CN109355273A - A kind of recombinant lysozyme derived from the gene of Trichinella sinensis - Google Patents
A kind of recombinant lysozyme derived from the gene of Trichinella sinensis Download PDFInfo
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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Abstract
The invention discloses a kind of recombination lysozyme from hydriopsis cumingii gene, the coded sequence of the recombination lysozyme is nucleotide sequence shown in SEQ ID No.1, and the amino acid sequence of the recombination lysozyme is shown in SEQ ID No.2.Recombination lysozyme of the invention has wider pH value tolerance range, and all has fungistatic effect to Gram-positive and Gram-negative bacteria, becomes apparent from, is with a wide range of applications to the fungistatic effect of gram-positive bacteria.
Description
Technical field
The present invention relates to a kind of lysozyme, in particular to a kind of recombination lysozyme from hydriopsis cumingii gene.
Background technique
Prawn is the most dominant Important Economic aquiculture animal in shallow sea.Culture of Penaeus Chinensis industry is quickly grown, however,
Current cultivation average success rate only has 20-30%.Wherein, prawn Deaths syndrome EMS (Early Mortality
Syndrome) and the generation of Acute Hepatic Pancreatic Necrosis to shrimp culture industry causes tremendous influence, seriously restricts its duration hair
Exhibition.At past 20 years, people used various antibiotic treatments to the infectious diseases of shrimps, and achieve certain effect.
However there are the hidden danger of safety and drug resistance for abuse of antibiotics.Firstly, in livestock products antibiotic residual and breeding process
It is medium-term and long-term to will lead to immunologic function of livestock and birds decline using antibiotic.Secondly, aquatic products intensive culture results in antibiotic residue, resistance to
The problems such as pharmacological property, not only inhibits the development of culture fishery, but also also has certain damage to human body.Meanwhile in aquatic products
The chemical preservative of addition also results in the vigilance of people.Therefore, the substitute for finding antibiotic is that solve the problems, such as one is non-
Normal effective approach.
Lysozyme (lysozyme) is also known as muramidase (muramidase), is a kind of alkali water-soluble protease.It leads
To make cell wall not by the glycosidic bond between the -acetylmuramic acid and N-Acetyl-D-glucosamine in cutting whole cell peptidoglycan
Dissolubility sticks polysaccharide and resolves into soluble glycopeptide, leads to cell wall rupture, bacteria lysis.Lysozyme can be with negatively charged viral egg
It is white to bind directly, double salt is formed with DNA, RNA apoprotein, is made virally inactivated.Most of lysozymes are for Gram-positive
The cell wall damage of bacterium is stronger, and acts on Gram-negative bacteria very weak.Lysozyme is as the nospecific immunity factor, in itself
It is a kind of native protein, pathogen will not have both been made to generate drug resistance, animal will not be made to generate residue problem, be a kind of safety
The very high feed enzyme preparation of property.
Hydriopsis cumingii is commonly called as freshwater mussel, pearl freshwater mussel, fresh water pearl freshwater mussel, triangle freshwater mussel, scientific name Hyriopsis cumingii.Fresh water
Bivalve belongs to Bivalvia, Unionidae, sail freshwater mussel category.It is distributed widely in Hunan, Hubei, Anhui, Jiangsu, Zhejiang, Jiangxi etc.
It saves, it is especially more with China's Dongting Lake and the distribution of medium-sized lake.Shell is big and flat, shell surface black or sepia, thick and hard, long
Nearly 20 centimetres, rear back edge stretches out a sail rear wing upwards, makes freshwater mussel shape in triangular shape.Notalia is made of several tubercle protrusion
The thick rib of diagonal.Pearl thickness, gloss are strong.Hinge is flourishing, and left housing has 2 pieces of anisometric pseudocardinal tooths and 2 pieces of side teeth, and right shell has 2
Piece pseudocardinal tooth and 1 piece of side tooth.Hydriopsis cumingii is the distinctive freshwater mussel resource in China, and is the good material grown cultured pearls.The treasure being bred as with it
Pearl is high-quality, and 80~120 freshwater mussels can be bred as 500 grams of seedless pearl, and the grains such as also fertile nucleated pearl, color bead, legendary luminous pearl are big
Sparkling and crystal-clear brilliant rare pearl.Meat is edible；Meat and shell powder can make the feed of domestic animal, poultry.So far, there has been no about utilization
Hydriopsis cumingii Data mining recombinates the research report of lysozyme and its antibacterial activity.
