TW200811196A - An anti-microorganism peptide - Google Patents

Abstract

This invention discloses an anti-microorganism peptide that have the peptide sequence described as sequence table SEQ ID NO: 1. Those peptides can be used to effectively inhibit and kill microorganism, decrease pathogenic bacteria and cure diseases that may improve health statue of animals body. Furthermore, the immunity and against disease capabilities of animals can be enhanced for decreasing animal death and increasing animal livability and birth rate by inducing and coding the animal immune gene behaves.

Description

200811196 IX. Description of the Invention: [Technical Field] The present invention relates to an antimicrobial peptide, which particularly refers to an antimicrobial peptide which inhibits and kills microorganisms and serves as a therapeutic purpose and increases the health of an individual. [Prior Art]

Grouper is an important breeding record in the Asia-Pacific region. Because of its beauty, it has long been the top seller of aquatic products and is widely loved by consumers in Taiwan, China, mainland China, Japan and Southeast Asia. Taiwan's stone reduction and breeding began in the past 5 years, and the style of raising money can be divided into the sea cage network to raise the inland salt water group. In order to promote the reliance on the Asia-Pacific Aquatic Seedlings Center, the Jiannian Agricultural Casting Department has already promoted various types of stone _ as the primary policy project is also the focus of science and technology farming (Cai, 2_b Taiwan grouper farming has nearly 2 〇 In the history of the year, in 1999, the interest rate was as high as 20%, and the age stone shouted as a high-turning fish species, and in Taiwan it is a mature and stable profitable industry. a. Grouper disease is mainly Divided into two types of viruses and fine _ sex, the virus is currently a serious disease of the grouper Φ, including iridescent silk (iridGvims) and the gods turn dead silk (Li __ also _ is two viruses susceptible to grouper; among them, Nervous necrosis virus ((5) 诵s η fiber & ^(10) 黯) is the most troubled virus of the current grouper. The virus has a rapid infection ability and is extremely virulence, allowing a large number of fish to die in a short period of time. Made in the knot virus __ marriage ten it has no external fine, the configuration is twenty a #合二^有钱股叫在鱼苗_most prosthetic alkali dyeing, infection when ^ make", ready to lose balance ability, to spiral, Di mode swims ice, and it will collide in different directions; 2 strict 002)...* on the surface On the body side, the body floats upwards, and when there is stimulation, it will swell and swim at a speed (Liu, fine halogen poisoning includes: love to wait for the smashing of the slabs... this 4~^ garden, _, Aeromonas, streptococci and grapes Ball halogen, some fine _ class has always been a problem with the breeding case, the grouper is also vulnerable to 5 200811196 silk 'caused the loss of the industry. There are many reasons for the fish body to be infected with bacteria, the most easy to issue * job: Feeding density is too high, fish size difference is large, residual food, "inappropriate, etc., is a factor that makes it easy for fish to produce wounds, which leads to bacterial infection; bacterial infection: king body (four) and complicated infection; systemic silk For the bacterial parts throughout each group =, local infection county _ only infected body surface or _. When the grouper is dyed by the arc thief, white spots appear on the surface of the body. As the condition is serious, the wound is ulcerated, red and swollen, scales fall off Bleeding with the internal organs eventually leads to the death of the fish (Liu, 2002).

The immune response in the fish body, so the vaccine can not effectively make the fish body _ increase immunity. Not only that, these vaccines have more than 50% of the disease, which has so far allowed the industry-grouper to be a farmed fish species in Taiwan, but it is easily plagued by diseases during the fry, resulting in an unstable annual output. Various studies use vaccines to increase the immunity of fish. 'However, the immune power generated by the use of the epidemic is not good, and additional vaccines are often needed to question the safety of the vaccine. It can be seen that it is necessary to study the fish immune system and to find the fish's own antibacterial substances. Antimicrobial peptides (AMPs) are ubiquitous in plants and animals and are part of the endogenous immune system. Antimicrobial peptides are typically small molecule cations composed of a positively charged amino acid (Hancock and Lehrer, 1998). The anti-microbial peptide of fish, which was discovered from shark squalamine (Moore et al., 1993), is also published in many fish. Antimicrobial traits are classified into three major categories according to their chemical structure. The first type is amphipathic (x-helix), because the structural relationship is also called linear peptides, and there is no cysteine in the structure. Cysteine residues, thus no disulfide bond formation; the second type is the formation of intramolecular disulfide bonds, such antibacterial peptides contain cysteine residues, Producing a disulfide bond to form a hairpin-like β-sheet and an a-helix-p-sheet mix 6 200811196 Bulet et al., 1999), the second class is the overpronation and/or glycine residues, which are highly pr〇line-rich. Peptides can be divided into short-chain (sh gentleman chain) and long-chain from the size of the molecule. In the current research on antimicrobial peptides, the antimicrobial peptide can be positive for Gram*. Gram-negative cockroaches, real brines and protozoa Bad ability (Powers and Hancock, .2003); in addition to fighting microbes, antimicrobial peptides may also be associated with development, animal molting, and reproduction; but in current research There are no related possibilities for the regulation of anti-microbial peptides and immune factors of animals and plants. The antimicrobial mechanisms of antimicrobial peptides fall into two broad categories, the first being peptide-lipid interactions, in which It can be subdivided into a ba3Tei-stave mode and a carpet-like cover pattern (Shai, 1999). The tubular piercing mode is rolled into a tubular shape with an amphoteric α-helix (α-helix) antimicrobial peptide. Insert into the membrane, use the hydrophobic peptide hydr〇ph〇bic end to contact the cell membrane of the bacteria, and the other hydrophilic (hydr〇philic) end face to face, then the hydrophilic end of the antimicrobial peptide is centered Point, in the form of a polymer to form holes in the membrane, causing the osmotic pressure inside and outside the bacteria to be non-parallel, resulting in bacterial death (Qiu, 2001). In addition, the carpet-like coverage pattern Φ is positively charged with positive electricity. After the peptide binds to the negatively charged cell membrane, the hydrophilicity of the antimicrobial peptide: 3⁄43⁄4 reacts with the phospholipids head groups or water molecules, and the anti-micro w bio-success is turned over, resulting in a hydrophobic end portion. The membrane surrounding a part of the cells is removed, and the membrane structure is removed to destroy the structure of the bacterial membrane and cause the pathogen to die (Yang, 2, 4). The second category of antimicrobial mechanisms is Recept〇r_mediated reeognitioii processes. 'Some anti-microbial peptides may be associated with deoxyribonucleic acid and autolysin (aut〇iySins). And cell permeability (pemieabil milk is used to inhibit the germination of fungal spores and the elongation and branching of mycelium (Thevissenetal, 1997). There is also a literature pointing out that 'the ability of anti-microbial peptides to bind to prokaryotic nucleic acids, thus inhibiting Egg 7 200811196 White matter production (Powers and Hancock, 2003). It can be seen that there are still many defects in the anti-microbial methods of the above-mentioned animals, which is not a good designer, but needs to be improved. ° The inventor of the present invention The shortcomings of the ribs and scorpions are improved and innovated, and after years of painstaking research, they finally succeeded in research and development of an anti-microbial peptide. The purpose is to provide an anti-microbial peptide to reduce the pathogenic bacteria in the moving clock, to achieve therapeutic effects, and The health state of the animal. The second aspect of the present invention is a nucleotide fragment encoding an antimicrobial peptide such as the above-mentioned object of the invention. The other object of the present invention is a composition of a micro-recorded The composition comprises a pharmaceutically effective amount of a peptide fragment, and a pharmaceutically acceptable carrier. The invention further provides a method for providing an immune elicitors capable of increasing the immunity of an animal. 