TWI316944B - - Google Patents

Description

1316944 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to an antimicrobial peptide, and more particularly to an antimicrobial peptide which inhibits and kills microorganisms and which serves as a therapeutic purpose and increases the health of an individual. [Prior Art] Grouper is an important species of farmed fish in the Asia-Pacific region. Due to its delicious meat quality, it has long been the first in the sale of aquatic products, and is widely loved by consumers in Taiwan, Hong Kong, mainland China, Japan and Southeast Asia. Taiwan grouper breeding began in the simple year, the breeding method can be divided into the sea box network 2 and the inner series of water Yu sister. In 199, the Agriculture and Fisheries Commission of the Agriculture and Fisheries promoted Taiwan's Asia-Pacific aquatic crops in the field of +~~, which has listed all kinds of grouper breeding as the primary policy project and is also the focus of science and technology farming (Cai, 2000). Taiwan grouper farming has a history of nearly 2 years, and in 1999 2 (8) 2 years, the interest rate was as high as 2G%. The age of stone is called high economic record, and it is a mature and stable profitable industry in Taiwan. The disease of grouper is mainly divided into two types: virus and bacteria. The virus is a serious disease that is currently harmful to groupers, including iridavims and neuronecrosis virus plus vims. It is two viruses susceptible to grouper infection. Its scar, necrosis virus (NNV) is the most troubled virus of the grouper. This virus has a rapid infection ability and is extremely virulence. It can kill a large number of fry in a short time. Nervous necrosis virus belongs to the family of the genus Actinomy (N〇 (javiride), which has no mantle, and is configured as an icosahedron and a ball off' with double-stranded RNA. ^ Shouting in the fry, the most stomach is infected by the ship, when infected To make the fry lose its balance, swim in the open-loop mode, and it will collide in different directions; the serious sick fish will float on the water surface, float on the side of the body, and suddenly swim quickly when stimulated (Liu, 2002)〇', 'Tianmu I" raw mothers include, Edwards, Vibrio, Aeromonas, Streptococcus and Staphylococcus, which have long been a problem for breeders, and groupers are also susceptible to 1316944 bacterial infections. 'Cause the loss of the industry. There are many reasons for the fish to be dyed by the thief. The most common causes are: high density of feeding, large differences in fish size, residual food, improper water quality, etc. These are easy to make fish. The body produces wounds, which cause the bacteria to be easily infected; the bacterial infection is divided into the body of the body 1 and the sensational wood and local infection; the systemic infection is the part of the bacterial infection throughout each organization's partial sexy _ only « Table or _. When the grouper is infected with g, there will be white spots on the surface of the body. With the disease _ serious, injury σ ulceration, redness, stagnation and visceral bleeding, eventually leading to fish death (Liu, 2002 ), the stone-spotted Taiwanese farmed fish species, but the fish is rectified by the disease, resulting in an annual unstable production. Currently, various studies use vaccines to increase the immunity of the fish; 'The vaccine used to produce poor immunity, often requires additional vaccines to maintain the immune response in the fish, so the vaccine can not effectively increase the immunity of the fish. Not only that, more than 50% of these vaccines use death The pathogens, which have so far allowed the industry to question the safety of vaccines. It is therefore known that it is necessary to study the fish immune system and to find fish antibacterial substances. Antimicrobial peptides (AMPs) are ubiquitous in plants. Among animals, it is a part of the endogenous immune system. Antimicrobial peptides are usually small molecule cations, aged by a positively charged amino acid group. (Hancock and Lehrer, 1998). Antimicrobial peptides from fish, from the meal: fish horn meal: squalamine was discovered (Moore et al., 1993), and many of the fish's antimicrobial peptides have also been published. — Antimicrobial peptides are classified into three major categories according to their chemical structure. The first type is amphipathic α-hdix, because the structural relationship is also called Hnear peptides, and there are no cysts in the structure. Cysteine residues, thus no disulfide bond formation; the second is the formation of intramolecular disulfide bonds, which contain cysteine residues. It can produce a disulfide bond and thus form a hairpin-like β-sheet and an α-helix-p-sheet mix 1316944 ( Bulet et al., 1999); the third class is the proline and/or glycine residues, which contain a highly proline-rich peptide. 'From the size of the molecule can be divided into short keys (sh〇 Rt_chain) and long-chain 〇 In the current study of antimicrobial peptides, anti-microbial peptides have the ability to destroy Gram-positive bacteria, Gram-negative bacteria, fungi and protozoa ( P〇wers and Hanc〇ck, - 2003); in addition to anti-microbial, anti-microbial peptides may also be associated with development (devel〇pment), animal shelling (m〇lting) and reproduction (reproduction); The current study does not reveal any relevant possibilities for the regulation of anti-microbial peptides and immune factors in plants and animals. The antimicrobial mechanisms of antimicrobial peptides fall into two broad categories. The first is peptide-lipid interaction, which can be subdivided into a cylindrical chiseling mode (barrd_stave brain) and carpet-like coverage mode (carpet). Mode) (Shai, 1999). The cylindrical perforation mode is wound into a cylindrical insert with an amphoteric α_helix • (a_hellx) antibacterial peptide, and the hydrophobic peptide is contacted with the cell membrane of the fungus, and the other hydrophilic end is mutually Face-to-face, the hydrophilic end of the antimicrobial peptide faces the center point, and pores are formed on the membrane in the form of a polymer to cause the osmotic pressure inside and outside the bacteria to be non-parallel, resulting in bacterial death (Qiu, 2〇〇1). In addition, the carpet-like coverage pattern φ is the reaction between the hydrophilic end of the antimicrobial peptide and the phospholipids head groups or water molecules after the positively charged antimicrobial peptide binds to the negatively charged cell membrane. After the anti-microbial peptide is inverted, the hydrophobic end portion surrounds a part of the membrane of the bacterial cell, thereby removing the membrane structure and destroying the structure of the bacterial membrane to cause the pathogen to die (Yang, 2, 4). The second category of antimicrobial mechanisms is Receptor_mediated recognition processes. 'Some antimicrobial peptides may be transparent to deoxyribonucleic acid, autolysins, and cells in prokaryotes. The ability (permeabi (iv)) can inhibit the germination of fungal spores and the elongation and branching of mycelium (Thevi_etal, i997). It is also noted in the literature that the ability of antimicrobial peptides to bind to prokaryotic nucleic acids inhibits the production of egg 1316944 white matter ( Powers and Hancock, 2003). It can be seen that there are still many designers who lack the anti-microbial method of the above-mentioned ribs and need to be improved. The inventor of this case is based on the above-mentioned anti-microbial method of animal use. Although various thoughts have been improved and innovated, and after years of painstaking research and painstaking research, I finally succeeded in researching the county point, which is an anti-microbial peptide. [Inventive content] • The purpose of the present invention is to provide Microbial peptides to reduce the pathogens in animals, achieve therapeutic effects, and improve the health of animals The purpose of the present invention is to provide a nucleotide fragment encoding an antimicrobial peptide such as the above-mentioned object of the present invention. Another object of the present invention is to provide a composition for killing microorganisms, the combination - comprising a pharmaceutically effective amount of a peptide fragment, and - a pharmaceutically acceptable carrier. - A further object of the present invention is to provide an immune elicitors for increasing the immune immunity of a sieve vessel A further object of the present invention is to provide a method for increasing the disease resistance of an animal, which comprises orally administering an anti-infective agent to an animal. The antimicrobial peptide capable of achieving the above object of the invention comprises The amino acid sequence shown by SEQ ID N〇: i in the Sequence Listing. — Since the antimicrobial peptide of the present invention is an antimicrobial peptide (amp), the antimicrobial peptide is widely distributed in various animal and plant immune systems. The peptide peptide of the present invention; therefore, the antimicrobial peptide of the present invention can be obtained from a natural animal, especially an aquatic animal, or synthesized by a conventional chemical synthesis method, or a molecular molecule can be known. The present invention also encompasses a nucleotide fragment for encoding an antimicrobial peptide as described above, which has a nucleotide sequence as shown in SEQ ID NO: 2 of the Sequence Listing. 1316944 a pharmaceutical composition for killing a microorganism, the composition comprising: - a pharmaceutically effective amount of a peptide fragment; and a pharmaceutically acceptable carrier. wherein the fragment has the sequence of SEQ ID NO: 1 The amino acid sequence shown. The medium and composition may be in the form of an embedding, soaking liquid, feed, feed supplement, oral '/main shot field, patch, powder, lozenge, injection liquid, suspension Liquids, external liquids, drops, • liniments, paints, creams, ointments, ointments, pastes, wins, and gels. The '5 borne carrier can be an excipient, a diluent, a thickener, a filler, a binder, a disintegrant, a '闰α agent, a fat or non-greasy base, an surfactant, a suspending agent, Glue; suspects, adjuvants, preservatives, anti-rolling agents, stabilizers, colorants, perfumes, etc. The lubricant includes, but is not limited to, glycerin and oils (eg, peanut oil, onion oil). The ruthenium base comprises a hydrocarbon (hydr〇carb〇ns), which includes but is not limited to more rocky soil, soft stone worm, stone butterfly oil, glycerin, bee sting, metal soap, natural oil (such as : almond oil, - corn oil, peanut oil, castor oil or eucalyptus oil), lanolin and its derivatives, fatty acids (such as stearic acid or oleic acid) or combinations thereof. Wherein the surfactant comprises an anionic surfactant, a cationic surfactant, and a non-ionic surfactant. The surfactant includes, but is not limited to, sorbitan vinegar, polyoxyethylene, and derivatives thereof. (eg, polyoxyethylene fatty acid esters), carbopol-like materials (eg, carbopol) c. The suspending agent includes, but is not limited to, natural gums, cellulose ( Cellulose) Bio-inorganic substances (such as: silicace〇us silicas), polyethylene glycol 54 (p〇lyeth plus e-glycan 1 540), polyethylene glycol 3350 and propylene glycol (propyl glyC〇i). Wherein the gelling agent comprises any gelling agent for pharmaceutically forming a colloid, the gelling agent including but not limited to a cellulose (Cellulose) derivative (eg, methyl cdlul〇se, hydroxyl group B16944 B) Hydroxyethyl cellulose and carb〇xymethyl cellulose, vinyl polymers (vinyi p〇iymers) (eg, polylyl alcohols), olyvinyl pyrrolidones, polyethylamine (carboxypolymethylene) derivatives (such as carbopol), pectin, gums (gUms) (eg, gum arabic, tragacanth, alginates, agar, gelatin (gdatin) ) and carrageenates). Among them, 3 Xuan medicine compositions are administered to humans and animals by oral, soaking, injecting, smearing or patching.

