Anti-IHN virus affinity peptide and its application

Abstract

The present invention discloses affinity peptides for use against IHN viruses and their applications. The technical problem solved by the present invention is how to inhibit the reproduction of IHN viruses. Specifically disclosed are polypeptides or pharmaceutically acceptable salts or derivatives thereof, the polypeptides being as follows: a polypeptide having formula (I): SWWFPQWMAQYP‑X1‑X2(I), wherein X1 represents GGS or is absent, and X1 represents YPYDVPDYA or is absent. After chemically synthesizing the SWWFPQWMAQYPGGSYPYDVPDYA polypeptide, RTG‑2 cells were treated with YPYDVPDYA as a control, and then inoculated with IHN virus. The titer of IHN virus after treatment with SWWFPQWMAQYPGGSYPYDVPDYA was significantly lower than that after treatment with YPYDVPDYA. It can be seen that the SWWFPQWMAQYP polypeptide can be used to inhibit the reproduction of IHN viruses.

Description

Technical Field

[0001] The present application specifically relates to affinity peptides against IHN virus and their applications. Background Art

[0002] Rainbow trout (Oncorhynchus mykiss), a member of the salmonid family, is a major cold-water fish species farmed and consumed in my country. However, increasingly prominent diseases are hindering the sustainable development of my country's rainbow trout industry. Infectious Hematopoietic Necrosis Virus (IHNV), one of the most challenging rainbow trout diseases, is characterized by its long incubation period, sudden death, and high mortality rate.

[0003] Due to the lack of effective drugs to prevent and treat IHNV infection, my country's rainbow trout aquaculture industry is facing the dual threats of domestic strain prevalence and mutation, as well as the importation of foreign strains. Therefore, how to provide an effective means to fight IHNV is a problem faced by those skilled in the art. Summary of the Invention

[0004] The technical problem solved by the present invention is how to inhibit the reproduction of IHN virus and / or prevent or treat or assist in treating diseases caused by IHN virus, especially infectious hematopoietic necrosis.

[0005] In order to solve the above problems, the present application provides a polypeptide or a pharmaceutically acceptable salt or derivative thereof.

[0006] The amino acid sequence of the polypeptide is sequence 1, sequence 3, sequence 2 or sequence 4 in the sequence table.

[0007] In order to solve the above problems, the present application also provides a polypeptide derivative, which is a compound obtained by subjecting the above polypeptide to at least one of the following modifications:

[0008] A1) a compound obtained by attaching an amino-terminal protecting group to the amino-terminal of the polypeptide and / or attaching a carboxyl-terminal protecting group to the carboxyl-terminal of the polypeptide;

[0009] A2) a polypeptide capable of specifically binding to rainbow trout SOCS-1 and resisting IHN virus obtained by adding amino acid residues to the amino terminus and / or carboxyl terminus of the polypeptide;

[0010] A3) A polypeptide capable of specifically binding to rainbow trout SOCS-1 and resisting IHN virus is obtained by linking fatty acids to the amino terminus and / or carboxyl terminus of the polypeptide.

[0011] The amino acid sequence of rainbow trout SOCS-1 is sequence 6, as follows:

[0012] MVAHSAVEEQDTTTKPPSSSPSRPLGASSTFLSTPHSDHHTTSPSSSSPPSSSSSVSQSHRPRPSNQCVASPIPGPDLLDHQPPLPLLDPVPTHFPLFPCKVDFLLITRTAAMLERSGFYWGPLGV EEAHTRLKDVATGTFLIRDSRQTDVFFTLSYRAASGPVSVRIVYKGQRFSLAGSEHSFPCLFLLLLEHYINSSKKSLTVPYRKQRPTLQELCRKQVAESCGGEVERVARVPVNPVLKHFLLEFPYRI.

[0013] In the present invention, the amino-terminal protecting group or the carboxyl-terminal protecting group may be any one of an acetyl group, an amino group, a maleoyl group, a succinyl group, a tert-butyloxycarbonyl group, a benzyloxy group, or other hydrophobic groups or macromolecular carrier groups; the carboxyl terminus of the cyclic polypeptide of the present invention may contain a carboxyl-terminal protecting group, and the carboxyl-terminal protecting group may be any one of an amino group, an amide group, a carboxyl group, or a tert-butyloxycarbonyl group, or other hydrophobic groups or macromolecular carrier groups.

[0014] In order to solve the above problems, the present application also provides compounds.

[0015] The derivative is a compound of formula (I): F1-K-SWWFPQWMAQYP-X1-X2-F2 (Formula I), wherein F1 is a fatty acid, K is a group connecting F1 and SWWFPQWMAQYP, X1 represents GGS or does not exist, X2 represents YPYDVPDYA or does not exist, and F2 is a carboxyl terminal protecting group.

[0016] In the above compounds, K, S, W, F, Q, M, A, Y, G, D, V and P represent different amino acids.

[0017] In the above compounds, K may be an amino acid.

[0018] In the above compounds, K may be lysine.

[0019] In the above compounds, different amino acids may be linked by peptide bonds.

[0020] In the above compounds, the carboxyl terminal protecting group is an amino group.

[0021] In the above compounds, the fatty acid may be palmitic acid.

[0022] In the above text, the palmitic acid molecular formula is C 16 H 32 O2.

[0023] In the above, the carboxyl terminal protecting group may be amidation-modified.

[0024] In the above, the F1 can be palmitic acid, and K can be an amino acid. The palmitic acid carboxyl group and the N-terminal amino group of lysine undergo dehydration condensation.

[0025] In the above, the F2 may be an amino group. The C-terminal amino group of the derivative is modified.