Summary of the invention
The purpose of the present invention is to provide a kind of recombination lysozymes from hydriopsis cumingii gene, have wider pH value
Tolerance range, and fungistatic effect is all had to Gram-positive and Gram-negative bacteria, it is with a wide range of applications.For into one
Step research novel green drug provides reference and theoretical foundation, at the same for antibacterial therapy provide it is efficient, less toxic, be not likely to produce it is resistance to
The drug of pharmacological property lays the foundation to develop feed addictive with independent intellectual property rights.
The technical solution adopted by the present invention to solve the technical problems is:
The coded sequence of a kind of recombination lysozyme from hydriopsis cumingii gene, the recombination lysozyme is SEQ ID No.1
Shown nucleotide sequence.
The amino acid sequence of a kind of recombination lysozyme from hydriopsis cumingii gene, the recombination lysozyme is SEQ ID
Shown in No.2.
A kind of preparation method of the recombination lysozyme from hydriopsis cumingii gene, comprising the following steps:
(1) sample total serum IgE is extracted from hydriopsis cumingii tissue；
(2) reverse transcription is carried out as template using the sample total serum IgE of extraction and obtains cDNA library；
(3) using cDNA library as template carry out PCR amplification obtain pcr amplification product；
(4) pcr amplification product I digestion of EcoR I and Xho, is then connected with the pEQ-30 expression vector through same enzyme digestion
Form recombinant plasmid；
(5) by recombinant plasmid transformed into e. coli bl21 DE3, screening obtains recombinant bacterium after culture；
(6) then recombinant bacterium carries out ion-exchange chromatography through IPTG inducing expression, elution, collects eluent, and eluent uses
Molecular sieve is centrifuged desalination, obtains recombination lysozyme.
The present invention carries out Enzyme assay to it by Optimal Expression condition, a large amount of recombination lysozymes for obtaining purifying,
Inquire into the influences of the factors to enzymatic activity such as pH value, temperature, metal ion, inhibitor or detergent.Simultaneously to recombination lysozyme
PH value tolerance, temperature tolerance, antibacterial type are also probed into.Development of the invention will be helpful to answering for environmentally protective medicament
With, provide reference and theoretical foundation for further research novel green drug, while for antibacterial therapy provide it is efficient, less toxic,
It is not likely to produce the drug of drug resistance, is laid the foundation to develop feed addictive with independent intellectual property rights.
The primer that PCR amplification uses in step (3) is as follows:
Forward primer: 5 '-AGGAATTC ATGGCTGCATCTTCAAAC-3 ',
Reverse primer: 5 '-ACCCTCGAGATCAGCCAAGTTTCTTATTC TC-3 '.
PCR reaction condition in step (3) are as follows: 94 DEG C initial denaturation 5 minutes, subsequently into following circulation: 94 DEG C be denaturalized 30 seconds,
59 DEG C are annealed 30 seconds, and 72 DEG C extend 60 seconds, carry out 30 circulations altogether, last 72 DEG C extend 10 minutes.
Recombinant plasmid transformed is used to thermal shock method in step (5) into e. coli bl21 DE3.
The parameter of IPTG inducing expression in step (6) are as follows: IPTG concentration 0.1mM/L, shaking speed 200-220rpm, 37 DEG C
Cultivate 4h.
The beneficial effects of the present invention are: there is wider pH value tolerance range, and to Gram-positive and Gram-negative
Bacterium all has fungistatic effect, becomes apparent from, is with a wide range of applications to the fungistatic effect of gram-positive bacteria.
The present invention provides reference and theoretical foundation for further research novel green drug, while providing for antibacterial therapy
Efficiently, drug that is less toxic, being not likely to produce drug resistance, lays the foundation to develop feed addictive with independent intellectual property rights.
Detailed description of the invention
Fig. 1 is the SDS-PAGE testing result figure after recombination lysozyme purification of the invention.
M indicates albumen Marker；1 indicates the bacterium solution for being not added with inducer IPTG；After 2 indicate addition IPTG inducing expression
Bacterium solution；3 indicate that recombinant protein (recombination lysozyme) one, 4 indicates that recombinant protein two, 5 indicates that recombinant protein three, 6 indicates recombination egg
White six.
Fig. 2 is BCA protein quantification canonical plotting.
Fig. 3 is influence of the pH to enzymatic activity.
