyribonucleotides that induce interferon (IFN)-α in the pig and the phenotype of the IFN-α producing cells", 《VET IMMUNOL IMMUNOPATHOL. 》 * |
| QING CHENG ET AL.: "CpG oligodeoxynucleotide promotes protective immunity in the enteric mucosa and suppresses enterotoxigenic E. coli in the weaning piglets", 《INTERNATIONAL IMMUNOPHARMACOLOGY》 * |
| 于小娟等: "CpG-ODN作为动物疫苗佐剂的研究进展", 《黑龙江畜牧兽医》 * |
| 包木胜, 《中国优秀博硕士学位论文全文数据库 (博士)》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112322626A (en) * | 2020-10-26 | 2021-02-05 | 中国科学院昆明动物研究所 | CpG-ODN with specific immunostimulation effect on PRRSV and application thereof |
| CN112410344A (en) * | 2020-10-26 | 2021-02-26 | 中国科学院昆明动物研究所 | CpG-ODN with specific immunostimulation effect on PRRSV and application thereof |
| CN112626073A (en) * | 2020-10-26 | 2021-04-09 | 中国科学院昆明动物研究所 | CpG-ODN with specific immunostimulation effect on PRRSV and application thereof |
| CN112322626B (en) * | 2020-10-26 | 2021-06-15 | 中国科学院昆明动物研究所 | CpG-ODN with specific immunostimulation effect on PRRSV and application thereof |
| CN112410344B (en) * | 2020-10-26 | 2021-06-15 | 中国科学院昆明动物研究所 | CpG-ODN with specific immunostimulation effect on PRRSV and application thereof |
| CN112626073B (en) * | 2020-10-26 | 2021-06-15 | 中国科学院昆明动物研究所 | CpG-ODN with specific immunostimulation effect on PRRSV and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110042103B (en) | 2023-01-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110042103A (en) | A kind of pair of pig has CpG-ODN and its application of specific immunity stimulation | |
| Jørgensen et al. | CpG oligodeoxynucleotides and plasmid DNA stimulate Atlantic salmon (Salmo salar L.) leucocytes to produce supernatants with antiviral activity | |
| TWI351288B (en) | Cpg dna adjuvant in avian vaccines | |
| Thim et al. | Immunoprotective activity of a Salmonid Alphavirus Vaccine: comparison of the immune responses induced by inactivated whole virus antigen formulations based on CpG class B oligonucleotides and poly I: C alone or combined with an oil adjuvant | |
| CN1997391A (en) | Polyinosinic acid-polycytidylic acid-based adjuvant | |
| CN1810970B (en) | CpG-containing single-stranded deoxynucleotide, its vaccine composition and their application | |
| CN1307196C (en) | Modified CpG oligodeoxynucleotide with improved immunoregulatory function | |
| Carrington et al. | CpG oligodeoxynucleotides up-regulate antibacterial systems and induce protection against bacterial challenge in rainbow trout (Oncorhynchus mykiss) | |
| CN103405762A (en) | Polyinosinic acid-polycytidylic acid dominated adjuvant | |
| Wang et al. | A solution with ginseng saponins and selenium as vaccine diluent to increase Th1/Th2 immune responses in mice | |
| Yamamoto et al. | The discovery of immunostimulatory DNA sequence | |
| Wu et al. | A novel chitosan CpG nanoparticle regulates cellular and humoral immunity of mice | |
| Tousignant et al. | DNA sequences in cationic lipid: pDNA-mediated systemic toxicities | |
| CN101426514A (en) | HCV vaccines | |
| Yamamoto et al. | Activation of NK cell (human and mouse) by immunostimulatory DNA sequence | |
| Yu et al. | Effects of IBDV infection on expression of chTLRs in chicken bursa | |
| Patil et al. | Alpha-D-glucan-based vaccine adjuvants: Current status and future perspectives | |
| Yamamoto et al. | Oligodeoxyribonucleotides with 5′-ACGT-3′ or 5′-TCGA-3′ sequence induce production of interferons | |
| CN101559225B (en) | Meningococcus vaccine | |
| Yu et al. | The immune enhancement effect of CpG-ODNs on the vaccine of inactivated Vibrio harveyi in tiger puffer (Takifugu rubripes) | |
| CN101559224B (en) | Poliomyelitis vaccine | |
| CN104248754A (en) | Streptococcus suis vaccine composition, and preparation method and application thereof | |
| Canova et al. | Oligodeoxynucleotides CpGs increase silver catfish (Rhamdia quelen) resistance to Aeromonas hydrophila challenge | |
| CN101492672A (en) | Sulpho-oligodeoxynucleotide with immune stimulation activity and uses thereof | |
| CN101757018A (en) | Polyinosinic powder for livestock and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |