Targeted PepT1 nucleotide sequence for relieving fish enteritis and application of targeted PepT1 nucleotide sequence

Abstract

The invention discloses a PepT1-targeted nucleotide sequence for relieving fish enteritis and application of the PepT1-targeted nucleotide sequence, the nucleotide sequence comprises a positive-sense strand and an antisense strand, the sequence of the positive-sense strand is shown as SEQ ID NO.6, and the sequence of the antisense strand is shown as SEQ ID NO.7. The invention further discloses a preparation method of the PepT1-targeted nucleotide sequence for relieving fish enteritis. After the PepT1-targeted nucleotide sequence for relieving fish enteritis is injected into a fish body, expression of a fish intestinal PepT1 gene can be effectively knocked down, the knock-down efficiency reaches 90% or above, and the PepT1-targeted nucleotide sequence has the advantage of being high in knock-down efficiency, can completely inhibit the expression level of a fish intestinal inflammatory factor IL-1beta induced by bacteria MDP, remarkably relieves fish intestinal inflammatory response and has a good application prospect. The compound has a good fish enteritis treatment effect, and a new thought can be provided for research, development and preparation of fish bacterial enteritis prevention and treatment drugs (such as fish immunomodulators and fish bacterial enteritis treatment drugs).

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic molecular biology, and specifically relates to a nucleotide sequence targeting PepT1 and alleviating fish enteritis and an application thereof. Background Art

[0002] Grass carp (Ctenopharyngodon idella) is one of the four major carps in my country. It is rich in nutrition, delicious and has high economic value. At present, with the continuous improvement of the degree of aquaculture intensification and the continuous increase in aquaculture density, the original ecological balance of the aquaculture environment has been destroyed, the normal physiological functions of the fish have been disturbed, the disease resistance has decreased, and it is susceptible to infection by various pathogenic microorganisms. Large-scale disease problems are becoming increasingly prominent. Bacterial enteritis is a frequently occurring infectious disease in the large-scale aquaculture of grass carp, which seriously endangers the health of grass carp and even causes a large number of fish deaths, which can easily cause huge economic losses. For a long time, farmers have mostly used antibiotics or chemical drugs to prevent and treat bacterial enteritis in fish, but the effect is not satisfactory; and the long-term and excessive use of chemical drugs and antibiotics has destroyed the fish's own intestinal mucosal immune system, further reduced its disease resistance, and inevitably caused environmental pollution. Therefore, it is of great scientific significance and application value to carry out gene target and intervention research on bacterial enteritis in fish.

[0003] In recent years, the gene of PepT1 (peptide transporter) in the intestinal epithelial cells of fish has been cloned and identified in rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and grass carp (Ctenopharyngodonidella), and in-depth research has been done on its gene expression pattern, nutritional regulation and other aspects. In the preliminary research of the team of the inventor of the present application, it was found that the bacterial oligopeptide product Muramyl Dipeptide (MDP) can significantly induce intestinal inflammatory response of grass carp, and PepT1 may be involved in mediating the MDP-induced bacterial inflammatory response of the fish intestine, but the knockdown expression of the PepT1 gene and its function in treating bacterial enteritis in fish have not been paid attention to. In addition, there is no related report in the prior art on the application of RNA interference (RNAi) technology to the functional study of the PepT1 gene in fish. Therefore, obtaining a small interfering RNA (siRNA) targeting PepT1 to alleviate fish enteritis is of great significance for effectively reducing the bacterial intestinal inflammatory response in fish. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a nucleotide sequence and application thereof for targeting PepT1 to relieve fish enteritis.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions.

[0006] A nucleotide sequence targeting PepT1 to alleviate fish enteritis, comprising a sense chain and an antisense chain, the sequence of the sense chain is shown as SEQ ID NO.6, and the sequence of the antisense chain is shown as SEQ ID NO.7.

[0007] The above-mentioned nucleotide sequence targeting PepT1 to alleviate fish enteritis is further improved, and the nucleotide sequence of PepT1 is shown in SEQ ID NO.1.

[0008] The above-mentioned nucleotide sequence targeting PepT1 to alleviate fish enteritis is further improved, wherein the fish is grass carp.

[0009] As a general technical concept, the present invention also provides a use of the above-mentioned nucleotide sequence targeting PepT1 to alleviate fish enteritis in knocking down the expression of PepT1 gene in the fish intestine.