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 AIDS inhibitor to an animal. The antimicrobial peptide comprising the above object of the invention comprises an orderly List SEq id NO: amino acid sequence shown by 1 y. Since the antimicrobial peptide of the present invention is an antimicrobial peptide (AMP), the antimicrobial peptide is a broadly distributed in the immune system of various animals and plants. Peptide fragments; therefore, the antimicrobial peptides of the present invention can be obtained from natural animals, especially aquatic animals, or synthesized by conventional chemical synthesis methods, or can be prepared by conventional molecular biological methods. Also included is a nucleotide fragment encoding an antimicrobial peptide as described above, the nucleotide fragment having a nucleotide sequence as shown in SEQ ID NO: 2 of the Sequence Listing. 8 200811196 The present invention further provides a method for killing A pharmaceutical composition for dead microorganisms, the composition comprising: a pharmaceutically effective amount of a peptide fragment; and a pharmaceutically acceptable carrier, wherein the peptide fragment has an amino acid as shown in the sequence listing SEqIDN〇: The composition may be in the form of an embedding, soaking liquid, feed, feed additive, oral-liquid, vaccination, patch, powder, lozenge, injection liquid, suspension, external solution, drops, , liniments, paints, creams, ointments, ointments, pastes, gels, gels, etc. Wherein the carrier can be an excipient, a diluent, a thickener, a filler, a binder, a disintegrant, a # 'slip agent, a grease or a non-greasy base, an surfactant, a suspending agent, and a gel. Agents, adjuvants, preservatives, antioxidants, stabilizers, colorants, perfumes, and the like. The lubricant includes, but is not limited to, glycerin and oils (eg, peanut oil, castor oil). Wherein the base comprises hydrocarbons, including but not limited to hard paraffin, soft stone, stone oil, glycerin, bee bad, metal soap, natural oil (eg 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 including but not limited to sorbitanes, polyoxyethylene and derivatives thereof (eg: polyoxyethylene fatty acid ester Ρ〇 χ χ χ χ χ χ acid acid acid acid acid acid acid acid acid acid acid acid acid acid acid acid acid acid acid acid acid oxy oxy oxy oxy oxy oxy oxy oxy oxy oxy oxy oxy oxy oxy oxy oxy oxy oxy oxy oxy oxy But not limited to natural gums, cellulose (Cellul〇se) derivative inorganic substances (such as: silicaceous silicas), polyethylene glycol 540 (polyethylene glycol 540), polyethylene glycol 3350 and propylene glycol (propyl glycol) Wherein the gelling agent comprises any gelling agent for pharmaceutically forming a colloid, the gelling agent comprising, but not limited to, a cellulose (Cellulose) derivative (eg, methyl cdlul〇se, Meridian 9 200811196 hydroxyethyl cellulose, carboxymethyl cellulose (carbazole Xymethyl cellulose, etc.), vinyl polymer (vinyl p〇] lymers) (eg······························ Alcohds), dyvi Nyl pynOlid〇nes), carboxyp〇lymethyl_ (such as carbopol), pectin, gums (such as gum arabic, tragacanth ^ ^^ (alginates) > ^||(agar) > ^(gelatin)carrageenates) iIihai pharmaceutical composition is administered orally, immersed, injected, smeared or patched to humans and animals. It may be a livestock animal or an aquatic animal; when it is a livestock animal, it is preferably a cow, a sheep, a good pig. The better one is a chicken Hjc animal gf, preferably a fish, and more preferably a farm fish; The term "farmed fish" refers to fish that can be raised by hand, preferably halibut, salmon, squid, squid, goldfish, tilapia and zebra. Fish (zebraflsh), etc., but not limited to this. The present invention further provides a method for screening immune elicitors capable of increasing immunity of an animal, comprising: Step-mixing the immune inducer to be screened with a feed to obtain a mixture; • the two-to-one animal is orally administered with the mixture, and the mixture can also be administered by soaking; u. Step 3 detects the amount of the peptide in the animal as shown in SEQ ID NO: i of the Sequence Listing; The effect of the immune inducer on the disease resistance of the animal was judged by the amount of expression of the progenitor fragment. The type of the detection method of the peptide fragment in the third step is not particularly limited in the present invention, and those skilled in the art can detect the expression amount of the fragment by any method, for example, colloidal electrophoresis. Western dot method, immune response scale, not limited to 200811196. The detection of this peptide tablet can also be determined by detecting the amount of expression from the plate encoding the segment. The sequence table seqidn〇: 2 is the transcription of the nuclear Wei sequence; the check of the performance of the coffee eight, such as the northern ink dot method, but not limited to this. The animal of the present invention can produce the water and the secret of the water; when it is a livestock animal, the chicken or the pig is better; the chicken is better; when it is a aquatic animal, the fish is preferred. • Better known as farmed fish; the term “farmed fish” as used herein refers to fish that can be reared by aquaculture, such as: grouper (four) sinking), Lai (c〇 (four), Net • Fish (Seabream), Halibut (secret, fish (four) coffee), squid (10) M, #.(carp) > (goldfish) >^$P,#,(tilapia)i|M^/fx (zeb^, limited to this. The present invention still further provides a method for increasing disease resistance of an animal, comprising: Step A provides an immunologically inducing agent for pharmaceutically effective s, the immune inducing agent is capable of inducing production of an animal such as SEQ ID NO: 1 in the Sequence Listing The antimicrobial peptide is shown; Step B provides an animal; and Step C, the immune inducer is administered to the animal. • The cap is immune to the lining - the rib can increase the immunity of the animal. Method for immunization-inducing agents (immimeelidtors), in the sequence of SEQidn〇: 丨 peptide expression or sequence, SERI The DNO: 2 mRNA gene expression multi-screening method can increase the immunity-inducing agent, and the immune-inducing agent can be further selected. The immune-inducing agent can further comprise a pharmaceutically acceptable carrier. Peptide fragments can be obtained from natural animals, especially aquatic objects, or synthesized by conventional chemical synthesis methods, or can be prepared by conventional molecular biological methods. The molecular biological methods are as follows: Step a provides SEQ ID NO: a nuclear morphic acid fragment shown in Figure 2. Step b provides a performance vector; 200811196 Step C: Recombining the nucleotide fragment with the expression vector into a recombinant expression vector; Step d: delivering the recombinant expression vector into a host cell; Step e causes the host cell to express the peptide fragment. The main cell may be Escherichia coli (five (7)"), yeast and animal cells, but is not limited thereto. * Taking Escherichia coli (five co/z·) as an example After selecting the plastids of E. coli (five co/〇 system, « re-select the appropriate restriction enzyme site in the plastid, and the restriction enzyme male The expression plastid 'also cleaves the nucleotide fragment containing the nuclear bitter acid sequence encoding the peptide fragment of the present invention with the restriction enzyme, and performs the cleavage of the plastid and the nucleotide fragment by the restriction enzyme Engagement, followed by transformation of the Escherichia coli (f. w") plastid containing the nucleotide sequence encoding the peptide fragment of the present invention into Escherichia coli (five co") In the microbial cells, the Escherichia coli (five co/z·) may be pentacco/f BL21TM (DE3) pLysS, and any conventional