CN110013485A - Antisense nucleic acid AMO-miR-9875 is preparing the application in white spot syndrome virus resisting preparation - Google Patents

Antisense nucleic acid AMO-miR-9875 is preparing the application in white spot syndrome virus resisting preparation Download PDF

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CN110013485A
CN110013485A CN201910175058.2A CN201910175058A CN110013485A CN 110013485 A CN110013485 A CN 110013485A CN 201910175058 A CN201910175058 A CN 201910175058A CN 110013485 A CN110013485 A CN 110013485A
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CN110013485B (en
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龚燚
李升康
孔彤彤
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Shantou University
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    • A—HUMAN NECESSITIES
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Abstract

The present invention relates to antisense nucleic acid AMO-miR-9875 to prepare the application in wide spectrum white spot syndrome virus resisting preparation, the sequence of the tiny RNA antisense nucleic acid AMO-miR-9875 are as follows: 5 '-CUCCUCCCUUCCUCUUCC-3 '.Tiny RNA antisense nucleic acid AMO-miR-9875 of the invention can significantly inhibit WSSV in Scylla paramamosain, Marsupenaeus japonicus and the intracorporal proliferation of Procambius clarkii, and WSSV is greatly reduced to seawater and the infection of freshwater aquiculture Shrimp waste and lethality.The present invention provides a kind of potential drug for white spot syndrome virus resisting in aquaculture, it efficiently can quickly inhibit the duplication of WSSV, it is applied widely, it is applicable not only to the Marsupenaeus japonicus and Scylla paramamosain of sea-farming, can equally be well applied to the Procambius clarkii of freshwater aquiculture.Tiny RNA antisense nucleic acid AMO-miR-9875 molecular mass of the invention is small degradable, will not destroy the ecological balance of sea farming, and will not cause harm to the human body, and is a kind of not only environmental protection but also effective virus defense substance.

Description

Antisense nucleic acid AMO-miR-9875 is in preparing white spot syndrome virus resisting preparation Using
Technical field
The present invention relates to the viral disease prevention and control field of aquaculture more particularly to a kind of tiny RNA antisense nucleic acid AMO- MiR-9875 is preparing the application in wide spectrum white spot syndrome virus resisting preparation.
Background technique
Scylla paramamosain, abbreviation mud crab have nutritive value high, and delicious meat, growth is fast, and adaptable advantage, is me The important economic cultivated crabs class of state.Marsupenaeus japonicus is commonly called as spending shrimp, spot section shrimp etc., and distributed pole is wide, and individual is big, delicious meat, first Shell is thick and hard, resistance to dry dew, resistance to arrest, and is conducive to transport for long-distance, has great economic value.Procambius clarkii also known as small dragon Shrimp is Freshwater shrimps, is distributed widely in Different Waters that the speed of growth is fast, and breeding potential is high, adaptable, is China's fresh water Valuable source in shrimps.
With the popularization of high-density breeding mode, illness outbreak is frequent in aquaculture process, including bacterium, fungoid disease Disease and viral disease, have seriously affected the sustainable development of culture fishery.Wherein, white spot syndrome virus (white Spotsyndrome virus, WSSV) have it is highly pathogenic, the lethality of Marsupenaeus japonicus and Procambius clarkii is up to 90% or more, the death of mud crab is also resulted in after accumulating in Scylla paramamosain body to certain amount.After WSSV enters in host, Proliferation, and route of transmission multiplicity can be replicated rapidly, be one of the main pathogens of China's culture fishery viral disease outburst.
Scylla paramamosain, Marsupenaeus japonicus and Procambius clarkii belong to invertebrate, rely primarily on congenital immunity system It unites to resist the invasion of pathogenic microorganism.For a long time, it is mainly prevented and treated using antibiotic etc in aquaculture process feeding The disease broken out during growing.Largely using so that the drug resistance of pathogenic microorganism enhances for antibiotic, makes the prevention and treatment of disease more Add difficulty.In addition to this, the use of antibiotic can cause serious influence to the balance of breeding ecological environment, be unfavorable for aquatic products and support Grow the sustainable development of industry.Therefore, it finds not only environmental protection and is conducive to the sustainable development of culture fishery, but also disease can be effectively prevented Pharmaceutical methods it is particularly significant.
