CN112626073B

CN112626073B - CpG-ODN with specific immunostimulation effect on PRRSV and application thereof

Info

Publication number: CN112626073B
Application number: CN202011156263.3A
Authority: CN
Inventors: 冯璥; 李涛; 何文彬; 吕雪梅
Original assignee: Kunming Institute of Zoology of CAS
Current assignee: Kunming Institute of Zoology of CAS
Priority date: 2020-10-26
Filing date: 2020-10-26
Publication date: 2021-06-15
Anticipated expiration: 2040-10-26
Also published as: CN112626073A

Abstract

The invention relates to the technical field of biological engineering, in particular to a CpG-ODN with specific immunostimulation effect on PRRSV and application thereof, wherein the nucleotide sequence is shown as SEQ ID NO: 1, and the following components: the method comprises the following steps of randomly forming 10 groups of immune activation units by using 11 CpG-ODN fragments which are screened, wherein the position information of each CpG-ODN fragment in each group of immune activation units is different, inserting the 10 groups of immune activation units into a multi-copy cloning vector Puc19 derived vector FVX through two enzyme cutting sites of XbaI and EcoRI, constructing recombinant plasmid FVX-CpG by means of gene synthesis, and preparing a preparation containing the recombinant plasmid FVX-CpG, wherein the preparation is low in production cost, and overcomes the defects of instability, sequence diversity, individual difference and the like of the CpG-ODN in vivo; not only can induce the activation of the natural immune function in the pig body, but also can activate the acquired immune function of the pig body to PRRSV, improve the immune response inducing effect of the pig body to PRRSV, reduce the incidence rate of PRRSV and reduce the pig breeding cost.

Description

CpG-ODN with specific immunostimulation effect on PRRSV and application thereof

Technical Field

The invention relates to the technical field of biological engineering, in particular to CpG-ODN with specific immunostimulation effect on PRRSV and application thereof.

Background

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a pathogenic factor of Porcine Reproductive and Respiratory Syndrome (PRRS), and is a new RNA virus infectious disease virus causing sow reproductive failure and respiratory symptoms and high mortality of newborn piglets. PRRSV was first discovered in the united states in 1987, and in 1991 the virus was first isolated from diseased piglets and sows in Wensvoot in the netherlands, and subsequently in germany, the united states, the uk, etc. Currently, the virus is spread throughout north america and europe and is spread worldwide, and epidemic infections occur successively in various places, causing serious economic losses to the swine industry. Although the porcine reproductive and respiratory syndrome fire-fighting virus vaccine is widely used, the vaccine is accompanied by side effects and has only partial immune protection effect, so that the immune protection effect on the porcine reproductive and respiratory syndrome virus is not ideal.

CpG motifs (CpG motifs) refer to a class of specific structures that are composed of unmethylated cytosine guanine dinucleotides (CpG) as core sequences. On the surface of a great deal of research, CpG motif is a powerful nonspecific immunostimulation DNA sequence and can directly or indirectly activate various immune cells such as T cells, B cells, dendritic cells, macrophages and the like. The DNA containing CpG motifs is referred to as CpG-DNA, and artificially synthesized Oligodeoxyribonucleotides (ODNs) containing unmethylated CpG motifs are referred to as CpG-ODNs. Through experimental research of synthesizing CpG-ODN, the immunostimulation effect of bacterial DNA is found to be only related to the non-methylated CpG nucleotide with specific flank; unlike bacterial DNA, DNA molecules native to mammals do not have an immunostimulatory effect. CpG motifs occur only infrequently in mammalian and other vertebrate DNA molecules, and 60-90% of the cytosine fifth carbon atoms in CG motifs are methylated.