[0026] In the above text, the other letters in (Formula I) and the capital letters in X1 and X2 are all amino acids. Amino acids have meanings well known in the art, for example: C is cysteine, D is aspartic acid, E is glutamic acid, Y is tyrosine, F is phenylalanine, W is tryptophan, V is valine, H is histidine, etc. All amino acids in the cyclic polypeptide sequence can be L-type amino acids, and one or more (such as 2-5, 2-4 or 2-3) amino acids therein can also be replaced with amino acids with D-type conformation, artificially modified amino acids, rare amino acids existing in nature, etc., to improve the bioavailability, stability and / or antiviral activity of the cyclic polypeptide. Among them, D-type amino acids refer to amino acids corresponding to L-type amino acids that constitute proteins; artificially modified amino acids refer to common L-type amino acids that constitute proteins that have been modified by methylation, phosphorylation, etc.; rare amino acids existing in nature include uncommon amino acids that constitute proteins and amino acids that do not constitute proteins, such as 5-hydroxylysine, methylhistidine, γ-aminobutyric acid, homoserine, etc.

[0027] In the present application, the carboxyl terminal protecting group is an amino group, which is achieved by amidation modification of the carboxyl terminal.

[0028] The fatty acid may be F1, F2 or F3:

[0029] F1, saturated fatty acids or unsaturated fatty acids;

[0030] F2, saturated fatty acids containing 8 to 20 carbon atoms;

[0031] F3, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid or arachidic acid.

[0032] In the above-mentioned polypeptides, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0033] In the above-mentioned polypeptides, identity refers to amino acid sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, the identity of a pair of amino acid sequences can be calculated by searching in Advanced BLAST 2.1 using blastp as the program, setting the Expect value to 10, all filters to OFF, BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively. The identity value (%) can then be obtained.

[0034] In the above polypeptides, the above 80% identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0035] One or more amino acids in the polypeptides described herein may be replaced with amino acids in a D-form, artificially modified amino acids, or rare amino acids found in nature to enhance the bioavailability, stability, and / or anti-cancer activity of the polypeptides. D-form amino acids refer to amino acids corresponding to the L-form amino acids that make up proteins; artificially modified amino acids refer to common L-form amino acids that have been modified by methylation, phosphorylation, or other methods. Rare amino acids found in nature include uncommon amino acids that make up proteins and amino acids that do not form proteins, such as 5-hydroxylysine, methylhistidine, gamma-aminobutyric acid, and homoserine.

[0036] Use of the above polypeptide or its pharmaceutically acceptable salt or derivative of the above polypeptide or any of the compounds as described above in the preparation of drugs for preventing and / or treating and / or assisting in the treatment of infectious hematopoietic necrosis.

[0037] Use of the above polypeptide or its pharmaceutically acceptable salt or derivative of the above polypeptide or any of the compounds as described above in the preparation of drugs for preventing and / or treating and / or assisting in the treatment of diseases caused by infectious hematopoietic necrosis virus.

[0038] Use of the above polypeptide or its pharmaceutically acceptable salt or derivative of the above polypeptide or any of the above compounds in the preparation of anti-IHN virus drugs.

[0039] In order to solve the above problems, the present application also provides an anti-IHN virus drug.

[0040] The drug comprises the above polypeptide or a pharmaceutically acceptable salt thereof, or a derivative of the above polypeptide or the above compound. Summary of the invention:

[0042] In the above, the drug further includes a pharmaceutically acceptable carrier or excipient.

[0043] Carriers or excipients include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Among them, water-soluble carrier materials are preferred. These materials can be used to make a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, buccal tablets, suppositories, freeze-dried powder injections, etc. They can be conventional preparations, sustained-release preparations, controlled-release preparations, and various microparticle delivery systems. In order to make unit dosage forms into tablets, various carriers well known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dried starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate with citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further prepared as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets. To prepare the unit dosage form into a pill, a wide variety of carriers known in the art can be used. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, gelucine, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginates, sodium lauryl sulfate, methylcellulose, and ethylcellulose. To prepare unit dosage forms as suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, higher alcohol esters, gelatin, and semi-synthetic glycerides. To prepare unit dosage forms as injectable preparations, such as solutions, emulsions, lyophilized powder injections, and suspensions, all diluents commonly used in the art can be used, including water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters.In addition, to prepare an isotonic injection solution, an appropriate amount of sodium chloride, glucose, or glycerol may be added to the injectable formulation. Conventional solubilizers, buffers, pH adjusters, and the like may also be added. Furthermore, colorants, preservatives, fragrances, flavoring agents, sweeteners, or other materials may be added to the pharmaceutical formulation as needed. The above-described dosage forms can be administered via injection, including subcutaneous, intravenous, intramuscular, and intracavitary injections; cavitary administration, such as rectal and vaginal administration; and respiratory administration.

[0044] In order to solve the above problems, the present application also provides a method for preparing an anti-IHN virus drug.

[0045] The method comprises using the above polypeptide or its pharmaceutically acceptable salt or the above polypeptide derivative or any of the above compounds as a component of a drug to obtain an anti-IHN virus drug.

[0046] In the present application, the IHN virus may be infectious hematopoietic necrosis virus.

[0047] In the present application, the Latin name of the infectious hematopoietic necrosis virus is Infectioushematopoietic necrosis virus.

[0048] Beneficial effects

[0049] The present invention first utilizes phage display peptide library panning technology to screen and obtain polypeptide sequences capable of binding to rainbow trout SOCS-1, and based on this, chemical synthesis and modification of rainbow trout SOCS-1 affinity peptides are performed. The SWWFPQWMAQYPGGSYPYDVPDYA polypeptide is chemically synthesized, wherein YPYDVPDYA is an HA tag and GGS is a connecting peptide; the N-terminus is modified with fatty acids to enhance the cell permeability of the affinity peptide; the C-terminus is introduced with an HA tag and amidation modification to facilitate subsequent detection and ensure the stability of the affinity peptide. Using an HA control peptide (YPYDVPDYA) as a control, RTG-2 cells are pretreated with the SWWFPQWMAQYPGGSYPYDVPDYA polypeptide, and then inoculated with IHN virus. The titer of the IHN virus after treatment with SWWFPQWMAQYPGGSYPYDVPDYA is significantly lower than that of YPYDVPDYA, indicating that the SWWFPQWMAQYP polypeptide can be used to inhibit the reproduction of the IHN virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The relationship between SOCS-1 and IHNV infection.