Wherein the animal may be a livestock animal or an aquatic animal; when it is a livestock animal, preferably a cow, a sheep, a chicken or a pig is better as a chicken; when it is an aquatic animal, a preferred domain is a better one. For the culture of fish; the term "aquaculture fish" as used herein refers to fish that can be cultivated by humans, preferably fish cultured by aquaculture workers, such as grouper (gr〇uper), sea bream (c〇bia), Net fish (10) &_), halibut (hahbut) 'salmon (saimon), squid (tr〇m), remaining fish (catfi_fish (goldfish), tilapia (tilapia) and zebrafish (zebrafish), but Not limited to this. The present invention further provides a method for screening immune elicitors that increase the immunity of an animal, comprising:

Step-mixing the desired immunochemical agent with the mixture to obtain a mixture; the second step is to - the animal oral needle, and the mixture can be administered by soaking. The third step is to detect the animal. The expression amount of the peptide fragment shown in SEQ ID N〇: 序列 in the sequence table, and the y-throat: the secret of the amount of silk from the silk to the cause of the disease. In the third step, the detection method of the secret peptide fragment _ class is not particularly limited in this wealth, and the amount of performance of any green check is, for example, colloidal electrophoresis, western ink Point method, immunoreactivity method, etc., but not only ^ 1316944 • The detection of the peptide fragment can also be determined by detecting the amount of mRNA encoding the peptide fragment, and the sequence of the mRNA is A sequence transcribed from the acid sequence shown by SEQ ID NO: 2; a method for detecting the amount of mRNA expression, such as, but not limited to, the northern blot method. The animal of the present invention may be a silk animal or an aquatic animal; when it is a livestock animal, it is preferably a cow, a sheep, a chicken or a pig, and more preferably a chicken; when it is an aquatic animal, it is preferably Fish, * Better known as farmed fish; referred to herein as farmed fish, refers to fish that can be raised by hand, compared to fish cultured by aquaculture, such as grouper, sea otter ( C〇bia),调• ^(-bream) . tb a , (halibut). , (salm〇n) ^ ^ ^ # ^ squid (c ton), goldfish, goldfish (tilapia) and zebra The fish (small melon ring is limited to this. The present invention further provides a method for increasing the disease resistance of an animal, comprising: • Transfer A provides a fresh and effective amount of an immunosuppressant, the immune inducer is capable of inducing an animal - suspending the order The antimicrobial peptide shown in SEQ ID NO: 1 is listed; Step B provides an animal; and Step C is administered to the animal in the body. • wherein the immune inducer can also be used to screen A method for increasing the immunity of animal immunity by i_neelicitors, with the sequence listing SEQidn〇: i The expression of the winning form or the immunological-inducing agent capable of increasing the immunity of the animal by screening the amount of the SEQ ID NO: 2 mRNA gene, and the immune inducing agent selected. The immune inducing agent can further comprise - pharmacy An acceptable carrier. The peptide fragment can be obtained from a natural animal, especially a water-like object, or synthesized by a conventional chemical synthesis method, and is also known as a molecular biological method (4). The molecular biological method is as follows: Step a provides a nucleotide fragment as shown in SEQ ID NO: 2 of the Sequence Listing; Step b provides a representation vector; 11 D16944 Step c Recombines the nucleotide fragment with the expression vector into a recombinant expression vector; The recombinant expression vector is delivered to the host cell; and step e causes the host cell to express the peptide fragment, wherein the host cell can be E. coli (five c〇/〇, yeast, and animal cells, but is not limited thereto). Escherichia coli (where co/z·) is used as an example. After selecting the plastids of the E. coli delayed response system, the appropriate restriction enzyme site in the poor body is selected. Enzyme-clearing the plastid, and also cleavage of the nucleocapsid fragment containing the nucleotide sequence of the nucleotide sequence encoding the peptide of the present invention by the restriction enzyme And the nuclear bud fragment is subjected to ligation> followed by Escherichia coli containing a nucleotide sequence encoding the peptide fragment of the present invention (V. system expressing plastid transformation (transf〇rm) into Escherichia coli (five In pure bacterial cells, the large intestine rod can be used for £(7)"BL21TM (DE3) pLysS, and any conventional transformation technique such as electr〇-transf_ation or heat shock can be used. The transformation is carried out with the inducer _ and the induction of the large intestine rod _•(3)/〇 shows the peptide fragment of the present invention, and the inducer may be isopropyl thiohalfion _SGpmpylthiQgalaetoside, IPTQH stomach Μ _. Taking yeast sputum as an example, after the ( 酶 四 及 及 及 及 及 及 及 及 及 及 及 及 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 ; ; ; ; ; ; ; ; ; The nuclear welix-like british acid fragment of the peptide fragment of the present invention will be subjected to a ligation reaction with a cleavage-expressing plastid and a nucleotide acid fragment, followed by a (tetra) acid sequence which encodes the segment-segment of the present invention. In the expression of the yeast system, the transformation can be carried out by any conventional transformation technique. For example, the cell wall of the yeast can be removed first, and the yeast can be transformed into a protoplast spheroid (sphemplast). Or use a basic anion (such as Lici or Rbc'i)