In 1995, Arthur M.Krieg et al, by analyzing the immunostimulatory activity of different sequences of CpG-ODN that were artificially synthesized, found that: the CpG-ODN is required to have a certain length, one or more CpG motifs can be contained in one CpG-ODN, and the specific purine and pyrimidine on both sides of the CpG motif and the difference of the bases between the CpG motifs all influence the immune stimulation type and intensity of the CpG motif. Therefore, researchers have provided a CpG-ODN and a recombinant plasmid and a preparation containing the CpG-ODN for effectively improving the congenital and acquired immunity of the pig, for example, the CpG-ODN with specific immunostimulation effect on the pig and the report of the effect thereof are disclosed in the patent application No. 201910229571.5. No research report of an immunostimulation unit with immunostimulation effect on PRRSV is found.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention provides a CpG-ODN with specific immunostimulation effect on PRRSV and application thereof.

The method is realized by the following technical scheme:

one of the purposes of the invention is to provide CpG-ODN with specific immunostimulation effect on PRRSV, and the nucleotide sequence is shown as SEQ ID NO: 1, overcomes the defects of instability, sequence diversity, individual difference and the like of the traditional CpG-ODN in vivo, has specific immune stimulation effect on PRRSV, can induce the immune reaction effect on porcine reproductive and respiratory syndrome in a pig body, reduces the incidence rate of the porcine reproductive and respiratory syndrome and reduces the economic loss of the pig raising industry.

The invention also aims to provide a recombinant plasmid containing the CpG-ODN, wherein the recombinant plasmid is FVX-CpG, the FVX is a vector, and the nucleotide sequence is shown as SEQ ID NO: 2, the preparation cost is low, and the CpG-ODN is constructed by inserting the CpG-ODN into two enzyme cutting sites of XbaI and EcoRI and using a gene synthesis method.

The invention also aims to provide application of the CpG-ODN and the recombinant plasmid in preparation of preparations for treating and/or preventing PRRSV.

The fourth purpose of the invention is to provide a preparation for treating and/or preventing PRRSV, wherein the preparation contains 10-100ug/mL of PBS solution of the recombinant plasmid.

In order to achieve the above purposes, the invention is realized by the following operation scheme:

through screening 11 CpG-ODN fragments, the 11 CpG-ODN fragments are connected end to form a DNA sequence containing a plurality of CpG motifs which are the immune activation units and take-CG-dinucleotide as one CpG motif. In order to search whether the position information of each CpG-ODN fragment can affect the immune stimulation activity so as to screen out the optimal immune activation unit, the screened 11 CpG-ODN fragments are randomly arranged and combined for 10 times to obtain 10 groups of immune activation units, and the position information of each CpG-ODN fragment in each group of immune activation units is different.

Inserting 10 groups of immune activation units into a multi-copy cloning vector Puc19 derived vector FVX through two enzyme cutting sites of XbaI and EcoRI, constructing recombinant plasmid FVX-CpG containing 10 groups of immune activation units by means of gene synthesis, and verifying the recombinant plasmid by sequencing; a preparation containing recombinant plasmid FVX-CpG was prepared. The preparation can be used as an immune activator, effectively activates the natural immune function of pigs to PRRSV, and replaces or partially replaces antibiotics for PRRSV treatment and/or prevention; the vaccine can also effectively activate the acquired immune function of the PRRSV in a pig body, can be used as an immune adjuvant, improves the vaccine protection effect, reduces the morbidity of the PRRSV and reduces the breeding cost.

Preferably, when the preparation is used as the PRRSV immune activator, the injection dosage is 0.3-0.5 mL/kg; when in use, the injection is injected subcutaneously for one week before the onset peak of the pig, and is administered once after one week.

Preferably, when the preparation is used as a PRRSV immune adjuvant, the injection dose is 0.1-1 mL/kg. The vaccine is directly and uniformly mixed with each pig and then is administrated, and the administration is carried out once after one week.

Compared with the prior art, the invention has the technical effects that:

the CpG-ODN created by the invention has specific immune stimulation effect on PRRSV, can induce the activation of natural immune function in a pig body, can also activate the acquired immune function of the pig body on the PRRSV, improves the immune response induction effect of the pig body on the PRRSV, reduces the incidence rate of the PRRSV and reduces the pig breeding cost.