Fig. 4 is influence of the temperature to enzymatic activity.
Fig. 5 is the pH stability of enzyme.
Fig. 6 is the thermal stability of enzyme.
Fig. 7 is the influence of inhibitor or detergent to being recombination lysozyme activity.
Fig. 8 is to recombinate lysozyme to the inhibition zone size of subiilis and E.coli.
Fig. 9 is to recombinate lysozyme to the inhibitory effect of different bacterium.
In figure: relative activity relative activity, temperature temperature, time time.
Specific embodiment
Below by specific embodiment, technical scheme of the present invention will be further explained in detail.
In the present invention, if not refering in particular to, used raw material and equipment etc. are commercially available or commonly used in the art.
Method in following embodiments is unless otherwise instructed the conventional method of this field.
Reagent: peptone, yeast extract purchased from hundred think Biotechnology Co., Ltd, 1.5M Tris-HCl pH 8.8,
1M Tris-HCl pH 6.8, TEMED, kanamycins are purchased from green skies biotechnology research institute, IPTG, Tris-base, sweet ammonia
Acid, SDS, 30%Arc-Bis, Ni-IDA Resin are purchased from Hangzhou Nuo Yang Bioisystech Co., Ltd, and imidazoles is purchased from Beijing Suo Lai
Precious Science and Technology Ltd., Triton X-100 are purchased from Aladdin Industrial Co., Ltd., the upper ocean Protein Marker, SDS-PAGE
Buffer, G-250 albumen rapid dyeing reagent, BCA protein quantification kit are century Biotechnology Co., Ltd, mistake purchased from health
Ammonium sulfate, calcium chloride, magnesium chloride, lithium chloride, potassium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate are purchased from close europeanized of Tianjin section
Reagent Co., Ltd, SDS, Tween-20 are purchased from Aladdin Industrial Co., Ltd., and lysozyme enzyme activity determination kit is purchased from Nanjing
Bioengineering Research Institute is built up, sodium chloride is purchased from Gao Jing Fine Chemical Co., Ltd.
Hydriopsis cumingii is purchased from the Hangzhou poplar door market of farm produce.
Embodiment:
A kind of preparation method of the recombination lysozyme from hydriopsis cumingii gene, comprising the following steps:
(1) use RNA extracts kit (High Pure FFPET RNA Isolation Kit, Roche) from hydriopsis cumingii group
It knits and extracts sample total serum IgE in (musculature).
(2) reverse transcription (Reverse Transcriptase kit, PrimeScript RT are carried out by template of the sample total serum IgE of extraction
Reagent Kit Perfect Real Time, TaKaRa) obtain cDNA library.
(3) using cDNA library as template carry out PCR amplification obtain pcr amplification product (target gene):
PCR reaction system is as follows:
2 μ L of cDNA, primers F (10umol/L) 1 μ L, primer R (10umol/L) 1 μ L, ddH2O6 μ L, Mix (10 × Taq
Buffer、MgCl2, dNTP, Taq enzyme) 10 μ L；20 μ L of total volume.Mix (realtime PCR master mix, producer:
TOYOBO, model: QPK-101).
PCR reaction condition are as follows: 94 DEG C initial denaturation 5 minutes, subsequently into following circulation: 94 DEG C be denaturalized 30 seconds, 59 DEG C annealing
30 seconds, 72 DEG C extended 60 seconds, carried out 30 circulations altogether, last 72 DEG C extend 10 minutes.
Primer is as follows:
Forward primer: 5 '-AGGAATTC ATGGCTGCATCTTCAAAC-3 ' (SEQ ID No.3),
Reverse primer: 5 '-ACCCTCGAGATCAGCCAAGTTTCTTATTC TC-3 ' (SEQ ID No.4).
(4) pcr amplification product, pcr amplification product I enzyme of EcoR I and Xho are recycled by Tiangeng DNA QIAquick Gel Extraction Kit
It cuts, then is connected to form recombinant plasmid with the pEQ-30 expression vector through same enzyme digestion.
(5) recombinant plasmid is converted by chemical transfection thermal shock method into e. coli bl21 DE3, and is coated on LB solid
On culture plate, 30 DEG C are cultivated 12 hours；The bacterium colony grown is examined by PCR, gene successful connection is determined, chooses bacterium colony to Shanghai
Raw work sequencing, it is ensured that gene correctly sieves to obtain recombinant bacterium afterwards.Recombinant bacterium is seeded in LB liquid medium, and 37 DEG C of overnight incubations obtain
Bacterium solution.
