A nucleotide sequence targeting PepT1 to alleviate enteritis in fish and its application

Abstract

This invention discloses a nucleotide sequence targeting PepT1 to alleviate fish enteritis and its application. The nucleotide sequence includes a sense strand and an antisense strand, wherein the sequence of the sense strand is shown in SEQ ID NO. 6, and the sequence of the antisense strand is shown in SEQ ID NO. 7. When injected into fish, this nucleotide sequence targeting PepT1 to alleviate fish enteritis can effectively knock down the expression of the PepT1 gene in the fish intestine, with a knockdown efficiency of over 90%, demonstrating high knockdown efficiency. It can completely inhibit the expression level of the fish intestinal inflammatory factor IL-1β induced by bacterial MDP, significantly alleviating the intestinal inflammatory response in fish and exhibiting good therapeutic effects for fish enteritis. This provides new ideas for the research and preparation of drugs for the prevention and treatment of fish bacterial enteritis (such as fish immunomodulators and fish bacterial enteritis treatment drugs).

Description

Technical Field

[0001] This invention belongs to the field of aquatic molecular biology technology, specifically relating to a nucleotide sequence that targets PepT1 to alleviate fish enteritis and its application. Background Technology

[0002] Grass carp (Ctenopharyngodon idella) is one of the four major freshwater fish species in my country, rich in nutrients and with delicious meat, possessing high economic value. Currently, with the increasing intensification of aquaculture and the continuous increase in stocking density, the original ecological balance of the aquaculture environment is being disrupted, the normal physiological functions of the fish are being interfered with, their disease resistance is decreasing, and they are susceptible to infection by various pathogenic microorganisms, leading to increasingly prominent large-scale disease problems. Bacterial enteritis is a frequently occurring infectious disease in the large-scale farming of grass carp, seriously endangering the health of grass carp and even causing mass mortality, resulting in 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 effects are unsatisfactory; while long-term and excessive use of chemical drugs and antibiotics damages the fish's own intestinal mucosal immune system, further reducing their disease resistance and inevitably causing environmental pollution. Therefore, conducting research on gene targets and interventions for bacterial enteritis in fish has significant scientific and applied value.

[0003] In recent years, the PepT1 gene, a small peptide transporter in fish intestinal epithelial cells, has been cloned and identified in rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), and grass carp (Ctenopharyngodonidella), and its gene expression patterns and nutritional regulation have been studied in depth. In previous research by the inventors' team, it was found that the bacterial oligopeptide product muramyl dipeptide (MDP) can significantly induce intestinal inflammation in grass carp. PepT1 may be involved in mediating MDP-induced bacterial intestinal inflammation in fish, but the knockdown of the PepT1 gene and its function in treating bacterial enteritis in fish have not yet been studied. Furthermore, there are no reports in the existing technology of applying RNA interference (RNAi) technology to the functional study of the fish PepT1 gene. Therefore, obtaining a small interfering RNA (siRNA) that targets PepT1 to alleviate enteritis in fish is of great significance for effectively reducing 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 shortcomings of the prior art and provide a nucleotide sequence that targets PepT1 to alleviate fish enteritis and its application.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution.

[0006] A nucleotide sequence for targeting PepT1 to alleviate enteritis in fish, the nucleotide sequence for targeting PepT1 to alleviate enteritis in fish includes a sense strand and an antisense strand, the sequence of the sense strand is shown in SEQ ID NO.6, and the sequence of the antisense strand is shown in 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 enteritis in fish is further improved, wherein the fish is grass carp.

[0009] As a general technical concept, the present invention also provides the application 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] Further improvements to the above application include the following steps: injecting a nucleotide sequence that targets PepT1 to alleviate fish enteritis into the fish to specifically knock down the expression of the PepT1 gene in the fish intestine.

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

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

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

[0014] This invention discloses a nucleotide sequence (siRNA) targeting PepT1 to alleviate fish enteritis. When injected into fish, this nucleotide sequence can effectively knock down the expression of the PepT1 gene in the fish intestine with a knockdown efficiency of over 90%, demonstrating high knockdown efficiency. It can completely inhibit the expression level of IL-1β, an inflammatory factor in the fish intestine induced by bacterial MDP, significantly alleviating the inflammatory response in the fish intestine and showing good therapeutic effects on fish enteritis. This invention can provide new ideas for the research and development and preparation of drugs for the prevention and treatment of fish bacterial enteritis (such as fish immunomodulators and drugs for the treatment of fish bacterial enteritis). Attached Figure Description

[0015] Figure 1 This is a diagram showing the knockdown effect of different types of siRNA on the expression of the 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 on the expression of the PepT1 gene in the intestine of grass carp under MDP stimulation conditions in Example 2 of the present invention.