The drug invention of current anti-WSSV mainly includes the drug synthesized by multiple material, as prevented and treated in prawn white spot disease Western compound medicine (patent No.: 200810152729.5), the raw material of the Chinese and Western compound medicine include: the coptis, radix scutellariae, Cortex Phellodendri, Cape jasmine, astragalus polyose, Glucurolactone, vitamin C, mixing mix thoroughly into it is powdered after be added in feed to prawn feeding.Its institute The raw material types needed are various, and the material molecule quality for being included is relatively large, and degradation complexity is unknown, if will affect feeding It is also unknown to grow the ecological balance.Therefore, finding method is simple, and the control method of high-efficiency environment friendly is particularly important.
MiRNA is a kind of non-coding RNA being made of 18-22 nucleotide, by the 3 ' non-codings for acting on target gene Area (3 ' UTR) and then the expression for inhibiting target gene, play the function of post-transcriptional control.It has recently been demonstrated that miRNA is in place Crucial effect is played in the main Interaction with virus, miRNA has good as the drug candidate of antiviral therapy Application prospect.
Summary of the invention
The purpose of the present invention is to provide a kind of tiny RNA antisense nucleic acid AMO-miR-9875 to prepare the anti-hickie synthesis of wide spectrum Application in syndrome virus preparation, it is green in quasi- cave that tiny RNA antisense nucleic acid AMO-miR-9875 of the invention can significantly inhibit WSSV Crab, Marsupenaeus japonicus and the intracorporal proliferation of Procambius clarkii efficiently solve WSSV and infect seawater and freshwater aquiculture Shrimp waste And the problems such as lethal phenomenon.
A kind of efficient tiny RNA antisense nucleic acid AMO-miR-9875 for resisting WSSV, sequence are as follows: 5 '- CUCCUCCCUUCCUCUUCC-3’。
The above-mentioned efficient tiny RNA antisense nucleic acid AMO-miR-9875 for resisting WSSV is preparing wide spectrum white spot syndrome virus resisting Application in preparation.
Further, the wide spectrum white spot syndrome virus resisting preparations include the AMO-miR- of antisense nucleic acid containing tiny RNA 9875 feed addictive, the AMO-miR-9875 solid of antisense nucleic acid containing tiny RNA or liquid medicine.
Further, the wide spectrum white spot syndrome virus resisting preparation can be used for shellfish.
Further, the shellfish includes sea-farming crab class, sea-farming shrimps and freshwater aquiculture shrimps.
Further, the shellfish includes japonicus, Procambius clarkii and Scylla paramamosain.
A kind of preparation comprising the above-mentioned efficient tiny RNA antisense nucleic acid AMO-miR-9875 for resisting WSSV.
Compared with prior art, present invention has the advantage that
(1) tiny RNA antisense nucleic acid AMO-miR-9875 of the invention is quick, inhibits high-efficient to WSSV, is not only applicable in In prevention, the also applicable breeding process in the shrimp crab for having broken out white spot syndrome.
(2) tiny RNA antisense nucleic acid AMO-miR-9875 molecular weight of the invention is minimum, and safety is degradable, will not destroy sea The ecological balance during water and freshwater aquiculture is conducive to the sustainable development of culture fishery.
(3) present invention is applied widely, is applicable not only to the Scylla paramamosain and Marsupenaeus japonicus of sea-farming, is also applied for The Procambius clarkii of freshwater aquiculture is the drug of anti-WSSV of potential wide spectrum a kind of.
(4) white spot syndrome virus resisting preparation of the invention can be only mono- comprising tiny RNA antisense nucleic acid AMO-miR-9875 One chemicals, the present invention are easier to be applied to actual production.
Detailed description of the invention
The expression quantity that Fig. 1 is miR-9875 in Scylla paramamosain blood lymphocyte after WSSV is handled.
Fig. 2 is the expression quantity of miR-9875 in Scylla paramamosain body with PBS, miR-9875 analogies and miR-9875- The situation of change of scrambled (sequence that miR-9875 analogies are upset) processing.
