A recombinant bacteria for interfering with wsv108 gene expression and use thereof

Abstract

The application discloses a recombinant bacterium for interfering with wsv108 gene expression and application thereof. The application takes wsv108 gene in WSSV as a target gene, provides dsRNA capable of inhibiting wsv108 gene expression, namely wsv108-dsRNA, the dsRNA can effectively inhibit replication of WSSV and reduce mortality of shrimps after the shrimps are infected by WSSV. On this basis, the application further provides a recombinant bacterium capable of producing wsv108-dsRNA and used for interfering with wsv108 gene expression, the recombinant bacterium can produce wsv108-dsRNA after fermentation culture, and after the wsv108-dsRNA is mixed with shrimp feed and fed to shrimps, white spot syndrome of the shrimps can be effectively prevented and treated. The wsv108-dsRNA and the recombinant bacterium are harmless to shrimps, have high specificity and high biological safety, and are favorable for prevention and treatment of white spot syndrome of shrimps.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology. More particularly, it relates to a recombinant bacteria for interfering with the expression of wsv108 gene and its application. BACKGROUND

[0002] Litopenaeus vannamei is an important marine aquaculture species in China, and its annual output accounts for about 80% of the total output of shrimp aquaculture in China. In recent years, due to the frequent occurrence of infectious diseases in shrimp, it has caused huge economic losses to the shrimp aquaculture industry. White spot syndrome (WSS) in shrimp is caused by white spot syndrome virus (WSSV) infection, which is the most serious infectious disease to the shrimp aquaculture industry. The typical pathological features of WSS are white spots on the shell of shrimp, which can be accompanied by body redness, loss of appetite, slow movement, blood lymph dilution, hepatopancreas swelling and shell easy to peel, etc. The mortality rate is extremely high. At present, there is no drug or method that can effectively prevent and control WSS.

[0003] After the virus enters the host cell, it completes a virus replication cycle through genome replication and virus protein synthesis to release the progeny virus. It is a promising prevention and control strategy to intervene in the replication of the virus to inhibit the infection of the virus. Existing studies have found that the monoclonal antibody against the envelope protein (VP28 protein) of WSSV prepared by immunizing mice can neutralize WSSV in shrimp, inhibit the replication of WSSV, and delay the death of shrimp infected with WSSV. Although the development of monoclonal antibodies against WSSV proteins can prevent and control WSS, the price of monoclonal antibodies is high. Therefore, other products and methods that can prevent and control WSS by inhibiting protein expression need to be developed.

[0004] RNA interference (RNAi) technology refers to a technology for inhibiting expression of a homologous gene in vivo by using double-stranded RNA (dsRNA) of an exogenous or endogenous source. There are two ways in the process of RNAi technology; one is to use long-chain dsRNA, and the other is to use small interference siRNA (small interference RNA, siRNA). The synthesis of dsRNA or siRNA has two methods of chemical synthesis and biological synthesis; among them, the chemical synthesis method has high cost and low efficiency, and is difficult to popularize on a large scale. The biological synthesis method is divided into eukaryotic cell synthesis and prokaryotic cell synthesis, and both methods are to express the required infectious RNA in the host through the dsRNA or siRNA expression vector. However, due to technical limitations, at present, only specific interference RNA expressed in bacteria (prokaryotic cells) is used in insects, and it has not been effectively used in other organisms. SUMMARY

[0005] The present application aims at the deficiencies of the prior art, and provides a dsRNA capable of inhibiting the expression of wsv108 gene in WSSV, i.e. wsv108-dsRNA, which can effectively inhibit the replication of WSSV and reduce the mortality of prawns after WSSV infection. On this basis, the present application further provides a recombinant bacteria for interfering with the expression of wsv108 gene, which can efficiently express wsv108-dsRNA and is harmless to prawns, and is beneficial to the prevention and treatment of white spot syndrome of prawns.

[0006] The first object of the present application is to provide the use of a preparation for inhibiting the expression of wsv108 gene of white spot syndrome virus in the preparation of a product for preventing and / or treating white spot syndrome of prawns.

[0007] The second object of the present application is to provide the use of a preparation for inhibiting the expression of wsv108 gene of white spot syndrome virus in the preparation of a product for inhibiting white spot syndrome virus.

[0008] The third object of the present application is to provide a dsRNA for interfering with the expression of wsv108 gene.

[0009] The fourth object of the present application is to provide a recombinant plasmid for expressing a dsRNA targeting wsv108 gene.

[0010] The fifth object of the present application is to provide a recombinant bacteria for interfering with the expression of wsv108 gene.

[0011] The sixth object of the present application is to provide the use of the dsRNA, the recombinant plasmid or the recombinant bacteria in the preparation of a product for preventing and / or treating white spot syndrome of prawns.

[0012] A seventh object of the present application is to provide the use of the dsRNA, the recombinant plasmid or the recombinant bacteria in the preparation of a product for inhibiting white spot syndrome virus.