[0017] Figure 3 This is a diagram illustrating the effect of targeted intervention on the expression of PepT1 gene in grass carp under MDP stimulation on the expression of IL-1β gene in the intestine, as shown in Example 2 of this invention. Detailed Implementation

[0018] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention. All materials and instruments used in the following embodiments are commercially available.

[0019] Example 1:

[0020] A method for obtaining a nucleotide sequence that targets PepT1 to alleviate enteritis in fish includes the following steps:

[0021] (1) The PepT1 gene in the intestinal tract of grass carp was cloned, and the obtained grass carp PepT1 gene sequence is shown in SEQ ID NO.1. Testing showed that the expression level of the grass carp intestinal PepT1 gene was significantly upregulated 6 hours after injection of bacterial MDP, revealing a strong correlation between PepT1 gene expression and bacterial enteritis in grass carp. The PepT1 gene can serve as a biomarker for bacterial enteritis in grass carp, and the detection method of PepT1 gene expression level can be used for effective diagnosis of bacterial enteritis in grass carp.

[0022] Three specific PepT1-siRNAs were designed for different target sites: siRNA-1, siRNA-2, and siRNA-3, as well as a negative control siRNA-NC. The addition of tt (thymine deoxynucleotide) to the 3' end of the siRNA sequence enhances gene knockdown efficiency. Table 1 shows information on different types of siRNAs, synthesized by a biotechnology company in Shanghai.

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

[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 on different types of siRNA

[0061]

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

[0063] Figure 1 This image shows the knockdown effects of different types of siRNA on the expression of the PepT1 gene in the intestinal tract of grass carp in Example 1 of this invention. Figure 1 It can be seen that after 48 hours, siRNA-2 knocked down the grass carp PepT1 gene by more than 90%, which was more effective than siRNA-1 and siRNA-3. Therefore, siRNA-2 was selected as the siRNA to target the expression of the grass carp PepT1 gene for subsequent experiments.

[0064] Example 2:

[0065] An experiment investigating the intervention effect of PepT1 gene knockdown in the intestine of grass carp on muramyl dipeptide (MDP)-induced intestinal inflammatory response included the following steps:

[0066] (1) Healthy live grass carp (about 5g) were divided into 3 groups: NC group, NC+MDP group and siRNA-2+MDP group. NC was siRNA-NC. MDP was injected 48h after siRNA knockdown. The concentration of MDP injected was 10μg / g, and the concentration of siRNA-NC and siRNA-2 injected was 1μg / g.

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

[0068] The PCR reaction system consisted of: 5.68 μL ddH2O, 8.0 μL TB Green Premix Ex Taq (Tli RNase HPlus) (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: 94℃ pre-denaturation for 4 min; 94℃ for 10 s, 56℃ for 10 s, 72℃ for 10 s (40 cycles).

[0070] Table 2 Information on primers with different specificities

[0071]

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

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

[0074] In summary, the nucleotide sequence (siRNA) of the present invention that targets PepT1 to alleviate fish enteritis, when injected into fish, can effectively knock down the expression of the PepT1 gene in the fish intestine with a knockdown efficiency of over 90%, demonstrating high knockdown efficiency. It can completely inhibit the expression level of IL-1β, an inflammatory factor in the fish intestine induced by bacterial MDP, and can significantly alleviate the inflammatory response in the fish intestine, exhibiting good therapeutic effects on fish enteritis. This provides new ideas for the research and development and preparation of drugs for the prevention and treatment of fish bacterial enteritis (such as fish immunomodulators and drugs for the treatment of fish bacterial enteritis).

[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. The application of a nucleotide sequence targeting PepT1 to alleviate fish enteritis in the preparation of a therapeutic drug for fish bacterial enteritis, characterized in that, The nucleotide sequence targeting PepT1 to alleviate fish enteritis includes a sense strand and an antisense strand, the sequence of which is shown in SEQ ID NO.6 and the sequence of which is shown in SEQ ID NO.7; the fish is grass carp.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the PepT1 is shown in SEQ ID NO.1.

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