knowledge such as electro-transformation or heat shock may be used for transformation. The transformation technique is carried out, and after transformation, the E. coli (five (7)/ζ·) is induced by an inducer to express the peptide fragment of the present invention, and the inducer may be isopropylthiogalactoside (IPTG). Etc. Taking yeast as an example, the plastid of the yeast system and the restriction cleavage site of the appropriate φ in the plastid can be selected, and the plastid is cleaved by the restriction enzyme, and the restriction enzyme is also used to shear the plastid. A nucleotide fragment encoding a nucleotide sequence of the peptide fragment of the present invention, which will undergo a ligation reaction with a plastid and a nucleotide fragment after restriction enzyme cleavage, and then will contain a peptide encoding the present invention. The expression of the nucleotide sequence of the yeast acid sequence of the fragment is transformed into the yeast, and 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 form a protoplast spheroid. (spheroplast) is further transformed, or the yeast is treated with a basic anion (such as Licl or (3) plus heat shock for transformation, and after transformation, the yeast is induced by the inducer to express the peptide fragment of the present invention. The inducer may be a known inducer such as IP1X}. In the case of an animal cell, the expression plastid of the animal cell system and the restriction cleavage site of the plastid may be selected. Cut the quality And ligating the nucleotide fragment containing the nucleotide sequence encoding the peptide fragment of the present invention with the restriction enzyme, and performing the ligation reaction on the plastid and the nucleotide fragment after the restriction enzyme cleavage, and then An animal cell system containing a nucleotide sequence encoding a peptide fragment of the present invention is transfected into an animal cell, which may be an insect cell or a mammalian cell, and any transfection may be used. Conventional transfection techniques are carried out, such as by lipofectin (lip0fecti〇n) or by acid fishing (calcium method) to represent the peptide fragments of the present invention. Furthermore, in order to be treated by the antimicrobial method of the present invention The animal's ability to resist disease can be effective, and the immune-inducing agent can be administered to the animal by oral, soaking or injection; oral administration can be directly orally or added to the feed, orally. In the case of administration, in order to prevent the immune inducing agent from being destroyed by the digestive juice in the digestive tract when passing through the digestive tract of the animal, the immune inducing agent can be further microscopically carried out by a conventional technique. Buried to enhance its ability to resist digestive juice; the injection method includes intravenous (intravenous injecti〇n, iv), intramuscular injection (IM) and intraperitoneal injection (IP), but not only It is limited to this; when it is administered intravenously to animals, it can exert the maximum effect. φ The composition or peptide of the present invention can promote the animal to resist the invasion of pathogenic bacteria, thereby increasing the disease resistance of the animal. The health of the animal can be improved. The composition or peptide of the present invention can also be used as a preservative for external use and a fungicide. [Embodiment] Example 1 Preparation of the antimicrobial peptide (AMP) fragment of the present invention is firstly based on Chomczynski And Sacchi's method (1987) to isolate total RNA from the silk tissue of gentian grouper (EpzV^p/^/milk (9)/α plus); next, synthesize cDNA from 10pg total RNA using 200 U Molony murine leukemia virus reverse transcriptase, 1 mM dNTP, 160 U RNase inhibitor 13 200811196 (inhibitor) and 1·6 pg random primer IX reverse transcription buffer, 42. (: Transcription was carried out for 30 minutes. The anti-microbial peptide (AMP) gene was amplified (amplify) by polymerase chain reaction (p〇iymerase chain reacti〇n, PCR), and the PCR system used 20 μ§ reverse transcription product, 0.5 U 80μ11Χ PCR buffer of Taq DNA polymerase and 1 forward and reverse AMP primer, the forward AMP primer has the nucleotide sequence shown in SEQ ID NO: 3, and the reverse AMP primer has ^ as shown in SEQ iD: 4 The nucleotide sequence, pCR is in the DNA temperature cycle reactor • (thermal cycler) 'PCR step for annealing at 55 °C for 1 minute 30 seconds, at 72 C extension (extension) l minutes 30 seconds Denaturation for 1 minute at 94 °C for a total of _ 35 cycles. After the PCR reaction was terminated, 15 μΐ of the PCR product was analyzed by electrophoresis with 2% agarose gel, followed by vinyl bromide. (ethidimn bromide) staining. The purified PCR product will be cloned into the *Smal position of M13mpl8 or pBluescript, and will be sequenced by the dideoxynucleotide chain termination method. The AMP gene will be sequenced after sequencing. List the sequence shown in SEQ ID NO: 2; The sequence shown in SEQ ID NO: 2 shows that the anti-microbial peptide (AMp) cDNA sequence selected from the gentian scutellaria has 494 nucleotides, of which the translation region (c〇ding regi〇) n) consisting of 204 nucleotides, as shown in Figure 1, after derivation, an anti-micro-[[lambda]) peptide (AMP) with 67 amino acids is obtained, the sequence of which is SEQ ID NO On the other hand, the purified PCR product was cleaved with the expression vector pET31b(+) with restriction enzymes j/>vNI, and the cleavage PCR product and pET31b(+) were ligated. The PCR product-containing pET31b(+) obtained after the ligation reaction was transformed into the expression host E. cWBL21TM (DE3) pLysS, and cultured at 37 ° C with shaking 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 showed that the AMP peptide represented by E. coli BL2FM (DE3) pLysS was located in the insoluble protein fraction, and its molecular weight was about 2.7 kDa. As shown in the second, the Western ink collapse method also proves that it is indeed an AMP peptide. The obtained insoluble protein fraction was purified by histidine acid chromatography (His·Bind® chromatography) into 200811196, followed by shearing with cyanogen bromide (CNBr), whereby a purified AMp peptide was obtained. 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, while in other fish species, it is similar to squid and flounder, and in the classification of antimicrobial peptides, each species is a different type, but the types of anti-ii peptides are different in the same species, regardless of Vertebrates or invertebrates are hard to find a regular rule. The antimicrobial peptide of the present invention has 67 amino acids (as shown in the sequence listing SEQ ID N: j). It contains a signal peptide, a mature peptide and a prodomain, and its 3D stereo The structure is shown in Figure 4. The message peptide has an amino acid sequence as shown in the sequence table seq id n〇: 5, which mainly functions to cleave during intracellular message transmission, leaving the mature peptide and prodomain. The mature peptide has an amino acid sequence as shown in SEQ ID NO: 6 of the Sequence Listing, and is a position mainly interacting with the pathogen, and has a charge to bind to the cell membrane of the bacteria, and it is possible that the mode of action of p is cylindrical. The piercing mode (barrel_stave m〇de) is inserted into the thin membrane with the a_hdix antibacterial reference peptide, and the hydrophilic end of the antibacterial peptide is contacted with the cell membrane of the bacteria, and the other hydrophilic end faces each other, which is the hydrophilicity of the antibacterial peptide. The end-facing point, in the form of a polymer - takes into account the shape of the hole, causing the Hernite to be non-parallel, leading to gambling death (Qiu, „2001). Carpetmode (Shai, 1999). When the positively charged antimicrobial peptide binds to the negatively charged bacterial cell membrane, the hydrophilic end does not react, and the anti-g peptide turns over to cause a hydrophobic portion, which surrounds part of the bacterial cell membrane and then removes the lining, destroying the structure of the bacterial membrane. Let the pathogens die (Yang, 2GG4), cause the death of bacteria, and effectively resist the pathogens to produce internal immunity.

