CN103555726B - Unmethylated CpG contained DNA sequence (CpG-DNA) and its application - Google Patents
Unmethylated CpG contained DNA sequence (CpG-DNA) and its application Download PDFInfo
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- CN103555726B CN103555726B CN201310479972.9A CN201310479972A CN103555726B CN 103555726 B CN103555726 B CN 103555726B CN 201310479972 A CN201310479972 A CN 201310479972A CN 103555726 B CN103555726 B CN 103555726B
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Abstract
The invention relates to the field of immunology, specifically to an unmethylated CpG contained DNA sequence which has immune stimulation effect in Paralichthys olivaceus and its application in anti-virus. The unmethylated CpG contained DNA sequence (CpG-DNA) is a CpG-DNA sequence which has immune stimulation effect in Paralichthys olivaceus. The CpG-DNA sequence is as shown in the base sequence of sequence table SEQ ID No.1. The CpG-DNA provided by the invention can defense the infection of megalocytivirus, can be used as a vaccine adjuvant and can enhance specific immune effect of vaccine.
Description
Technical field
The present invention relates to field of immunology, a kind of specifically have the DNA sequence dna containing non-methylated CpG of immune-stimulating effect and the application in antiviral thereof in lefteye flounder.
Background technology
CpG-DNA is the DNA sequence dna containing non-methylated CpG, is a kind of feature of prokaryotic organism DNA sequence dna.During evolution; vertebrates obtains the Immune Recognition System for CpG-DNA; with this, protective immunological reaction being made to the invasion of microorganism cause of disease, comprising by acting on Toll-like receptor family member 9, the cellular immunization of activation Th1 induction and humoral immunization etc.There are some researches show, CpG-DNA has species specificity, and in different plant species, effective CpG-DNA sequence dna is different, and the immunne response that different CpG-DNA brings out in same species is also different.Lefteye flounder is the important cultivating seawater fishes of China, and its aquaculture is constantly subject to microorganism disease impact in recent years.Although present stage has found the CpG-DNA that can bring out bacterial-infection resisting in lefteye flounder.But for the report that the CpG-DNA that can bring out remarkable anti-virus infection in lefteye flounder is not relevant.
Summary of the invention
The object of the invention be to provide a kind of there is immune-stimulating effect in lefteye flounder the DNA sequence dna containing non-methylated CpG and application in antiviral.
For achieving the above object, the technical solution used in the present invention is:
A DNA sequence dna containing non-methylated CpG, contains the DNA sequence dna (CpG-DNA) of non-methylated CpG for having the CpG-DNA sequence of immune-stimulating effect in lefteye flounder, shown in the base sequence in sequence table SEQ IDNo.1.
The application of the DNA sequence dna containing non-methylated CpG, described CpG-DNA sequence can be prepared into the medicine of antiviral agent.Described CpG-DNA sequence can be prepared into the medicine of anti-swollen Magnocellular virokine.
The application of the DNA sequence dna containing non-methylated CpG, is characterized in that: described CpG-DNA sequence is as vaccine adjuvant.
Tool of the present invention has the following advantages:
1. virus infection can be suppressed.CpG-DNA of the present invention can make fish resist infecting of enlargement cell virus.
2. there is vaccine adjuvant effect.CpG-DNA of the present invention can use as vaccine adjuvant, can strengthen the specific immunity effect of vaccine.
Embodiment
Below in conjunction with embodiment, the invention will be further described.Embodiment is intended to carry out citing to the present invention and describes, but not limits the invention in any form.
Experimental technique routinely involved in embodiments of the present invention is all adopted with the following method:
1. plasmid extraction, DNA (PCR) product purification, DNA fragmentation reclaim from gel, bacterial genomes DNA extraction all uses the corresponding reagent box of " TIANGEN Biotech (Beijing) Co., Ltd. ".
2. plasmid, the conversion of DNA connecting fluid enter intestinal bacteria and all use Hanahan method (Sambrook andRussell:Molecular Cloning:A Laboratory Mannual.Cold Spring HarborLaboratory Press2001);
3. all restriction enzymes and ligase enzyme are all purchased from " Niu Yinglun Bioisystech Co., Ltd ", Beijing.
4. all oligonucleotide synthesis are all synthesized by " Shanghai Sangon Biological Engineering Technology And Service Co., Ltd ".
Embodiment 1
Base sequence in lefteye flounder specific C pG-DNA sequence tool sequence table SEQ ID No.1.
Sequence table SEQ ID No.1 is:
GGCGCGCGTCGCGCGCTA
(a) sequence signature:
● length: 18
● type: nucleotide sequence
● chain: strand
● topological framework: linear
(b) molecule type: DNA
C () is supposed: no
(d) antisense: no
E () is originated at first: engineer
Embodiment 1
CpG-DNA is as the application of antiviral agent
1) preparation of viral suspension.The concrete preparation method of enlargement cell virus RBIV-C1(is shown in ZhangM; Xiao Z; Hu Y; Sun L.Characterization of a megalocytivirus fromcultured rock bream; Oplegnathus fasciatus (Temminck & Schlege), inChina.Aquac Res.2012; 43:556 – 64) in PBS, be diluted to 5x10
6copies/ml, is viral suspension.
Described PBS moiety is by weight percentage: 0.8%NaCl, 0.02%KCl, 0.358%Na
2hPO
4.12H
2o, 0.024%NaH
2pO
4, 98.798% distilled water.
