CN109022439B - Application of small interfering RNA molecule in resisting enlarged cell virus - Google Patents
Application of small interfering RNA molecule in resisting enlarged cell virus Download PDFInfo
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- CN109022439B CN109022439B CN201810934632.3A CN201810934632A CN109022439B CN 109022439 B CN109022439 B CN 109022439B CN 201810934632 A CN201810934632 A CN 201810934632A CN 109022439 B CN109022439 B CN 109022439B
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Abstract
The invention relates to the field of molecular biology, in particular to application of a small interfering RNA molecule in resisting megalocytic viruses (megalocyticus). The small interfering RNA molecule has the following sequence shown as the sequence. The small interfering RNA molecules of the invention are capable of specifically inhibiting the expression of proteasome assembly chaperonin 2(Psmg 2). The small interfering RNA molecule can obviously inhibit the replication of megacytoviruses (megacytoviruses) after being injected into fish bodies.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to application of a small interfering RNA molecule in resisting megalocytic viruses (megalocyticus).
Background
The small interfering RNA (siRNA) is short double-stranded RNA (ribonucleic acid) which is composed of a sense strand and an antisense strand and has the length of 21-23 nt, has a strong gene closing function, and can cause the phenomenon that homologous mRNA is degraded to cause gene expression silencing. RNA interference (RNAi) is a powerful gene expression inhibition pathway mediated by siRNA, is a protective measure for inhibiting the expression of exogenous genes in the evolution process of most eukaryotic cells such as plants, nematodes, fungi, insects, protists and animals, and has very important effects on the aspects of the generation, development and defense systems of organisms and the like. With the continuous and intensive research on siRNA-mediated RNA interference mechanism, the research on siRNA against virus infection has been advanced primarily, and a large number of studies indicate that siRNA is very effective in antiviral therapy. At present, the specific siRNA is synthesized directly to degrade the virus RNA or mRNA, and RNAi inhibits the expression of some genes of host cells involved in the virus replication process to play the antiviral role. RNAi technology has great potential in the prevention and treatment of virus infectious diseases, provides a new way for the prevention and treatment of clinical virus infectious diseases, and siRNA-mediated antiviral ways become hot spots of domestic and foreign research and have wide application prospects.
Disclosure of Invention
The invention aims to provide a small interfering RNA molecule and application thereof in resisting a swollen cell virus.
In order to achieve the purpose, the invention adopts the technical scheme that:
a small interfering RNA molecule having the following sequence:
5'-CCUCAUCGUGUCAACUCUUTT-3' as sense strand;
antisense strand 5'-AAGAGUUGACACGAUGAGGTT-3'.
The application of a small interfering RNA molecule in resisting a large cell virus.
The application of the small interfering RNA molecule in preparing a preparation for inhibiting the expression of a virus infection related gene Psmg2 or inhibiting the virus replication.
The virus is a swelling cell virus.
The invention has the following advantages: after the small interfering RNA molecule is injected into cynoglossus semilaevis, the expression of Psmg2 protein and the replication of the enlarged cell virus can be obviously inhibited.
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FIG. 1 shows that small interfering RNA molecule siPsmg2 provided by the embodiment of the present invention inhibits the expression of cynoglossus semilaevis enlarged cell virus infection-related gene Psmg2 (. about.P < 0.01).
Detailed Description
The present invention will be further described with reference to the following examples. The examples are intended to illustrate the invention, but not to limit it in any way.
Example 1
Small interfering RNA molecule siPsmg2 inhibits expression of virus infection related gene Psmg2
Step 1) preparation of small interfering RNA molecule diluent
The sequence of the small interfering RNA molecule siPsmg2 is a sense strand 5'-CCUCAUCGUGUCAACUCUUTT-3'; antisense strand 5'-AAGAGUUGACACGAUGAGGTT-3'. The small interfering RNA molecule siPsmg2 was synthesized by Shanghai Genephrma, with the siCR sequence of the control small interfering RNA molecule being sense strand 5'-UUCUCCGAACGUGUCACGUTT-3'; antisense strand 5'-ACGUGACACGUUCGGAGAATT-3'. SiPsmg2 and siCR were diluted to 1. mu.g/. mu.l in PBS, i.e., small interfering RNA molecule and control small interfering RNA dilutions, respectively.
The PBS comprises the following components in percentage by weight: 0.8% NaCl, 0.02% KCl, 0.358% Na2HPO4.12H2O,0.024％NaH2PO4And the balance being water.
Step 2) preparation of small interfering RNA molecule mixed solution and control mixed solution
In vivo transfection reagent EnTransterTMIn vivo transfection reagent is diluted 4 times by PBS (phosphate buffer solution), and the small interfering RNA molecule diluent in the step 1 and the diluted in vivo transfection reagent are mixed in equal volume to obtain small interfering RNA molecule mixed solution; and (3) mixing the control small interfering RNA molecule diluent obtained in the step (1) with the in vivo transfection reagent diluent in the same volume to obtain a control mixed solution.
Step 3) injecting fish
10 cynoglossus semilaevis (weighing about 11.8g) were randomly divided into 2 groups of 5. These 2 groups were designated as a and B, respectively. Injecting 100 mul of the small interfering RNA molecule mixed solution obtained in the step 3) into each fish of the group A, and injecting 100ul of the control mixed solution obtained in the step 3) into each fish of the group B (control group).