The heat shock is used to treat the yeast for transformation, and after transformation, the yeast is used to induce the yeast to express the peptide fragment of the present invention, and the inducer may be a known inducer such as IPTG. X Take animal cells as an example. The sputum cell system can be smear and the plastid is 121316944. After the restriction enzyme cleavage position, the zygote is cut into the genus, and (10) the cut contains The stalk-like nuclear nucleus of the peptide-like fragment of the present invention can be subjected to a ligation reaction with the current negative and nucleotide fragments after cleavage by a shut-off enzyme, followed by containing a peptide fragment encoding the peptide of the present invention. The expression of the animal cell system of the nuclear sequence is transfected into the animal cell. The secret cell can be an insect cell or a mammalian cell, and the transfection can be performed by a female transfection technique, such as a silky feeling on the lipGfeetiQn. __(iv) ium (four) method - to represent the peptide fragment of the present invention. In addition, in order to effectively improve the disease resistance of the animal treated by the antimicrobial method of the invention, the aforementioned immune inducer can be administered to the animal by oral, immersion or injection; Directly orally or added to the feed, when administered orally, in order to prevent the immune inducer from being destroyed by the digestive juice in the digestive tract when passing through the digestive tract of the animal, the immunologically decomposing agent can further advance It is micro-(four) treated by conventional techniques to enhance its ability to resist digestive juices; the injection methods include intravenous injection (IV), intramuscular injection (Intram Na·injection, IM), and intraperitoneal injection (intraperit〇). Neal et al, but not limited to this; when administered intravenously to animals, in order to maximize its effect. 隹 The composition or the triumph of the invention can promote the animal against pathogen invasion, so Increasing the disease resistance of the animal and improving the health state of the animal. The composition or peptide of the present invention can also be used as a preservative for external use and a bactericide. Example 1 Preparation of the antimicrobial peptide (AMP) fragment of the present invention First, total RNA was isolated from the total silk tissue of gentian grouper/iOTceo/a plus according to the method of Chomczynski and Sacchi (1987); Down, synthesize cDNA from the total leg of the 1st net, using 200UMolony leukemia virus reverse transcriptase, 1 mM dNTP, 160 U RNase inhibitor 13 1316944 (inhibitor) and 1.6 Pg p machine primer (rand〇m primei^ ιχ reverse transcription buffer, transcribed at 42 〇c for 30 knives and then amplified by & gestation chain reaction (must coffee: take this reacti〇n, pcr) to enlarge ( Amplify) the anti-microbial peptide (AMp) gene, pCR uses _ χ χ pCR buffer containing 2 〇 positive reverse transcription product, 0.5 UTaq DNA polymerase and 1 forward and reverse AMp primer, and the forward AMP primer has SEQ ID NO: The nucleotide sequence shown in 3, the reverse AMP primer has the nucleotide sequence shown in SEQ ID NO: 4, the pCR is carried out in a DNA temperature cycle reactor, and the pCR step is at 55 ° C. Slow cooling pairing (annealing) l minutes 30 seconds, 'in 72 C extension (^ Lang 1011) 1 minute 30 seconds At 94. (: denaturation 1 minute, total • 35 cycles (Cyde); after the PCR reaction is terminated, 15 μΐ of the PCR product is analyzed by electrophoresis of 21⁄4 agarose gel, followed by vinyl bromide_ ____dyeing. The purified PCR product will be cloned into the M13mpl8 or pBluescript position and

Will be sequenced by the dideoxynucleotide chain termination method, the AMP group 'in & sequence will be followed by the sequence shown in SEQ ID NO: 2 of the Sequence Listing; from the sequence listing SEQ ID. N〇 The sequence shown in 2 shows that the anti-microbial peptide (AMP) cDNA sequence selected from the gentian group filament has 494 nucleotides, of which the translation region ((;〇(1丨叩regi〇) n) consisting of 2〇4 nucleotides', as shown in Figure 1, after derivation, an anti-microbial φ peptide (AMP) with 67 amino acids is obtained. The sequence is as shown in the sequence table seq. ID NO: 1. On the other hand, the purified PCR product was cleaved with the expression vector pET31b(+) with restriction enzyme d/vvNI, and the cleavage PCR product and pET31b(+) were ligated. The PCR product-containing pET31 b(+) obtained after the ligation reaction was transformed into the expression host five co/z_BL21TM (DE3) pLysS 'in a shaking culture at 37 ° C at 200 rpm' and induced with different concentrations of IPTG (induce The results were best induced by 1 mM IPTG. The 12% SDS-PAGE analysis revealed that the AMP peptide represented by E. coll BL21TM (DE3) pLysS was not located. The lysin fraction has a molecular weight of about 2.7 kDa, as shown in Figure 2. The Western ink-crush method also proves to be an AMp peptide. The obtained insoluble protein fraction is subjected to histidine-acid chromatography (His. Bind® chromatography). )

14 1316944 Purification followed by shearing with cyanogen bromide (CNBr) to obtain a purified AMp peptide. Example 2 Ratio of Antimicrobial Peptide (AMP) Sequences of Different Animals In the known species, 'antimicrobial peptides are all peptides of small molecules, and the antibiotics selected in the first embodiment of the present invention are The peptide is compared with the antimicrobial peptide amino acid sequence of other fishes, and the alignment result is shown in FIG. 3, and the antimicrobial peptide and the spotted grouped antimicrobial peptide amino acid sequence of the present invention are the most Similar in 'and in other species, carp and flounder are similar' and in the classification of antimicrobial peptides, 'the species are different, but in the same species - the types of antimicrobial peptides are different, regardless of Vertebrates or invertebrates are hard to find a regularity. The antimicrobial agent of the present invention has 67 amino acids (as shown in the sequence table seq id NO·1), which contains a signal peptide, a mature peptide (_coffee peptide) and a prodomain' 3D stereo The structure is shown in Figure 4. The message peptide has an amino acid sequence as shown in the Sequence Listing SEQ m N〇: .5, which produces a function to cleave mainly during intracellular signaling, leaving the mature peptide and Pr〇d〇main. The mature peptide has an amino acid sequence as shown in the sequence table SEqidn〇: 6, which is a position mainly acting on the pathogen, and has a charge to bind to the cell membrane of the bacteria. Currently, the AMP mode of action is a cylindrical perforation mode ( The barrel_stave m〇de) is inserted into the membrane of the cell with a strong antibacterial peptide; the hydrophobic end of the antibacterial peptide is contacted with the cell membrane of the bacteria, and the other hydrophilic end faces each other, and the hydrophilic end of the antibacterial peptide is faced. At the center point, pores are formed on the membrane in the form of a polymer, so that the osmotic pressure inside and outside the bacteria is not parallel, resulting in the death of the cells (Qiu-2001). Carpet-like coverage mode (caiPetmocie) (Shai, 1999). After the positively charged antimicrobial peptide is combined with the negatively charged bacterial cell membrane, the hydrophilic end reacts with the membrane, and the antimicrobial peptide is turned over to cause a hydrophobic portion, which surrounds part of the bacterial cell membrane to remove the membrane structure and destroy the bacterial membrane. The structure makes the pathogen® die (Yang, Shi 4). Causes the death of bacteria, effectively resisting the pathogens to produce endogenous immunity.