Prodomain has an amino acid sequence as shown in the Sequence Listing SEQ m N〇:7, and its function is 15 200811196 is to neutralize the charge of the mature peptide, so that when the mature peptide does not need to interact with the foreign pathogenic bacteria, the mature peptide is not It has activity 'to avoid harm to the host itself (Yin βία/_, 2(10)6). The positive peptide will positively interact with the negatively charged bacterial cell membrane, while the cell membrane of the organism itself is also negatively charged. The antimicrobial peptide cannot be uniquely identified. It is a foreign source and the organism itself, so it needs prodomain. And the charge of the antibacterial peptide, so as not to cause damage to the organism. ^ Example 3 gentian zebra antibacterial peptide structure and polarity analysis. Analysis of SEQ ID NO: 6 using Kyte-Doolittle hydropathy blots program AMP mature Shengyuetai 'shows 7 to 16 amino acids with significant hydrophobicity and hydrophilicity • (hydrophilic) staggered areas (as shown in Figure 4, Figure 5 and Annex 1). The alpha helix formed by the gentian grouper antimicrobial peptide can be found by analyzing the distribution of hydrophobic and hydrophilic amino acids and the 3-D structure diagram using the HelicalWheel instruction of protein analysis in the National Institutes of Health macromolecule sequence analysis software. a helix) has a distinctly hydrophobic and hydrophilic fraction (as shown in Figure 5 and Annex 1), which belongs to the amphipathic molecule and is a traditional antimicrobial peptide (Hancock and Lehrer, 1998). 〇 From this example, it can be inferred that the bactericidal mechanism of the gentian zebra microbial peptide is a cylindrical puncturing mode or a carpet-like covering mode, because the gentian plaque antimicrobial peptide structure is α-helix positively charged, _ 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, ExpASy-Proteomics software analysis after the 30th to 35th amino acid The position of the N-myristoylation site _ mainly contributes to the binding of protein deuteration to C-14 saturated fatty acids, presumably as antibacterial peptides and erythrocytes on the cell membrane. Combined use (Towler et aL, 1998). It can be known from the 3D stereogram prediction that gentian plaque AMP is divided into three parts, while the part of the mature peptide is α_Μχ structure (as shown in Figure 4). The first type of amphoteric antimicrobial peptide, presumably may be the position of the antimicrobial peptide of the present invention mainly with the cell membrane of the bacteria. Example 4 Distribution of the mRNA of the antimicrobial peptide (AMP) in the grouper 200811196 Real-time quantitative polymerase chain reaction (real-time quantitative PCR) technique was used to analyze the expression pattern of the antimicrobial peptide (AMP) gene in various tissues of gentian grouper. The SYBR green I can be embedded in the DNA double-strand groove. Fluorescence is generated by halogen lamp emission, and the amount of fluorescence is measured by debt. When SYBRgreenI is not embedded in double-stranded DNA, the background value of fluorescence is very low; when SYBRgreenI starts to be embedded in the amplified gene fragment of PCR amplification On the B-keeper, the fluorescent signal will also be relatively raised. If there is no combination of non-specific primers or the interference of genomic DNA, the PCR reaction synthesizes the DNA of the target gene. The state of synthesis can be distinguished as: (!) twice the multiplication of the geometric progression doubling period (ge〇metric • phase); (7) when the reactants are insufficient, the gene synthesis is not a linear multiplication period of double doubling; (3) The last reactant is depleted and fails, reaching the plateau phase of the reaction end point. Therefore, to detect the amount of the target gene in the tissue and organ, it must be quantified in the geometric phase of the PCR reaction. significance. The principle of Reai-time quantitative pCR is to use different concentration templates under the same PCR reaction conditions, the reaction containing high concentration template will reach the geometric multiplication period faster, and the lower concentration template will be reached slowly, which will be defined. The number of critical PCR cycles at the midpoint of the geometric progression multiplication period is determined as CT (thresh〇ldcyde), that is, the CT value increases as the Ik template is reduced. Using the 10 values of the simultaneous quantitative PCR reaction of different concentration standards, the standard curve and the regression formula were calculated by software, and the absolute expression of the gene containing 軚 in the sample to be tested was converted by interpolation. At the same time, if a melting curve (MeltingCurve) is to be obtained, the melting temperature of the target temperature is 66°c-99°c after the target base is amplified. The analysis of the curve _ results can be _ (four) is The sand itself has a mutual lion phenomenon, or the specificity of the primer, to more accurately help to understand the correctness of the experimental quantitative. 41 extraction of total RNA (tote丨RNA) from the various spots of the grouper. For the brain, total silk, eye, heart, head kidney, liver, spleen, intestine, stomach, muscle fluid skin tissue of the group 1〇 volume of Trjz〇i reagent (such as life called usA), use the knife to first cut the tissue and then use a homogenized rod to break, add 1 / s volume of chloroform (four) 〇r〇f_), 17 200811196 strong shaking After 30 seconds, let stand at room temperature for 1 minute, centrifuge at i2〇〇〇Xg for 15 minutes at 4 °C, aspirate the supernatant into a new microcentrifuge tube, add TRIzol reagent equal volume of isopropanol (isopropanol) was uniformly mixed, allowed to stand at room temperature for 1 minute, centrifuged at 12000 x g for 15 minutes at 4 ° C; the RNA was dissolved in 20 μl of DEPC water, dissolved at 55 ° C for 10 minutes, and then stored at -80 The °C refrigerator was used to measure the ratio of RNA concentration to purity. 4·2 Reverse Transcription The extracted RNA was quantified to 5 pg of total RNA per tube and subjected to reverse transcriptase reaction. 4. 3 real-time quantitative PCR specific primer (Primer) designed AMP gene partial cDNA sequence according to SEQ ID NO: 1, using LightCyclerProbeDesignprogmm to design real-time quantitative polymerase chain reaction specificity primer The AMP gene was subjected to real-time quntitative PCR using real-time qmititative PCR AMP specific primers A3 and A4 (the sequences of which are shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively). 4·4 real-time quantitative polymerase chain reaction (reai_time qmititative PCR) 4·4·1 standard curve preparation 4·4·1·1 small amount of plastid preparation (Mmi-preparaticm) SEQ ID NO: 1 is sequenced and determined The strain containing the partial cDNA sequence of the Mx gene and the AMP gene was taken out in a -80 ° C refrigerator, cultured in 30 ml of LB/Ampicillin medium (Luria-Bertanimedium), and cultured at 37 ° C for about 16 hours overnight. , remove the bacterial solution and prepare a small amount of plastid. Take 1 ml of the overnight culture solution and place it in a 1.5〇11 microcentrifuge tube at 4. (: Centrifuge at 12,000 xg for 5 minutes, pour off the supernatant, resuspend the cells by adding 150 μM Solution I (50 mM glucose, 25 mM Tris-HCl pH 8·0, 10 mM EDTA pH 8.0), and add 200 μΐ. Freshly prepared Solutionll (0.:2NNaOH, 1% SDS), placed on ice for 10 minutes, added 150μ1 Solution III (3 M potassium acetate, 10% glacial acetic acid), placed on ice for 5 minutes 18 200811196 clock, Centrifuge at 12,000 xg for 5 minutes at 4 °C, collect the supernatant into a new microcentrifuge tube, add an equal volume of saturated phenol and chloroform, shake vigorously for 30 seconds, and let it stand at room temperature. Allow to stand for 10 minutes, centrifuge at 12,000 xg for 15 minutes at 4 ° C. Collect the supernatant into a new microcentrifuge tube, add twice the volume of 95% iced alcohol, mix and mix at -20 °C for 20 minutes. Centrifuge at 12,000xg for 5 minutes at 4 °C, pour off the supernatant*, leave the sinking material, wash the sediment twice with 70% alcohol, then dry the precipitate by vacuum drying, and dissolve the precipitate in 20μ1 In sterile water, store at -20 °C for later use. 4·4·1·2 Calculation of plastid DNA concentration and purity Ρ Splitting The photometer measures OD260, and the standard for calculating the concentration of plastid DNA is 1 OD260 = 50 pg of DNA / ml, which is 50 pg X dilute factor X A260 = pg / ml. The purity of plastid DNA is based on the ratio of OD260 / OD280. Between 1·6 and 1·8 is a relatively pure DNA. After the plastid DNA concentration is calculated, the plastid DNA concentration is adjusted to 1 μ§/μ1, and serially diluted from ΗΤ1 to 1 by 10-fold dilution. (Τ9, after dilution, store the plastid DNA at -20 °C for use. 4.4.1.3 Prepare the standard curve by taking 3 μΐ of the diluted plastid solution of 10_4-104 into the reaction capillary, and then add 2·4 respectively. Μΐ MgCl2, 1 μΐ SYBR, real-time quntitative PCR AMP specific primers φ A3 and A4 (the sequences are shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively) 〇·5 μΐ, 12.6 μΐ sterile The final total volume of water was 20 μΐ; the reaction capillary was placed in a timely quantitative analysis. In the instrument (LightCycler 1.2), the following conditions were set: 95°C dissociation for 10 minutes; 95°C dissociation for 0 seconds; 64°C for 10 seconds. ; synthesis at 72 ° C for 15 seconds; dissociation, bonding, synthesis steps repeated 40 cycles, then cooled to 40 ° C and continued to heat up to 95 C, to the reaction product after dissociation and continuously detect fluorescence value. After the reaction, the results of the measurement 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 The reverse transcriptase reaction (RT) product obtained in Example 4.2 was placed in the reaction capillary, and was added to 200811196. · 4 μΐ MgCl2, 1 μΐ SYBR, real-time qimtitative PCR AMP-specific primers A3 and A4 (the sequences are shown in SEQ ID NO: 8 & SEQ ID N: 9 respectively) 〇·5 μ Bu 12.6 Μΐ sterile water, the final total volume is 2〇μΐ. The reaction capillary was placed in a timely quantitative analyzer (LightCyderl. 2), and the following conditions were set: dissociation at 95 ° C for 10 minutes; bonding at 64 ° C for 5 seconds; synthesis at 72 ° C for 15 seconds; repetition of dissociation, bonding, and synthesis steps After 4 cycles, the temperature is lowered. • After 40 °C, the temperature is continuously raised to 95 °C to dissociate the product after the reaction and continuously detect the fluorescence value. After the reaction - the detection results were analyzed with LightCycler Software 3·5 program in conjunction with the standard curve.