[0051] Figure 2Figure 3 is the diagram of gene cloning, recombinant protein expression and purification of SOCS-1; A is the cloning of SOCS-1 gene, M: Trans2K DNA marker, 1: SOCS-1 gene PCR product; B is the prokaryotic expression of recombinant SOCS-1, M: Protein marker, 1: product after empty vector induction; 2: supernatant after ultrasonic lysis of SOCS-1 induced expression product; 3: precipitate after ultrasonic lysis of SOCS-1 induced expression product; C is the purification and renaturation of recombinant SOCS-1 protein, M: Protein marker, 1: final product of recombinant SOCS-1 protein.

[0052] Figure 3 This is a diagram of the screening and identification of positive phages.

[0053] Figure 4 Figure 2 is a validation diagram of the binding of SW affinity peptide to SOCS-1.

[0054] Figure 5 This figure shows the inhibitory effect of C16-SW affinity lipopeptide on IHNV.

[0055] Figure 6 The graph shows the inhibitory effect of SW-encoding plasmid on IHNV after cells were transfected. DETAILED DESCRIPTION

[0056] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0057] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0058] The data in the following examples were processed using SPSS 11.5 statistical software. The experimental results were expressed as mean ± standard deviation and tested using One-way ANOVA. P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated an extremely significant difference.

[0059] Example 1 Analysis of SOCS-1 Function During HNV Infection

[0060] Rainbow trout RTG-2 cells were infected with infectious hematopoietic necrosis virus (IHNV) at two challenge doses of 100 MOI and 10 MOI, or RTG-2 cells were treated with recombinant rainbow trout interferon. Total RNA was extracted from the cells and the expression level of SOCS-1 was detected by real-time PCR. It was found that IHNV infection caused a significant upregulation of SOCS-1 expression and that the expression of this molecule increased accordingly with viral replication ( Figure 1 A), while the expression of interferon effector protein Mx1 was negatively correlated with SOCS-1 ( Figure 1 This suggests that IHNV infection can regulate SOCS-1 expression, and SOCS-1 expression may be related to the body's antiviral interferon response.

[0061] Example 2 Screening and Identification of Rainbow Trout SOCS-1 Affinity Peptides

[0062] 2.1 Amplification of target gene and construction of recombinant prokaryotic expression plasmid

[0063] The SOCS-1 amplification primer pair (5'- GGA TCC ATGGTCGCTCACAGTGCTGTG-3' and 5'- AAGCTT TTATATCCTGTACGGGAACTCT-3', the restriction endonuclease sites are underlined, with BamHI upstream and HindIII downstream), RTG-2 cells (rainbow trout gonadal cell line) were stimulated with the previously prepared recombinant rainbow trout interferon riIFN1a, RNA was extracted, and reverse transcription was performed to obtain cDNA. PCR reaction was performed using SOCS-1 amplification primers as primers and cDNA as template, and a fragment of 774 bp was finally obtained ( Figure 2 A). The amplified product was digested and ligated with the prokaryotic expression vector pET-32a(+), and the ligated system was introduced into Escherichia coli Rosetta (DE3). TM After culturing the competent cells, single colonies were picked for sequencing, and the vector with correct sequencing was named the recombinant vector pET-32a(+)-SOCS-1.

[0064] The recombinant vector pET-32a(+)-SOCS-1 is obtained by replacing the fragment between the restriction endonuclease BamHI and HindIII recognition sites in the prokaryotic expression vector pET-32a(+) with sequence 5, while keeping the other sites of the prokaryotic expression vector pET-32a(+) unchanged. The recombinant vector pET-32a(+)-SOCS-1 was transformed into Escherichia coli Rosetta (DE3) TM Escherichia coli Rosetta (DE3) was obtained in competent cells TM / recombinant vector pET-32a(+)-SOCS-1 (also known as Escherichia coli / recombinant vector pET-32a(+)-SOCS-1).

[0065] Sequence 5 is as follows:

[0066] Atggtcgctcacagtgctgtggaagaacaagacacaacaacaaaaccaccatcgtcgtcaccttctagacctctaggcgcctcctccaccttcctctcgactccacactcggaccaccacacaacctcaccatcatcatcatcaccaccatcgtcatcatcatcatcagtctcccagtcacaccgtccgcgcccatccaaccagtgtgtggcgtcccctatcccaggaccagacctgttagaccaccagccccctcttcccctcctggacccagtgcccacccactttcctctgttcccctgcaaggtagacttcctgttgatcactcggacggccgccatgttggaacgttccggcttctactggggcccgctaggagtggaggaggctcacactcgactgaaggacgttgccacgggaacattcttgatccgggacagtcgtcagacagacgtcttcttcacgctgtcctatcgcgcggccagcggcccggtcagcgtacgcatcgtctataagggacaacggttcagcctggcaggaagtgagcactctttcccctgcctcttcctcctgctcgaacactacataaattcctctaagaaaagcctgaccgttccgtacaggaagcagcgccctacgctccaggaactgtgcaggaaacaggtcgcggagtcgtgtggcggtgaggtggaacgggtcgccagggtccccgtcaacccagtcctaaaacacttcctgttagagttcccgtacaggatatga。

[0067] The amino acid sequence of rainbow trout SOCS-1 is Sequence 6, as follows:

[0068] MVAHSAVEEQDTTTKPPSSSPSRPLGASSTFLSTPHSDHHTTSPSSSSPPSSSSSVSQSHRPRPSNQCVASPIPGPDLLDHQPPLPLLDPVPTHFPLFPCKVDFLLITRTAAMLERSGFYWGPLGV EEAHTRLKDVATGTFLIRDSRQTDVFFTLSYRAASGPVSVRIVYKGQRFSLAGSEHSFPCLFLLLLEHYINSSKKSLTVPYRKQRPTLQELCRKQVAESCGGEVERVARVPVNPVLKHFLLEFPYRI.

[0069] 2.2 Prokaryotic expression and purification of recombinant protein

[0070] The E. coli / recombinant vector pET-32a(+)-SOCS-1 was induced to express with 1.0 mM IPTG, and the recombinant SOCS-1 protein was obtained with a size of approximately 46 kDa ( Figure 2 Middle B). Recombinant SOCS-1 protein was purified by Ni-NTA affinity chromatography system ( Figure 2 After dialyzed and concentration determined, the suspension was stored at -80°C.