Prodomain has an amino acid sequence as shown in SEQ ID SEQ.7 of the Sequence Listing, and its function is 15 1316944. The film is widely replaced with a charge that neutralizes the mature peptide. When the foreign disease (4) acts, the mature peptide is not recorded in the lungs to avoid injury # to the host peach Yin heart, 2_). The silk bond is positively charged with the negatively charged _ cell membrane side, while the cell membrane of the organism itself is also negatively charged, and the antibacterial condition is specifically identified as the foreign disease M and the organism itself, so it is necessary to (4) (10) blood to neutralize the antibacterial The charge of the peptide so as not to cause damage to the organism. Example 3 Structure and polarity analysis of gentian plaque antimicrobial peptides SEQ ID NO: 6 was analyzed using Kyte-Doolittle hydropathy blots. AMP mature peptide showed significant hydrophobicity and hydrophilicity after 7th to 16th amino acid ( Intersecting areas (as shown in Figure 4, Figure 5 and Figure 14). Analysis of hydrophobic and hydrophilic silk-distribution and 3-D structure maps using the HeUeal Whed instruction of the National Institutes of Health's giant tissue analysis pjOtein analysis can reveal the α-helix formed by the 5-spot antimicrobial peptide ( a helix) has a distinctly hydrophobic and hydrophilic fraction (as shown in Figures 5 and 14). The antimicrobial peptide belongs to the amphipathic molecule and is a traditional antibacterial peptide (Hancock and Lehrer, 1998) ° It can be inferred from this example that the bactericidal mechanism of the gentian plaque antimicrobial peptide is a cylindrical chiseling mode or a carpet-like covering mode, because the gentian plaque antimicrobial peptide structure is α-helix positively charged, when When killing the cell membrane of the pathogen, the antibacterial peptide needs to be an amphiphilic molecule in order to smoothly create pores in the membrane of the pathogenic bacteria, resulting in an imbalance of osmotic pressure to cause the pathogen to die (Shai, 1999). The main function of N_myrist〇ylati〇n ske at the 30th to 35th amino acid position after ExPASy Proteomics software analysis is to combine protein deuteration with C-14 saturated fatty acid, presumably to bind antibacterial peptide to lipid filling on cell membrane ( Towleretal., 1998). It can be known from the 3D stereogram prediction that the gentian grouper AMP is divided into two parts' and the part of the mature triumph is a_heiix structure (as shown in Fig. 4), which belongs to the first class of amphoteric antimicrobial peptides. It is speculated that the antimicrobial peptide of the present invention may be mainly located at the cell membrane with bacteria. Example 4 Distribution of Antimicrobial Peptide (AMP) mRNA in Grouper 16 1316944 This experiment analyzes the antimicrobial peptide (AMP) gene in gentian grouper by real-time quantitative PCR. The performance patterns of each organization. Using the fluorescent dye SYBR green I can be placed on the DNA double-strand groove and excited by a halogen lamp to produce a fluorescent property's measure of the amount of fluorescence. When SYBRgreenI is not embedded in double-stranded DNA, the background value of the fluorescence is very low; when SYBR green I is first embedded in the target gene fragment amplified by PCR, the fluorescence signal will increase relatively. If there is no binding of non-specific primers, or the interference of gene-group DNA (g(10)mic DNA), the synthesis state of DNA synthesized by PCR reaction can be distinguished as: (1) Double multiplication of geometric progression multiplication (geometric φ phase); (2) The linear phase of non-double doubling of gene synthesis when the reactant is insufficient; and (3) the last reactant depletion, failure, and the plateau phase of the reaction endpoint. Therefore, in order to detect the amount of expression of the target gene in tissues and organs, it must be quantified in the geometric phase of the pcR reaction. The principle of Real4ime quantitative pCR is that • with different concentration templates, under the same PCR reaction conditions, the reaction with high concentration of template will be faster than the doubling period of the series, and the relative 'low concentration template will be slower. The number of critical PCR cycles defined to reach the midpoint of the geometric progression 彳σ period is determined as (threshold cycle), that is, the C!T value increases as the template concentration decreases. Using the different concentrations of standard products to simultaneously quantify the pCR response of the CT call #, the software calculates the standard curve and the regression formula, and interpolates the absolute expression of the gene containing the heart in the sample to be tested. At the same time, if a Meiting curve is to be obtained, a melting temperature 66 is performed after the target gene amplification cycle.匸_99. The result of the continuous fluorescence detection of the 萤 析 熔 熔 可以 可以 可以 可以 可以 可以 可以 可以 可以 可以 可以 可以 可以 可以 可以 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 41 extraction of total RNA from all tissues of the group of plaques (total RNA) knives take the spotted brain, silk, eye, heart, head kidney, liver, spleen, intestine, stomach, muscle blood, skin tissue, join 10 times the volume of TRIzol reagent (Invitrogen, USA), use the knife to first cut the tissue and then use a homogenized rod to break, add 1/5 volume of air imitation, 17 1316944 vigorously shake for 30 seconds, let stand at room temperature 10 minutes, centrifugation at uoooxg for 15 minutes' Aspirate the supernatant into a new microcentrifuge tube, add TRIzol reagent in equal volumes of isopropanol, mix evenly, and let stand at room temperature for 1 minute, at 4 . Centrifuge at 12000 xg for 1 minute; dissolve the RNA in 20 μl DEPC water, dissolve at 55 ° C for 10 minutes, and store in a -80 ° C freezer to measure the ratio of rna concentration to purity. 4.2 Reverse Transcriptase Reaction (ReverseTranscripti〇n) The extracted RNA was quantified to 5 pg of total RN A per tube and subjected to reverse transcriptase reaction. 4.3 Real-time quantitative polymerase chain reaction (reai_time qUantitative pcr) The specific primer (Primer) designed the AMP gene partial cDNA sequence according to SEQ ID NO: 1 'Designing an instant quantitative polymerase chain reaction-specific primer 'AMP gene using LightCyclerProbeDesignprogram Real-time quntitative PCR was performed using real-time quntitative PCR AMP specific primers A3 and A4 (the sequences are shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively). 4·4 real-time quantitative polymerase chain reaction (rea _time qUntitative PCR) 4·4·1 standard curve preparation of mini-preparation (Mini-preparation) • SEQ ID NO: 1 sequence and identified containing Mx gene and The AMP gene partial cDNA sequence was removed from the -8 (TC refrigerator), cultured in 30 ml of LB/Ampicillin medium (Luria-Bertanimedium), and cultured at 37 ° C for about 16 hours overnight, and the bacterial solution was taken out. _ and prepare a small amount of plastids. Take 1 ml of the overnight culture solution and put it into a 1.5 ml microcentrifuge tube, and centrifuge at 5 °C for 1 minute at 12,000 xg. Add the supernatant and add 150 μΐ. Resuspend the cells in Solution I (50 mM glucose, 25 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0), add 200 μl of freshly prepared Solution II (0.2 N NaOH, 1% SDS), place on ice for 10 minutes, add 150μ1 Solution III (3 M potassium acetate, 10% glacial acetic acid), placed on ice for 5 minutes 18 1316944 hrs. Centrifuge at 12,000 xg for 5 minutes at 4 °C, collect the supernatant into a new microcentrifuge tube. Add an equal volume of phenol and chloroform, shake vigorously 30 For one second, let stand at room temperature for 10 minutes, centrifuge at 12,000 xg for 15 minutes at 4 t. Collect the supernatant into a new microcentrifuge tube, add twice the volume of 95% iced alcohol, mix evenly - Centrifuge at H,000xg for 5 minutes at 20 °C for 20 minutes at 20 °C, pour off the supernatant to leave a precipitate. Wash the precipitate twice with 70% alcohol, then dry the precipitate by vacuum drying. Dissolved in 20μ1 sterile water, stored in _2〇. 〇 spare. 4.4.1.2 Calculation of plastid DNA transcript and purity OD260 is measured by spectrophotometer, and the standard for calculating the concentration of plastid DNA is 1 OD260 = 50 pg of DNA / ml, the formula is 50 pg X dilute factor X A260= pg / ml. The purity of plastid DNA is between 0.01 and 1.8 as the pure DNA in the ratio of OD260/OD280. After the plastid DNA concentration is calculated, The plastid DNA concentration was adjusted to 1 μ§/μ1, and serially diluted from 10·1 to 10-9 in a 10-fold dilution manner. After dilution, the plastid DNA was stored in _2 〇. 〇. 4.4.1.3 Preparation standard The curve is taken from the diluted plastid solution of 10_4-1〇_8 and placed in the reaction capillary, and then added 2_4 μΐ MgCl2. 1 μΐ SYBR, real-time quntitative PCR AMP specific primers A3 and A4 (the sequences are shown in sequence table SEq ID no·· 8 and SEQ ID NO: 9 respectively) 0·5 μΐ, 12.6 μΐ sterile water' last The total volume was 2 〇μ1; the reaction capillary was placed in a timely quantitative analyzer (LightCycler 1.2), and the following conditions were set: 95 ° C dissociation for 10 minutes; 95. (: dissociation leap seconds; 64 ° C bonding 10 seconds; 72 ° C synthesis 15 seconds; dissociation, bonding, synthesis steps repeated 40 cycles, after cooling to 40 ° C, continued to heat up to 95T: to dissociate the reaction product and continue Fluorescence values were detected. After the reaction, the results were analyzed by LightCycler Software 3.5 program to prepare a standard curve. 4.4.2.1 gentian grouper tissue real-time quantitative polymerase chain reaction detection 3 μΐ obtained in Example 4.2 The reverse transcriptase reaction (RT) product was placed in a reaction capillary, subdivided into 19 1316944, 2.4 μΐ MgCl2, 1 μΐ SYBR, real-time quntitative PCR AMP specific primers A3 and A4 (the sequences are shown in SEQ ID NO: 8 and SEQ ID NO: 9) 0.5 μΐ, 12.6 μΐ sterile water 'final total volume is 2〇μΐ. Place the reaction capillary in a timely quantitative analyzer (LightCyclerl.2) 'Set the following conditions to react · 95 °C dissociation for 10 minutes; 95. (: dissociation leap seconds; 64 C bonding for 5 seconds; 72 C synthesis for 15 seconds; dissociation, bonding, synthesis steps repeated 4 cycles and then cooled to 40 ° C and continued to heat up to 95. : 'To dissociate the reaction product and continuously detect Fluorescence value. The post-reaction detection results were analyzed with LightCycler Software 3.5 program and standard curve. The unit of data obtained after analysis was μβ/μΐ, then divided by the number of pairs of plastids, and then divided by the basis weight of 66〇dalton. The data obtained by multiplying the mole/μb by 6.02x1023 to obtain the copies/μb experiment, using the (Statistic Alalysis System) software, using univariate analysis (〇ne-way anaiysis 〇f variance), using Duncan's MultipleRange Tes to compare each The degree of significant difference between factors (p < 0.05), the data obtained is expressed as mean soil standard deviation (Mean soil SD); all methods of AMP gene expression analysis in this invention are used. See Figure 6 'from AMP gene in each organization Experimental results show that gentian grouper has AMP gene expression in brain, heart, liver, spleen, intestine, stomach, muscle, fluid, total silk, eyes, head kidney, skin and fin; The highest amount of performance, followed by the spleen, silk, blood. These tissues have blood flow related tissues, further proof that the AMP gene may be secreted by blood cells. The AMP study of the fish pointed out that it is also an important immune organ in fish (Murray ei α/·, 2003); and the results of this example show that the AMP gene has obvious performance in silk (see Figure 6). Therefore, in the next embodiment of the present invention, the total filament is used as an indexing tissue for analyzing the expression of the AMP gene. Example 5 Injection of a virus-like nucleic acid drug polyinosinic-polycytidylic acid (polyI:C) into a gentian group to analyze the performance of the antimicrobial peptide (AMP) gene. The gentian grouper was divided into four groups, each group was injected differently. Concentration of the virus-like nucleic acid drug polyinosinic-polycytidylic acid (polyI: C), in grams per gram of fish body weight (BW), 20 1316944 grams per gram of fish body weight of injected drugs is 200μ1, the concentration of poly I: C injection They were Opg/gBW (control group), lpg/gBW, 2pg/gBW, 5pg/gBW, and the solvent was grouper PBS, which was injected intramuscularly and injected into the back muscles. After 24 hours, 15 〇ppm of MS-222 (Ethyl 3-amino-benzoate methanesulfonate solt) was used to reduce the stimulation of fish anesthesia; seven tissues including silk, head kidney, spleen, brain, blood, liver and skin were taken. The total RNA was extracted with TRIzol reagent and stored in a -80 °C refrigerator for real-time quantitative PCR analysis of AMP gene expression patterns. The AMP gene expression of each tissue after injection of different concentrations of poly I:C for 24 hours is shown in Figure 7. The total silk is used as an indicator of the performance of the AMP gene. 2pgpoly I:C is injected per gram of fish body weight. The treatment group had the highest AMP gene expression. For the treatment of 2 pg of poly I:C per gram of fish, the total RNA of the head kidney, brain, spleen, liver, blood, skin and silk was taken at 12 hours, 24 hours, 48 hours, and 72 hours after injection. 'Using immediate expectation; polymerase chain reaction (reai_tjme quantitatjve pcr) analysis of AMP gene expression pattern, the results shown in Figure 8, under the long-term treatment of 72 hours, the total and silk AMP gene performance compared with the control group High, it is speculated that because the organism-induced antimicrobial peptide is the first line of defense, when the foreign substance enters the organism, the antimicrobial peptide (AMp) gene may also be induced to fight against foreign objects. Example 6 'Hp〇p〇丨y Saccharide (LPS) with different stimuli to the gentian plaque, and analyzed the performance of the antimicrobial peptide (AMP) gene. The gentian grouper was divided into four groups. Each group was injected with different concentrations of Lps, and the weight per gram of fish body weight (BW) was injected into the sputum (control group), 5 咫, 1 ton, 15 咫 LPS' per gram of fish body weight per gram of fish body weight. 2 〇〇 μιη incomplete injection adjuvant was added to the injection. 'Injection method is intraperitoneal injection. After 24 hours, i5〇ppm MS-222 (Ethyl 3-amino-benzoate methanesulfonate s〇lt) was used to reduce the stimulation of fish anesthesia; the liver, brain and spleen liver blood and total silk were taken separately and extracted by TRjzoi reagent. Total RNA,