[0010] The above application is further improved and comprises the following steps: after injecting the nucleotide sequence targeting PepT1 to relieve fish enteritis into the fish, specific knockdown of PepT1 gene expression in the fish intestine is achieved.

[0011] As a general technical concept, the present invention also provides a use of the above-mentioned nucleotide sequence targeting PepT1 to alleviate fish enteritis in the preparation of a fish immunomodulator.

[0012] As a general technical concept, the present invention also provides a use of the above-mentioned nucleotide sequence targeting PepT1 to alleviate fish enteritis in the preparation of a drug for treating fish bacterial enteritis.

[0013] Compared with the prior art, the advantages of the present invention are:

[0014] The present invention discloses a nucleotide sequence (siRNA) targeting PepT1 to relieve fish enteritis. The nucleotide sequence can be injected into fish to effectively knock down the expression of the fish intestinal PepT1 gene, and the knockdown efficiency is more than 90%. It has the advantage of high knockdown efficiency, can completely inhibit the expression level of the fish intestinal inflammatory factor IL-1β induced by bacterial MDP, significantly relieve the fish intestinal inflammatory response, has a good fish enteritis treatment effect, and can provide new ideas for the research and development and preparation of fish bacterial enteritis prevention and treatment drugs (such as fish immunomodulators, fish bacterial enteritis treatment drugs). BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a diagram showing the knockdown effect of different types of siRNA on the expression of PepT1 gene in the intestine of grass carp in Example 1 of the present invention.

[0016] Figure 2 This is a diagram showing the effect of targeted intervention of PepT1 gene expression in the intestine of grass carp under MDP stimulation conditions in Example 2 of the present invention.

[0017] Figure 3 This is a diagram showing the effect of targeted intervention of grass carp PepT1 gene expression on intestinal IL-1β gene expression under MDP stimulation conditions in Example 2 of the present invention. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with the accompanying drawings and specific preferred embodiments, but the protection scope of the present invention is not limited thereby. The materials and instruments used in the following embodiments are all commercially available.

[0019] Embodiment 1:

[0020] A method for obtaining a nucleotide sequence that targets PepT1 and relieves fish enteritis comprises the following steps:

[0021] (1) The grass carp intestinal PepT1 gene was cloned and the grass carp PepT1 gene sequence was obtained as shown in SEQ ID NO. 1. After testing, the expression level of the grass carp intestinal PepT1 gene was significantly upregulated 6 hours after the injection of bacterial MDP, revealing that the expression of the PepT1 gene was well correlated with bacterial enteritis in grass carp. The PepT1 gene can be used as a biomarker for bacterial enteritis in grass carp. The detection method of the PepT1 gene expression level can be used to effectively diagnose bacterial enteritis in grass carp.

[0022] Three specific PepT1-siRNAs were designed for different target sites, namely siRNA-1, siRNA-2, siRNA-3, and siRNA negative control siRNA-NC; tt (thymidine deoxynucleotide) added to the 3' end of the siRNA sequence is conducive to enhancing the gene knockdown efficiency. Table 1 shows the information of different types of siRNA, which was synthesized by a Shanghai Biotechnology Co., Ltd.

[0023] The nucleotide sequence of the grass carp PepT1 gene is shown in SEQ ID NO.1, specifically:

[0024] atggcagaca aagagggaaa aaatgcgagg aagcaagggc caagttgctt gggatatcct

[0025] gtcagtatat ttttcattgt ggtgaatgag ttctgtgagc gattctccta ctatggcatg

[0026] aaagcggtgc tggtgttgta cttcaagtac tttattggtt gggacaatga tttatccact

[0027] accatctacc acacgttcgt ggctctgtgc taccttacac ccatcatggg ggccataatt

[0028] gcagattcgt ggctgggcaa gttcaagact atcgtgtacc tgtccatagt gtacaccatt

[0029] ggccaggtga tcatggctgt cagttccatc catgacatca cagatttcaa taaagacggt

[0030] gttcctgaca acatgaccct ccatattgcc ttgtccatgc tgggactcat cctcattgcc

[0031] ttgggaaccg gaggtattaa accctgtgtg gcagcgtttg gaggagatca gtttgaagaa

[0032] caccaggaaa aacaaagaag tactttcttc tccatatttt atctgtccat taacgctggc

[0033] agtcttctct ccactctcat cacaccaatt cttagatctc aggagtgtgg tatctactca

[0034] aagcagagct gtttcccgct ggcttttggg gtccctgcag ctctcatggt ggttgctctt

[0035] gttgtgttca tcgcaggcca tagcatgtac attatggaat cccccaaggg caacatccta

[0036] ctacgagtca tgaattgcat tatatttgct cttagtaacc gttttgaaca ccgcggtaag

[0037] caatacccca agagagagca ctggatggac tgggcggaag agaaatatga taaactcctg

[0038] attgctcagg tgaagatggt attgaaggtg ctgttcctct atatccctct gccaatgttc

[0039] tgggccttat ttgaccagca gggctcccgt tggactattc aggccaccac tatggacggt

[0040] aactttggag cgtttataat tcagccagat cagatgcaga tcgtgaaccc catactgatt

[0041] gtgattatgg tgccgatcat ggactctgca gtgtacccac ttatcaaaat gtgccgtatt

[0042] aacttcacgc ccctgcggaa gatgactgtc ggtatgctgc tcgcggcgtt agcatttgtg

[0043] gctgctgctc ttttgcaaat tcaaattgac caaacaatgc cccagttccc atcaagctct

[0044] caaacccagg tgaagttcct gaatttggag aaaacatcgc tgcctgtttt agtggagggg

[0045] caagaacaat tttttgttcc tggttttaat tcctcaaatg agtatatgac acttgacacc

[0046] atgaacgtta cagtctctgt ggggggaaga aacgagacag gttattccct gaataaaatg

[0047] aggcacacgg ttttaataaa tgctggtgga atgatacaga ctctcaatga cataactgac

[0048] aaaccgagtc aaggcgggaa tgccatcagg tttgtgaatg gttttaggac tcagctgaac

[0049] atcacagtaa agtctggtga cctgggtttc ctctctccct tagaaggatc atcatactta

[0050] atggttcccc atggcaaagc caactttact attgggaatg aaaatggaca gaactgcaca

[0051] tacatcatgc agctgggctt tggaagctcc tacacagtcc tgatccccag cacctttagt

[0052] ttcggacaaa attgtaacaa caccattaca gcagtacagg atattgagcc gaacagcgtc

[0053] cacatggcct ggcagatcat tcagtacttc ttgatgacct gtggagaagt ggtcttct

[0054] gtcaccggtc tggacttctc ctatgcacag gcaccaagca atatgaagtc agtcctccag

[0055] gctggttggc tgttgactgt tgctgtgggg aacatcatcg tgctcattgt ggctgaggca

[0056] ggctcacttc cagatcagtg ggcagaatac gtgctgttct cgtctctcct ggtggctgtg

[0057] agtgcgcct ttgccatcat ggcttatttc tacacctaca ctgaccctgc ggaaatcgag

[0058] tctaaattta tggagctcaa gccacaagat aagacaaaga aagtgcttgc aatgatgacg

[0059] aaagacaatg tggcgtacat tcagagcgag aacacagacg tcaaacaaac gaagatctag

[0060] Table 1 Information of different types of siRNA

[0061]

[0062] (2) Healthy live grass carp (about 5 g) were selected and randomly divided into 4 groups and placed in an indoor recirculating culture system. Different types of siRNA (1 μg / g) were injected into the abdominal cavity of each group of grass carp, namely siRNA-1, siRNA-2, siRNA-3, and negative control siRNA-NC. Intestinal tissue samples were taken 48 hours after injection, and qRT-PCR was used to screen a pair of siRNAs with the best interference effect. The results are shown in the figure. Figure 1 shown.

[0063] Figure 1 This is a diagram showing the knockdown effect of different types of siRNA on the expression of PepT1 gene in the intestine of grass carp in Example 1 of the present invention. Figure 1 It can be seen that after 48 hours, siRNA-2 knocked down the grass carp PepT1 gene by more than 90%, and its knockdown effect was better than that of siRNA-1 and siRNA-3 groups. Therefore, siRNA-2 was selected as the siRNA targeted intervention in the expression of grass carp PepT1 gene for subsequent experiments.

[0064] Embodiment 2:

[0065] The intervention experiment of PepT1 gene knockdown in the intestine of grass carp on the intestinal inflammatory response induced by muramyl dipeptide (MDP) includes the following steps:

[0066] (1) Healthy living grass carp (about 5 g) were divided into three groups: NC group, NC+MDP group and siRNA-2+MDP group. NC was siRNA-NC, and MDP was injected 48 hours after siRNA knockdown. The injection concentration of MDP was 10 μg / g, and the injection concentrations of siRNA-NC and siRNA-2 were both 1 μg / g.