[0013] The above objects of the present application are achieved by the following technical solutions.

[0014] The present application finds that the dsRNA specifically targeting wsv108 can knock down the expression of wsv108 by in vitro synthesis, which can effectively reduce the replication of WSSV in prawns, reduce the level of apoptosis of blood cells caused by WSSV, and reduce the mortality of prawns infected with WSSV. In view of the high cost of in vitro synthesis of dsRNA, it needs to be injected into the prawn body when used, which is time-consuming, laborious and low in efficiency. Therefore, the present application uses the mRNA of the functional region of wsv108 as a dsRNA synthesis template to construct a recombinant plasmid recombinant bacteria capable of expressing dsRNA specifically interfering with wsv108, and finds that the recombinant bacteria can knock down the expression of wsv108, reduce the replication of WSSV in prawns, and reduce the mortality of prawns infected with WSSV, which is beneficial to the prevention and treatment of white spot syndrome of prawns.

[0015] The present application claims the use of a preparation for inhibiting the expression of white spot syndrome virus wsv108 gene in the preparation of a product for preventing and / or treating white spot syndrome of prawns.

[0016] The present application also claims the use of a preparation for inhibiting the expression of white spot syndrome virus wsv108 gene in the preparation of a product for inhibiting white spot syndrome virus.

[0017] Specifically, the amino acid sequence of the wsv108 protein encoded by the wsv108 gene is shown as SEQ ID NO. 1.

[0018] The nucleotide sequence of the ORF region of the wsv108 gene is shown as SEQ ID NO. 2.

[0019] As an optional implementation manner, the preparation is an RNAi fragment for interfering with the expression of the wsv108 gene.

[0020] Alternatively, the RNAi fragment is a dsRNA targeting the wsv108 gene.

[0021] The present application also provides a dsRNA for interfering with the expression of the wsv108 gene, which is prepared based on the nucleotide sequence shown in SEQ ID NO. 3.

[0022] Specifically, the dsRNA is obtained by reverse transcription based on the nucleotide sequence shown in SEQ ID NO. 3.

[0023] Optionally, the dsRNA is based on the nucleotide sequence shown in SEQ ID NO. 3, using T7 RiboMAX TM The express RNAi system is generated by in vitro transcription.

[0024] The present application also provides a recombinant plasmid for expressing the dsRNA targeting the wsv108 gene, wherein the recombinant plasmid contains the nucleotide sequence shown in SEQ ID NO. 3.

[0025] Optionally, the plasmid used for constructing the recombinant plasmid is L4440.

[0026] The present application also provides a recombinant bacteria for interfering the expression of the wsv108 gene, wherein the recombinant bacteria contains the recombinant plasmid described above.

[0027] The present application also claims the use of the dsRNA, the recombinant plasmid or the recombinant bacteria in preparing a product for preventing and / or treating the white spot syndrome of prawn.

[0028] The present application also claims the use of the dsRNA, the recombinant plasmid or the recombinant bacteria in preparing a product for inhibiting the white spot syndrome virus.

[0029] The present application also provides a prawn feed containing the recombinant bacteria described above.

[0030] Specifically, the method for preparing the prawn feed comprises the following steps:

[0031] S1. mixing the recombinant bacteria and the prawn feed in a ratio of 50g: 0.8-1.2kg;

[0032] S2. adding sodium alginate powder to the mixture obtained in step S1 in a ratio of 20g: 0.8-1.2kg and mixing uniformly;

[0033] S3. adding 0.04 mol / L CaCl2 solution to the mixture obtained in step S2 in a ratio of 200mL: 0.8-1.2kg, and mixing uniformly until the sodium alginate forms a gel coating on the surface of the feed.

[0034] Preferably, the ratio of the recombinant bacteria and the prawn feed is 50g: 1kg.

[0035] Preferably, the ratio of the mixture obtained in step S1 and the sodium alginate powder is 20g: 1kg.

[0036] Preferably, the ratio of the CaCl2 solution and the mixture obtained in step S2 is 200mL: 1kg.

[0037] Specifically, the shrimp feed is Penaeus vannamei feed, which is purchased from Haide Group.

[0038] The present application has the following advantages:

[0039] The present application provides a dsRNA (wsv108-dsRNA) targeting wsv108 gene in WSSV and inhibiting the expression of the gene, and the replication of WSSV can be effectively inhibited by using the wsv108-dsRNA, and the mortality of shrimp infected by WSSV can be reduced. In order to overcome the problem of high cost and low efficiency of synthesizing dsRNA by chemical synthesis, the present application further provides a recombinant bacteria capable of expressing wsv108-dsRNA and interfering with the expression of wsv108 gene, a large amount of the recombinant bacteria can be obtained by fermentation culture, and the wsv108-dsRNA can be efficiently produced by induction culture, which has the advantages of simple culture, low cost of dsRNA production, and easy large-scale use in factory farming system. After the recombinant bacteria after fermentation induction culture are made into WSSV inhibitor and mixed with shrimp feed, the shrimp is fed, and the wsv108-dsRNA in the WSSV inhibitor will also be absorbed by the intestinal tract with the digestion of the feed, the wsv108-dsRNA in the intestinal tract will function in the intestinal tract, and will be transported to other tissues and organs through the open circulation system of the shrimp, and finally cause systemic RNA interference, when the wsv108 gene in the shrimp is interfered, the replication of WSSV virus is blocked, thereby reducing the death of the shrimp infected by WSSV, and achieving the prevention and treatment of white spot syndrome of shrimp.

