CN110013485B - Application of antisense nucleic acid AMO-miR-9875 in preparation of anti-white spot syndrome virus preparation - Google Patents
Application of antisense nucleic acid AMO-miR-9875 in preparation of anti-white spot syndrome virus preparation Download PDFInfo
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Abstract
The invention relates to an application of antisense nucleic acid AMO-miR-9875 in preparation of a broad-spectrum white spot syndrome virus resistant preparation, wherein the sequence of the small RNA antisense nucleic acid AMO-miR-9875 is as follows: 5'-CUCCUCCCUUCCUCUUCC-3' are provided. The small RNA antisense nucleic acid AMO-miR-9875 can obviously inhibit the proliferation of WSSV in the bodies of Scylla paramamosain, Japanese Pacific shrimp and Procambrus clarkii, and greatly reduce the infection and death phenomenon of WSSV on seawater and freshwater aquaculture shrimps and crabs. The invention provides a potential medicine for resisting white spot syndrome virus in aquaculture, can efficiently and quickly inhibit the duplication of WSSV, has wide application range, is not only suitable for sea water cultured marsupenaeus japonicus and Scylla paramamosain, but also suitable for freshwater cultured Procambarus clarkii. The small RNA antisense nucleic acid AMO-miR-9875 is small in molecular weight, easy to degrade, free of damage to the ecological balance of marine culture and harm to a human body, and is an environment-friendly and effective virus defense substance.
Description
Technical Field
The invention relates to the field of viral disease prevention and treatment of aquaculture, in particular to application of a small RNA antisense nucleic acid AMO-miR-9875 in preparation of a broad-spectrum anti-white spot syndrome virus preparation.
Background
The Scylla paramamosain, called blue crab for short, has the advantages of high nutritive value, delicious meat, quick growth and strong adaptability, and is an important economic crab culture in China. The marsupenaeus japonicus, commonly called as flower shrimp, spotted shrimp and the like, has extremely wide distribution, large individuals, delicious meat, thick and hard shell, dew resistance, catching resistance, contribution to long-distance transportation and great economic value. The procambarus clarkia, also called crayfish, is a freshwater shrimp, is widely distributed in various water bodies, has high growth speed, high reproduction rate and strong adaptability, and is an important resource in freshwater shrimps in China.
With the popularization of the high-density culture mode, diseases including bacteria, fungal diseases and viral diseases frequently occur in the aquaculture process, and the sustainable development of the aquaculture industry is seriously influenced. Wherein, White Spot Syndrome Virus (WSSV) has high pathogenicity, the lethality rate of the WSSV to the Japanese cyst prawn and the Procambrus clarkii is more than 90 percent, and the death of the blue crab can be caused after a certain amount of WSSV is accumulated in the body of the Scylla paramamosain. After entering a host body, the WSSV can be rapidly replicated and proliferated, has various transmission ways, and is one of main pathogens of viral disease outbreaks in the aquaculture industry in China.
Scylla paramamosain, Pacific crayfish and Procambrus clarkii belong to invertebrates and rely mainly on the innate immune system to resist the invasion of pathogenic microorganisms. For a long time, antibiotics and other medicines are mainly used in the aquaculture process to prevent and treat outbreak diseases in the aquaculture process. The use of a large amount of antibiotics enhances the drug resistance of pathogenic microorganisms, and makes the prevention and treatment of diseases more difficult. In addition, the use of antibiotics can cause serious influence on the balance of the aquaculture ecological environment, and is not beneficial to the sustainable development of aquaculture industry. Therefore, it is very important to find a drug method which is not only environment-friendly and beneficial to the sustainable development of the aquaculture industry, but also can effectively prevent and treat diseases.