(6) recombinant bacterium expression and purification:
6.1 prepare good material, and super-clean bench carries out sterilizing works；
6.2 take 50 μ L of bacterium solution to be connected in 5mL culture medium (LB liquid medium), add 5 μ L kanamycins, are uniformly mixed, set
It is stayed overnight in 200-220rpm, 37 DEG C of shaking table cultures；
5mL bacterium solution after 6.3 shaking table cultures accesses in new 250mL LB liquid medium, 250 μ L kanamycins of addition, and 37
DEG C culture；
6.4 cultures 3 hours are to bacterium solution OD600=0.6, the 50 μ final concentration of 0.1mM/L of L 500mM/L IPTG to IPTG are added,
37 DEG C of culture 4h of 200-220rpm；
The bacterium solution of 6.5 inducing expressions is in 12000g, 4 DEG C of centrifugation 3min；
6.6 take precipitating, are added ice-cold 15mLbuffer A, ultrasound cracking bacterium under condition of ice bath (70%, 5s/5s,
10min)；
6.7 18000g, 4 DEG C of centrifugation 5min；
6.8 media in ion exchange column (nickel column, Novagen company, GE) should be balanced first with buffer, be then added from
Supernatant after the heart, 4 DEG C slowly combine 1-2h；
6.9 wash column with 20mL buffer B, divide 5 times, each 4mL, abandon；
6.10 wash column with 20mL buffer C again, divide 5 times, each 4mL, abandon；
6.11 are added 0.5mL buffer D, close flow liquid (discarding) after liquid is close to be flow to end, 0.5mL is then added
Buffer D impregnates 5min (can blow afloat medium herein to be allowed to suspend), collects eluent, the eluent of the collection is exactly purpose egg
It is white；
6.12 repeat 6.11 steps 2 time；
6.13 protein eluates are centrifuged desalination through over-molecular sieve (genome company of the U.S.), spare.
Solution needed for protein purification
Buffer A (pH=7.9): 0.5mol/LNaCl, 10mmol/L imidazoles, 0.5%TritonX-100,20mmol/
LTris-HCl；Buffer B (pH=7.9): 0.5mol/L NaCl, 20-40mmol/L imidazoles, 0.5%TritonX-100,
20mmol/LTris-HCl；Buffer C (pH=7.9): 0.5mol/L NaCl, 20-40mmol/L imidazoles, 20mmol/
LTris-HCl；
Buffer D (pH=7.9): 0.1mol/L NaCl, 250mmol/L imidazoles, 20mmol/LTris-HCl.
Through being sequenced, the coded sequence that the present invention recombinates lysozyme is nucleotide sequence shown in SEQ ID No.1, recombinates bacteriolyze
The amino acid sequence of enzyme is shown in SEQ ID No.2.
Experimental method
1.1 recombination lysozymes are quantitative
The recombination lysozyme of purifying detects purity of protein by polyacrylamide gel electrophoresis (SDS-PAGE).It determines in product and contains
After having recombination lysozyme, the albumen of purifying is quantified using Microdilution plate method using BCA protein quantification kit.
BCA protein quantification is kit specification referring to health.
The Activity determination of 1.2 recombination lysozymes
1.2.1 the optimum pH and pH value tolerance of lysozyme are recombinated
Under conditions of pH value is followed successively by 3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0, using micrococcus lysodeikticus as substrate,
Carry out the measurement of enzymatic activity, the optimum pH of analysis recombination lysozyme.In addition, being separately added into the different solution of above-mentioned pH
The recombination lysozyme of amount measures enzymatic activity after being resistant to half an hour.
1.2.2 the optimum temperature and temperature tolerance of lysozyme activity are recombinated
Using micrococcus lysodeikticus as substrate, in the optimum pH solution of enzymatic activity, (10 DEG C, 20 DEG C, 30 at a temperature of different
DEG C, 40 DEG C, 50 DEG C, 60 DEG C), detection recombination lysozyme optimum temperature.Equally, at different temperatures, recombination lysozyme tolerance
Enzymatic activity is detected after 10min, 20min, 30min, 40min, 50min.