The CpG-ODN created by the invention randomly forms 10 groups of immune activation units by using 11 CpG-ODN fragments screened out, the position information of each CpG-ODN fragment in each group of immune activation units is different, the 10 groups of immune activation units are inserted into a multi-copy cloning vector Puc19 derived vector FVX through two enzyme cutting sites of XbaI and EcoRI, recombinant plasmid FVX-CpG is constructed by means of gene synthesis, and the preparation containing the recombinant plasmid FVX-CpG is prepared.

The CpG-ODN-containing preparation provided by the invention has small toxic and side effects on organisms, has great potential, can be widely applied to the live pig breeding industry, reduces the production cost, reduces the incidence of PRRSV, and improves the survival rate and the breeding efficiency.

Drawings

FIG. 1 is a CpG-ODN restriction enzyme cleavage site map.

FIG. 2 is a restriction enzyme map of FVX vectors.

FIG. 3 is a flow chart of construction of CpG-ODN.

FIG. 4 is a flow chart of recombinant plasmid construction.

FIG. 5 is a graph showing the results of double restriction enzyme digestion.

Detailed Description

The technical solution of the present invention is further defined below with reference to the specific embodiments, but the scope of the claims is not limited to the description. The test methods used in the following examples are, unless otherwise specified, conventional methods, and materials, reagents, and the like used therein are, unless otherwise specified, reagents or materials which can be directly obtained from commercial sources.

The CpG-ODN fragments are joined end-to-end to form a DNA sequence containing a number of CPG motifs of-CG-dinucleotides as "Multi-CpG", i.e.immune activating units.

EXAMPLES screening of specific CpG-ODN fragments

(1) The screening method comprises the following steps: the CpG-ODN fragments were selected in a targeted manner according to the conventional techniques, and the results are shown in Table 1 below.

TABLE 1 CpG-ODN fragments selected

The CpG-ODN fragment selected above is based on the screening of the related sequence research by the related personnel, such as:

the C274 reference is "Marshall, J.D., Higgins, D., Abbat, C., et al (2004.). Polymyxin B industries ISS-mediated immunity access multiple reactions, cellular Immunology,229, 93-105." and "Teleshova, N., Kenney, J., Williams, V., et al. (2006.). CpG-C ISS-ODN activation of blood-derived B cells from and of chlorine immunity viruses.

The BCG-A4a reference is "Yamamoto, S., Yamamoto, T., Nojima, Y., et al (2002). Discovery of immunological CpG-DNA and its application to clinical details maintenance. Japanese Journal of Infectious diseases Disease,55, 37-44.".

The ODN1681 reference is "Jorgensen, J.B., Johansen, L.H., Steiro, K., et al (2003. CpG DNA indeces protective anti viral antigens responses in the alkaline salmon (Salmo salar L.). Journal of Virology,77, 11471-.

Reference is made to ODN K3 and ODN2006P as "Smith, r.l., Chong, t.w., Hughes, m.g., et al (2004.) Impact of immunomodulatory oligodynamics oligonucleotides on cytokine production in the lipid polysaccharide-fused human serum module.

The ODN2395 reference is "Vollmer, J., Weratna, R., Payette, P., et al (2004),. Characterisation Soft eye CpG oligodeoxyncleotide classes with discrete immunological activities. European Journal of Immunology, 34, 251-.

ODN1018P is referenced as "Marshall, j.d., Higgins, d., Abbate, c., et al (2004.) Polymyxin B enterprises ISS-mediated immunity experiments, cellular Immunology 229, 93-105.

The AACGTC-30 reference is "Iho, S., Yamamoto, T., Takahashi, T., et al (1999) Oligoxynated compounds relating to palindrome sequences with internal 50CpG-30act direct on human NK and activated T cells to induced IFN-gamma production in video. journal of Immunology,163, 3642 and 3652.