21 1316944 was stored in a -80 ° C refrigerator for real-time quantitative polymerase chain reaction (4) plus q_titative PCR) analysis of AMP gene expression patterns. The AMp gene expression of each tissue after injection of different concentrations of LPS for 24 hours is shown in Figure 9. The AMP gene in the silk tissue and the head kidney tissue at each concentration of Lps showed a significant increase in the performance compared with the control group (ρ&lt ;0·〇5) 'Shows that the amount of AMP gene induced in different tissues is different. LPS was injected intraperitoneally with 15 ng of body weight per gram of fish, and samples were taken at 24 hours, 48 hours, 96 hours and 132 days, respectively, to investigate the gene expression of the head kidney, brain, spleen, liver, blood and silk. As shown in Figure 10, the results showed that the AMP gene of silk tissue was significantly increased in 48 hours. The spleen and head kidneys showed a significant increase in expression at 24 hours. It can be inferred that the immune response rate of various organs of the organism is not the same, and the tissue produced by the immunity is changed because of the location of the stimulation. In Kim (2〇〇5), it was found that the time to produce an immune response in the different tissues of L. sinensis was different, and • The expression of some tissues decreased over time. - Example 7 Analysis of the performance of gentian grouper anti-microbial peptide (AMP) gene after changing the environmental factor salinity The gentian grouper was divided into a control group and an experimental group, and the control group was pure seawater (salinity _ 35 %.) 'And experiment _ daily salinity reduction brain treatment three angels experimental group salinity is $ placed 24 】, after the 'use 15 〇 ppm of MS-222 (Ethyl 3-amino-benzoate methanesulfonatesolt) 'Let the fish anesthesia reduced Stimulation; taking total silk, head kidney, spleen, brain,