Fig. 3 is the change that WSSV copy number is handled with miR-9875, miR-9875-scrambled and PBS in Scylla paramamosain body Change situation；Above-mentioned processing carried out to the metainfective Scylla paramamosain of WSSV, detection WSSV infection 0h, for 24 hours with Scylla paramamosain body after 48h The copy number of interior WSSV.
Fig. 4 is the expression quantity of miR-9875 in Scylla paramamosain body with PBS, AMO-miR-9875 and AMO-miR-9875- The situation of change of scrambled (sequence that AMO-miR-9875 upsets) processing.
Fig. 5 is WSSV copy number in Scylla paramamosain body with AMO-miR-9875, AMO-miR-9875-scrambled and PBS The situation of change of processing；Above-mentioned processing carried out to the metainfective Scylla paramamosain of WSSV, detection WSSV infection 0h, is intended for 24 hours with after 48h The copy number of WSSV in the mud crab body of cave.
Fig. 6 is influence of the AMO-miR-9875 to blood lymphocyte vigor in Scylla paramamosain body；After WSSV infection, experimental group AMO-miR-9875 is injected, control group injects AMO-miR-9875-scrambled, and detection Scylla paramamosain is intracorporal after handling 48h Blood lymphocyte vigor.
Fig. 7 is the influence of AMO-miR-9875 Scylla paramamosain blood lymphocyte apoptosis, by AMO-miR-9875 or AMO- It is injected in mud crab body after miR-9875-scrambled mixing WSSV, control group only injects PBS, uses flow cytomery The apoptosis of Scylla paramamosain blood lymphocyte after WSSV infects 48 hours.
Fig. 8 be in Marsupenaeus japonicus body WSSV copy number with AMO-miR-9875, AMO-miR-9875-scrambled and The situation of change of PBS processing；Above-mentioned processing carried out to the metainfective Marsupenaeus japonicus of WSSV, detection WSSV infection 0h, for 24 hours and After 48h in Marsupenaeus japonicus body WSSV copy number.
Fig. 9 be in Procambius clarkii body WSSV copy number with AMO-miR-9875, AMO-miR-9875-scrambled and The situation of change of PBS processing；Above-mentioned processing carried out to the metainfective Procambius clarkii of WSSV, detection WSSV infection 0h, for 24 hours and After 48h in Procambius clarkii body WSSV copy number.
Figure 10 is influence of the AMO-miR-9875 to blood lymphocyte vigor in Marsupenaeus japonicus and Procambius clarkii body； After WSSV infection, experimental group injects AMO-miR-9875, and control group injects AMO-miR-9875-scrambled, after handling 48h Detect Marsupenaeus japonicus and the intracorporal blood lymphocyte vigor of Procambius clarkii.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, the present invention is made into one below in conjunction with attached drawing Step ground detailed description.
Embodiment 1:
The detection of miR-9875 expression quantity is tested in Scylla paramamosain body after WSSV infection
By weight 30-40g, it is negative Scylla paramamosain indoors in seawater water tank in 10 ‰ salt that qRT-PCR, which detects WSSV, Degree is raised and train under 25 DEG C of water temperature conditions after a week, in injecting 100 μ L/ on the 4th appendage only, 106The WSSV of copy number infects liquid.Point 0h not after injection, for 24 hours and 48h collects mud crab blood lymphocyte, carries out cDNA reverse transcription after extracting total serum IgE, used draws Object are as follows: 5-GTCGTATCCAGTGCAGGGTCCGAGGTCACTGGATACGACCTCCTCCC-3 ' is finally examined using qPCR technology MiR-9875 expression quantity is surveyed, the primer is P1 and P2.
P1:5'-CGCCGGGAAGAGGAAGGG-3'；
P2:5’-TGCAGGGTCCGAGGTCACTG-3’。
The result is shown in Figure 1, data show that Scylla paramamosain is infected for 24 hours and after 48h miR- in its blood lymphocyte by WSSV 9875 expression quantity significantly reduces, and shows that miR-9875 may take part in the Interaction between host's virus, therefore, miR-9875 It is the novel targets of potential WSSV prevention and treatment.