The traditional WSSV resisting medicine mainly comprises a medicine synthesized by a plurality of materials, such as a Chinese and western compound medicine (patent number: 200810152729.5) for preventing and treating white spot disease of prawns, and the raw materials of the Chinese and western compound medicine comprise: coptis root, scutellaria baicalensis, phellodendron, gardenia, astragalus polysaccharide, glucurolactone and vitamin C are mixed and stirred uniformly into powder, and then the powder is added into feed to feed prawns. The variety of the required raw materials is various, the molecular weight of the contained materials is relatively large, the degradation difficulty and the degradation degree are unknown, and whether the ecological balance of the culture is influenced is also unknown. Therefore, the search for a simple, efficient and environment-friendly prevention and treatment method is extremely important.
miRNA is a non-coding RNA composed of 18-22 nucleotides, and can inhibit the expression of target genes by acting on 3 'non-coding regions (3' UTR) of the target genes, thereby playing a role in post-transcriptional regulation. Recent research shows that miRNA plays a key role in the interaction process of host and virus, and miRNA has good application prospect as candidate drug for antiviral therapy.
Disclosure of Invention
The invention aims to provide application of a small RNA antisense nucleic acid AMO-miR-9875 in preparation of a broad-spectrum anti-white spot syndrome virus preparation, and the small RNA antisense nucleic acid AMO-miR-9875 can obviously inhibit the proliferation of WSSV in bodies of Scylla paramamosain, Japanese marsupenaeus japonicas and Procambrus clarkii, and effectively solves the problems of infection and death of WSSV to seawater and freshwater cultured shrimps and crabs and the like.
A small RNA antisense nucleic acid AMO-miR-9875 for efficiently resisting WSSV has a sequence as follows: 5'-CUCCUCCCUUCCUCUUCC-3' are provided.
The small RNA antisense nucleic acid AMO-miR-9875 capable of efficiently resisting WSSV is applied to preparation of a broad-spectrum anti-white spot syndrome virus preparation.
Further, the broad-spectrum anti-white spot syndrome virus preparation comprises a feed additive containing the small RNA antisense nucleic acid AMO-miR-9875, a solid or liquid medicine containing the small RNA antisense nucleic acid AMO-miR-9875.
Further, the broad-spectrum anti-white spot syndrome virus preparation can be used for crustaceans.
Further, the crustaceans include seawater-cultured crabs, seawater-cultured shrimps, and freshwater-cultured shrimps.
Further, the crustaceans include penaeus japonicus, procambarus clarkii, and scylla paramamosain.
A preparation containing the small RNA antisense nucleic acid AMO-miR-9875 with high WSSV resistance.
Compared with the prior art, the invention has the following advantages:
(1) the small RNA antisense nucleic acid AMO-miR-9875 has quick response and high WSSV inhibition efficiency, and is not only suitable for prevention, but also suitable for the culture process of shrimps and crabs with outbreak of white spot syndrome.
(2) The small RNA antisense nucleic acid AMO-miR-9875 has extremely small molecular weight, is safe and easy to degrade, does not damage the ecological balance in the seawater and freshwater aquaculture process, and is beneficial to the sustainable development of the aquaculture industry.
(3) The invention has wide application range, is not only suitable for the sea water cultured Scylla paramamosain and the Japanese marsupenaeus but also suitable for the freshwater cultured Procambarus clarkii, and is a potential broad-spectrum anti-WSSV medicine.
(4) The anti-white spot syndrome virus preparation can only contain a single chemical compound of the small RNA antisense nucleic acid AMO-miR-9875, and the anti-white spot syndrome virus preparation is more easily applied to actual production.
Drawings
FIG. 1 shows the expression level of miR-9875 in scylla paramamosain hemolymph cells after WSSV treatment.
FIG. 2 shows the variation of the expression level of miR-9875 in Scylla paramamosain with the treatment of PBS, miR-9875 simulant and miR-9875-scrambled (sequence disturbed by miR-9875 simulant).
FIG. 3 shows the variation of the copy number of WSSV in Scylla paramamosain with the treatment of miR-9875, miR-9875-scrambled and PBS; the above treatment is carried out on the Scylla paramamosain infected by the WSSV, and the copy number of the WSSV in the Scylla paramamosain is detected after the WSSV is infected for 0h, 24h and 48 h.
FIG. 4 shows the variation of the expression level of miR-9875 in Scylla paramamosain with PBS, AMO-miR-9875 and AMO-miR-9875-scrambled (sequence scrambled by AMO-miR-9875).