The liver tissue of the night liver was extracted with TRIzol reagent and stored in the -8 ° ° C 'ice phase' to analyze the AMP gene expression pattern by real-time quantitative PCR. As shown in Figure 10, the experimental group had an increased amount of AMP gene in the tissues of the head kidney, skin, blood, and total silk in a low salinity environment. It can be seen that when the salinity changes, the immune gene will be induced, which will increase the immunity of the fish and change the salinity to 5%. When speculated, it will stimulate

22 1316944 The genital plague system of the gentian grouper makes the gentian grouper susceptible to infection or onset by pathogens, making gentian grouper less susceptible to bacterial and viral infections. Fish immunization urgency includes: changes in salinity, temperature changes, insufficient dissolved oxygen, chemical pollution, etc., causing many physiological changes in the fish's response to the environment. When the environment is urgent, the physiology, appearance, tissues and cells of the fish will produce an immune response (Bart〇n Blanchard, 2001). The results of this example are further confirmed by changing the salinity of the culture environment. The amount of performance further enhances the ability of anti-viral and anti-bacterial infections of gentian grouper; this concept can be applied to aquaculture to improve the immunity and disease resistance of fish, and to reduce the mortality rate of death and death. With the purpose of production and so on. Example 8 In vitro susceptibility test of chemically synthesized grouper antimicrobial peptide to each pathogen The antibacterial peptide was synthesized by Coroine International Technology Co., Ltd., and the purity was tested by high performance liquid chromatography (HPLC). Purity of more than 90%; • Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) test for bacteria using the synthetic antimicrobial peptide. 8.1 Preparation of bacterial solution ❿ Escherichia c〇li mi5a, Vibrio harveyi, Vibrio alginofyticus, Vibrio anguilhrum, Vibrid Salmonicida), 轰气^ A 岚 岚 A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A ), after 16 hours 37. (: After culture, scrape the colony and dissolve it in the designated culture solution, so that OD54 is 1 (concentration is about 1X109), and 500μ1 of bacteria is added to 5〇〇μ1 of LB or TSB (+1.5% NaCl). The liquid concentration is ΐχ108 bacteria/πύ. 8.2 Susceptibility test Using 96-well culture alone, add 130 μM of bacteria solution to each groove. The concentration of the bacteria solution is lxl〇8 bacteria 23 1316944 number/m b in the addition of 20μ1 Different concentrations (0.075 μΜ-0.675 μΜ) of chemically synthesized gentian sarcophagus antimicrobial peptide, cultured at 37 ° C for 16 hours, observed the lowest antimicrobial peptide concentration of clarified bacterial liquid without bacterial growth, this is Minimum inhibitory concentration (MIC); a group of clarified bacterial liquids will be presented, and the complete bacterial solution will be applied to culture on LBA (£· co/z· DH5a) or TSA (+1.5% NaCl;) After incubation at 37 ° C for an hour, the minimum bactericidal concentration (MBC) was observed for the colonies, and each experiment group had three replicates and a control group. The test results are shown in Table 1. The minimum inhibitory concentration (MIC) of the seven strains were respectively For: E. coli cc»" DH5a) 0.075 μΜ, Vibrio harveyi 0.15 μΜ ' Bathing fox fungus (ί^· π·ο 0_15 μΜ,, Vibrio anguillarum (Ρ%η_ο 0.15 μΜ, Vibrio cholerae (volume η·ο sa/wom'c/iia) 0_ 15 μΜ, Aeromonas aeruginosa (Jero inflation y / y; Ορ/π./α) 0.375 μΜ, Staphylococcus epidermidis ep/i/erm/ifo) 0.075 μΜ; and the minimum bactericidal concentration (MBC) is 0.15 μΜ, 0.225 μΜ, 0.375 μΜ, 0.45 μΜ, 0.3 μΜ, 0.675 μΜ, 0.525 μΜ. Table 1 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of gentian plaque antimicrobial peptide (AMP) for different pathogenic bacteria Minimum bacteriostatic concentration (MIC) Minimum sterilization Concentration (MBC) Gram-negative bacteria Aeromonas hydrophila 0.375 μΜ 0.675 μΜ Vibrio an^uillarum 0.15 μΜ 0.45 μΜ Vibrio salmonicida 0.15 μΜ 0.30 μΜ Harvey's arc Vibrio harveyi 0.15 μΜ 0.225 μΜ Vibrio al^inolyticus 0.15 μΜ 0.375 μΜ Escherichia coli DH5a 0.075 μΜ 0.15 μΜ Gram-positive Staphylococcus epidermidis 0.075 μΜ 0.525 μΜ 24 1316944 The results of this embodiment can be The antimicrobial peptide of the present invention can kill the Gram-positive bacteria and the Gram-negative bacteria, and has the effect of killing and inhibiting g to different microorganisms. Therefore, the antimicrobial peptide of the present invention can also be used as a broad-spectrum effect. Bactericide or preservative. Example 9 Protective effect of antimicrobial peptide (AMp) on grouper The gentian grouper was divided into four groups 'injected pBS (control group), Vibrio (technical), /Ixio cfb/fish, and antimicrobial The peptide cecr0pin (concentration i _/flsh) + Vibrio (concentration lxlO6 cfb/fish), the antimicrobial peptide of the invention (gAMP, concentration 丨pM/fish) + Vibrio (concentration lxl06ciWflsh), After 24, 48, and 72 hours, the mortality of each group was observed. The results are shown in Fig. 12. The mortality rate was 〇% after 72 hours in the PBS group (control group) and 72 hours after the injection in the Vibrio group. 100%; the injection of the antimicrobial peptide cecr〇pin+Vibrio group had a mortality rate of 80% after 72 hours; the injection of the antimicrobial peptide (gAMp) + Vibrio of the present invention had a mortality rate of 60% after 72 hours; The experiment only injected various AMPs once, and the results showed that 'injection of the anti-microbial peptide (gAMP) of the present invention can significantly reduce the mortality of gentian grouper 40%, and it can be seen that the anti-microbial peptide (gAMP) of the present invention has reached the fish. Only provide protection. The test for the effect of injecting the antimicrobial peptide (AMP) of the present invention against virulence can directly prove that the use of the antimicrobial peptide (AMP) of the present invention does increase the resistance of the grouper. Since the AMP has a broad-spectrum bactericidal ability, the use of the antimicrobial peptide (AMp) of the present invention can protect animals and improve their disease resistance. The animal should not be restricted to aquatic animals, but also livestock animals and humans. Example 10 Analysis of the performance of the gentian grouper anti-microbial peptide (AMP) gene after immersing the Chinese herbal medicine The gentian grouper was divided into four groups, which were respectively a control group in which no Chinese medicine was placed in pure sea water, and each contained 50 ppm of radix isatidis ( / Like Yi Jia <% such as α〇, 黄耆 (4) about the job _ _ such as α biliary;; e (10)), licorice plus wra / emzi) seawater treatment group; with strong air to make the Chinese medicine evenly mixed In 30 liters of water, after soaking for 24 hours, use 150ppm of MS-222 (Ethyl 25 1316944 S-amino-benzoatemethanesulfonatesolt) to reduce the stimulation of fish anesthesia; take silk, head kidney, spleen, brain, blood, liver, Tissues such as skin, using TM Ζ〇ΐ 明 加 加 加 加 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , It is shown that the treatment of the soaked Astragalus membranaceus has a significant difference in the expression of AMp gene in the gray liquor (ρ<0.〇5); it can enhance cell production due to the induction of scutellaria interferon.