MB-4531-F14, MB-5519 and MB-4531 are referenced as "Lee, K.W., Jung, J., Lee, Y., et al (2006.). Immunostimulative oligomeric isocyanate from genome with screening of Mycobacterium bovis, molecular immunology,43, 2107. Anschonosomal 2118".

(2) The selected CpG-ODN fragments are joined end to form a DNA sequence containing a plurality of CPG motifs of-CG-dinucleotide as "Multi-CpG", i.e., an immune activating unit.

In order to investigate whether the positional information of CpG-ODN fragments affects the immunostimulatory activity, the present investigators randomly arranged and combined the selected CpG-ODN fragments 10 times during the study to form 10 groups of "Multi-CpG", and the positional information of the CpG-ODN fragments in each group was different. The recombinant plasmid FVX-CpG is constructed by a genetic engineering synthesis method through inserting two restriction enzyme sites of XbaI and EcoRI into a high copy cloning vector Puc19 derived vector FVX (the sequence is shown as SEQ ID NO: 2), and numbering.

Randomly obtained 10 groups of 'Multi-CpG' are inserted into a high-copy cloning vector Puc19 derived vector FVX (the sequence is shown as SEQ ID NO: 2) through two enzyme cutting sites of XbaI and EcoRI, and recombinant plasmids FVX-CpG containing 10 groups of 'Multi-CpG' (the sequence is shown as SEQ ID NO: 1) are constructed by a genetic engineering synthesis method and numbered.

The numbering results are shown in table 2 below:

preparing the recombinant plasmids with the numbers obtained in the table 2 and a sterile PBS solution into preparations corresponding to the plasmid numbers, wherein the content of the recombinant plasmids is 40 ug/mL; the sterile PBS solution contains 0.4 percent of NaCl, 0.05 percent of KCl and 0.0213 percent of Na in percentage by weight₂HPO₄， 0.029％KH₂PO₄And the balance of deionized water. The sterile PBS solution is prepared by adding sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate into water, stirring to dissolve, adding water to 1L, sterilizing at 120 deg.C for 30min in a normal pressure sterilizing pot.

(2.1) toxicity study:

55 healthy mice of 10 days old are selected and randomly divided into 11 groups, each group comprises 5 mice, each mouse is injected with 0.5mL/kg of preparation containing FVX-CpG recombinant plasmid, the mice are fed according to a conventional feeding method, and after the mice are fed for 50 days, the mortality and tissue lesion of the mice are observed, and the results are shown in the following table 3:

TABLE 3

The results in table 3 show that: the mortality rates of the mice in the recombinant plasmid FVX-CpG containing any group of 'Multi-CpG' and the recombinant plasmid FVX-CpG containing 10 groups of 'Multi-CpG' are zero, no obvious lesion is found through tissue dissection, and the mice in each group are active and alive, which indicates that the mice in each group are safe and non-toxic.

(2.2) cytological experiments:

the results of analyzing the expression of porcine immune activity factors IP-10 and IFN- γ using lymphocytes from a 10-20 day old white pig breed as a test cell (PBMC), a recombinant plasmid preparation with the numbers of FVX-CpG-1 to FVX-CpG-11 as an immunostimulating agent, and porcine reproductive and respiratory syndrome virus as a pathogenic bacterium are shown in Table 4:

TABLE 4 FVX-CpG preparation induced effects on IP-10, IFN-gamma after PRRSV infection of porcine PBMC

The results in Table 4 show that, for different CpG-ODN fragment position information, the recombinant plasmid FVX-CpG constructed by the same CpG-ODN fragment and PRRSV co-culture pig PBMC can promote the expression of immune activity factors IFN-gamma and IP-10, and the expression of each group tends to be stable; and for the nucleotide sequence of SEQ ID NO: the recombinant plasmid FVX-CpG preparation shown in 1 can remarkably improve the expression of immune activity factors IFN-gamma and IP-10, and shows that the nucleotide sequence is shown as SEQ ID NO: the recombinant plasmid FVX-CpG shown in 1 has a good effect of resisting PRRSV infection and has a good innate immunity enhancing capability.