FIG. 5 shows the variation of WSSV copy number in Scylla paramamosain with AMO-miR-9875, AMO-miR-9875-scrambled and PBS treatment; the above treatment is carried out on the Scylla paramamosain infected by the WSSV, and the copy number of the WSSV in the Scylla paramamosain is detected after the WSSV is infected for 0h, 24h and 48 h.
FIG. 6 shows the effect of AMO-miR-9875 on the activity of blood lymphocytes in Scylla paramamosain; after WSSV infection, AMO-miR-9875 is injected into an experimental group, AMO-miR-9875-scrambled is injected into a control group, and the activity of blood lymphocytes in the scylla paramamosain body is detected after 48h treatment.
FIG. 7 shows the effect of AMO-miR-9875 Scylla paramamosain hemolymphocyte apoptosis, AMO-miR-9875 or AMO-miR-9875-scrambled mixed WSSV is injected into a scylla paramamosain body, PBS is only injected into a control group, and the apoptosis of Scylla paramamosain hemolymphocyte is detected by a flow cytometer after WSSV is infected for 48 hours.
FIG. 8 shows the variation of WSSV copy number in Penaeus japonicus with AMO-miR-9875, AMO-miR-9875-scrambled and PBS treatment; WSSV infected Penaeus japonicus was treated as described above, and the number of WSSV copies in the bodies of Penaeus japonicus was measured at 0h, 24h and 48h after WSSV infection.
FIG. 9 shows the variation of WSSV copy number in Procambrus clarkii with AMO-miR-9875, AMO-miR-9875-scrambled and PBS treatment; the above treatment was performed on procambarus clarkii after WSSV infection to detect the number of WSSV copies in procambarus clarkii after 0h, 24h and 48h of WSSV infection.
FIG. 10 is a graph of the effect of AMO-miR-9875 on the viability of blood lymphocytes in Japanese Palaemon japonicus and Procambrus clarkii; after WSSV infection, AMO-miR-9875 is injected into an experimental group, AMO-miR-9875-scrambled is injected into a control group, and the activity of blood lymphocytes in the bodies of the Japanese marsupenaeus japonicus and the procambarus clarkii is detected after 48 hours of treatment.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.
Example 1:
detection test for miR-9875 expression quantity in scylla paramamosain body after WSSV infection
Cultivating Scylla paramamosain with weight of 30-40g and WSSV negative by qRT-PCR in indoor seawater tank at salinity of 10 ‰ and water temperature of 25 deg.C for one week, and culturing in water tankInjection on the fourth appendage of 100. mu.L/one, 106Copy number of WSSV infection. Collecting blue crab blood lymphocytes at 0h, 24h and 48h after injection, extracting total RNA, and performing cDNA reverse transcription by using primers as follows: 5-GTCGTATCCAGTGCAGGGTCCGAGGTCACTGGATACGACCTCCTCCC-3', and finally detecting the miR-9875 expression quantity by utilizing a qPCR technology, wherein the used primers are P1 and P2.
P1:5’-CGCCGGGAAGAGGAAGGG-3’；
P2:5’-TGCAGGGTCCGAGGTCACTG-3’。
The result is shown in figure 1, and data show that the expression quantity of miR-9875 in the hemolymph cells of the scylla paramamosain is obviously reduced after the scylla paramamosain is infected by WSSV for 24h and 48h, which indicates that the miR-9875 possibly participates in the interaction process between host viruses, so that the miR-9875 is a potential new target point for WSSV prevention and treatment.
Example 2:
overexpression test of miR-9875 in scylla paramamosain
Scylla paramamosain was randomly divided into 3 groups as in example 1, and 100. mu.L/body of either miR-9875 mimic or miR-9875-scrambled or 100. mu.L/body of PBS control were injected, respectively. And detecting the expression quantity of miR-9875 in the blue crab blood lymphocytes by the method in reference example 1 48h after injection.
The result is shown in figure 2, and data show that the miR-9875 mimic injected can remarkably up-regulate the expression quantity of miR-9875 in the scylla paramamosain body, and can be used for subsequent functional tests.