Interferon capacity (segment, 2〇〇5). In addition, in the mouse study, the saponin component of the astragalus extract contains cydoastmgenol and cyd〇galegenin, which can increase the ability of immunoglobulin (lgG) (Yangd虬, 2005). The results of this example show that Astragalus membranaceus has significant immunoregulatory functions in animals (including aquatic animals), including non-specific immunity, and can induce the production of interferon, which is a strong biological regulator, which is very clinically ill. Effective drugs. The anti-microbial assay provided by the present invention has the following advantages when compared with (4) Lai Sui: 1. The anti-microbial peptide gene of the present invention is in the immune system as described above. Analysis of performance, antibiotic resistance, physical factor (salt), immunostimulant (polyl.C and LPS), Chinese herbal medicine, etc., all confirmed that the antimicrobial peptide gene expression of the present invention and the immunity of grouper were positive. Relatedly, when the amount of the antimicrobial peptide (AMp) mRNA is increased, the resistance of the grouper is also increased; and the effect of injecting the antimicrobial peptide (AMp) of the present invention against the disease force is more directly proved when using the present The invention of the antimicrobial peptide (AMp) does increase the resistance of groupers. 2. From the special chemical structure of the antimicrobial peptide (AMp) of the present invention, it can be known that the antimicrobial peptide (AMP) has no biological thief as a force, indicating that the antimicrobial peptide has a special sterilization force and confrontation The biological ability of the bacterium is non-specific (n〇n_specific) and broad-spectrum; therefore, the antimicrobial peptide (AMp) provided by the present invention does have an antibacterial and disease-resistant function. The detailed description above is a detailed description of one of the possible embodiments of the present invention, but the embodiment 26 1316944 is not intended to be used in the scope of the invention. It should be included in the patent scope of this case. In summary, this case is not only a novel anti-microbial peptide, but also has many of the above-mentioned effects. It should have fully complied with the statutory invention patent requirements of novelty and progressiveness, and apply for it according to law. You are requested to approve the patent application for this invention. The case, in order to invent, to the sense of virtue. [Simple description of the schema] - Figure 1 shows the full-length cDNA sequence of the gentian grouper antimicrobial peptide gene. A total of 494 nucleosides, an acid composition consisting of a 5-terminal untranslated region (5, UTR) consisting of 92 nucleotides, a translational region consisting of 204 nucleotides (codinS region) and 198 nucleotides. 3-terminal untranslated area (3, UTR). The start code (startcocjon) ATG is shown in bold, and the stop code (st〇pc〇d〇n) TGA is indicated by an asterisk (*). The translational region is presumably translated into 67 amino acids, which contain 22 amino acids of the signal peptide (marked by the bottom line) and 25 amino acids of the mature peptide (marked by the box). And prodomain 20 amino acids (marked by wavy bottom lines). i Figure 2 shows the SDS_PAGE electrophoresis pattern of recombinant antibiotic AMP (rAMP) protein. The evoked results were examined using a 15% SDS-PAGE; the arrows indicate rAMP. Figure 2 shows the alignment of the antimicrobial peptide amino acid sequences of each species. Epinephehs lanceohtus, Epirjephehs coioides, grinning mjSiniperca —'), European wolf owls, golden eyes, squid, and squid (Moro«e oil) 7's 'Bigfish Island ec such as gallbladder (10) s), mere- (Hippoglossus hippoglossus) 'Daxi Xu Hou fisherman Hipp0gi〇ssuspiatess〇ides). Figure 4 shows the 3D structure of the gentian grouper antimicrobial peptide. The protein structure modelling molecule is used to simulate the 3D structure diagram of gentian grouping antibacterial peptide, which contains signal peptide, mature peptide, prodomain °. Figure 5 is the edge of biliary plaque AMP mature peptide to pull the ^ peptide) hydrophobic (hydrophobic) And prediction of hydrophilicity. The polar regions of the 27 1316944 'amp mature peptide were analyzed by the Kyte-Doolittle hydropathy plots program. Figure 6 shows the performance of the AMP gene in various tissues of gentian grouper. Analyze the antimicrobial, real-time quantitative RT-pcR for brain, heart, liver, spleen, intestine, stomach, muscle, blood, total silk, eyes, head kidney, skin and fin tissue of gentian grouper The pattern of peptide gene expression in various tissues. Figure 7 shows the effect of AMP gene expression on various tissues of gentian grouper at different concentrations of poly I:C. The injection concentration of each fish was 0 pg/g BW (control group), 1 pg/g BW, 2 pg/g

r BW, 5 pg/g BW p〇iyI:c; gentian plaque treatment 24 hours after taking the kidney, brain, spleen, Φ liver, blood and total silk total RNA' using real-time quantitative polymerase chain reaction analysis The effect of AMP gene mRNA expression was statistically analyzed by Duncan, s Multiple Range. A, b, c showed significant differences (^ < U 〇 5); experimental data are expressed as mean ± SD. Figure 8 shows the AMp gene expression for each tissue of gentian grouper at different time points under poly I:c treatment. After the female fish was injected with poly I:C at a concentration of 2 pg/g BW, the gentian zebra head kidney, brain, spleen, liver, blood, and skin were taken at 12 hours, 24 hours, 48 hours, and 72 hours, respectively. And Lin's total RNA, Xiang instant quantitative polymerase chain reaction analysis of AMp gene mRNA expression 'and Duncan, s Multiple Range Dingcheng statistical analysis &, b, c indicates that φ is significantly different (Ρ <0·〇5) The experimental data is represented by the mean soil SD. Nine is the effect of AMp gene expression in female bilirubin (4) under the treatment of LPS without concentration. The concentration of LPS injected into each fish was 〇_BW (control group - please, 1 〇pg/g BW, 15 ug/g BW, 24 hours after taking gentian zebra head kidney, brain, spleen, liver, dirty, Blood and total silk of her! RNA, using the real-time quantitative polymerase chain reaction to analyze the effect of AMp gene mRNA expression, and the Dunca heart-like stalk as statistical analysis a, ^, c, d indicates significant difference (ρ < 0·05), the experimental data is expressed as mean ± sd. Figure 10 shows the effect of LPS treatment on the A· gene expression of each tissue of the gentian. 15 _ listening to the processed% less 28 1316944 aagagctgca agacctggac caacgtgcct ttgaacgaga gaaagctttt gcctgagtcc 300 atgatagcct agtgaaggag ccactcattg ttaacacaaa aagaaaaagt ttttgttttt 360 gagtatagga agtattggtt caattgggta accaaaatat tttacactga tctaattgat 420 tttggaaaaa tgttagttat ttgaaataaa tctggaatct gtgttacaca caaaaaaaaa 480 aaaaaaaaaa aaaa 494 < 210 > 3 < 211 & gt 21 <212> DNA <213>Artificial sequence<220><223> PCR primer, forward AMP primer <400> 3 atgaggtg Ca tcgccctctt t 21 <210> 4 <211> 21 <212> DNA <213> artificial sequence <220><223> PCR primer, reverse AMP primer <400> 4 tcaggcaaaa gctttctctc g 21