The data unit is μ#μ1, and then divided by the number of base pairs of the plastid, divided by the base pair molecular weight of 66〇 _ dalton to be converted into mole/μΐ, multiplied by 6.02Χ1023 to obtain copies/μΐ. The data obtained from the experiment, using SAS (Statistic Alalysis System) software, using univariate analysis (〇ne_way analysis Qf variance) 'Using Duncan's MultipleRange Tes to compare the significant difference between the factors (p < 0.05), the data obtained by the average soil standard The deviation (Mean soil SD) is indicated; this method is used for all amp gene expression analyses in the present invention. Please refer to Figure 6. The results of the AMP gene expression pattern between tissues show that gentian grouper is in the brain, heart, liver, spleen, intestine, stomach, muscle, blood, total silk, eyes, head kidney, skin and The fins have the expression of AMP gene; the head kidney has the highest performance, followed by the spleen, silk, and φ blood. These tissues all have blood flow related tissues, further demonstrating that the AMP gene may be secreted by blood cells. In the AMP study of flounder, it is pointed out that cockroaches are also an important immune organ in fish (Murray et al., 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 silkworm is used as the index structure for analyzing the expression of the AMP gene. Example 5 Injecting a virus-like nucleic acid drug polyinosinic-polycytidylic acid (po丨yI:C) into a gentian plaque, and analyzing the performance of the antimicrobial peptide (AMP) gene, the gentian grouper was divided into four groups, each group separately Injecting different concentrations of the virus-like nucleic acid drug polyinosinic-polycytidylic acid (polyI:C) in units of fish body weight (BW) per gram of fish, and the volume of the injected drug is 2.0 μl per kg of fish, and the volume of each group of poly I:C is injected. The concentrations were Opg/gBW (control), ipg/gBW, 2 pg/g BW, 5 pg/g BW, and the solvent was grouper PBS, which was injected intramuscularly and injected into the back muscles. After 24 hours, 150 ppm of MS-222 (Ethyl 3-amino-benzoatemethanesulfonatesolt) was used to reduce the stimulation of fish anesthesia; seven tissues including silk, head kidney, spleen, brain, blood, liver and skin were taken and extracted with TRIzol reagent. T〇tal 'Cai Ba' was stored in a -80 ° C refrigerator for real-time quantitative polymerase chain reaction (reai_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 VII. Using total silk as an indicator of the performance of the amp gene, 2pgpolyI:C was injected per gram of fish body weight. The treatment group had the highest AMP gene expression. The 2 μ§ poly I:C treatment group was injected into the mother's body weight, and the total kidney, brain, spleen, liver, blood, skin and silk were taken at 12 hours, 24 hours, 48 hours, and 72 hours after the injection. RNA 'analysis of AMP gene expression pattern by real-time quantitative polymerase chain reaction (reaptime qUantitative pcR), the results shown in Figure 8. Under the 72-hour long-term treatment, the AMP gene expression of silk and head kidney was higher than that of the control group. 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) • the gene may also be directed against foreign objects. Example 6 was injected with different concentrations of lipopolysaccharide (LPS) into gentian plaque ♦ to analyze the performance of the antimicrobial peptide (AMP) gene. The gentian grouper was divided into four groups, each group was injected differently. The concentration of LPS is measured in grams per gram of fish body weight (BW), and the weight of each gram of fish is injected into the injection of 〇 (control group), 5 、, 1 〇, 15 tons of LPS 'mother gram fish body weight 2 FμΐFreund's inc〇mpiete adjuvant, the injection method is intraperitoneal injection. After 24 hours, 15 〇ppm of MS_222 (Ethyl 3_amino_benzc>atemethanesulfonatesolt) was used to reduce the stimulation of fish anesthesia; tissues such as kidney, brain, spleen, liver, blood and silk were taken separately, and t〇tal (10) was extracted with TRIz〇1 reagent. , 21 200811196 Stored in a _80 °C refrigerator' for real-time quantitative polymerase chain reaction (real_time ah - 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 ; 0·05), showing that the amount of AMP gene induced in different tissues is different. LPS was injected intraperitoneally with 15 μ§ of the weight of the mother fish, and samples were taken at 24 hours, 48 hours, 96 hours, and 132 hours to investigate the AMP gene expression in the head kidney, brain, spleen, liver, blood, and silk. As shown in Figure 10, the results show that the A^p gene of the total silk tissue has a significant increase in the amount of φ in your hour; while the spleen and head kidney have 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_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 the gentian grouper antimicrobial 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 (the salinity was 35%). The experimental group reduced the salinity by 10% per day. The salinity of the three angel experimental group was 5%. After 24 hours, use 150 ppm of MS_222 (Ethyl - methanesulfonate solt) to reduce the stimulation of fish anesthesia; take the total silk, head kidney, spleen, brain, blood, liver, skin and other tissues, extract with TRIzol reagent 1 (^1 inverse^8, stored at -8〇.0 ice phase, for real-time quantitative polymerase chain reaction (real_time qUantitatiVe PCR) analysis of AMP gene expression pattern. As shown in Figure 11, the experimental group at low salinity Under the environment, its amp gene has a significant increase in the expression of head kidney, skin, blood, and total silk. It can be seen that when the salinity changes, the immune gene will be induced, thereby increasing the immunity of the fish and changing the salinity. When it is 5%, it is speculated that it will stimulate the endogenous immune system of gentian grouper, which makes the gentian grouper susceptible to pathogen infection or disease, making gentian grouper less susceptible to bacterial and viral infection. Including: changes in salinity, temperature changes, insufficient dissolved oxygen, chemical pollution, etc., causing many physiological changes in the fish, responding to the urgency of the environment. At the time, the physiology, appearance, tissues and cells of the fish produce an immune response (Barton and .Blanchard, 2〇〇ι). The results of this example further confirm that the AMP gene expression can be increased by changing the salinity of the culture environment. Further enhance the anti-viral and anti-bacterial infection of gentian grouper; this concept can be applied to aquaculture to improve the immunity and disease resistance of fish, to reduce death, death and yield. 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 passed through high performance liquid chromatography (HPLC). The purity of the test was over 90%; the minimum inhibitory concentration (minima丨 ibjt〇ry concentration, MIC) and the minimum bactericidal concentration (4) of the bacteria were determined by using the δ hai synthetic anti-bio-peptide to test the bactericidal conce blood-MBC. 8.1 Preparation of bacterial solution _ Escherichia coli (five to a / / DH5c 〇, Vibrio harveyi (pine > η · 〇 / ^ noisy), dissolved bath (Vibrio alginolyticus), slow fungus (Vibrio anguiHarum ), 孤- salmonicida, Aeromonas (^eromonas 'Staphylococcus aureus • (plus; %fccoc Gaya), these seven strains were cultured in LBA DH5a) or TSA (+ On 1.5% NaCl), after 16 hours of incubation at 37 °C, the colonies were scraped and dissolved in the designated culture medium to make OD54 11 (concentration is about ^1〇9), and 5 〇(^1 bacterial solution) Add 5 〇〇 1 of [; 3 or TSB (+1.5% NaCl) to reduce the bacterial concentration to ixio8. 8. 