Example 3:
test for promoting WSSV to replicate in scylla paramamosain by miR-9875
The Scylla paramamosain is randomly divided into 3 groups according to the method in example 1, the experimental group is injected with the mixed solution of WSSV and miR-9875, and the control group is injected with the mixed solution of WSSV and miR-9875-scrambled or PBS. Genomic DNA of leg muscle tissues of the Scylla paramamosain after injection for 0h, 24h and 48h is respectively extracted, and then virus copy number detection is carried out by qPCR technology, wherein the primers are P3 and P4.
P3:5’-CAAATCTCCCCTTCATCTACTCAAC-3’；
P4:5’-AATAATTTTCCCGTTTCTGAATAGA-3’。
The results are shown in figure 3, and the data show that after the scylla paramamosain is infected by WSSV, miR-9875 simulant can promote the replication of the virus in a host, so that in order to screen medicines for resisting the WSSV, the subsequent experiment uses the antisense nucleic acid AMO-miR-9875 of miR-9875 to carry out antiviral experiment.
Example 4:
test for inhibiting miR-9875 expression in scylla paramamosain body by AMO-miR-9875
Scylla paramamosains were randomly divided into 3 groups as in example 1, and 100. mu.L/individual of AMO-miR-9875 or AMO-miR-9875-scrambled or 100. mu.L/individual of PBS control were injected, respectively. And detecting the expression quantity of miR-9875 in the blue crab blood lymphocytes by the method in reference example 1 48h after injection.
The result is shown in fig. 4, and data show that the expression level of miR-9875 in the scylla paramamosain body is remarkably reduced after AMO-miR-9875 is injected compared with that of a control group, namely AMO-miR-9875 can remarkably inhibit the expression level of miR-9875 in the scylla paramamosain body and can be used for subsequent functional tests.
Example 5:
AMO-miR-9875 test for inhibiting WSSV replication in scylla paramamosain
The experimental group was injected with the mixture of WSSV and AMO-miR-9875, and the control group was injected with the mixture of WSSV and AMO-miR-9875-scrambled or PBS, as described in example 3. The virus copy number was determined as in example 3 after 0h, 24h and 48h of injection, respectively.
The result is shown in figure 5, and the result shows that AMO-miR-9875 can obviously inhibit the replication of virus in vivo and increase the antiviral capacity of Scylla paramamosain after the Scylla paramamosain is infected by WSSV.
Example 6:
AMO-miR-9875 test for enhancing activity of hemolymph cells in scylla paramamosain body
Dividing healthy Scylla paramamosain into 4 groups, injecting WSSV only in the first group, injecting WSSV and AMO-miR-9875 in the 2 nd group, injecting WSSV and AMO-miR-9875-scrambled in the 3 rd group, and not treating the 4 th group. Blue crab hemolymphocytes were collected 48h after injection, and the Viability of the hemolymphocytes was examined using the Cell viatility Assay Kit from Abnova.
The result is shown in figure 6, and data shows that the activity of blood lymphocytes in the scylla paramamosain body is sharply reduced after WSSV infection, the activity of the blood lymphocytes after virus infection can be obviously improved by injecting AMO-miR-9875, and the virus resistance of the scylla paramamosain is effectively enhanced.
Example 7:
AMO-miR-9875 test for promoting apoptosis of scylla paramamosain blood lymphocytes
Referring to example 5, the experimental group was injected with a mixture of WSSV and AMO-miR-9875, and the control group was injected with a mixture of WSSV and AMO-miR-9875-scrambled or PBS alone. Hemolymph cells from blue crabs were collected 48 hours after injection, and the collected cells were stained with FITC Annexin V Apoptosis Detection kit (BD Pharmingen TM), and Apoptosis of the cells was detected by flow cytometry.
The result is shown in fig. 7, and data show that compared with other control groups, the blue crab blood cell apoptosis rate of the AMO-miR-9875 treatment group is remarkably increased, which indicates that the action mechanism of the AMO-miR-9875 is to inhibit the replication of the WSSV in the scylla paramamosain body by inducing apoptosis of host cells.