<210> 5 <211> 22 <212> PRT <213> Epinephelus lanceolatus <400> 5

Met Arg Cys lie Ala Leu Phe Leu Val Leu Ser Leu Val Val Leu Met 1 5 10 15

Ala Glu Pro Gly Glu Gly 20 2 15 1316944 <223> PCR primer, real-time quantitative PCR AMP specificity bow | sub A4 <400> 9 caaaggcacgt tggtc 4

Claims (1)

:1316944 : X. Patent application scope: 1. An antimicrobial peptide isolated from gentian grouper, having a peptide sequence as shown in SEQ ID NO: 1 of the Sequence Listing for bacteriostatic and bactericidal, This strain contains Escherichia coli Dli5H), Vibrio harveyi, fibrio aigino (cus), Vibrio cmguillarum, Vibrio sabnonicida, and I. Aeromonas hydrophila), Staphylococcus epidermidis. 2. A nucleotide fragment isolated from a gentian group, having a nucleotide sequence as shown in SEQ ID NO: 2 of the Sequence Listing, for encoding an antibody as described in claim 1 A nucleotide fragment of a microbial peptide. 3. A pharmaceutical composition comprising: a pharmaceutically effective amount of a peptide fragment' wherein the peptide fragment has an amino acid sequence as set forth in SEQ ID NO: J of the Sequence Listing; and a pharmaceutically acceptable carrier. 4. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition can be in the form of an embedding, a soaking liquid, a feed, a feed additive, an oral liquid, an injection vaccine, a patch, a powder, a key. Agents, injection liquids, suspensions, external solutions, drops, liniments, paints, creams, ointments, ointments, pastes, gels, and gels. 5. The pharmaceutical composition according to claim 3, wherein the carrier is an excipient, a diluent, a thickener, a filler, a binder, a disintegrant, a lubricant, a grease or a non-fat. Bases, surfactants, suspending agents, gelling agents, adjuvants, preservatives, antioxidants, stabilizers, colorants, perfumes. 6. The pharmaceutical composition of claim 5, wherein the agent comprises, but is not limited to, glycerin and oil. 7. The pharmaceutical composition of claim 6, wherein the oil comprises, but is not limited to, flower oil and castor oil. The pharmaceutical composition of claim 5, wherein the base comprises a hydrocarbon, and the hydrocarbon includes, but is not limited to, hard money, soft stone sacrifice, stone oil, Glycerin, bee soil, metal lanolin and its derivatives, lipid thieves or combinations thereof. The pharmaceutical composition according to Item 8, wherein the natural and oily oils include, but are not limited to, almond oil, corn oil, peanut oil, secret oil or eucalyptus oil. The pharmaceutical composition of claim 8, wherein the fatty acid comprises, but is not limited to, stearic acid or oleic acid. The n composition of the fifth aspect of the invention is characterized in that it comprises an anionic surfactant, a cationic surfactant, a nonionic surfactant, and the surfactant comprises, but is not limited to, sorbitol vinegar. Polyoxyethylene oxime and its derivatives, carbamazepine and spermatozoa. 12. The composition of claim 4, wherein the derivative of the polyoxyethylene includes, but is not limited to, a polyoxyethylene fatty acid ester. 13. The pharmaceutical composition of claim 5, wherein the silk sigmoid derivative comprises, but is not limited to, carbopol. The pharmaceutical composition according to claim 5, wherein the suspending agent comprises, but is not limited to, natural tree vinegar, cellulose fragrant inorganic substance f, polyethylene glycol 54 (), polyethylene glycol side and propylene derivative Inorganic substance 15. The pharmaceutical composition according to claim 14, wherein the cellulosic material comprises, but is not limited to, siIicace〇us s out of cas. 6. The pharmaceutical composition according to claim 5, wherein the gelling agent comprises any of the formulas for the use of a squamous scale-eating agent, the Polymer, polyethylene derivative, pectin, gum.如. For example, the application of the patented 5% of the medicinal composition of the medicinal compound includes, but is not limited to, f-based cellulose, silk ethyl cellulose, and succinyl cellulose. The pharmaceutical composition of claim 16, wherein the vinyl polymer comprises, but is not limited to, polyethylene glycol, olyvinylpyrrolidones. The pharmaceutical composition according to claim 16, wherein the carboxyvinyl derivative comprises, but is not limited to, carbopol. 20. The pharmaceutical composition of claim 16, wherein the gum comprises, but is not limited to, gum arabic, scutellaria, sputum, loam, gelatin, and carrageenates. 21. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is administered to humans and animals by oral, soaking, injecting, smearing or patching. 22. The pharmaceutical composition of claim 21, wherein the animal is a livestock animal. The pharmaceutical composition according to claim 22, wherein the animal animal is selected from the group consisting of cattle, sheep, chicken, pig, and a group thereof. 24. The pharmaceutical composition of claim 21, wherein the animal is an aquatic animal. The pharmaceutical composition according to claim 24, wherein the aquatic animal is a fish. 26. The pharmaceutical composition of claim 25, wherein the fish is a farmed fish. 27. The pharmaceutical composition according to claim 26, wherein the farmed fish is selected from the grouper, the sea, the net fish, the flounder, the shoes: the fish, the fish, the fish, the fish, the goldfish, the Wu Guo fish, zebrafish and their group. 28. A method for screening an immune inducer (immune eiicit〇rs) capable of increasing gentian plaque immunity, comprising: step 1 mixing an immune inducer to be screened with a feed to obtain a mixture; Step 2: Oral administration of the mixture to a gentian grouper, and the mixture may be administered by soaking; Step 3: detecting the performance of the peptide fragment as shown in SEQ ID NO: 1 of the sequence table in the gentian group And the fourth step is to judge the immune inducer to the gentian grouper 1316944 by the amount of the peptide fragment The effect of bactericidal antibacterial activity includes Enterobacter aeruginosa (Escherichia coH DH5a), Vibrio cholerae (P. serrata, Alginolyticus), Vibrio angui Harum, Vibrio salmonicida, Aeromonas hydrophila 'Staphylococcus epidermidis. 29_ The method for screening for an immune inducing agent capable of increasing the immunity of gentian grouping, as described in claim 28, wherein in the third step, the method for detecting the peptide fragment is, but not limited to, colloidal electrophoresis 'Western ink point method, immune response method. 30. The method for screening for an immune inducing agent capable of increasing the immunity of gentian grouping according to claim 28, wherein the amount of the peptide fragment in the step 3 can also be encoded by detection. The expression amount of the mRNA of the peptide fragment is determined by the sequence transcribed from the nucleotide sequence shown in SEQ ID NO: 2 of the Sequence Listing. 31. A method for screening for an immune-inducing agent capable of increasing the immunity of gentian plaque as described in claim 30, wherein the method for detecting the amount of mRNA expression is, but not limited to, northern blotting, polymerase Chain reaction, in situ hybridization. 1316944 _________ * ’ card correction replacement poverty Figure 14