[0040] The wsv108-dsRNA and the recombinant bacteria expressing the wsv108-dsRNA are harmless to the shrimp, and since the wsv108-dsRNA is derived from WSSV, no DNA sequence homologous to the wsv108-dsRNA has been found in other organisms at present, according to the principle of RNA interference, the dsRNA of wsv108 will not interfere with the non-target genes of other organisms, and has high specificity. Escherichia coli also widely exists in the intestinal tracts of various organisms, and is also a component bacterium in the intestinal tract of shrimp, and therefore, feeding the Escherichia coli will not adversely affect the function of the intestinal tract of the shrimp, that is, the WSSV inhibitor has high biological safety, and can be used for preventing and treating white spot syndrome of shrimp. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1Effect of targeted inhibition of wsv108 gene of WSSV on mortality of prawns infected with WSSV; A in the figure is the knockdown efficiency of dsRNA on wsv108; B in the figure is the change in copy number of WSSV after wsv108 is knocked down; C in the figure is the mortality of prawns infected with WSSV after wsv108 is knocked down; D in the figure is the change in mRNA level of a key gene (wsv249) of WSSV after wsv108 is knocked down; E in the figure is the change in mRNA level of a key gene (VP28) of WSSV after wsv108 is knocked down.

[0042] Figure 2 Effect of targeted inhibition of wsv108 gene of WSSV on apoptosis rate of prawns infected with WSSV and expression amount of apoptosis-related genes of prawns; A and B in the figure are the effect of WSSV infection on apoptosis rate of prawn blood cells after wsv108 is knocked down, wherein B is the corresponding statistical result; C in the figure is the effect of WSSV infection on expression amount of apoptosis-related genes of prawns after wsv108 is knocked down.

[0043] Figure 3 Effect of IPTG induction at different concentrations on expression of wsv108-dsRNA.

[0044] Figure 4 Effect of feeding WSSV inhibitor (wsv108-dsRNA expression strain) on anti-WSSV infection ability of prawns; A in the figure is a flow chart of construction principle of wsv108-dsRNA expression strain; B in the figure is detection result of wsv108-dsRNA expression of wsv108-dsRNA expression strain; C in the figure is mortality of prawns infected with WSSV after feeding wsv108-dsRNA expression strain; D and E in the figure are, in sequence, relative expression amount of wsv108 in muscle and gill tissues of prawns after feeding wsv108-dsRNA expression strain; F and G in the figure are, in sequence, relative expression amount of mRNA of wsv108 in intestinal and stomach tissues of prawns after feeding wsv108-dsRNA expression strain; H-K in the figure are, in sequence, copy amount of WSSV in different tissues of prawns infected with WSSV after feeding wsv108-dsRNA expression strain; L-O in the figure are, in sequence, expression of apoptosis-related genes ATF, Bax, Bcl2 and Caspase3 in stomach of prawns infected with WSSV after feeding wsv108-dsRNA expression strain.

[0045] *P<0.05; **P<0.01; ns represents no significant difference; single factor analysis of variance is used, different letters represent significant difference, P<0.05, and same letters represent no significant difference. DETAILED DESCRIPTION

[0046] The present application will be further described by the following description of the drawings and specific examples, which are not intended to limit the present application in any way. Unless otherwise defined, the reagents, methods and apparatuses used in the present application are those conventional in the art.

[0047] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0048] Example 1 Effect of Targeted Inhibition of wsv108 Gene on WSSV Replication and WSSV-Infected Prawn

[0049] The present application uses wsv108 gene of WSSV as the target gene, and uses T7 RiboMAX TM Express RNAi (Promega, USA) system to generate double-stranded RNA (dsRNA) specific to wsv108 by in vitro transcription, which is named as dsRNA-wsv108. After injecting the dsRNA specific to wsv108 into prawns, it is found that it can effectively reduce the replication of WSSV in prawns, reduce the level of apoptosis of blood cells caused by WSSV and reduce the mortality of prawns infected with WSSV. The amino acid sequence of the wsv108 protein, the nucleotide sequence of the ORF region of the gene encoding the wsv108 protein and the test process are as follows:

[0050] The amino acid sequence of the wsv108 protein is as shown below (SEQ ID NO. 1):