[0071] 2.3 Screening of positive phages

[0072] Refer to Ph.D.-12 TM The operating manual of the Phage Display 12-Peptide Library Kit v2 (NEB product, #E8210S) is summarized as follows: a. Coat the recombinant SOCS-1 protein diluted in carbonate buffer on the ELISA plate, block overnight, wash, add the diluted phage library (reagents in the #E8210S kit) and incubate. After thorough washing, add the eluent and shake, transfer the supernatant to a 1.5mL centrifuge tube, and add the neutralization solution.

[0073] b. Preparation of medium A: Add 10 g tryptone, 5 g yeast extract, and 5 g NaCl per 1 L of double-distilled water, sterilize at 121°C for 30 min, and cool to room temperature to obtain medium A.

[0074] Preparation of host strain culture: The phage host strain ER2738 was streaked (the phage host strain ER2738 is a component of the phage library in the commercial kit; Ph.D.-12 TM Phage Display 12 Peptide Library Kit v2 (NEB product, #E8210S) was used to pick a single colony and culture it in 20 ml of medium A. The culture was shaken at 37°C and 200 rpm for 4-8 h until the early logarithmic growth stage (OD600nm is 0.01-0.05), and a host strain culture is obtained.

[0075] Neutralized phage was added to a culture of the phage host strain ER2738. After shaking at 37°C for 5 hours, the supernatant was collected by centrifugation and precipitated overnight with PEG-NaCl at 4°C. The supernatant was discarded by centrifugation and the pellet was resuspended in an appropriate volume of TBS for titer determination. c. The serially diluted phage eluate was mixed with ER2738, the mixture was added to the melted top agar layer, and the mixture was poured onto the bottom agar layer. The number of plaques formed was counted to calculate the phage titer.

[0076] According to the above steps, 5 rounds of panning were performed.

[0077] 2.4 Identification of positive phages

[0078] a. Coat the recombinant SOCS-1 protein diluted in carbonate buffer onto an ELISA plate. Use the phage amplification solution obtained by panning as the primary antibody and HRP-labeled M13 antibody as the secondary antibody to screen positive phages.

[0079] b. Extract the phage genome according to the method in the third edition of the "Molecular Cloning Experimental Guide". Design primers for the empty vector M13KE and perform PCR reaction. Identify positive phage by analyzing whether there is gene insertion. Send the extracted phage double-stranded DNA to a sequencing company for sequencing. Ultimately, obtain the polypeptide sequence information displayed by the phage with high affinity.

[0080] The sequence of the affinity peptide (SW affinity peptide) finally obtained is: SWWFPQWMAQYP (SEQ ID No. 1).

[0081] c. Coat the recombinant SOCS-1 protein on the ELISA plate and add 10 12 and 10 10 The positive phage displaying SW affinity peptide was added with HRP-labeled M13 antibody for color development and the OD was read. 450nm The results showed that the phage displaying SW affinity peptide had a 12 It can bind to the target molecule SOCS-1 but not to the non-target molecule BSA ( Figure 3 ).

[0082] 3. Anti-IHN virus effect of rainbow trout SOCS-1 affinity peptide

[0083] 3.1 Preparation and identification of affinity peptides

[0084] Nanjing Jiepeptide Biotechnology Co., Ltd. was commissioned to chemically synthesize C16-SW affinity lipopeptide and C16-HA lipopeptide. The N-terminus of the C16-SW affinity lipopeptide was modified with fatty acids (a lysine was introduced into the N-terminus of the SW affinity peptide, and palmitic acid was covalently bound to the lysine residue to form a fatty acid-modified polypeptide, i.e., a lipopeptide) to enhance the cell permeability of the affinity peptide. An HA tag was introduced into the C-terminus (to facilitate subsequent detection, the amino acid sequence of the HA tag is YPYDVPDYA), and amidation modification was performed (to enhance the stability of the affinity peptide) to obtain the C16-SW affinity lipopeptide. The structural formula of the C16-SW affinity lipopeptide is:

[0085] C 16 -K-SWWFPQWMAQYPGGSYPYDVPDYA-NH2, among which C 16 is palmitic acid, K is lysine, and the amino acid sequence of the HA tag is YPYDVPDYA; GGS is a connecting peptide; NH2 (amino) is a carboxyl terminal protecting group - amino, that is, the carboxyl terminal is amidated; the lysine connecting modification group (C 16 ) and the amino acid (SWWFPQWMAQYPGGSYPYDVPDYA) of the polypeptide, the palmitic acid carboxyl group and the amino group (NH2) at the N-terminus of lysine are dehydrated and condensed, and the lysine is peptide-bonded to the above polypeptide ((SWWFPQWMAQYPGGSYPYDVPDYA)); the C 16 (palmitic acid) molecular formula is C 16 H 32 O2, CAS number is 57-10-3, and other capital letters are amino acids.

[0086] A control lipopeptide was synthesized: C 16 -K-GGSYPYDVPDYA-NH2, where C 16 is palmitic acid, K is lysine, and the amino acid sequence of the HA tag is YPYDVPDYA; GGS is a connecting peptide; NH2 (amino) is a carboxyl terminal protecting group - amino, that is, the carboxyl terminal is amidated; the lysine connecting modification group (C 16 ) and the amino acid (GGSYPYDVPDYA) of the polypeptide, the palmitic acid carboxyl group and the amino group (NH2) at the N-terminus of lysine are dehydrated and condensed, and the lysine is peptide-bonded to the above polypeptide ((GGSYPYDVPDYA)); the C 16 (palmitic acid) molecular formula is C 16 H 32 O2, CAS number is 57-10-3, and other capital letters are amino acids.

[0087] Sequence 1 is as follows: SWWFPQWMAQYP.

[0088] Sequence 2 is as follows: SWWFPQWMAQYPGGSYPYDVPDYA.

[0089] Sequence 3 is as follows: KSWWFPQWMAQYP.