(2.3) immunoprotection animal assay:

(2.31) innate immunity test

Taking the body weight and healthAbout 5kg of the growing white pig was bred into 77 pigs, randomly divided into 11 groups of 7 pigs, and fed in the same manner to each group, and the pigs were used as test subjects. The FVX-CpG-1 to FVX-CpG-11 formulations were used as immune stimulators, and injected subcutaneously and intramuscularly at 0.3mL/kg, after 7 days, again with the same formulations. After the end of the second injection, each piglet was inoculated subcutaneously with virus using PRRSV and, one week after inoculation, the titer value log of the specific antibody of each group was determined₂ ^(X)The results are shown in table 5 below:

specific antibody titer value log after Table 57 d₂ ^(X)Value of

(2.32) acquired Immunity test

33 healthy and long white pig species weighing about 5kg were randomly divided into 11 groups of 3 animals, and the raising condition was the same for each group, and the animals were used as test subjects. Adopting FVX-CpG-1 to FVX-CpG-11 preparation as vaccine adjuvant, purchasing the existing PRRSV live vaccine (VR2332 strain vaccine) from the market as the vaccine for test, injecting FVX-CpG + vaccine preparation, injecting 0.3mL/kg subcutaneously and intramuscularly, after 7 days, drawing 5mL of blood sample, diluting positive serum, performing ELISA reaction, determining the dropping value log of each group of specific antibody₂ ^(X)The results are shown in table 6 below:

TABLE 6

(2.33) conclusion

As can be seen from the results in tables 5 and 6, the nucleotide-containing sequences are shown in SEQ ID NO: 1, compared with the position information of different CpG-ODN fragments, the recombinant plasmid FVX-CpG preparation group constructed by the same CpG-ODN fragment has better effect in the aspects of prevention and treatment.

Finally, the invention is based on the existing biological genetic engineering technology, the CpG-ODN fragments are manually screened, 11 CpG-ODN fragments which are screened are randomly connected and combined into an immune activation unit, the position information of each CpG-ODN fragment in the combined immune activation unit is completely different, simultaneously, 10 groups of immune activation units which are randomly connected are inserted into a high-copy cloning vector Puc19 derived vector FVX (the sequence is shown as SEQ ID NO: 2) through two enzyme cutting sites of XbaI and EcoRI, recombinant plasmid FVX-CpG containing 10 groups of 'Multi-CpG' (the sequence is shown as SEQ ID NO: 1) is constructed by a genetic engineering synthesis method, and the recombinant plasmid is obtained by experimental research. To the extent that nothing else is left, it is sufficient for a person skilled in the art to make reference to technical means disclosed in the prior art, or to common general knowledge or conventional technical means known to a person skilled in the art.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Sequence listing

<110> Kunming animal research institute of Chinese academy of sciences

<120> CpG-ODN with specific immunostimulation effect on PRRSV and application thereof