Embodiment 2:
The overexpression test of miR-9875 in Scylla paramamosain body
Scylla paramamosain is divided into 3 groups at random as described in Example 1, injects the miR-9875 analogies of 100 μ L/ only respectively Or the PBS control of miR-9875-scrambled or 100 μ L/ only.Method after injection 48h referring to embodiment 1 detects mud crab blood The expression quantity of miR-9875 in lymphocyte.
As a result see that Fig. 2, data display injection miR-9875 analogies can significantly raise miR-9875 in Scylla paramamosain body Expression quantity, can be used for follow-up function test.
Embodiment 3:
The test that miR-9875 promotes WSSV to replicate in Scylla paramamosain body
Scylla paramamosain is randomly divided into 3 groups by method in embodiment 1, experimental group injects the mixing of WSSV and miR-9875 Liquid, control group inject the mixed liquor of WSSV and miR-9875-scrambled or PBS.Injection 0h is extracted respectively, for 24 hours and after 48h Then the genomic DNA of Scylla paramamosain leg muscle tissue carries out the detection of viral copy number, the primer by qPCR technology For P3 and P4.
P3:5'-CAAATCTCCCCTTCATCTACTCAAC-3'；
P4:5’-AATAATTTTCCCGTTTCTGAATAGA-3’。
As a result after seeing that Fig. 3, data show that Scylla paramamosain is infected by WSSV, injection miR-9875 analogies can promote virus In the intracorporal duplication of host, therefore, for the drug for screening anti-WSSV, follow-up test uses the antisense nucleic acid AMO- of miR-9875 MiR-9875 carries out antivirus test.
Embodiment 4:
AMO-miR-9875 inhibits the test of miR-9875 expression in Scylla paramamosain body
Scylla paramamosain is divided into 3 groups at random as described in Example 1, inject respectively 100 μ L/ AMO-miR-9875 only or The PBS control of AMO-miR-9875-scrambled or 100 μ L/ only.Method after injection 48h referring to embodiment 1 detects mud crab The expression quantity of miR-9875 in blood lymphocyte.
As a result see Fig. 4, the expression quantity of miR-9875 is compared in Scylla paramamosain body after data display injection AMO-miR-9875 It is significantly reduced in control group, i.e. AMO-miR-9875 can significantly inhibit the expression quantity of miR-9875 in mud crab body, after can be used for Continuous function test.
Embodiment 5:
AMO-miR-9875 inhibits the test of WSSV duplication in Scylla paramamosain body
As described in Example 3, experimental group injection WSSV and AMO-miR-9875 mixed liquor, control group inject WSSV with The mixed liquor of AMO-miR-9875-scrambled or PBS.It is examined as described in Example 3 respectively at injection 0h, for 24 hours with after 48h Survey viral copy number.
As a result see Fig. 5, the results showed that after Scylla paramamosain is infected by WSSV, injection AMO-miR-9875 can significantly inhibit disease The duplication of poison in vivo, increases the anti-virus ability of Scylla paramamosain.
Embodiment 6:
AMO-miR-9875 enhances the test of blood lymphocyte vigor in Scylla paramamosain body
The Scylla paramamosain of health is divided into 4 groups, first group is only injected WSSV, the 2nd group of injection WSSV and AMO-miR-9875, 3rd group of injection WSSV and AMO-miR-9875-scrambled, the 4th group is not handled.Collection mud crab hemolymph is thin after injecting 48h Born of the same parents, and utilize the vigor of the Cell Viability Assay Kit kit of Abnova company detection blood lymphocyte.
As a result see Fig. 6, blood lymphocyte vigor drastically reduces in Scylla paramamosain body after data display WSSV infection, injects AMO-miR-9875 can significantly improve the vigor of blood lymphocyte after virus infection, effectively enhance Scylla paramamosain to virus Resistivity.
