CN112080505A - CG island oligonucleotide with piglet immunostimulation capacity and application thereof in feed additive - Google Patents

CG island oligonucleotide with piglet immunostimulation capacity and application thereof in feed additive Download PDF

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CN112080505A
CN112080505A CN202010716395.0A CN202010716395A CN112080505A CN 112080505 A CN112080505 A CN 112080505A CN 202010716395 A CN202010716395 A CN 202010716395A CN 112080505 A CN112080505 A CN 112080505A
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island
oligonucleotide
piglets
piglet
feed
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姜啸风
胡智奕
吕正兵
周天琼
周正兵
何缤霞
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Hangzhou Huadi Group Co ltd
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Abstract

The invention discloses a CG island oligonucleotide with piglet immunostimulation capacity, the sequence of which is shown as SEQ ID NO.1, and also discloses a strain expressing the CG island oligonucleotide vector, which is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is 13337. The bacillus expressing CG island oligonucleotide is prepared into dry powder, is added into feed in the form of additive, and is tested for function and effect in animal body, so that a new solution is provided for disease treatment and prevention of piglets.

Description

CG island oligonucleotide with piglet immunostimulation capacity and application thereof in feed additive
Technical Field
The invention belongs to the technical development field of nucleic acid anti-infection preparations, and particularly relates to preparation of CG island oligonucleotide and application thereof in the field of piglet feed additives.
Technical Field
With the rapid development of the breeding industry in China, the diseases of piglets frequently and increasingly serious, and huge economic losses are caused. Meanwhile, due to the use of a large amount of antibiotics and pesticides, the problems of environmental pollution, pathogen resistance, food safety and the like are caused, and the sustainable development of the breeding industry and the body health of people are seriously influenced. With the national emergence of a series of strict food safety standards, a large amount of antibiotics are prohibited to be applied to piglet cultivation, and the breeding industry faces the difficult situation of no medicine application. Therefore, development and development of a safe, pollution-free and environment-friendly novel and efficient disease-resistant technology and disease-resistant preparation are urgently needed. Among them, immune defense techniques represent an important direction of development.
A large number of researches prove that the artificially synthesized CG island oligonucleotide and CG island oligonucleotide of some bacteria have good immune enhancement effect. The CG island refers to a dinucleotide composed of cytosine (C) and guanine (G) connected via a phosphodiester bond (p), and the CG island dinucleotide and two bases at the 5 'end and the 3' end of the CG island constitute a CG island motif (CG island nucleotide sequence). CG island motifs are also known as immunostimulatory sequences (ISS), while CG island oligonucleotides refer to oligodeoxynucleotides containing unmethylated CG island motifs. The CG island dinucleotide has a low frequency of occurrence in the genome of vertebrates and is mostly methylated, while the CG island dinucleotide has a high frequency of occurrence in the genome of bacteria and is mostly unmethylated. The immune system of vertebrates recognizes this feature in the bacterial genome as a danger-stimulating signal through Pattern Recognition Receptors (PRRs), thereby stimulating the body to produce an immune protective response. The CG island oligonucleotide can promote the maturation and activation of dendritic cells, macrophages and B cells, up-regulate the expression of CD80, CD86, CD40 and MHC-II molecules, promote the secretion of Thl-type cytokines such as IL-6, IL-12, IFN-gamma and the like, and induce an organism to generate Thl-type immune response.
In 1995, Arthur M.Krieg et al analyzed the immunostimulatory activity of artificially synthesized CG island oligonucleotides of different sequences to derive the essential rules that the structure of CG island oligonucleotides with stimulatory ability must have: (1) the stimulatory sequence must contain an unmethylated CG island motif. (2) The CG island motif sequence with two purine nucleotides at the 5 'end and two pyrimidine nucleotides at the 3' end has stronger stimulation effect. The basic structure is 5'-X1X2CGY1Y2-3', wherein X1 represents a purine, X2 represents a purine or a thymine, and Y1 and Y2 represent pyrimidines. (3) The CG island oligonucleotide is required to have a certain length, one or more CG island motifs can be arranged in one CG island oligonucleotide, and the specific purine and pyrimidine on the two sides of the CG island motif and the difference of the bases between the CG island motifs can influence the type and the strength of the immune stimulation of the CG island motif.
After the CG island oligonucleotide is taken into immune cells by endocytosis, the CG island oligonucleotide is recognized by TLR9 on endosome/lysosome in the cells and combined with the same, so that TLR9 is dimerized, and then a dimerized compound recruits medullary differentiation factor 88(Myeloid differentiation factor 88, MyD88) and IL-1receptor-associated kinase (IL-1receptor-associated kinase 4, IRAK4) at the downstream, so that IRAK4 is phosphorylated and interacts with tumor necrosis factor receptor-associated factor 6(TNF-receptor-associated factor 6, TRAF6), and further activates IkB kinase, releases NF-kB into the nucleus, activates a series of nuclear transcription factors, finally promotes related immune cells to secrete a series of cytokines and chemotactic factors, triggers a bactericidal mechanism in the cells or induces an inflammatory response, and eliminates or eliminates pathogenic microorganisms invading the body.