2 Susceptibility test Using a 96-well culture dish, add ΐ30μ1 to each groove. The bacterial liquid, the concentration of the bacterial liquid is lxl〇8 bacteria 23 200811196 number / m Bu after adding 20μ1 different concentrations (0.075 μΜ-0·675 μΜ) of chemically synthesized gentian grouper antimicrobial peptide, cultured at 37 C 16 Hours, observe the lowest antimicrobial peptide concentration of the clarified bacterial liquid without bacterial growth, which is the minimum inhibitory concentration (MIC); then the group of clarified bacterial liquid will be presented. The bacterial solution was applied to LBA (£· cW DH5a) or TSA (+1.5% NaCl). After 16 hours of 37 C culture, the colonies were observed to know the minimum bactericidal concentration (interest). Each experimental group had 2. Repeat and control group. • The test results are shown in Table 1. The minimum inhibitory concentration (MIC) of the seven strains are: Escherichia coli (and co" DH5a) 0·075 μΜ, Harvey's Vibrio buckle) · 15 μΜ, φ 弧 弧 ( 脱 脱 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( 0.375 μΜ, Staphylococcus epidermidis (Kang Khan (7) c (10) printed / as served, also) 0.075 μΜ; and its 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 grouper antimicrobial peptide (AMP) for different pathogenic bacteria Minimum inhibitory concentration (MIC) Minimum bactericidal concentration (MBC) Gram-negative bacteria Aeromonas hydrophila 0.375 μΜ 0.675 μΜ Vibrio an^uil Larum) 0.15 μΜ 0.45 μΜ Vibrio salmonicida 0.15 μΜ 0.30 μΜ 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 bacteria Staphylococcus epidermidis 0.075 μΜ 0.525 μΜ 24 200811196 It can be seen from the results of this example that the antimicrobial peptide of the present invention can kill Gram-positive bacteria and Gram-negative bacteria 'The bactericidal and bacteriostatic effects of different microorganisms, therefore, the antimicrobial peptide of the present invention can also be used as a broad-acting fungicide or preservative. Example 9 Protective effect of antimicrobial peptide (AMP) on grouper • The gentian grouper was divided into four groups and injected with PBS (control group), Vibrio, • / Chencheng 1x100 cfil/flsh), antimicrobial peptide Cecropin (concentration of 1 _fish) + Vibrio (concentration of IxlO6 cfb / fish), the antimicrobial peptide of the invention (gAMp, concentration of 1 __) + Vibrio (concentration of lxl06cfo / flsh), after 24, 48, After 72 hours, the mortality of each group was observed. The sin is shown in Fig. 12. The mortality rate is 〇% after 72 hours in the PBS group (control, group) and 100% in the Vibrio group after 72 hours. 〇/〇; 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 of the invention (gAMp) + Vibrio had a mortality rate of 60% after a small day This experiment only injected various AMP-times, and the results showed that the 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 antimicrobial peptide (gAMP) of the present invention has been Achieve the protective effect on fish. 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. ® Because of the broad-spectrum bactericidal ability of AMP, 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 and animals. Humanity. Example 10 Analysis of the performance of the gentian grouper anti-microbial peptide (ΑΜΡ) gene after immersion of Chinese herbal medicine The gentian grouper was divided into four groups, respectively, a control group in which pure water was not placed in any Chinese medicine, and each containing 50 ppm of plate i Roots (Isatis indigotica), Astragalus membrcmace (10), licorice seawater treatment group; strong airing to make the Chinese medicine evenly mixed in 30 liters of water, soak for 24 hours, use 150ppm MS-222 (Ethyl 25 200811196 B-amino-benzoatemethanesulfonatesolt),. Kidney, spleen, brain, blood, liver, skin 胄 m M TRIz〇l Wgent H t〇tal RNA > Saved in Russian refrigerator 'Jane's foot 4 polymerase Lian Ying (4)· Time _ such as ive PCR) analysis of AMP gene expression patterns. As shown in Figure 11, the treatment group of the pericardium sputum has a significant difference in the expression of 鸠p gene in the blood (4). 〇5); because of the scutellaria sinensis, it can enhance the ability of cells to produce interferon. (Section, 2005). In addition, in the mouse study, the saponin component of the astragalus extract contains cydoastmgenol and cyclogalegenin, which can increase the immunoglobulin (purchasing

(Yang βία/·, 2〇〇5). The results of this example show that Astragalus membranaceus has significant immunoregulatory effects on animals (including aquatic animals), including non-specific sputum, and can induce interferon production. It is a potent biological agent and is very useful for clinical diseases. Utility drugs. The antibacterial peptide provided by the present invention has the following advantages when compared with other conventional techniques: 1. The above description and examples show that the antimicrobial peptide gene of the present invention is expressed in the immune system. , antibacterial peptide bactericidal species and concentration, physical factors (salt), immunostimulants (P〇lyI: C and LPS), Chinese herbal medicine, etc., all confirmed the performance of the antimicrobial peptide gene of the present invention and the immunity of grouper Positively related, when the amount of the antimicrobial peptide (AMp) mRNA is increased, the resistance of the grouper is also increased; the effect of injecting the antimicrobial peptide (AMP) of the present invention on the disease resistance is more directly proved when used. The antimicrobial peptide (AMP) of the present invention does increase the resistance of groupers. 2. The specific chemical structure of the antimicrobial peptide (AMP) of the present invention, the ability of the antimicrobial peptide (AMP) to interact with the microbial cell membrane is known, and it is shown that the antimicrobial peptide has a special bactericidal monthly b force' and The bactericidal ability is non-specific (n〇I>Specigc) 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, and the embodiment 26 200811196 is not intended to be used in the context of the present invention. Both should be included in the scope of the patent in this case. In summary, this case is not only a novel anti-microbial peptide, but also has a number of functions. The n-point conforms to the statutory invention patent requirements of novelty and progress, and is recommended according to law. (4) Please ask your office to approve the invention patent. Apply for the case, in order to invent the invention, to the sense of virtue.

^ [Simple description of the schema] H is the cDna sequence of the full length of the anti-microbial peptide gene of the gallstone group. A total of 494 nucleotide compositions consisting of a 5-terminal untranslated region consisting of 92 nucleotides (5, UTR), 2〇4 nucleotides # (codinS regi〇n) and 198 nucleotides The 3-terminal untranslated region (3, UTR) is composed. The start code (startc〇d〇n) ATG is shown in bold, and the stop code is (9)_)Τ (3Α is represented by an asterisk (*). The translated region can be translated into 67 amino acids, which contain the message peptide (signal) Peptide 22 amino acids (indicated by the bottom line) 'mature wins oil ^^) 25 amino acids (marked by box), and pr〇 (i〇main 20 amino acids (with wavy bottom line) Figure 2 shows the SDS-PAGE electrophoresis pattern of recombinant antibiotic AMP (rAMP) protein. The results were detected by 15% SDS-PAGE; the arrow indicates the collision. Figure 2 shows the antimicrobial resistance of each species. Amino acid sequence alignment. Epinephelus hnceohtus", Epinephelus coioides, turtle, Che (JSMperca, European wolfberry (p/e(9) plus /α) ), Golden Eyed Wolverine (Morc^, spot. Moine saxatilis, BA (Pleuronectes americanus), Green (Hippoglossus hippoglossus), Great West End Green (Hippoglossusplatessoides) Figure 4 is gentian stone Speckle anti-microbial peptide 3D stereostructure. Use protein structure model The ling molecule mimics the 3D structure of the gentian grouping antibacterial peptide, which contains the signal peptide, the mature peptide, and the prodomain. The figure 5 is the gentian plaque AMP mature peptide (mature peptide) hydrophobic and hydrophilic (hydrophilic) Prediction: The polar region of AMP mature peptide was analyzed by Kyte-Doolittle hydropathy plots. Figure 6 shows the performance of AMP gene in gentian grouper. The brain, heart and liver of gentian grouper were taken respectively. The spleen, intestine, stomach, muscle, blood, silk, eyes, head kidney, skin and fin tissue were analyzed by real-time quantitative RTpCR to analyze the pattern of antimicrobial gene expression in various tissues.