[0067] (2) Six hours after MDP challenge, intestinal tissues of grass carp were collected and qRT-PCR analysis of PepT1 and IL-1β was performed. Figure 2 , Figure 3The specific primers for detecting the expression level of PepT1 gene include PepT1-F and PepT1-R, the specific primers for detecting the expression level of IL-1β gene include IL-1β-F and IL-1β-R, and the specific primers for detecting the expression level of internal reference β-actin gene include β-actin-F and β-actin-R, as shown in Table 2.

[0068] PCR reaction system: 5.68 μL ddH 2 O, 8.0 μL TB Green Premix Ex Taq (Tli RNaseHPlus) (2×, TaKaRa), 0.32 μL ROX Reference Dye II (50×), 0.5 μL forward and reverse primers (10 μM), and 1 μL cDNA template.

[0069] The PCR reaction program was as follows: pre-denaturation at 94°C for 4 min; 94°C for 10 s, 56°C for 10 s, and 72°C for 10 s (40 cycles).

[0070] Table 2 Information of primers with different specificity

[0071]

[0072] Figure 2 This is a diagram showing the effect of targeted intervention of PepT1 gene expression in the intestine of grass carp under MDP stimulation conditions in Example 2 of the present invention. Figure 2 It can be seen that bacterial MDP can significantly upregulate the expression level of PepT1 in the intestine of grass carp, and siRNA-2 can significantly reduce the expression level of PepT1 gene in the intestine of grass carp induced by MDP.

[0073] Figure 3 This is a diagram showing the effect of targeted intervention of grass carp PepT1 gene expression on intestinal IL-1β gene expression under MDP stimulation conditions in Example 2 of the present invention. Figure 3 It can be seen that bacterial MDP can significantly upregulate the expression level of inflammatory factor IL-1β in the intestine of grass carp, and siRNA-2 can significantly reduce the expression level of IL-1β gene in the intestine of grass carp induced by MDP.

[0074] Based on the above results, it can be seen that the nucleotide sequence (siRNA) targeting PepT1 for alleviating fish enteritis of the present invention can be injected into fish, which can effectively knock down the expression of PepT1 gene in the fish intestine, and the knockdown efficiency is more than 90%. It has the advantage of high knockdown efficiency, can completely inhibit the expression level of fish intestinal inflammatory factor IL-1β induced by bacterial MDP, can significantly alleviate the intestinal inflammatory response of fish, has a good therapeutic effect on fish enteritis, and can provide new ideas for the research and development and preparation of fish bacterial enteritis prevention and treatment drugs (such as fish immunomodulators, fish bacterial enteritis treatment drugs).

[0075] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as above in the preferred embodiment, it is not used to limit the present invention. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention, still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A nucleotide sequence targeting PepT1 to relieve fish enteritis, characterized in that: The nucleotide sequence targeting PepT1 to alleviate fish enteritis includes a sense chain and an antisense chain. The sequence of the sense chain is shown in SEQ ID NO.6, and the sequence of the antisense chain is shown in SEQ ID NO.

7.

2. The nucleotide sequence targeting PepT1 for alleviating fish enteritis according to claim 1, characterized in that: The nucleotide sequence of PepT1 is shown in SEQ ID NO.

1.

3. The nucleotide sequence targeting PepT1 for alleviating fish enteritis according to claim 2, characterized in that: The fish is grass carp. 4 . Use of a nucleotide sequence targeting PepT1 for alleviating fish enteritis as claimed in any one of claims 1 to 3 in knocking down the expression of PepT1 gene in the intestine of fish.

5. The use according to claim 4, characterized in that: The following steps are involved: After the nucleotide sequence targeting PepT1 to alleviate fish enteritis was injected into the fish, specific knockdown of PepT1 gene expression in the fish intestine was achieved.

6. Use of a nucleotide sequence targeting PepT1 for alleviating fish enteritis as claimed in any one of claims 1 to 3 in the preparation of a fish immunomodulator.

7. Use of a nucleotide sequence targeting PepT1 and alleviating fish enteritis as claimed in any one of claims 1 to 3 in the preparation of a drug for treating fish bacterial enteritis.

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