The existing CG island oligonucleotide products are nucleic acid products, have linear double-chain structures on the premise of lacking base modification, have water solubility, are not beneficial to long-term storage and transportation due to simple structures, and are not high in temperature resistance and humidity resistance. In order to solve the problems, the CG island oligonucleotide sequence commonly used at present is generally subjected to thio modification on all bases of the CG island oligonucleotide sequence, but the optimization scheme can cause that the immunostimulatory capacity of the CG island oligonucleotide sequence is weakened. The plasmid is DNA with a double-chain closed-loop structure, has a stable topological structure, can be obtained in large quantity by a boiling/ethanol precipitation method, and is safe and convenient to produce. The problem of unstable structure of the CG island oligonucleotide can be effectively solved.
In addition, the CG island oligonucleotide is generally used as an immune adjuvant to be absorbed by a body injection mode at present, during the injection process, the condition of piglet incompatibility is easy to occur, and the problems of tissue injury, pain, potential complications, rapid adverse reaction and the like exist.
Therefore, the market needs to provide an oligonucleotide capable of ensuring that the CG island can be safely and conveniently absorbed by piglets, and the oligonucleotide can be used for replacing antibiotics in the daily feeding process of the piglets.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a CG island oligonucleotide with piglet immunostimulation capacity, wherein the sequence of the CG island oligonucleotide is shown as SEQ ID NO. 1.
The invention also provides a strain expressing the CG island oligonucleotide vector, which is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 13337. The Bacillus methylotrophicus is classified as Bacillus methylotrophicus, the preservation date is 2016, 11 and 24 days, and the preservation address is No. 3 of Xilu No.1 Beijing Hokko, Kogyo, Tokyo, Yangyo.
The invention also provides a preparation method of the strain expressing the CG island oligonucleotide vector, which comprises the following steps:
(1) connecting CG island oligonucleotide (SEQ ID NO.1) with immunostimulating capacity and pUC18 empty vector by ligase;
(2) transforming the ligation product into bacillus, and culturing to obtain the strain expressing the CG island oligonucleotide vector.
The invention also provides application of the strain in preparation of feed additives. The method specifically comprises the following steps: the strain is put into a fermentation tank for amplification and culture, and the fermentation product bacterial liquid is collected and freeze-dried to prepare bacterial powder.
The invention has the following advantages:
the preparation of the bacterial powder containing CG island oligonucleotide provided by the invention is artificially synthesized, and the sequence is accurate and the purity is high;
the bacillus powder provided by the invention can be obtained in large quantity by fermentation, is low in generation cost, small in toxic and side effects on organisms, environment-friendly and widely applicable to the piglet breeding industry;
the bacterial powder provided by the invention can effectively activate the natural immune function of piglets, can be used as an immune activator to successfully replace or partially replace antibiotics, is beneficial to reducing the production cost, reducing the morbidity of piglets and improving the survival rate and the breeding efficiency of the piglets;
the bacterial powder provided by the invention enters the bodies of piglets in an oral way, and the effective components are safely absorbed.
Based on the reasons, the inventor connects a plurality of CG island oligonucleotides which are not modified by base sequences to a cloning vector pUC18 with high specific replication capacity, converts a recombinant vector into bacillus, finally prepares the modified bacillus with immunostimulation into dry powder, mixes the dry powder into feed in the form of additive, and tests the functions and effects of the dry powder in animal bodies, thereby providing a new solution for treating and preventing infectious diseases of piglets.
Drawings
FIG. 1 is a schematic representation of the immunostimulatory effect of CG island oligonucleotides in this patent;
FIG. 2 is a graph of the change in body weight of piglets fed with different feeds;
FIG. 3 shows that the IL-6 expression of piglets fed with different ingredients varies;
FIG. 4 shows that the IL-8 expression of piglets fed with different ingredients varies;
FIG. 5 shows the change of IFN-gamma expression of piglets fed with different ingredients.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The present invention will be described in detail with reference to the following specific examples, but the present invention is not limited to these examples, and any modifications or substitutions in the basic spirit of the examples are included in the scope of the present invention as claimed in the claims.
EXAMPLE 1 piglet feeding
Healthy piglets are selected, all the piglets are purchased by a biochemical research institute of a life institute and a medical institute of university of Zhejiang science and technology, the weight of the piglets is 20-40 kg, and all the piglets are bred in a No. 6 building breeding room of the university of Zhejiang science and technology. Feeding 3 times daily, wherein the feed is selected from logistics support Limited of university of physical Engineers in Zhejiang. The breeding room is ventilated daily, so that the breathing health of the piglets is ensured.
Example 2 piglet infection model construction
And (3) constructing a piglet bacterial infection model by using the porcine enterotoxigenic escherichia coli. Piglets were divided into two groups, one group was the disease group and the other group was the normal group. Each piglet in the disease group was injected subcutaneously with 1mL of porcine enterotoxigenic E.coli at a concentration of 10E5/mL, and the normal group was controlled by injecting the same dose of PBS.
Example 3pUC18-CG island oligonucleotide Bacillus bacteria powder preparation