Example 8:
AMO-miR-9875 test for inhibiting WSSV replication in Penaeus japonicus and Procambrus clarkii
Japanese Palaemon japonicus and Procambarus clarkii with the weight of 10-12g are temporarily kept in a laboratory for more than one week, and the injection method of example 5 is referred to, wherein the experimental group is injected with the mixed solution of WSSV and AMO-miR-9875, and the control group is injected with the mixed solution of WSSV and AMO-miR-9875-scrambled or PBS. The virus copy number was determined as in example 3 after 0h, 24h and 48h of injection, respectively.
The results are shown in fig. 8 and fig. 9, and indicate that AMO-miR-9875 can remarkably inhibit the replication of viruses in vivo and increase the antiviral capacity of the marsupenaeus japonicus and the procambarus clarkii after the marsupenaeus japonicus and the procambarus clarkii are infected by WSSV.
Example 9:
AMO-miR-9875 test for enhancing activity of hemolymph cells in marsupenaeus japonicus and procambarus clarkii
After a healthy marsupenaeus japonicus and procambarus clarkii having a weight of 10 to 12g were temporarily kept in an indoor culture pond for one week, the method according to example 6 was divided into 4 groups, the first group was injected with WSSV only, the 2 nd group was injected with WSSV and AMO-miR-9875, the 3 rd group was injected with WSSV and AMO-miR-9875-scrambled, and the 4 th group was not treated. Hemolymphocytes from Penaeus japonicus and Procambrus clarkii were collected 48 hours after injection, and the Viability of the collected cells was examined using the Cell viatility Assay Kit from Abnova.
The results are shown in FIG. 10, and data show that the activity of hemolymph cells in the marsupenaeus japonicus and the procambarus clarkii is sharply reduced after WSSV infection, the activity of the hemolymph cells after virus infection can be obviously improved by injecting AMO-miR-9875, and the virus resistance of the marsupenaeus japonicus and the procambarus clarkii is effectively enhanced.
SEQUENCE LISTING
<110> Shantou university
Application of <120> antisense nucleic acid AMO-miR-9875 in preparation of white spot syndrome virus resistant preparation
<130> 2019
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> RNA
<213> unknown
<400> 1
cuccucccuu ccucuucc 18
Claims (7)
1. A small RNA antisense nucleic acid AMO-miR-9875 for efficiently resisting WSSV is characterized in that the sequence is as follows: 5'-CUCCUCCCUUCCUCUUCC-3' are provided.
2. The application of the small RNA antisense nucleic acid AMO-miR-9875 with high WSSV resistance in preparing a broad-spectrum anti-white spot syndrome virus preparation according to claim 1.
3. The use according to claim 2, wherein the formulation comprises a feed additive, solid or liquid medicament comprising AMO-miR-9875.
4. Use according to claim 2, wherein the formulation is for crustaceans.
5. Use according to claim 4, wherein the crustaceans comprise marine cultured crabs, marine cultured shrimps and freshwater cultured shrimps.
6. The use according to claim 4, wherein the crustaceans include marsupenaeus japonicus, procambarus clarkii and scylla paramamosain.
7. A formulation comprising the small RNA antisense nucleic acid AMO-miR-9875 highly effective against WSSV according to claim 1.
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| CN1437654A (en) * | 2000-09-15 | 2003-08-20 | 阿克佐诺贝尔公司 | Antigenic proteins of shrimp white spot syndrome virus uses thereof |
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| CN1437654A (en) * | 2000-09-15 | 2003-08-20 | 阿克佐诺贝尔公司 | Antigenic proteins of shrimp white spot syndrome virus uses thereof |
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| Experimental infection of European crustaceans with white spot syndrome virus (WSSV);V Corbel等;《Journal of Fish Diseases》;20011231;第24卷;第377-382页 * |
| RNA干扰在对虾抗病毒免疫中的作用;黄添枝;《中国博士学位论文全文数据库基础科学辑》;20130115(第01期);A006-36 * |
| WSSV部分基因序列分析及防控WSS免疫增强剂的筛选;黄建华;《中国优秀硕士学位论文全文数据库农业科技辑》;20140415(第04期);D052-27 * |
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| 对虾白斑综合症病毒（WSSV）膜蛋白VP12和VP150的功能研究以及病毒极早期基因RNA干扰的初步研究;李建波;《中国博士学位论文全文数据库农业科技辑》;20140515(第05期);D052-8 * |
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