[0051] MSHINSTSAATTSSNTLPICTTTAPMIAAARAAAIASRTSASAVTSINSNSTSSSAMFRVPQGISVTAMPPVPALTSLTESTGTRMSSTPNVDVIPVPGPKNKSKSKKKDSKRKKNQNGNRSSDEDEPSLVIDDGSGRQSKNKKYSWVTSLATTTAERNNDTLAPPRPFLPTPEEGNMSEIDAGLSNPVTRQITGEVYSAALTSGVGDNGLYPSHFTVADTSYGDCETPIPGPAFVLDDGTVSRGTSLLHREEAEFLNDGSKVIHTVKPRNSKYSNIQRAASCMAYAVDLLNNHNITSDQFDFMAMTAWAARQRCGEMAKFFEKRDKDIGEYRNKVVQYNRGIFTRTTEMNKRAKIILEQQQRREAAAAAAATGATAPIPTTSAAGVGATSSATTNSLEYQEIRYQ

[0052] The nucleotide sequence of the ORF region of the gene encoding the wsv108 protein is shown below (SEQ ID NO. 2):

[0053]

[0054] Specifically, the dsRNA targeting the wsv108 gene (wsv108-dsRNA) is reverse transcribed based on the nucleotide sequence shown as follows (SEQ ID NO. 3).

[0055] AGTGACGAGGACGAACCATCTCTTGTTATCGACGAGTTCTGGAAGACAGTCTAAGAACAAGAAATATTCTTGGGTCACATCTCTTGCTACTACTACGGCTGAAAGAAACAACGACACTCTCGCCCCACCTAGGCCCTTCCTTCCCACACCCGAAGAAGGAAATATGTCTGAAATTGACGCAGGGCTAAGTAATCCAGTCACTCGCCAAATCACCGGAGAAGTTTATAGCGCTGCACTCACTTCTGGAGTTGGAGATAATGGACTATATCCTTCCCACTTCACGGTTGCTGACACTTCTTACGGAGATTGCGAAACACCCATACCTGGACCTGCTTTTGTCCTCGACGACGGGACAGTTAGCAGAGGCACATCTCTTCTGCACAGAGAAGAGGCAGAATTCTTGAATGATGGAAGTAAGGTGATCCATACCGTTAAACCAAGAAACAGCAAGTACTCCAATATTCAACGTGCCGCTAGCTGTATGGCCTACGCTGTGGACCTTCTAAACA

[0056] The present application uses the obtained dsRNA-wsv108 to test its influence on the replication of WSSV and the like, and the specific process is as follows:

[0057] Based on the nucleotide sequence shown as SEQ ID NO. 3, T7 RiboMAX TMExpress RNAi (Promega, USA) system, dsRNA specific to wsv108 (dsRNA-wsv108) and green fluorescent protein (dsRNA-GFP as control) were generated by in vitro transcription. The shrimp were divided into experimental and control groups (n = 25 per group), and the experimental group was injected with PBS buffer containing dsRNA-wsv108 to silence wsv108 with dsRNA-wsv108, 50 μL of PBS buffer containing 10 μg of dsRNA-wsv108 was injected into each shrimp; the control group was injected with the same amount of PBS buffer containing 10 μg of dsRNA-GFP. After silencing wsv108 with dsRNA-wsv108, the blood cells were collected by centrifugation at 1000 x g for 3 min at 4°C, washed twice with PBS, and the apoptotic cells were detected using Annexin V-fluorescein isothiocyanate (FITC) / propidium iodide (PI) apoptosis detection kit (Sigma-Aldrich), and the percentage of apoptotic cells was determined using BD C6 software.

[0058] At 24 hours post injection (hpi), the shrimp were injected with WSSV (10 6 copies / shrimp) and the cumulative mortality rate was recorded, and the RNA interference (RNAi) efficiency of wsv108 was detected at 48 h and 96 h post infection (WSSV challenge) (hpi), respectively, and the WSSV load was determined by real-time PCR from the blood cells of 6 shrimp, and the expression levels of several genes related to apoptosis in shrimp (AIF, Bax, Caspase3 and Bcl2) and wsv249, VP28, EF-1ɑ genes in the blood cells were detected by real-time PCR. Among them, EF-1ɑ is a housekeeping gene, which is used as an internal control gene here, and the specific expression is measured by relative quantification, while wsv249 is a key early gene of WSSV, and VP28 is an important capsid protein of WSSV, and the expression of these two genes can indirectly reflect the overall replication level of WSSV. In addition, the number of WSSV genomic DNA was detected by absolute real-time quantitative PCR using primers WSSV32678-F and WSSV32753-R and Taqman probe WSSV32706. The standard curve was generated using 10-fold serially diluted plasmid pMD19-T (Takara, Japan) containing the corresponding WSSV genomic DNA fragment, which was used to calculate the number of WSSV genome copies in 1 μg of muscle template DNA.