Figure 7 shows the effect of amp gene expression on various tissues of gentian grouper under different concentrations of poly I:c. The mother fish injection concentration was 〇pg/g BW (control group), 1 pg/g BW, 2 pg/g BW, 5 pg/g BW poly I:C; gentian grouper treatment after 24 hours The total of kidney, brain, spleen, Φ liver, gold liquid and total silk, the effect of AMP gene mRNA expression was analyzed by real-time quantitative polymerase chain reaction, and the statistical analysis of Duncan's Multiple Range Test a, b, c showed significant difference ( ρ <0·05); Experimental data is expressed as an average value. Figure 8 shows the effect of poly I:C treatment on the AMp gene expression in various tissues of gentian grouper at different time points. After the mother fish was injected with poly l:C at a concentration of 2 pg/g BW, the gentian zebra head kidney, brain, spleen, liver, blood, skin and sputum were taken at 12 hours, 24 hours, 48 hours, and 72 hours, respectively. The total RNA of silk was analyzed by real-time quantitative polymerase chain reaction, and the expression of AMp gene mRNA was analyzed by Duncan's Multiple Range Test. A, b, c were expressed. • There was a significant difference (Ρ<〇·〇5), and the experimental data was averaged. The value of soil Sd is indicated. Figure 9 shows the effects of different concentrations of immune inducer LPS on the gill-gene performance of various tissues of gentian grouper. The concentration of LPS injected into each fish was Opg/gBW (control group), 5 pg/g • BW, 10 yang/gBW, 15 ng/g BW. After 24 hours, gentian zebra head kidney, brain, spleen and liver were taken. , total RNA of blood and silk, using the quantitative quantitative polymerase chain reaction analysis _ gene mRNA performance, and Duncan's Multiple Range Test statistical analysis a, b, c, d indicates significant difference (ρ < Ό · 〇 5), experimental data is expressed as mean ± SD. Figure 10 shows the effect of AMp gene expression on the tissues of gentian grouper at different time points. The concentration of LPS injected into each fish was 15 _ Na, 24 hours after treatment 28 200811196 Βτ said that the gentian zebra head kidney, brain, spleen, blood, skin and silk coffee RNA, _ (four) quantitative polymerase Chain reaction analysis of broad gene expression. And a, b, and statistical analysis of the Du surface 'phase pleple Range Test. , d, e indicate a significant difference (ρ < Ό · 05) 'Experimental data is expressed as mean soil SD. Figure 10 - shows the effect of salinity on the gene expression of the tissues of gentian grouper. After the salinity was treated for three days, it was adjusted to 5 PPt to take the gentian stone spot guess, brain, spleen, liver, blood, skin and secret UotdRNA ’ 攸 聚 聚 聚 补 礼 礼 礼

The current amount of 'Duncan's Multiple Range Test statistical analysis a, b indicates that there is a significant difference <Ό·05) 'Experimental data is expressed as the mean sd. Figure 12 shows the protective side of the grouper. The grouper was separately injected with PBS (control group), 5 melon bacteria, microbial peptide cecropin + Vibrio, and the antimicrobial peptide + bacterium of the present invention. Figure 13 shows the effect of Chinese herbal medicine soaking on the expression of AMp gene in various tissues of gentian grouper. Banlangen (/ Rong hW coffee (four), Huang Qi " price (four) - Tengyi r_ce W ten licorice (kiss listen to coffee torn) 50PPm soak for 24 hours, take gentian stone head kidney, brain, spleen, liver, blood, skin and The total RNA of the silk was analyzed by the real-time quantitative polymerase chain reaction, and the influence of AMp gene 10 mRNA expression was analyzed by Dimcan's Multiple Range Test &, 1), (: indicates significant difference (ρ<0·05) The experimental data is expressed as the average value. • Annex I is the prediction of the hydrophobicity and hydrophobicity of the gentian plaque AMP mature peptide (Mature peptide). The HelicalWheel analysis of the alpha helix (ahelix) The distribution of hydrophobic and hydrophilic amino acids in the region, blue represents hydrophobicity, and red represents hydrophilicity. [Main component symbol description]

Claims (1)

200811196 X. Patent application scope: 1. An antibiotic biopeptide has a peptide sequence as shown in SEQ ID NO: 1 of the Sequence Listing, which is used for bacteriostasis and sterilization. A nucleotide fragment having a nucleotide sequence as shown in the sequence table SEqidn〇: 2, which is a nucleotide fragment for encoding an antimicrobial peptide as described in the scope of claim [i]. 3. A pharmaceutical composition comprising: - a pharmaceutically effective peptide fragment - the peptide fragment having an amino acid sequence as shown in the sequence listing SEqidn::; and a pharmaceutically acceptable carrier. 4. The pharmaceutical composition according to item 3 of the special machine g, the form of the pharmaceutical composition may be an embedding substance, a soaking liquid, a feed, a feed additive, an oral liquid, an injection green seed, a patch, a powder. , recording agents, injection liquids, suspensions, external liquids, drip wipes, 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, and the like. 6. If the pharmaceutical composition described in item 5 is specifically claimed, the lining agent comprises, but is not limited to, glycerin and oil (e.g., peanut oil, castor oil). 7. The pharmaceutical composition according to claim 5, wherein the fine comprises carbonitrides, including but not limited to hard rock, soft rock, rocky oil, glycerin, bee, metal Soap, natural oil (such as: almond oil, corn oil, peanut oil, brain oil or withdrawal oil), wool month s and its street organisms, fat (such as: stearic acid or oleic acid) or a combination thereof. 8. The pharmaceutical composition according to claim 5, wherein the interface activity comprises an anionic surfactant, a cationic surfactant, a nonionic surfactant, and the surfactant 200811196 comprises, but is not limited to, sorbitol liver. Vinegar, polyethylene oxide and its derivatives (such as: polyethylene oxide fatty acid scorpion), polytetraethylene and its derivatives (such as: Carberhan resin carb〇p〇1). 9. The pharmaceutical composition as described in detail in Patent Application No. 5, wherein the buoyant comprises, but is not limited to, natural tree vinegar, fiber residual inorganic matter f (eg: silieae_ying (four), polyethylene glycol 54 〇, polyethylene 2 The pharmaceutical composition according to claim 5, wherein the gelling agent comprises any gelling agent for a pharmaceutical apical colloid, the gelling agent including but not limited to Cellulose derivatives (such as methyl cellulose, base ethyl cellulose, and carboxymethyl cellulose), ethylene-based polymers (such as: _polyethylene glycol, 〇1γν¥ P^lidones), Carbopol derivatives (such as: Kappahan resin (10) b_), pectin, gums (such as: arabina, yellow, algin, loam, gelatin and carrageenates). 11. If the patent = patent scope 3 The pharmaceutical composition, wherein the pharmaceutical composition is administered to the human body and the animal in an oral/recurrent/package/main shot, smear or patch. 12·as described in the U a pharmaceutical composition, wherein the animal is a livestock animal, such as the pharmaceutical group described in claim 12 The animal, wherein the animal is selected from the group consisting of a cow, a sheep, a chicken, a pig, and a group thereof. #M. The pharmaceutical composition according to claim 13, wherein the animal is an aquatic animal. The pharmaceutical composition of claim 14, wherein the aquatic animal is a preparation. The pharmaceutical composition according to claim 15, wherein the fish is a farmed fish. The pharmaceutical composition according to claim 16, wherein the farmed fish is selected from the grouper, sea bream, squid, flounder, squid, squid, squid, squid, goldfish, squid zebrafish And the group formed by the same. 18. - The method for increasing the immunity of the animal (IV) The agent (10) The method of the coffee (4) comprises the steps of: mixing the desired immune inducer with a feed, thereby Obtaining a mixture; 31 200811196 Step 2: Oral administration of the mixture to an animal, and the mixture may be administered by soaking; Step 3: detecting the peptide fragment of the animal as shown in SEQ ID NO. Performance; and steps The effect of the immuno-inducing agent on the disease resistance of the animal is judged by the amount of expression of the peptide fragment. 