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TCTAGAAGCA GCGTTCGTGT CGGCCTTCGT TTTGTCGTTT GACTGTGAAC 50

GTTCGAGAAC CGATAACGTC GCCGGTGACG GCACCACGCT GGCGTAGCGC 100

CTCGGCCTAC CGATGTCGTT GCGGTGACGA TCGACTCTCG AGCGTTCTCT 150

CGTCGAACGT TCGAGATGAT TGACTCGTTC GTGTCGCATG TCGTCGTTTT 200

CGGCGCGCGC CGACCGATGA CGTCGCCGGT GACGGCACCA CGAGCAGCGT 250

TCGTGTCGGC CTTCGTCGAA CGTTCGAGAT GATTGACTGT GAACGTTCGA 300

GACTGGCGTA GCGCCTCGGC CTTCGTTTTG TCGTTTGACT CGTTCGTGTC 350

GCATGACCGA TGACGTCGCC GGTGACGGCA CCACGACCGA TAACGTCGCC 400

GGTGACGGCA CCACGTCGTC GTTTTCGGCG CGCGCCGACC GATGTCGTTG 450

CGGTGACGAT CGACTCTCGA GCGTTCTCTC GTTTTGTCGT TCTGGCGTAG 500

CGCCTCGGCC TACCGATGTC GTTGCGGTGA CGTCGTCGTT TTCGGCGCGC 550

GCCGACCGAT AACGTCGCCG GTGACGGCAC CACGTGACTC GTTCGTGTCG 600

CATGAGCAGC GTTCGTGTCG GCCTTCGTCG AACGTTCGAG ATGATATCGA 650

CTCTCGAGCG TTCTCTGACT GTGAACGTTC GAGAACCGAT GACGTCGCCG 700

GTGACGGCAC CACGACCGAT GACGTCGCCG GTGACGGCAC CACGTGACTC 750

GTTCGTGTCG CATGAGCAGC GTTCGTGTCG GCCTACCGAT AACGTCGCCG 800

GTGACGGCAC CACGTCGTCG AACGTTCGAG ATGATATCGA CTCTCGAGCG 850

TTCTCTCGTC GTTTTCGGCG CGCGCCGTGA CTGTGAACGT TCGAGAACCG 900

ATGTCGTTGC GGTGACGCTG GCGTAGCGCC TCGGCCTTCG TTTTGTCGTT 950

TGACTCGTTC GTGTCGCATG AGCAGCGTTC GTGTCGGCCT ATCGACTCTC 1000

GAGCGTTCTC CTGGCGTAGC GCCTCGGCCT TCGTCGTTTT CGGCGCGCGC 1050

CGACCGATGA CGTCGCCGGT GACGGCACCA CGACCGATAA CGTCGCCGGT 1100

GACGGCACCA CGTCGTTTTG TCGTTTCGTC GAACGTTCGA GATGATACCG 1150

ATGTCGTTGC GGTGACGTGA CTGTGAAC GTTCGAGATC GTCGTTTTCG 1200

GCGCGCGCCG TGACTGTGAA CGTTCGAGAA TCGACTCTCG AGCGTTCTCA 1250

CCGATGTCGT TGCGGTGACG ACCGATAACG TCGCCGGTGA CGGCACCACG 1300

TGACTCGTTC GTGTCGCATG AGCAGCGTTC GTGTCGGCCT CTGGCGTAGC 1350

GCCTCGGCCT TCGTTTTGTC GTTACCGATG ACGTCGCCGG TGACGGCACC 1400

ACGTCGTCGA ACGTTCGAGA TGATTCGTTT TGTCGTTACC GATGTCGTTG 1450

CGGTGACGAC CGATGACGTC GCCGGTGACG GCACCACGAG CAGCGTTCGT 1500

GTCGGCCTTG ACTGTGAACG TTCGAGAATC GACTCTCGAG CGTTCTCTGA 1550

CTCGTTCGTG TCGCATGCTG GCGTAGCGCC TCGGCCTACC GATAACGTCG 1600

CCGGTGACGG CACCACGTCG TCGTTTTCGG CGCGCGCCGT CGTCGAACGT 1650

TCGAGATGAT ACCGATAACG TCGCCGGTGA CGGCACCACG ACCGATGTCG 1700

TTGCGGTGAC GTGACTGTGA ACGTTCGAGA ATCGACTCTC GAGCGTTCTC 1750

CTGGCGTAGC GCCTCGGCCT TCGTTTTGTC GTTAGCAGCG TTCGTGTCGG 1800

CCTTGACTCG TTCGTGTCGC ATGTCGTCGT TTTCGGCGCG CGCCGTCGTC 1850