[0059] The sequences of the primers for detecting WSSV genomic DNA are as follows:

[0060] WSSV32678-F: TGTTTTCTGTATGTAATGCGTGTAGGT

[0061] WSSV32753-R: CCCACTCCATGGCCTTCA

[0062] TaqMan probe WSSV32706: CAAGTACCCAGGCCCAGTGTCATACGTT

[0063] The sequences of the primers for detecting the wsv249, VP28, EF-1a, AIF, Bax, Caspase3 and Bcl2 genes are as follows:

[0064] wsv249-F: CCCGGACGGAGACGTGATAA

[0065] wsv249-R: ATGATGATGGGCCTTTCTTCTCT

[0066] VP28-F: AACACCTCCTCCTTCACCC

[0067] VP28-R: GGTCTCAGTGCCAGAGTAGGT

[0068] EF-1a-F: CCTATGTGCGTGGAGACCTTC

[0069] EF-1a-R: GCCAGATTGATCCTTCTTGTTGAC

[0070] AIF-F: TCAGGACGGTTGGCTGGTGAA

[0071] AIF-R: GCAAACACGCCTACGGTTGGT

[0072] Bax-F: CGACCAGCAGTACGACCAGT

[0073] Bax-R: GTCTCCTTATGGAATATTCA

[0074] Caspase3-F: AGCGAGACTACCGCCTCCTAC

[0075] Caspase3-R: CCAAATCTGAGCCACTCCTGTT

[0076] Bcl2-F: GGTGGAATCACAAGAGAGCGA

[0077] Bcl2-R: TCTCCACGGTGTCTCACTTGG

[0078] The effects of targeted inhibition of the wsv108 gene of WSSV on WSSV replication and shrimp mortality after WSSV infection, such as Figure 1 As shown; Figure 1 In this context, A represents the knockdown efficiency of dsRNA against wsv108. Figure 1 In this context, B represents the change in the copy number of WSSV infection after wsv108 knockdown; Figure 1 In this context, C represents the mortality rate of shrimp infected with WSSV after wsv108 knockdown; Figure 1 D in the figure represents the change in mRNA levels of the key gene (wsv249) for WSSV infection after wsv108 knockdown; Figure 1 In the figure, E represents the change in mRNA levels of the key gene (VP28) in WSSV infection after wsv108 knockdown. Figure 1 It was found that, compared with the control group, the wsv108 mRNA levels after WSSV infection at 48 and 96 h after injection of dsRNA-wsv108 decreased to 0.04% and 21.56%, respectively. Figure 1 There were significant differences in (A) among the groups. After wsv108 knockdown, compared to the control group, the copy number of WSSV decreased by 31.41% and 44.73% at 48 and 96 hours after injection of dsRNA-wsv108, respectively. Figure 1 (B in the original text). Simultaneously, this invention also recorded the cumulative survival rate of shrimp from 4 hours to 184 hours after WSSV infection. Within 184 hours, the final cumulative survival rates of the wsv108 group and the GFP knockout group were 48% and 0%, respectively. Figure 1 In the C group, the cumulative survival rate of the wsv108 group was significantly higher than that of the GFP control group (Kaplan-Meier log-rank χ2: 47.98, p<0.01). Compared with the dsRNA-GFP group, the mRNA levels of the key WSSV genes wsv249 and VP28 were significantly decreased 96 h after injection of dsRNA-wsv108. Figure 1 (D and E in the above results). The results indicate that wsv108 plays a key role in WSSV replication.

[0079] The effects of targeted inhibition of the wsv108 gene of WSSV on the apoptosis rate and expression levels of apoptosis-related genes in shrimp infected with WSSV, as follows: Figure 2 As shown; Figure 2 A and B in the figure represent the effect of WSSV infection on the apoptosis rate of shrimp hemocytes after WSSV knockdown; Figure 2 In the figure, C represents the effect of WSSV infection on the expression levels of apoptosis-related genes in shrimp after knockdown of wsv108. Figure 2It can be seen that, compared with the dsRNA-GFP group, the number of blood cell apoptosis 48 hours after WSSV challenge following injection of dsRNA-wsv108 was significantly reduced. Figure 2 The results (A and B in the original text) indicate that wsv108 may be involved in WSSV-induced apoptosis. Compared with the dsRNA-GFP group, after WSSV challenge with dsRNA-wsv108 for 96 h, the mRNA levels of apoptosis-related genes AIF, Bax, caspase-3, and Bcl2 in shrimp were significantly decreased. Figure 2 (C in the text). Downregulation of AIF, Bax, caspase-3, and Bcl2 suggests that wsv108-induced apoptosis may be related to the expression profile of apoptosis-related genes.

[0080] The above results indicate that wsv108 can induce apoptosis in the host after WSSV infection, and inhibiting the expression of wsv108 can effectively reduce WSSV replication in shrimp, reduce the level of apoptosis in hematopoietic cells induced by WSSV, and reduce the mortality rate of shrimp infected with WSSV.