Step 5) Gene expression detection
Taking fish spleen tissue on the 3 rd day after the injection in the step 4). Total RNA was extracted using EZNA Total RNA kit (purchased from Omega Bio-tek, Doraville, USA) for cDNA preparation and quantitative fluorescent PCR (qRT-PCR) to detect expression of virus infection-associated gene Psmg 2. See in particular the literature Zheng WJ, Sun l.evaluation of housekeeping genes as reagents for quantitative time RT-PCR analysis of gene expression in Japanese floor (principles) Fish Shellfish immunol.2011; 30:638-45. The results showed that the expression level of Psmg2 gene was significantly lower in group a fish (P <0.01) than in the control group (see fig. 1). These results indicate that the small interfering RNA molecule siPsmg2 can significantly inhibit the expression of the virus infection-associated gene Psmg 2.
Example 2
Anti-swollen cell virus effect of small interfering RNA molecule siPsmg2
Step 1) preparation of small interfering RNA molecule mixed solution and control mixed solution
Mixing the small interfering RNA molecule diluent obtained in the step 1 of the example 1 and the in vivo transfection reagent diluted by 4 times in the step 3 of the example 1 in the same volume to obtain a small interfering RNA molecule mixed solution; the control small interfering RNA molecule diluent is mixed with the in vivo transfection reagent diluted 4 times in the step 3 of the above example 1 in the same volume, and the mixture is the control mixture.
Step 2) preparation of viral suspension
The tumor cell virus RBIV-C1 (see Zhang M, Xiao Z, Hu Y, Sun L. Characterisation of a megacytic virus from cultured rock virus, Oplegnathus fasciatus (Temminck)&Schlege), in china. aqua res.2012; 43: 556-64) in PBS to 106The copies/ml is the virus suspension.
Step 3) injecting fish
10 cynoglossus semilaevis (weighing about 11.8g) were randomly divided into 2 groups of 5. These 2 groups were designated as a and B, respectively. Injecting 100ul of the small interfering RNA molecule mixed solution obtained in the step 3) into each fish of the group A, injecting 100ul of the control mixed solution into each fish of the group B (control group), and injecting 100ul of the virus suspension obtained in the step 2) into each fish after 1 day.
Step 4) Virus replication detection
On day 3 after the virus injection in step 4 above, fish spleen tissue was taken. DNA was extracted from the tissues using a DNA extraction kit (purchased from Tiangen Biochemical technology, Beijing, Ltd.), and the virus content in the tissues was detected by an absolute quantitative PCR method. Specific methods are described in Zhang M, Xiao Z, Hu Y, Sun L. Characterisation of a megacytivirus from a pooled rock break, Oplegnathus fasciatus (Temminck)&Schlege), in china. aqua res.2012; 43:556-64. The results showed the number of viruses in spleen of group A fish (3.5X 10)3) Is significant (P)<0.01) lower virus count than spleen of group B fish (1.0X 10)4). These results indicate that the small interfering RNA molecule sipstm 2 is capable of significantly inhibiting viral replication.
Claims (1)
1. Use of a small interfering RNA molecule characterized by: the application of the small interfering RNA molecule in the preparation of a preparation for inhibiting the virus replication in the cynoglossus semilaevis;
the virus is a swelling cell virus RBIV-C1;
the small interfering RNA molecule has the following sequence:
5'-CCUCAUCGUGUCAACUCUUTT-3' as sense strand;
antisense strand 5'-AAGAGUUGACACGAUGAGGTT-3'.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101926990A (en) * | 2010-09-21 | 2010-12-29 | 中山大学 | Swelling cell virus vsocs gene-deleted attenuated live vaccine and preparation method and application thereof |
| CN103497243A (en) * | 2013-10-14 | 2014-01-08 | 中国科学院海洋研究所 | High-mobility fish group protein and application thereof |
| CN104623689A (en) * | 2015-01-09 | 2015-05-20 | 中国科学院海洋研究所 | Megalocytivirus DNA vaccine |
| CN105734060A (en) * | 2016-02-24 | 2016-07-06 | 中国科学院海洋研究所 | Small RNA molecule and application of small RNA molecule in antiviral |
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- 2018-08-16 CN CN201810934632.3A patent/CN109022439B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101926990A (en) * | 2010-09-21 | 2010-12-29 | 中山大学 | Swelling cell virus vsocs gene-deleted attenuated live vaccine and preparation method and application thereof |
| CN103497243A (en) * | 2013-10-14 | 2014-01-08 | 中国科学院海洋研究所 | High-mobility fish group protein and application thereof |
| CN104623689A (en) * | 2015-01-09 | 2015-05-20 | 中国科学院海洋研究所 | Megalocytivirus DNA vaccine |
| CN105734060A (en) * | 2016-02-24 | 2016-07-06 | 中国科学院海洋研究所 | Small RNA molecule and application of small RNA molecule in antiviral |
Non-Patent Citations (2)
| Title |
|---|
| Development of siRNA expression vector utilizing rock bream β-actin promoter: a potential therapeutic tool against viral infection in fish;Kosuke Zenke et al.;《APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY》;20090815;第679-690页 * |
| NCBI Reference Sequence: XM_008331510.3;GenBank;《GenBank》;20180516;序列部分 * |
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