19. The method for screening an immune-inducing agent capable of increasing animal immunity as described in claim 18 In the third step, the method for detecting the peptide fragment can be, but is not limited to, colloidal electrophoresis, Western blotting, immunoreaction, etc. _ 2G. The screening energy can be used as described in item 18 of the patent scope. A method for increasing an immunity-inducing agent for animal immunity, wherein in the third step, the expression amount of the peptide fragment can also be determined by detecting the expression amount of the mRNA encoding the peptide fragment, and the sequence of the mRNA is as follows. The sequence transcribed from the nucleotide sequence shown in SEQ ID NO: 2 is shown. 21. The method for screening for an immune inducing agent capable of increasing immunity of an animal according to claim 20, wherein the method for detecting the mRNA expression amount is, but not limited to, the northern ink dot method 'polymerase chain reaction, In situ hybridization and the like. 22. A method of increasing the disease resistance of an animal comprising: a reckless sputum providing a pharmaceutically effective amount of an immunoinducing agent which induces an animal to produce an antimicrobial agent as shown in SEQ ID NO: 1 of the Sequence Listing Peptide; - Step B provides an animal; and • Step C. The immune inducing agent is administered to the body of the animal. 23. The method of increasing the disease resistance of an animal according to the scope of claim 帛n, wherein the immunostimulant is also an immune inducer selected by the method of claim 18 of the patent application. For example, if you apply for the method of increasing the disease resistance of animals in the 22nd pub, the immune seduce is Chinese herbal medicine (Blagen, Astragalus, Licorice), ΐφ叩 (Lps), 32 200811196 β-glucan, pepdiglycan 〇 25. If you apply for the special product \f22_红增鸠 her Wei-guide agent can be further step-by-step - pharmaceutically acceptable 编 其中 其中 其中 其中 其中 其中 其中 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如The towel is contained as an excipient, a diluent, a granule, a filler, a binder, a disintegrant, a lubricant, a base of oil or a non-greasy agent, an surfactant, a suspending agent, a gelling agent, Adjuvants, preservatives, oxidizing agents, stabilizers, colorants, perfumes, and the like. a few 27. The composition of claim 26, wherein the lubricant comprises, but is not limited to, glycerin and oils (e.g., peanut oil, castor oil). 28. The pharmaceutical composition as described in claim 26, wherein the base comprises a chlorocarbon compound, including but not limited to hard money, soft stone butterfly, stone butterfly oil, glycerin, honey bee, genus soap Natural oils (such as almond oil, corn oil, peanut oil, lotus oil or scented oil), lanolin and its touch, linonic acid (such as hard oleic acid or oleic acid) or combinations thereof. The pharmaceutical composition according to claim 26, wherein the surfactant comprises an anionic surfactant, a cationic surfactant, and a nonionic surfactant, and the surfactant is not limited to mountain # alcohol A medicinal herb, polyoxyethylene acetophenone and its derivatives (such as: polyethylene oxide fatty acid vinegar), polytetraethylene and its derivatives (such as · Carbourne resin carb〇p〇1). 30. The pharmaceutical composition according to claim 26, wherein the suspending agent comprises, but is not limited to, a day;;, a tree 1 曰, a cellulose bamboo bio-inorganic substance (such as silieace〇us silicas), The medicinal composition of claim 26, wherein the gelling agent comprises any gelling agent for pharmaceutically forming a colloid, wherein the gelling agent comprises a gelling agent for pharmaceutically forming a colloid. Gelling agents include, but are not limited to, cellulose derivatives (eg, methylcellulose, transethylcellulose, and carboxymethylcellulose, etc.), vinyl polymers (eg, polyvinyl alcohol, olyvinyl pyrrolid〇nes) , carboxyvinyl derivatives (such as: carbopol resin), pectin, gums (such as: gum arabic, tragacanth, algin, agar, gelatin to 33 200811196 and cairageenates) 〇 32. The method for increasing the disease resistance of an animal according to Item 22, wherein the peptide fragment can be obtained from a natural animal, especially a water vivid object, or synthesized by a conventional chemical synthesis method, or can be prepared by a conventional molecular biological method. 33·If you apply for a special The method for increasing the disease resistance of the lining according to Item 22, wherein the immune inducing agent is administered orally to the animal. 34. Increasing the disease resistance of the animal as described in claim 22 The method, wherein the immune inducing agent is micro-embedded. / X 35. If the method of increasing the anti-mystery of the animal is described in detail, the immuno-inducing agent is administered to the animal by injection. " 36. A method for increasing the disease resistance of an animal according to Item 22, wherein the immunologically inducing agent is administered to the animal by intravenous injection. 37. =Special: The increase of _^^^ V诏 described in item 22 is administered to the body of the animal by the method of adding I human water. 38. A method of increasing the disease resistance of an animal according to claim 22, wherein the immunologically inducing agent is administered to the animal by soaking. Eight " 34 200811196 Sequence Listing SEQ ID NO: 1 Length: 67 Type: amino acid Bacteria gentian plaque Additional information: Antimicrobial peptide (AMP) 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 Phe lie Phe His Val lie Lys Gly Leu Phe 20 25 30 His Ala Gly Lys Met lie His Gly Leu Val Thr Arg Arg Arg His Gly 35 40 45 Met Glu Glu Leu Gin Asp Leu Asp Gin Arg Ala Phe Glu Arg Glu Lys 50 55 60 Ala Phe Ala 65 SEQ ID NO: 2 Length: 494 Type: Nucleotide (DNA) Creature: Gentiana Plaque Additional Information: Antimicrobial Peptide (AMP) agagacttgc agcatctgta gatctcacac tgctcgattg gccctcttca gtaacagctt 60 tttgacattc acgctcagtc actggaaaga ggatgaggtg catcgccctc tttcttgtgt 120 tgtcgctggt ggtcctcatg gctgaacccg gggagggttt tatcttccac gtcatcaaag 180 gactctttca cgctggcaag atgatccatg gacttgtcac caggagacga catggcatgg 240 aagagctgca agacctggac caacgtgcct ttgaacgaga gaaagctttt gcctgagtcc 300 atgatagcct agtgaaggag ccactcattg ttaacacaaa aagaaaaagt ttttgttttt 360 gagtatagga agtattggtt caattgggta accaaaatat tttacactga tctaattgat 420 tttggaaaaa tgttagttat tctggaatct gtgttacaca caaaaaaaaa 480 aaaaaaaaaa aaaa 494 35 200811196 SEQIDNO ttgaaataaa: 3 length: 21 type : Nucleotide (DNA) Creature: Artificial sequence Additional information: PCR primer, forward AMP primer atgaggtgca tcgccctctt t 21 SEQIDNO: 4 Length: 21 Type: Nucleotide (DNA) Creature: Artificial sequence Additional information: PCR primer, reverse Lead to AMP tcaggcaaaa gctttctctc g 21 SEQ ID NO: 5 Length: 22 Type: amino acid Bio: gentian grouper Additional information · Antimicrobial peptide (AMP) message peptide (Legal Met 15 Met Arg Cys lie Ala Leu Phe Leu Val Leu Ser Leu Val 1 5 10 Ala Glu Pro Gly Glu Gly 20 SEQ ID NO: 6 Length :25 Type: amino acid Biology: gentian grouper Other data: mature peptide of antimicrobial peptide (AMP) 36 200811196 Phe 1 lie Phe His Val 5 lie Lys Gly Leu Phe His 10 Ala Gly Lys Met lie 15 His Gly Leu Val 20 Thr Arg Arg Arg His 25 SEQ ID NO: 7 Length: 20 Type: amino acid Bio: gentian grouper Other data: Antimicrobial peptide (AMP) )prodomain Gly Met Glu Glu Leu Gin Asp Leu Asp Gin Arg Ala Phe Glu Arg Glu Lys Ala Phe Ala 20 SEQ ID NO : 8 Length: 15 Type: Nucleotide (DNA) Creature: Artificial sequence Additional information: PCR forward primer, real-time quntitative PCR AMP specific primer A3 ttgtgttgtcg ctggt 15 SEQ ID NO: 9 Length: 15 Type: Nucleotide (DNA) Biology: Artificial sequence Additional information: PCR reverse primer, real-time quntitative PCR AMP specific primer A4 caaaggcacgt tggtc 15 37