[0090] Sequence 4 is as follows: KSWWFPQWMAQYPGGSYPYDVPDYA.

[0091] The pcDNA3.1-Flag-SOCS1 recombinant plasmid was constructed and transiently transfected into RTG-2 cells. After 24 hours, the cells were treated with WB&IP cell lysate and immunoprecipitated using anti-Flag agarose. The final eluate (i.e., eukaryotically expressed SOCS-1 protein) was collected. The prokaryotically expressed recombinant SOCS-1 protein and the eukaryotic expressed SOCS-1 protein were coated on ELISA plates, and then the synthetic affinity peptide was added. After incubation, HA antibody was added, color was developed, and the values ​​were read. The final results showed that the C16-SW affinity lipopeptide could bind to SOCS-1 outside the cell ( Figure 4 Middle A).

[0092] RTG-2 cells were transiently transfected with the pcDNA3.1-Flag-SOCS1 recombinant plasmid. The cells were then incubated with 20, 6.7, and 2.3 μM C16-SW affinity lipopeptide. After 24 hours, the cells were harvested and lysed, immunoprecipitated with anti-Flag agarose gel, and the final eluate was collected. The eluate was directly coated onto an ELISA plate, and Flag or HA antibodies were added. After color development, the plate was read. Detection with the Flag antibody indicated that immunoprecipitation with the anti-Flag antibody was possible after pcDNA3.1-Flag-SOCS1 transfection. Detection with the HA antibody demonstrated that the synthesized C16-SW affinity lipopeptide penetrated the cells and bound to SOCS-1. The ELISA plate was coated with prokaryotically expressed SOCS-1 protein, then incubated with the eluate, and then HA antibody was added. The color was developed and the values ​​were read. The results showed that the affinity peptide that was co-precipitated by binding to SOCS-1 in the cell could be competitively bound by recombinant prokaryotically expressed SOCS-1, further confirming that the affinity peptide could bind to SOCS-1 both inside and outside the cell ( Figure 4 Middle B).

[0093] 3.2 Antiviral effects of affinity peptides

[0094] Nanjing Jiepeptide Biotechnology Co., Ltd. was commissioned to chemically synthesize C16-SW affinity lipopeptide and C16-HA lipopeptide. The N-terminus of C16-SW affinity lipopeptide was modified with fatty acids to enhance the cell permeability of the affinity peptide. An HA tag was introduced into the C-terminus (to facilitate subsequent detection) and amidation modification was performed (to enhance the stability of the affinity peptide) to obtain C16-SW affinity lipopeptide. The structural formula of C16-SW affinity lipopeptide is:

[0095] C 16 -K-SWWFPQWMAQYPGGSYPYDVPDYA-NH2, among which C 16 is palmitic acid, K is lysine, and the amino acid sequence of the HA tag is YPYDVPDYA; GGS is a connecting peptide; NH2 (amino) is a carboxyl terminal protecting group - amino, that is, the carboxyl terminal is amidated; the lysine connecting modification group (C 16 ) and the amino acid (SWWFPQWMAQYPGGSYPYDVPDYA) of the polypeptide, the palmitic acid carboxyl group and the amino group (NH2) at the N-terminus of lysine are dehydrated and condensed, and the lysine is peptide-bonded to the above polypeptide ((SWWFPQWMAQYPGGSYPYDVPDYA)); the C 16 (palmitic acid) molecular formula is C 16 H 32 O2, CAS number is 57-10-3, and other capital letters are amino acids.

[0096] A control lipopeptide was synthesized: C 16 -K-GGSYPYDVPDYA-NH2, where C 16 is palmitic acid, K is lysine, and the amino acid sequence of the HA tag is YPYDVPDYA; GGS is a connecting peptide; NH2 (amino) is a carboxyl terminal protecting group - amino, that is, the carboxyl terminal is amidated; the lysine connecting modification group (C 16 ) and the amino acid (GGSYPYDVPDYA) of the polypeptide, the palmitic acid carboxyl group and the amino group (NH2) at the N-terminus of lysine are dehydrated and condensed, and the lysine is peptide-bonded to the above polypeptide ((GGSYPYDVPDYA)); the C 16 (palmitic acid) molecular formula is C 16 H 32 O2, CAS number is 57-10-3, and other capital letters are amino acids.

[0097] Preparation of IHNV (Infectious hematopoietic necrosis virus, Latin name Infectious hematopoietic necrosis virus, abbreviated as IHNV): One day before virus inoculation, EPC cells (source ATCC, catalog number CRL-2872) were subcultured in a T25 cell culture flask. The cell confluence on the day of inoculation was estimated to be 80-90%. IHNV (IHNV is a clinical isolate and is described in the following literature: "Host miR-146a-3pFacilitates Replication of Infectious Hematopoietic Necrosis Virus byTargeting WNT3a and CCND1, which is named IHNV in this document, was diluted 1:1000 with M199 medium and allowed to adsorb cells for 2 hours. Fresh M199 medium containing 4% fetal bovine serum was then replaced and cultured at 15°C until obvious cytopathic effects occurred (approximately 5-7 days). The cell culture containing the virus was frozen and thawed three times and centrifuged at 1000 rpm to collect the supernatant, which was the IHN virus solution. The obtained virus solution was analyzed by TCID using the Reed-Muench method. 50 The virus solution was then stored in a -80°C freezer.

[0098] 50TCID 50 Preparation of IHN virus solution: Dilute the above-prepared IHN virus solution with Eagle's Minimum Essential Medium (ATCC, catalog number 30-2003) to 2×10 5.53 times to obtain 50TCID 50 IHN virus fluid.

[0099] Preparation of 20 mg / mL C16-SW affinity lipopeptide: The C16-SW affinity lipopeptide (4 mg) chemically synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd. was dissolved in 0.2 mL of DMSO, and the concentration of the C16-SW affinity lipopeptide was diluted to 20 mg / mL to obtain 20 mg / mL C16-SW affinity lipopeptide mother solution (also known as C16-SW affinity lipopeptide mother solution).