Embodiment 7:
The test of AMO-miR-9875 promotion Scylla paramamosain blood lymphocyte apoptosis
Referring to embodiment 5, experimental group injects the mixed liquor of WSSV and AMO-miR-9875, and control group injects WSSV and AMO- The mixed liquor of miR-9875-scrambled only injects PBS.The blood lymphocyte that mud crab is collected after injection 48h, uses FITC Annexin V Apoptosis Detection kit (BD Pharmingen TM) dyes the cell of collection, and Pass through the apoptosis situation of flow cytomery cell.
As a result see Fig. 7, data are shown compared to other control groups, the mud crab blood cell apoptosis of AMO-miR-9875 processing group Rate significantly increases, and shows that the mechanism of action of AMO-miR-9875 is apoptosis to occur by inducing host cell WSSV is inhibited to exist The intracorporal duplication of Scylla paramamosain.
Embodiment 8:
AMO-miR-9875 inhibits the test of WSSV duplication in Marsupenaeus japonicus and Procambius clarkii body
Weight is taken temporarily to support one week or more for the Marsupenaeus japonicus of 10-12g and Procambius clarkii in laboratory, referring to embodiment 5 injecting method, experimental group inject the mixed liquor of WSSV and AMO-miR-9875, and control group injects WSSV and AMO-miR- The mixed liquor of 9875-scrambled or PBS.Respectively at injection 0h, for 24 hours with virus detected after 48h as described in Example 3 copy Shellfish number.
As a result see Fig. 8 and Fig. 9, the results showed that after Marsupenaeus japonicus and Procambius clarkii are infected by WSSV, inject AMO- MiR-9875 can significantly inhibit the duplication of virus in vivo, increase the anti-virus ability of Marsupenaeus japonicus and Procambius clarkii.
Embodiment 9:
AMO-miR-9875 enhances the test of blood lymphocyte vigor in Marsupenaeus japonicus and Procambius clarkii body
By the healthy Marsupenaeus japonicus of weight 10-12g and Procambius clarkii, aquaculture pond is temporarily supported after a week, referring to real indoors The method for applying example 6 is divided into 4 groups, and first group is only injected WSSV, the 2nd group of injection WSSV and AMO-miR-9875, the 3rd group of injection WSSV And AMO-miR-9875-scrambled, the 4th group is not handled.The blood of Marsupenaeus japonicus and Procambius clarkii is collected after injection 48h Lymphocyte, and utilize the vigor of the Cell Viability Assay Kit kit of Abnova company detection collection cell.
The result is shown in Figure 10, data show after WSSV infection blood lymphocyte vigor in Marsupenaeus japonicus and Procambius clarkii body It drastically reduces, injection AMO-miR-9875 can significantly improve the vigor of blood lymphocyte after virus infection, effectively enhance day This capsule prawn and Procambius clarkii are to viral resistivity.
SEQUENCE LISTING
<110>University Of Shantou
<120>antisense nucleic acid AMO-miR-9875 is preparing the application in white spot syndrome virus resisting 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 kind of tiny RNA antisense nucleic acid AMO-miR-9875 for efficiently resisting WSSV, which is characterized in that sequence are as follows: 5 '- CUCCUCCCUUCCUCUUCC-3’。
2. according to claim 1 efficiently resist WSSV tiny RNA antisense nucleic acid AMO-miR-9875 prepare wide spectrum resist it is white Application in spot syndrome virus preparation.
3. application according to claim 2, which is characterized in that the preparation includes the feed addition containing AMO-miR-9875 Agent, solid or liquid medicine.
4. application according to claim 2, which is characterized in that the preparation can be used for shellfish.
5. application according to claim 4, which is characterized in that the shellfish includes sea-farming crab class, seawater Shrimps in culture and freshwater aquiculture shrimps.
6. application according to claim 4, which is characterized in that the shellfish includes Marsupenaeus japonicus, kirschner original Crayfish and Scylla paramamosain.
7. a kind of preparation comprising efficiently resisting the tiny RNA antisense nucleic acid AMO-miR-9875 of WSSV according to claim 1.
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CN112342212A (en) * 2020-09-25 2021-02-09 浙江理工大学 AMO-miRNA for resisting WSSV infection of penaeus japonicus
CN112342212B (en) * 2020-09-25 2022-03-25 浙江理工大学 AMO-miRNA for resisting WSSV infection of penaeus japonicus

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