[0081] Example 2: Construction of a strain that specifically interferes with wsv108 dsRNA expression

[0082] Given the high cost of in vitro synthesis of dsRNA and the fact that it requires injection into shrimp, which is time-consuming, labor-intensive, and inefficient, this invention uses the mRNA of the wsv108 functional region as a template for dsRNA synthesis to construct a recombinant bacterium capable of expressing dsRNA that specifically interferes with wsv108, namely the wsv108-dsRNA expression strain. The flowchart illustrating the construction principle of the wsv108-dsRNA expression strain is shown below. Figure 4 As shown in A in the diagram.

[0083] The specific process is as follows:

[0084] 1. Construction of the L4440-wsv108 recombinant plasmid

[0085] (1) Primers were designed using the mRNA of the wsv108 functional region as a template for dsRNA synthesis. The corresponding DNA fragment was obtained by PCR cloning using WSSV genomic DNA as a template. The cloned DNA fragment and the L4440 plasmid (Qiyunbio, catalog number: L4440) were double-digested using restriction endonucleases (Xba I and Kpn I). The digested DNA fragment and the linearized plasmid were ligated overnight at 16°C using T4 ligase (Thermo). The primer sequences designed using the mRNA of the wsv108 functional region as a template for dsRNA synthesis are shown below:

[0086] L4440-wsv108-Xba IF:

[0087] L4440-wsv108-Kpn I-R:

[0088] Note: The bold part is the enzyme cutting site, and the 3 bases before the bold part are the protection bases.

[0089] The reaction system of PCR cloning is shown in Table 1:

[0090] Table 1 Reaction system of PCR cloning

[0091]

[0092] The reaction procedure of PCR cloning is as follows: 95℃ for 5 min; 95℃ for 10 s, 60℃ for 1 min, 35 cycles; 72℃ for 10 min; 4℃ storage.

[0093] (2) The obtained ligation product in (1) is mixed with E. coli DH5a competent cells, incubated on ice for 30 min, heat shocked at 42℃ for 90 s, and plated on LB solid plate containing 100 μg / mL ampicillin sodium (Amp), and cultured overnight in a 37℃ bacterial incubator; a single colony is picked up in 1 mL of LB liquid medium containing 100 μg / mL Amp, and cultured at 37℃ and 200 rpm for 3 h, then the recombinant plasmid containing the target fragment is screened by PCR (L4440-wsv108-Xba I-F and L4440-wsv108-Kpn I-R), and sent to Tianyihui Biotech Co., Ltd. for sequencing; the bacterial liquid with correct sequencing is selected, the plasmid is extracted for standby, and the obtained plasmid is the L4440-wsv108 recombinant plasmid constructed.

[0094] After sequencing, the sequence inserted into L4440 is as follows, same as SEQ ID NO. 3:

[0095] AGTGACGAGGACGAACCATCTCTTGTTATCGACGACGGTTCTGGAAGACAGTCTAAGAACAAGAAATATTCTTGGGTCACATCTCTTGCTACTACTACGGCTGAAAGAAACAACGACACTCTCGCCCCACCTAGGCCCTTCCTTCCCACACCCGAAGAAGGAAATATGTCTGAAATTGACGCAGGGCTAAGTAATCCAGTCACTCGCCAAATCACCGGAGAAGTTTATAGCGCTGCACTCACTTCTGGAGTTGGAGATAATGGACTATATCCTTCCCACTTCACGGTTGCTGACACTTCTTACGGAGATTGCGAAACACCCATACCTGGACCTGCTTTTGTCCTCGACGACGGGACAGTTAGCAGAGGCACATCTCTTCTGCACAGAGAAGAGGCAGAATTCTTGAATGATGGAAGTAAGGTGATCCATACCGTTAAACCAAGAAACAGCAAGTACTCCAATATTCAACGTGCCGCTAGCTGTATGGCCTACGCTGTGGACCTTCTAAACA

[0096] 2. Construction and expression verification of wsv108-dsRNA expression strain

[0097] (1) 500 ng of the constructed L4440-wsv108 recombinant plasmid was mixed with 100 μL of HT115(DE3) competent cells, incubated on ice for 30 min, then heat shocked at 42°C for 90 s, and plated on LB solid medium (containing 100 μg / mL Amp and 15 μg / mL tetracycline hydrochloride), and cultured overnight in a 37°C bacterial incubator;

[0098] (2) A single colony was picked and inoculated in 1 mL of LB liquid medium (containing 100 μg / mL Amp and 15 μg / mL tetracycline hydrochloride), and cultured at 37°C and 200 rpm for 4 h;

[0099] (3) The bacterial solution obtained in (2) was inoculated in LB liquid medium (containing 100 μg / mL Amp and 15 μg / mL tetracycline hydrochloride) at a ratio of 1:1000 (v / v), and cultured overnight at 37°C and 200 rpm to obtain a seed solution;