[0100] 5 μg / mL C16-SW affinity lipopeptide dilution: Dilute 20 mg / mL of C16-SW affinity lipopeptide stock solution (also referred to as C16-SW affinity lipopeptide mother solution) with medium B containing 0.2% (v:v) DMSO to a final C16-SW affinity lipopeptide concentration of 5 μg / mL to obtain a 5 μg / mL C16-SW affinity lipopeptide dilution.

[0101] 10 μg / mL C16-SW affinity lipopeptide dilution: Dilute 20 mg / mL of C16-SW affinity lipopeptide stock solution (also referred to as C16-SW affinity lipopeptide mother solution) with medium B containing 0.2% (v:v) DMSO to a final C16-SW affinity lipopeptide concentration of 10 μg / mL to obtain a 10 μg / mL C16-SW affinity lipopeptide dilution.

[0102] 20 μg / mL C16-SW affinity lipopeptide dilution; 20 mg / mL C16-SW affinity lipopeptide stock solution (also known as C16-SW affinity lipopeptide stock solution) is diluted with B culture medium containing 0.2% (V:V) DMSO to a final C16-SW affinity lipopeptide concentration of 20 μg / mL to obtain a 20 μg / mL C16-SW affinity lipopeptide dilution.

[0103] HA control lipopeptide stock solution: The control lipopeptide (4 mg) chemically synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd. was dissolved in 0.2 mL of DMSO, and the concentration of the HA control lipopeptide was diluted to 20 mg / mL to obtain the HA control lipopeptide stock solution.

[0104] RTG-2 cells are rainbow trout gonadal cell lines (RTG-2): purchased from ATCC, catalog number CCL-55.

[0105] Preparation of B culture medium: 10% fetal bovine serum (Wisent, Catalog No. 086-150) and 1% penicillin-streptomycin double antibody solution (HyClone, Catalog No. SV30010) were added to Eagle's Minimum Essential Medium (ATCC, Catalog No. 30-2003) to obtain B culture medium.

[0106] Preparation of C culture medium: 4% fetal bovine serum (Wisent, cat. no. 086-150) and 1% penicillin-streptomycin double antibody solution (HyClone, cat. no. SV30010) were added to Eagle's Minimum Essential Medium (ATCC, cat. no. 30-2003) to obtain C culture medium.

[0107] RTG-2 cell culture medium: RTG-2 cells were inoculated into medium B and cultured at 20°C and 5% CO2 until a confluent monolayer was obtained to obtain RTG-2 cell culture medium. The culture was repeated multiple times in parallel.

[0108] Antiviral detection experiment of affinity peptide:

[0109] The experiment was divided into 6 groups, namely 2.5 μg / mL C16-SW affinity lipopeptide experimental group, 5 μg / mL C16-SW affinity lipopeptide experimental group, 10 μg / mL C16-SW affinity lipopeptide experimental group, 20 μg / mL C16-SW affinity lipopeptide experimental group and control group.

[0110] 2.5 μg / mL C16-SW affinity lipopeptide experimental group: Add 1 ml of 5 μg / mL C16-SW affinity lipopeptide diluent to 1 ml of RTG-2 cell culture medium to obtain 2.5 μg / mL C16-SW affinity lipopeptide RTG-2 cell culture medium. Repeat the experiment five times.

[0111] 5 μg / mL C16-SW affinity lipopeptide experimental group: Add 1 ml of 10 μg / mL C16-SW affinity lipopeptide diluent to 1 ml of RTG-2 cell culture medium to obtain 5 μg / mL C16-SW affinity lipopeptide RTG-2 cell culture medium. Repeat the experiment five times.

[0112] 10 μg / mL C16-SW affinity lipopeptide experimental group: Add 1 ml of 20 μg / mL C16-SW affinity lipopeptide diluent to 1 ml of RTG-2 cell culture medium to obtain 10 μg / mL C16-SW affinity lipopeptide RTG-2 cell culture medium. Repeat the experiment five times.

[0113] 20 μg / mL C16-SW affinity lipopeptide experimental group: Add 0.002 ml of C16-SW affinity lipopeptide stock solution to 2 ml of RTG-2 cell culture medium to obtain 20 μg / mL C16-SW affinity lipopeptide RTG-2 cell culture medium. Repeat the experiment five times.

[0114] HA control lipopeptide control group: Add 0.002 ml of HA control lipopeptide stock solution to 2 ml of RTG-2 cell culture medium to obtain HA control lipopeptide RTG-2 cell culture medium. Repeat the experiment 5 times.

[0115] Untreated control group: Add 0.002 ml of DMSO to 2 ml of RTG-2 cell culture medium to obtain untreated control RTG-2 cell culture medium. Repeat the experiment 5 times.

[0116] The above-mentioned 2.5 μg / mL C16-SW affinity lipopeptide RTG-2 cell culture medium, 5 μg / mL C16-SW affinity lipopeptide RTG-2 cell culture medium, 10 μg / mL C16-SW affinity lipopeptide RTG-2 cell culture medium, 15 μg / mL C16-SW affinity lipopeptide RTG-2 cell culture medium, 20 μg / mL C16-SW affinity lipopeptide RTG-2 cell culture medium, HA control lipopeptide RTG-2 cell culture medium and untreated control RTG-2 cell culture medium were cultured at 20°C and 5% CO2 for 24 h, and then the cell culture medium was replaced with 2 ml of the above-prepared 50TCID 50 The IHN virus solution was replaced with 2 ml of C medium 2 hours later. After further culture at 15°C and 5% CO2 for 24 hours, the expression of IHNV N gene, the titer of virus in the cell supernatant and the expression of Mx1 gene were detected.