CG island oligonucleotide (SEQ ID NO.1) with immunostimulating ability and pUC18 empty vector (Hongheyou creative antibody technology GmbH) were connected by T4 ligase at 16 deg.C overnight at a mass ratio of 1: 3. The following day the ligation products were transformed into B.methylotrophicus (CMCC (B)13337) competence and plated on Amp plates. After 12h, selecting bacterial plaque with obvious single bacterial colony for sequencing identification, putting the qualified pUC18-CG island oligonucleotide bacillus strain into a fermentation tank for amplification and culture, collecting the fermentation product bacterial liquid, and freeze-drying to prepare bacterial powder. When in use, the pig feed is mixed according to the weight ratio of 1: 1000.
The qualified pUC18-CG island oligonucleotide bacillus strain is simultaneously sent to the common microorganism center (CGMCC) of China Committee for culture Collection of microorganisms, CGMCC No.13337, for preservation. The classification name is Bacillus cereus.
Example 4 piglet weight detection
The piglets of the affected group and the piglets of the normal group are separately fed and are respectively fed with common feed, CG island-containing oligonucleotide bacillus powder feed and antibiotic-containing feed. The weight of 6 groups of piglets was recorded weekly by a platform scale and recorded as shown in fig. 2. It can be seen from the figure that in 6 groups of piglets, the weight of piglets in 5 groups increased with time, except for the affected group fed with normal feed, which continued to decrease. The weight of the normal group fed with the feed containing the CpG oligonucleotide bacterium powder is increased most, which shows that the feed used in the patent has better promotion effect on the growth of piglets than the common feed. In addition, the weight of the infected group fed with the feed disclosed by the patent is not reduced, which shows that the feed additive added in the patent plays a certain role in inhibiting piglet diseases.
Example 5 piglet blood inflammatory factor qPCR assay
Blood from 6 groups of piglets in example 4 was separately extracted through veins, and piglet blood mRNA was extracted. mRNA extraction Using Trizol reagent (available from Takara Bio Inc.) and reverse transcription of RNA to cDNA using reverse transcriptase (available from Samera Federation) as described, was performed according to Trizol reagent instructions. Primer design software Primer Premier 5.0 was used to design primers for fluorescent quantitative RT-PCR experiments, which were synthesized by Shanghai Biotech, Inc. The primer sequences are shown in table 1:
TABLE 1 quantitative primer sequences used in the present invention
Figure BDA0002598332830000051
Note: of the primers for amplifying each corresponding gene, the first row primer is an upstream primer, and the second row primer is a downstream primer.
The quantifier model is Roche fluorescence quantitative Light Cycler 480.
And (3) detecting the expression quantity of IL-6(genebank accession number AF518322), IL-12(genebank accession number) and IFN-gamma (genebank accession number) genes of the piglets by fluorescent quantitative qPCR.
The qPCR system was 4. mu.l c DNA template (concentration 50 ng/. mu.l), 0.2. mu.l forward primer (see Table 1 for the forward primer for amplifying the corresponding gene), 0.2. mu.l reverse primer (see Table 1 for the reverse primer for amplifying the corresponding gene), 3.1. mu.l deionized water, and 7.5. mu.l ultra SYBR Green fluorescent dye mixture (purchased from CWBIO).
The qPCR program was pre-denatured at 95 ℃ for 2min, then the following cycles were performed: denaturation at 94 ℃ for 15s, annealing at 60 ℃ for 15s, and extension at 72 ℃ for 20s, for a total of 40 cycles, melting curve preparation at 65-95 ℃, and finally cooling at 40 ℃.
Example 6 piglet fecal pathogenic microorganism detection
The genome of the feces of the 6 groups of piglets was extracted, and the gene expression level of the porcine enterotoxigenic escherichia coli of the piglets in example 4 was detected by qPCR, as shown in fig. 3-5. As can be seen from the figure, compared with the normal group, the expression levels of IL-6, IL-12 and IFN-gamma of the piglets in the affected group are all improved, wherein the IL-6 and IFN-gamma of the piglets fed with the CG island oligonucleotide feed are obviously improved, which shows that the CG island oligonucleotide feed has the capability of promoting the immune system of the piglets. In addition, the CG island oligonucleotide feed and the antibiotic feed have the same ability of promoting IL-12 expression on the affected piglets, which shows that the CG island oligonucleotide feed and the antibiotic feed have better anti-inflammatory effect compared with the common feed. According to the above, the CG island oligonucleotide used in the patent has strong immune activation capability, can promote the expression of anti-inflammatory factors in piglets, has important and positive effects on the healthy growth of piglets, and can meet the dual requirements of agricultural cultivation and environmental management in China.
The foregoing is merely a preferred embodiment of the invention, which is intended to be illustrative and not limiting. Those skilled in the art will recognize that many changes, modifications, and equivalents may be made thereto without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> Hangzhou Huazhi group Limited
<120> CG island oligonucleotide with piglet immunostimulation capacity and application thereof in feed additives
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 504
<212> DNA
<213> Unknown (Unknown)
<400> 1
tcgtcgtcga ccggtgcagt cgtttcgtcg tcgaccggtg cagtcgtttc gtcgtcgacc 60
ggtgcagtcg tttcgtcgtc gaccggtgca gtcgtttcgt cgtcgaccgg tgcagtcgtt 120
tcgtcgtcga ccggtgcagt cgtttcgtcg tcgaccggtg cagtcgtttc gtcgtcgacc 180
ggtgcagtcg tttcgtcgtc gaccggtgca gtcgtttcgt cgtcgaccgg tgcagtcgtt 240
tcgtcgtcga ccggtgcagt cgtttcgtcg tcgaccggtg cagtcgtttc gtcgtcgacc 300
ggtgcagtcg tttcgtcgtc gaccggtgca gtcgtttcgt cgtcgaccgg tgcagtcgtt 360
tcgtcgtcga ccggtgcagt cgtttcgtcg tcgaccggtg cagtcgtttc gtcgtcgacc 420
ggtgcagtcg tttcgtcgtc gaccggtgca gtcgtttcgt cgtcgaccgg tgcagtcgtt 480
tcgtcgtcga ccggtgcagt cgtt 504