[0100] (4) The seed culture obtained in (3) was inoculated into 100 mL of LB liquid medium (100 μg / mL Amp and 15 μg / mL tetracycline hydrochloride) at a ratio of 1:100 (v / v). The culture was shaken at 37℃ and 200 rpm until the OD600 (OD is the absorbance value and 600 is the wavelength) was 0.5. Then, the culture was dispensed into 50 mL tubes at a ratio of 20 mL / tube. IPTG was added to the final concentrations of 0 mM, 0.1 mM, 0.2 mM, 0.5 mM and 1 mM, respectively. The culture was shaken at 37℃ and 200 rpm for 5 h to induce expression.

[0101] (5) Centrifuge at 5000g for 10 min to obtain bacterial cells. Use the Trizol method to extract RNA and use agarose gel electrophoresis to detect the expression of wsv108-dsRNA.

[0102] The effects of different concentrations of IPTG on wsv108-dsRNA expression are as follows: Figure 3 As shown. By Figure 3 It can be seen that wsv108-dsRNA was successfully induced to express in the presence of IPTG, and the effect was best at IPTG concentrations of 0.5 mM and 1 mM.

[0103] The results of wsv108-dsRNA expression detection in the wsv108-dsRNA-expressing bacteria are as follows: Figure 4 As shown in B, the wsv108-dsRNA-expressing bacteria described in this invention successfully expressed wsv108-dsRNA after IPTG induction, and subsequent experiments were conducted using the constructed wsv108-dsRNA-expressing bacteria.

[0104] Example 3: Preparation and efficacy testing of WSSV inhibitors

[0105] This invention provides a recombinant *E. coli* strain capable of expressing wsv108-dsRNA (i.e., wsv108-dsRNA-expressing bacteria) in Example 2. This strain can be cultured in large quantities via fermentation and its dsRNA expression can be induced using IPTG. This invention prepared a WSSV inhibitor through fermentation culture of the wsv108-dsRNA-expressing bacteria. The obtained WSSV inhibitor was coated onto the surface of shrimp feed using the sodium alginate coating method. After feeding shrimp continuously for 5 days, they were challenged with WSSV, and the effectiveness of the WSSV inhibitor was tested. The specific process is as follows:

[0106] 1. Preparation of WSSV inhibitors

[0107] The obtained wsv108-dsRNA expression bacteria were expanded and induced for 8h at 0.5mM IPTG concentration to obtain a large amount of wsv108-dsRNA expression bacteria culture liquid, and the wsv108-dsRNA expression bacteria culture liquid was centrifuged at 5000xg for 10min to remove the supernatant to obtain the bacterial body of the wsv108-dsRNA expression bacteria; sterile water was added in a weight ratio of 1:1 to resuspend the bacterial body to obtain a bacterial suspension, which was the shrimp WSSV inhibitor.

[0108] (1) The constructed wsv108-dsRNA expression bacteria were picked into 1mL of LB liquid medium (containing 100μg / mL Amp and 15μg / mL tetracycline hydrochloride) and shaken at 37℃, 200rpm for 4h;

[0109] (2) The bacterial liquid in (1) was inoculated into LB liquid medium (containing 100μg / mL Amp and 15μg / mL tetracycline hydrochloride) at a ratio of 1:1000 (v / v) and shaken at 37℃, 200rpm overnight to obtain a seed liquid;

[0110] (3) The seed liquid was inoculated into LB liquid medium (containing 100μg / mL Amp and 15μg / mL tetracycline hydrochloride) at a ratio of 1:100 (v / v), and after shaking at 37℃, 200rpm until OD=0.5, IPTG was added to a final concentration of 0.5mM, and shaking induction expression was performed at 37℃, 200rpm for 5h;

[0111] (4) Centrifugation at 5000g for 10min, discarding the culture medium, and weighing the solid bacterial block, then adding PBS (pH 7.4) at a ratio of 1mL / g (1g of bacterial body added with 1mL of PBS) to resuspend the bacterial body.

[0112] 2. Bacterial body coated by sodium alginate embedding method

[0113] (1) The bacterial body resuspension prepared above was mixed with shrimp feed (South American white shrimp compound feed (Haida Group feed)) uniformly at a ratio of bacterial body:shrimp feed=50g:1kg;

[0114] (2) Sodium alginate powder was added and mixed uniformly at a ratio of sodium alginate powder:shrimp feed=20g:1kg;

[0115] (3) 0.04mol / L CaCl2 solution was added at a ratio of CaCl2 solution:shrimp feed=200mL:1kg, and mixed uniformly until the sodium alginate formed a gel coating on the surface of the feed.