GAACGTTCGA GATGATACCG ATGACGTCGC CGGTGACGGC ACCACGTCGT 1900

TTTGTCGTTA GCAGCGTTCG TGTCGGCCTT CGTCGTTTTC GGCGCGCGCC 1950

GACCGATAAC GTCGCCGGTG ACGGCACCAC GTCGTCGAAC GTTCGAGATG 2000

ATACCGATGA CGTCGCCGGT GACGGCACCA CGTGACTGTG AACGTTCGAG 2050

ATGACTCGTT CGTGTCGCAT GCTGGCGTAG CGCCTCGGCC TACCGATGTC 2100

GTTGCGGTGA CGATCGACTC TCGAGCGTTC TCCTGGCGTA GCGCCTCGGC 2150

CTACCGATGA CGTCGCCGGT GACGGCACCA CGAGCAGCGT TCGTGTCGGC 2200

CTTCGTCGTT TTCGGCGCGC GCCGACCGAT GTCGTTGCGG TGACGATCGA 2250

CTCTCGAGCG TTCTCTCGTC GAACGTTCGA GATGATACCG ATAACGTCGC 2300

CGGTGACGGC ACCACGTGAC TCGTTCGTGT CGCATGTCGT TTTGTCGTTT 2350

GACTGTGAAC GTTCGAGAGA ATTC 2374

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<400>2

AAAAATAAAC AAATAGGGGT TCCGCGCACA TTTCCCCGAA AAGTGCCACC 50

TGACGTCTAA GAAACCATTA TTATCATGAC ATTAACCTAT AAAAATAGGC 100

GTATCACGAG GCCCTTTCGT TGTAAAACGA CGGCCAGTCG AACCACGCAA 150

TGCGTCTCGA TCCGCAGTGT CTTGCGTCTC TTCTAGATCG TCGAACGTTC 200

GAGATGATAC CGATGACGTC GCCGGTGACG GCACCACGAC CGATGTCGTT 250

GCGGTGACGT CGTCGTTTTC GGCGCGCGCC GTGACTGTGA ACGTTCGAGA 300

TCGTTTTGTC GTTACCGATA ACGTCGCCGG TGACGGCACC ACGTCGTCGT 350

TTTGTCGTTT TGTCGTTTTG TCGTTTTGTC GTTCTGGCGT AGCGCCTCGG 400

CCTATCGACT CTCGAGCGTT CTCTCGTCGA ACGTTCGAGA TGATACCGAT 450

GACGTCGCCG GTGACGGCAC CACGACCGAT GTCGTTGCGG TGACGTCGTC 500

GTTTTCGGCG CGCGCCGTGA CTGTGAACGT TCGAGATCGT TTTGTCGTTA 550

CCGATAACGT CGCCGGTGAC GGCACCACGT CGTCGTTTTG TCGTTTTGTC 600

GTTTTGTCGT TTTGTCGTTC TGGCGTAGCG CCTCGGCCTA TCGACTCTCG 650

AGCGTTCTCT CGTCGAACGT TCGAGATGAT ACCGATGACG TCGCCGGTGA 700

CGGCACCACG ACCGATGTCG TTGCGGTGAC GTCGTCGTTT TCGGCGCGCG 750

CCGTGACTGT GAACGTTCGA GATCGTTTTG TCGTTACCGA TAACGTCGCC 800

GGTGACGGCA CCACGTCGTC GTTTTGTCGT TTTGTCGTTT TGTCGTTTTG 850

TCGTTCTGGC GTAGCGCCTC GGCCTATCGA CTCTCGAGCG TTCTCGAATT 900

CAGAGACGGA GTCACTGCCA ACCGAGACGG TCATAGCTGT TTCCTGTGTG 950

CCGCTTCCTC GCTCACTGAC TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA 1000

GCGGTATCAG CTCACTCAAA GGCGGTAATA CGGTTACCCA CAGAATCAGG 1050

GGATAACGCA GGAAAGAACA TGTGAGCAAA AGGCCAGCAA AAGGCCAGGA 1100

ACCGTAAAAA GGCCGCGTTG CTGGCGTTTT TCCATAGGCT CCGCCCCCCT 1150

GACGAGCATC ACAAAAATCG ACGCTCAAGT CAGAGGTGGC GAAACCCGAC 1200

AGGACTATAA AGATACCAGG CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT 1250