[0117] The specific method is as follows:

[0118] Detection of IHNV N gene expression: cells were scraped with a scraper, and total cellular RNA was extracted using an RNA extraction kit (Axygen, catalog number AP-MN-MS-RNA-250). TM RT Master Mix Kit (Takara, Cat. No. RR036A) was used for reverse transcription reaction to obtain cDNA. Premix Ex Taq TM II (Tli RNase H Plus) kit instructions (Takara, catalog number RR820A), and fluorescent quantitative PCR reactions were performed using an Applied Biosystems 7500. The 20 μL reaction system was 10 μL Premix Ex Taq TM II, 0.8 μL upstream primer (10 μM), 0.8 μL downstream primer (10 μM), 0.4 μL ROX Reference Dye II (50×), 2 μL cDNA template and 6 μL sterile deionized water. The reaction conditions were 95°C for 30 seconds, then 95°C for 5 seconds, 60°C for 34 seconds, for a total of 40 cycles, and finally a melting curve was drawn. The internal reference selected the β-actin gene (primers were GCCGGCCGCGACCTCACAGACTAC and CGGCCGTGGTGGTGAAGCTGTAAC), the IHNV N gene detection primers were TGTGCATGAAGTCAGTGGTGG and CCTGCTCATCATGACACCGTA, and 2 -ΔΔCTThe relative changes of IHNV N gene in the experimental group were determined by the method compared with the untreated control group. Figure 5 Middle A( Figure 5 In Figure A, 20 μg / mL, 10 μg / mL, 5 μg / mL and 2.5 μg / mL represent the 20 μg / mL C16-SW affinity lipopeptide experimental group, the 10 μg / mL C16-SW affinity lipopeptide experimental group, the 5 μg / mL C16-SW affinity lipopeptide experimental group and the 2.5 μg / mL C16-SW affinity lipopeptide experimental group, respectively; the HA control lipopeptide represents the HA control lipopeptide control group, and the untreated control represents the untreated control group. As shown in Figure 2, the IHNV N gene expression level in the 20 μg / mL C16-SW affinity lipopeptide experimental group was significantly decreased compared with the HA control lipopeptide control group and the untreated control group, which was 0.35 times lower than that in the HA control lipopeptide group and 0.53 times lower than that in the untreated control group. Figure 5 2.5 μg / mL (C16-SW affinity lipopeptide 2.5 μg / mL) was the 2.5 μg / mL C16-SW affinity lipopeptide experimental group, 5 μg / mL was the 5 μg / mL C16-SW affinity lipopeptide experimental group, 10 μg / mL was the 10 μg / mL C16-SW affinity lipopeptide experimental group, 20 μg / mL was the 20 μg / mL C16-SW affinity lipopeptide experimental group, HA control lipopeptide was the HA control lipopeptide control group, and the untreated control was the untreated control group.

[0119] Detection of IHNV virus titer in cell culture supernatant: Use a pipette to aspirate all the cell culture supernatants of the 20 μg / mL C16-SW affinity lipopeptide experimental group, HA control lipopeptide control group and untreated control group, and add 100 μL of the supernatant of the cells to be tested at a 10-fold dilution to a 96-well cell culture plate where RTG-2 cells have grown a monolayer. Set up 8 replicate wells for each dilution, place in a CO2 incubator, and culture at 15°C. At the same time, set up a negative control group without virus addition, observe and record the cell pathological changes under a microscope every day, count the number of wells with cell pathological changes, and calculate TCID using the Reed-Muench method. 50 .

[0120] The results are as follows Figure 5 As shown in Figure B (20 μg / mL C16-SW affinity lipopeptide is the 20 μg / mL C16-SW affinity lipopeptide experimental group, HA control lipopeptide is the HA control lipopeptide control group, and the untreated control is the untreated control group), compared with the untreated control group, the virus titer of the HA control lipopeptide group did not change significantly, while the IHN virus titer of the 20 μg / mL C16-SW affinity lipopeptide experimental group was significantly reduced, with a reduction of 0.25 times.

[0121] Detection of Mx1 gene expression: cells were picked up with a scraper, and total cellular RNA was extracted using an RNA extraction kit (Axygen, catalog number AP-MN-MS-RNA-250). TM RT Master Mix Kit (Takara, Cat. No. RR036A) was used for reverse transcription reaction to obtain cDNA. Premix Ex Taq TM II (Tli RNase H Plus) kit instructions (Takara, catalog number RR820A), and fluorescent quantitative PCR reactions were performed using an Applied Biosystems 7500. The 20 μL reaction system was 10 μL Premix Ex Taq TM II, 0.8μL upstream primer (10μM), 0.8μL downstream primer (10μM), 0.4μL ROX Reference Dye II (50×), 2μL cDNA template and 6μL sterile deionized water. The reaction conditions were 95℃ 30sec, then 95℃ 5sec, 60℃ 34sec, for a total of 40 cycles, and finally a melting curve was drawn. The internal reference selected the β-actin gene (primers were GCCGGCCGCGACCTCACAGACTAC and CGGCCGTGGTGGTGAAGCTGTAAC). The detection primers for the Mx1 gene were AGCGTCTGGCTGATCAGATT and AGCTGCTCGATGTTGTCCTT. 2 -ΔΔCT The relative changes of Mx1 gene in the experimental group were determined by the method compared with the untreated control group.

[0122] The results are as follows Figure 5 Middle C( Figure 5 In C, 20 μg / mL, 10 μg / mL, 5 μg / mL and 2.5 μg / mL represent the 20 μg / mL C16-SW affinity lipopeptide experimental group, the 10 μg / mL C16-SW affinity lipopeptide experimental group, the 5 μg / mL C16-SW affinity lipopeptide experimental group and the 2.5 μg / mL C16-SW affinity lipopeptide experimental group, respectively; the HA control lipopeptide represents the HA control lipopeptide control group, and the untreated control represents the untreated control group. As shown in Figure 3, compared with the untreated control group, there was no significant difference in the expression of Mx1 gene in the HA control lipopeptide group, while the 20 μg / mL C16-SW affinity lipopeptide experimental group, the 10 μg / mL C16-SW affinity lipopeptide experimental group, the 5 μg / mL C16-SW affinity lipopeptide experimental group, and the 2.5 μg / mL The C16-SW affinity lipopeptide experimental groups could significantly enhance the expression of Mx1 gene, with the enhancement times being 1.28 times, 2.19 times, 1.46 times and 1.42 times respectively.

[0123] The results showed that C16-SW affinity lipopeptide could significantly inhibit the replication of viral genes at a concentration of 20 μg / mL ( Figure 5 A) and virus release ( Figure 5 B), but only caused a slight increase in the expression of the interferon effector protein Mx1, without causing an excessive interferon response ( Figure 5 Middle C).