[0116] 3. Effect test of WSSV inhibitor

[0117] After the shrimp feed coated with WSSV inhibitor was prepared according to the above process, the feed was fed to the shrimp twice a day, and the total amount of feed was 3% of the body weight of the shrimp, and the feed without coating WSSV inhibitor was used as a control; after 5 days of continuous feeding, the shrimp were artificially infected with WSSV (10 6 copies / fish), and samples were taken at different time points (24h and 48h); 6 shrimp were randomly selected from each group, and muscle, gill, stomach and intestinal tissues were collected, DNA was extracted, RNA was extracted and reverse transcribed into cDNA, and the content of wsv108 and the transcription level of wsv108 in the collected tissues were detected by qRT-PCR; in addition, the release amount of wsv108 in the shrimp and the knockdown level of the target gene after the shrimp were fed with WSSV inhibitor and infected with WSSV were detected.

[0118] Among them, the reagents used for real-time fluorescence quantitative PCR (qRT-PCR) detection are Premix Ex TaqTMⅡ (Perfect Real Time, purchased from Takara), and the sequences of the primers used are as follows (5'-3'):

[0119] wsv108-F: ACGCAGGGCTAAGTAATCCAG

[0120] wsv108-R: TCGCAATCTCCGTAAGAAGTGT

[0121] The reaction system is shown in Table 2 below:

[0122] Table 2 qRT-PCR reaction system

[0123]

[0124] The qRT-PCR reaction amplification program is: 95℃ 5min; 95℃ 10s, 60℃ 1min, 35 cycles; 72℃ 10min; 4℃ storage.

[0125] After feeding the feed containing wsv108 expression bacteria for one week, the shrimp were injected with WSSV (10 6 copies / fish) (n=25 per group), and the cumulative mortality was recorded. At 24h and 48h after infection (hpi), muscle, gill, intestine and stomach were taken to detect the RNA interference (RNAi) efficiency of wsv108, and real-time PCR was used to determine the load of WSSV, and the expression amounts of VP28 and shrimp apoptosis-related genes AIF, Bax, Caspase3 and Bcl2 were detected.

[0126] The effect of feeding WSSV inhibitor (wsv108-dsRNA expression bacteria) on the ability of shrimp to resist WSSV infection is as follows Figure 4As shown; Figure 4 In the diagram, A represents the construction principle of the wsv108-dsRNA expression bacteria; Figure 4 B in the figure represents the result of wsv108-dsRNA expression detection in the wsv108-dsRNA-expressing strain; Figure 4 In this context, C represents the mortality rate of shrimp infected with WSSV after being fed with the wsv108-dsRNA-expressing strain. Figure 4 In the figure, D and E represent the relative DNA expression levels of wsv108 in shrimp muscle and gill tissue after feeding with the wsv108-dsRNA-expressing strain, respectively. Figure 4 F and G in the figure represent the relative expression levels of wsv108 mRNA in the intestinal and gastric tissues of shrimp after feeding with the wsv108-dsRNA-expressing strain. Figure 4 H to K in the table represent the copy number of WSSV in different tissues of shrimp after feeding with the wsv108-dsRNA-expressing strain and subsequent infection with WSSV. Figure 4 In the table, L to O represent the expression levels of the stomach apoptosis-related genes ATF, Bax, Bcl2, and Caspase3 in shrimp infected with WSSV after being fed with the wsv108-dsRNA-expressing strain.

[0127] Depend on ​ It was found that feeding with WSSV inhibitors effectively reduced shrimp mortality caused by WSSV infection and downregulated the expression of apoptosis-related genes. Furthermore, in different shrimp tissues, both DNA and mRNA levels demonstrated that feeding with WSSV inhibitors followed by WSSV infection successfully knocked down wsv108 and inhibited WSSV replication. These results indicate that the wsv108-dsRNA-expressing bacteria constructed in this invention can effectively inhibit WSSV replication and reduce the mortality rate of WSSV-infected shrimp, making it suitable for the prevention and treatment of white spot syndrome in shrimp.

[0128] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A dsRNA for interfering with the expression of a wsv108 gene, characterized in that, The dsRNA is prepared based on the nucleotide sequence shown in SEQ ID NO.

3.

2. A recombinant plasmid for expressing a dsRNA targeting the wsv108 gene, characterized in that, The recombinant plasmid contains the nucleotide sequence shown in SEQ ID NO.

3.

3. A recombinant bacteria for interfering with the expression of a wsv108 gene, characterized in that, The recombinant bacteria contain the recombinant plasmid of claim 2.

4. Use of the dsRNA of claim 1, the recombinant plasmid of claim 2 or the recombinant bacteria of claim 3 in the preparation of a product for preventing and / or treating white spot syndrome in prawns.

5. Use of the dsRNA of claim 1, the recombinant plasmid of claim 2 or the recombinant bacteria of claim 3 in the preparation of a product for inhibiting white spot syndrome virus.

6. A feed for prawns, characterized in that, The feed contains the recombinant bacteria of claim 3.

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