CTCCTGTTCC GACCCTGCCG CTTACCGGAT ACCTGTCCGC CTTTCTCCCT 1300

TCGGGAAGCG TGGCGCTTTC TCATAGCTCA CGCTGTAGGT ATCTCAGTTC 1350

GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG TGTGCACGAA CCCCCCGTTC 1400

AGCCCGACCG CTGCGCCTTA TCCGGTAACT ATCGTCTTGA GTCCAACCCG 1450

GTAAGACACG ACTTATCGCC ACTGGCAGCA GCCACTGGTA ACAGGATTAG 1500

CAGAGCGAGG TATGTAGGCG GTGCTACAGA GTTCTTGAAG TGGTGGCCTA 1550

ACTACGGCTA CACTAGAAGA ACAGTATTTG GTATCTGCGC TCTGCTGAAG 1600

CCAGTTACCT AAAAAGAGTT GGTAGCTCTT GATCCGGCAA ACAAACCACC 1650

GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA CGCGCAGAAA 1700

AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC 1750

AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA 1800

AGGATCTTCA CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT 1850

CTAAAGTATA TATGAGTAAA CTTGGTCTGA CAGTTACCAA TGCTTAATCA 1900

GTGAGGCACC TATCTCAGCG ATCTGTCTCT TTCGTTCATC CATAGTTGCC 1950

TGACTCCCCG TCGTGTAGAT AACTACGATA CGGGAGGGCT TACCATCTGG 2000

CCCCAGTGCT GCAATAATAC CGCGGGACCC ACGCTCACCG GCTCCAGATT 2050

TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT 2100

GCAACTTTAT CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG 2150

AGTAAGTAGT TCGCCAGTTA ATAGTTTGCG CAACGTTGTT GCCATCGCTA 2200

CAGGCATCGT GGTATCACGC TCGTCGTTTG GTATGGCTTC ATTCAGCTCC 2250

GGTTCCCAAC GATCAAGGCG AGTTACATGA TCCCCCATGT TGCGCAAAAA 2300

AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT AAGTTGGCCG 2350

CCGTGTTATC ACTCATGGTT ATGGCAGCAC TACATAATTC TCTTACTGTC 2400

ATGCCATCCG TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC 2450

ATTCTGAGAA TAGTGTATGC GGCGACCGAG TTGCTCTTGC CCGGCGTCAA 2500

TACGGGATAA TACCGCGCCA CATAGCAGAA CTTTAAAAGT GCTCATCATT 2550

GGAAAACGTT CTTCGGGGCG AAAACTCTCA AGGATCTTAC CGCTGTTGAG 2600

ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT TCAGCATCTT 2650

TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC 2700

GCAAAAAAGG GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTT 2750

CCTTTTTCAA TATTATTGAA GCATTTATCA GGGTTATTGT CTCATGAGCG 2800

GATACATATT TGAATGTATT TAG 2823

Claims

1. CpG-ODN with specific immunostimulation effect on PRRSV, which is characterized in that the nucleotide sequence is shown as SEQ ID NO: 1 is shown.

2. A recombinant plasmid comprising the CpG-ODN according to claim 1.

3. The recombinant plasmid of claim 2, wherein the recombinant plasmid is FVX-CpG, wherein the FVX is a vector and has a nucleotide sequence as set forth in SEQ ID NO: 2, respectively.

4. Use of the CpG-ODN of claim 1, the recombinant plasmid of claim 2 or 3 for the preparation of a formulation for the treatment and/or prevention of PRRSV.

5. A formulation for the treatment and/or prophylaxis of PRRSV, said formulation comprising 10-100ug/mL of the recombinant plasmid of claim 2 or 3 in PBS.

6. The formulation of claim 5, wherein the formulation, when used as a PRRSV immune activator, is injected at a dose of 0.3 to 0.5 mL/kg; when the preparation is used as a PRRSV immune adjuvant, the injection dose is 0.1-1 mL/kg.