[0124] In order to further determine the function of SW polypeptide and expand its application methods, based on the pEGFP-N1 vector (product of Miaoling Biotechnology Co., Ltd., P0133), the gene sequence encoding the SW polypeptide sequence 5'-GCCACCATGAGCTGGTGGTTTCCTCAGTGGATGGCCCAGTACCCTGGCGGCAGC-3' (SEQ ID NO: 7) was inserted into the pEGFP-N1 vector via the HindIII and BamHI restriction sites to ensure the correct expression of the gene sequence encoding the SW polypeptide sequence. The recombinant eukaryotic expression plasmid PEGFP-SW (abbreviated as SW) encoding the affinity peptide was constructed.

[0125] When the RTG-2 cells grow to about 60%, the RTG-2 cells in the logarithmic growth phase are plated at 2×10 5 The amount of cells was added to a 6-well plate, and cultured overnight at 20°C under 5% CO2 to obtain a 6-well plate of RTG-2 cells with a growth rate of about 60%.

[0126] The eukaryotic expression plasmid PEGFP-SW was obtained by replacing the fragment between the restriction endonuclease HindIII and BamHI recognition sites in the pEGFP-N1 vector with 5'-GCCACCATGAGCTGGTGGTTTCCTCAGTGGATGGCCCAGTACCCTGGCGGCAGC-3', while keeping the other sites of the pEGFP-N1 vector unchanged. It was named eukaryotic expression plasmid PEGFP-SW.

[0127] The eukaryotic expression plasmid PEGFP-SW was transfected into 6-well plates of RTG-2 cells grown to about 60%, with a dose gradient of 5 μg eukaryotic expression plasmid PEGFP-SW, 2 μg eukaryotic expression plasmid PEGFP-SW, 1 μg eukaryotic expression plasmid PEGFP-SW and 1 μg empty vector plasmid (pEGFP-N1 vector) / well. The empty vector plasmid (pEGFP-N1 vector) was used as the empty vector transfection control in the experiment. Each experiment was repeated 3 times. The specific operation was as follows: referring to the instruction manual of Lipofectamine 3000 (product of Thermo Fisher Scientific, L3000008), two sterile EP tubes were prepared, and 125 μl Opti-MEM culture medium (product of Thermo Fisher Scientific, 11058021) and 5 μl Lipofectamine were added to tube 1. 3000 transfection reagent, add 125μl of Opti-MEM medium, the plasmid to be transfected and the corresponding dose of P3000 reagent (2μl for every 1μg of plasmid) (reagent in the #L3000008 kit) to tube 2, add the reagent in tube 2 dropwise to tube 1, mix well and perform transfection at room temperature. 24h after transfection, replace the cell culture medium with 2ml of the above-prepared 50TCID 50 The IHN virus solution was replaced with 2 ml of C medium after 2 h and culture was continued. The titer of the cell supernatant and the expression level of the IHNV N gene in the cells were detected 24 h later (the specific steps are the same as the above-mentioned "Detection of IHNV virus titer" step and "Detection of IHNV N gene expression level" step).

[0128] The results showed that compared with the control group, SW plasmid transfection significantly reduced the expression of viral genes, and the 5, 2, and 1 μg SW plasmid transfection groups reduced the expression of viral genes by 0.49, 0.54, and 0.57 times, respectively ( Figure 6 In middle A, the vertical axis represents the relative expression of IHNV genes. The 5 μg SW plasmid transfection, 2 μg SW plasmid transfection, 1 μg SW plasmid transfection, and empty vector transfection controls correspond to the above-mentioned 5 μg eukaryotic expression plasmid PEGFP-SW, 2 μg eukaryotic expression plasmid PEGFP-SW, 1 μg eukaryotic expression plasmid PEGFP-SW, and 1 μg empty vector plasmid, respectively. SW plasmid transfection significantly reduced the virus titer, with the 5, 2, and 1 μg SW plasmid transfection groups all reducing the titer by 0.02 times ( Figure 6In Figure B, the vertical axis represents IHNV titer. The 5 μg SW plasmid transfection, 2 μg SW plasmid transfection, 1 μg SW plasmid transfection, and empty vector transfection controls correspond to the aforementioned 5 μg eukaryotic expression plasmid PEGFP-SW, 2 μg eukaryotic expression plasmid PEGFP-SW, 1 μg eukaryotic expression plasmid PEGFP-SW, and 1 μg empty vector plasmid, respectively. This indicates that cells transfected with plasmids encoding SW polypeptide sequences can still exert significant anti-IHNV effects.

[0129] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A polypeptide derivative, characterized in that: The polypeptide derivative is a C16-SW affinity lipopeptide, and its structural formula is C16-K-SWWFPQWMAQYPGGSYPYDVPDYA-NH2, wherein C16 is palmitic acid and K is lysine.

2. Use of a polypeptide or a pharmaceutically acceptable salt thereof or a polypeptide derivative according to claim 1 in the preparation of a drug for preventing and / or treating and / or assisting in the treatment of infectious hematopoietic necrosis; the amino acid sequence of the polypeptide is Sequence 1 in the sequence listing.

3. Use of a polypeptide or a pharmaceutically acceptable salt thereof or a polypeptide derivative according to claim 1 in the preparation of a drug for preventing and / or treating and / or assisting in the treatment of diseases caused by infectious hematopoietic necrosis virus; the amino acid sequence of the polypeptide is Sequence 1 in the sequence listing.

4. Use of a polypeptide or a pharmaceutically acceptable salt thereof or a polypeptide derivative according to claim 1 in the preparation of an anti-IHN virus drug; the amino acid sequence of the polypeptide is Sequence 1 in the sequence listing.

5. An anti-IHN virus drug, characterized in that: The drug comprises the polypeptide derivative according to claim 1.

6. A method for preparing an anti-IHN virus drug, characterized in that: The method comprises using the polypeptide derivative according to claim 1 as a component of a drug to obtain an anti-IHN virus drug.

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