Claims (3)

1. A CG island oligonucleotide with piglet immune stimulation capability has a sequence shown in SEQ ID NO. 1.
2. The strain expressing the CG island oligonucleotide vector is characterized by being preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of 13337.
3. Use of the strain according to claim 2 for the preparation of a piglet feed additive.
CN202010716395.0A 2020-07-23 2020-07-23 CG island oligonucleotide with piglet immunostimulation capacity and application thereof in feed additive Pending CN112080505A (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103045603A (en) * 2012-12-13 2013-04-17 河南德邻生物制品有限公司 Novel synthesized swine antibacterial peptide gene and application
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
CN106636115A (en) * 2016-11-08 2017-05-10 浙江大学 Fish Hepcidin expression accelerant, and applications thereof
CN106957805A (en) * 2017-01-21 2017-07-18 南京钱塘洪泰生物科技有限公司 It is a kind of with the bacterial strains of bacillus GBacillus 9 and its separation method of fungistatic effect and application
CN110004150A (en) * 2018-08-01 2019-07-12 中国农业科学院兰州兽医研究所 A kind of CpG oligonucleotide sequences and its application with immune-enhancing activity

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Publication number Priority date Publication date Assignee Title
CN103045603A (en) * 2012-12-13 2013-04-17 河南德邻生物制品有限公司 Novel synthesized swine antibacterial peptide gene and application
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
CN106636115A (en) * 2016-11-08 2017-05-10 浙江大学 Fish Hepcidin expression accelerant, and applications thereof
CN106957805A (en) * 2017-01-21 2017-07-18 南京钱塘洪泰生物科技有限公司 It is a kind of with the bacterial strains of bacillus GBacillus 9 and its separation method of fungistatic effect and application
CN110004150A (en) * 2018-08-01 2019-07-12 中国农业科学院兰州兽医研究所 A kind of CpG oligonucleotide sequences and its application with immune-enhancing activity

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Title
S KAMSTRUP等: "Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides", 《VET MICROBIOL》 *
SHUXIAZHANG等: "Recombinant plasmids containing CpG with porcine host defense peptides (PR-39/pBD-1) modulates the innate and adaptive intestinal immune responses (including maternal-derived) in piglets", 《INTERNATIONAL IMMUNOPHARMACOLOGY》 *
司兴奎等: "CpG寡脱氧核苷酸的免疫刺激特性及应用研究进展", 《动物医学进展》 *
张玲华等: "CpG ODN对仔猪淋巴细胞增殖和细胞因子诱生的影响:在体和离体试验", 《中国兽医学报》 *

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