2) 10 lefteye flounders (every bar heavily about 8g) are divided into 2 groups at random, often organize 5.By these 2 groups difference called after A and B group.Every bar fish tissue injection 100ul of A group is contained the PBS of 10ug CpG-DNA, by every bar fish injection 100ul PBS of B group (control group).By A and B group fish every bar abdominal injection 100ul above-mentioned steps 1 after 1 day) viral suspension.3d and 5d after infection, gets fish spleen tissue, extracts DNA, detects viral level (concrete grammar is shown in above-mentioned reference) in tissue by absolute quantitation PCR method.Result shows that the viral number of A group fish 3d and 5d after infection (is respectively 1.1x10
3/ mg and 3.8x10
3/ mg) all significantly (P<0.01) lower than the viral number (1.8x10 of B group fish
4/ mg and 4.3x10
4/ mg).Therefore, CpG-DNA has obvious antiviral effect.
Embodiment 2
CpG-DNA is as the application of vaccine adjuvant
Step 1) vaccine immunity is injected
Its building process of DNA vaccination plasmid pCN86(is shown in Zhang M; Hu Y; Xiao Z, Sun Y, Sun L.Construction and analysis of experimental DNA vaccines againstmegalocytivirus.Fish Shellfish Immunol.2012; 33:1192 – 8.) in PBS, be diluted to 200ug/ml.CpG-DNA is diluted to 200ug/ml in PBS.PCN86 and CpG-DNA diluent equal-volume is mixed, is pCN86+CpG-DNA mixed solution.PCN86 and PBS equal-volume is mixed, is pCN86 vaccine liquid.15 lefteye flounders (the same) are divided into 3 groups at random, often organize 5.By these 3 groups difference called afters A, B and C.By every bar fish tissue injection 0.1mlpCN86 vaccine liquid of A group, by every bar fish tissue injection 0.1ml pCN86+CpG-DNA mixed solution of B group, by every bar fish tissue injection 0.1ml PBS of C group (control group).
Step 2) preparation of viral suspension, record accordingly see in embodiment 1.
Step 3) is attacked malicious infection and immunity protective effect and is detected
In above-mentioned steps 1) immunity after the 30th day, the every bar fish abdominal injection 100ul above-mentioned steps 2 by 3 groups) viral suspension.In afterwards 20 days, observe every day and record the death condition of each group of fish.After 20 days, add up total mortality of each group of fish: A group, 20; B group, 11; C group, 28.Utilize following formulae discovery premunition protective efficacy (RPS):
RPS=100x (total Percent mortality of the total Percent mortality/control group fish of 1-immune group fish)
Show that the RPS of immune pCN86 and pCN86+CpG-DNA mixed solution group fish is respectively 29% and 61% thus, wherein the RPS remarkable (P<0.01) of immune pCN86+CpG-DNA mixed solution group fish is higher than immune pCN86 group.Therefore, CpG-DNA significantly improves the immunoprotective effec of vaccine pCN86.
Conclusion: CpG-DNA of the present invention had both had antiviral effect and also had vaccine adjuvant effect.
Claims (4)
1. the DNA sequence dna containing non-methylated CpG, is characterized in that: contain the DNA sequence dna (CpG-DNA) of non-methylated CpG for having the CpG-DNA sequence of immune-stimulating effect in lefteye flounder, shown in the base sequence of SEQID No.1.
2. an application for the DNA sequence dna containing non-methylated CpG according to claim 1, is characterized in that: described CpG-DNA sequence is for the preparation of the medicine of antiviral agent.
3. the application of the DNA sequence dna containing non-methylated CpG according to claim 2, is characterized in that: described CpG-DNA sequence is for the preparation of the medicine of anti-enlargement cell virus.
4. an application for the DNA sequence dna containing non-methylated CpG according to claim 1, is characterized in that: described CpG-DNA sequence is for the preparation of vaccine adjuvant.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851621A (en) * | 2010-04-16 | 2010-10-06 | 中国科学院海洋研究所 | Microbial molecular marker and construction and application thereof |
| US7892569B2 (en) * | 2005-08-31 | 2011-02-22 | The United States Of America As Represented By The Department Of Health And Human Services | Methods of altering an immune response induced by CpG oligodeoxynucleotides |
| CN102399784A (en) * | 2010-09-10 | 2012-04-04 | 中国科学院海洋研究所 | CpG oligonucleotide possessing immune enhancement activity to aquatic livestock, its preparation method and its application |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7892569B2 (en) * | 2005-08-31 | 2011-02-22 | The United States Of America As Represented By The Department Of Health And Human Services | Methods of altering an immune response induced by CpG oligodeoxynucleotides |
| CN101851621A (en) * | 2010-04-16 | 2010-10-06 | 中国科学院海洋研究所 | Microbial molecular marker and construction and application thereof |
| CN102399784A (en) * | 2010-09-10 | 2012-04-04 | 中国科学院海洋研究所 | CpG oligonucleotide possessing immune enhancement activity to aquatic livestock, its preparation method and its application |
Non-Patent Citations (4)
| Title |
|---|
| A C. Carrington,C J. Secombes.A review of CpGs and their relevance to aquaculture.《Veterinary Immunology and Immunopathology》.2006,第112卷87-101. * |
| C7: A CpG oligodeoxynucleotide that induces protective immuneresponse against megalocytivirus in Japanese flounder (Paralichthys olivaceus) via toll-like receptor 9-mediated signaling pathway;Zhi-xia Zhou et al.;《Developmental and Comparative Immunology》;20131212;第44卷;124-132 * |
| Yue Jai Kang, Ki Hong Kim.Effect of CpG-ODNs belonging to different classes on resistance of olive flounder (Paralichthys olivaceus) against viral hemorrhagic septicemia virus (VHSV) and Miamiensis avidus (Ciliata;Scuticociliatia) infections.《Aquaculture》.2011,39-43. * |
| 免疫佐剂CpG-DNA的研究进展;范媛 等;《中国动物保健》;20091031;44-49 * |
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