1.2.3 the influence of inhibitor or detergent to recombination lysozyme activity
Using micrococcus lysodeikticus as substrate, suitable recombination lysozyme is separately added into inhibitor lauryl sodium sulfate (SDS), is gone
In dirty agent Tween-20 and TritionX-100, it is placed in 10min under room temperature, to be added without inhibitor in optimum pH buffer
Recombination lysozyme with detergent is as blank determination enzymatic activity.
1.2.4 influence of the metal ion to recombination lysozyme activity
With the micro- substrate of micrococcus lysodeikticus, lysozyme will be recombinated and be separately added into the metal ion that concentration is 1M, 100mM, 10mM, 1mM
(Na in solution+、Mg2+、K+、Li+、Fe3+), it is placed in 10min under room temperature, the weight of metal ion to be not added in optimum pH buffer
Group lysozyme is as blank determination enzymatic activity.
1.2.5 the bacteriostatic experiment of lysozyme is recombinated
Mainly by Gram-negative bacteria Escherichia coli (E.coli) and gram-positive bacteria bacillus subtilis in this experiment
(B.subiilis) bacteriostatic experiment is carried out, Escherichia coli (E.coli), staphylococcus glucose coccus (S.aureus), withered grass are selected
Bacillus (B.subiilis), vibrio parahemolyticus (V.Parahemolyticus), Pseudomonas aeruginosa (P.aeruginosa) etc.
The scope of restraining fungi of observation recombination lysozyme.
2 results and analysis
2.1 recombinate lysozymes and quantify
As shown in Figure 1, being about to have at 27kD obviously in molecular weight of albumen size after the bacterial strain addition IPTG induction containing recombinant plasmid
Expression, i.e. recombination lysozyme obtain recombination lysozyme by affinity chromatography nickel medium purification.In order to be carried out to recombination lysozyme
It is quantitative, quantitative (Fig. 2) is carried out using BSA method, the albumen that we are purified into can be calculated by BCA protein quantification standard curve
Concentration is 1.45mg/mL.
The measurement of the optimal pH and optimum temperature of 2.2 recombination lysozymes
Opposite enzyme activity of the recombination lysozyme under different pH buffers and different temperatures is measured by substrate of micrococcus lysodeikticus, as a result
See Fig. 3 and Fig. 4.Enzyme activity reaches highest when recombination lysozyme pH=6 as shown in Figure 3, when pH=3 and pH=10 is opposite
Enzyme activity is respectively 26% and 37%, remains higher activity in the glucose-6-phosphate dehydrogenase of pH=5 and pH=8.As shown in Figure 4 with respect to enzyme activity
Reach highest at 30 DEG C, still keeps the 29% of enzyme activity at 5 DEG C, the characteristic feature with cold-adapted enzyme, and in 20 DEG C to 40 DEG C
Keep greater activity.
The pH stability and thermal stability of 2.3 recombination lysozymes
Enzyme solution is respectively placed in a series of 30 DEG C of heat preservation 30min in different pH buffers, then measures enzyme activity (Fig. 5) again,
The result shows that: enzyme shows extensive stability in pH5-9.Liquid is placed under 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C (Fig. 6)
Different time is handled, when pH=6.0 measures enzyme activity, the results showed that, enzyme has preferable stability when less than 40 DEG C.
The influence of 2.4 inhibitor or detergent to recombination lysozyme activity
Using micrococcus lysodeikticus as substrate, it is separately added into different inhibitor and detergent, sets by suitable recombination lysozyme
The 10min under room temperature is surveyed using the recombination lysozyme for being added without inhibitor and detergent as control in optimum pH buffer
Determine enzymatic activity.The result shows that: SDS, EDTA, TEMD, Trition X-100 and Tween-20 have facilitation to enzymatic activity,
And DTT has inhibiting effect (Fig. 7) to enzymatic activity.
Influence of 2.5 metal ions to recombination lysozyme activity
With the micro- substrate of micrococcus lysodeikticus, lysozyme will be recombinated and be separately added into the metal ion that concentration is 1M, 100mM, 10mM, 1mM
K in solution+、Mg2+、Fe3+、Na+), it is placed in 10min under room temperature, the recombination of metal ion is not added in optimum pH buffer
Lysozyme is as blank determination enzymatic activity.The result shows that (table 1) metal ion Na+、Fe3+、Li+There is facilitation to enzymatic activity,
And K+、Mg2+There are inhibiting effect, and increasing with concentration of metal ions to enzymatic activity, the inhibition or promotion to enzymatic activity
Effect is stronger.
Influence of 1 metal ion of table to recombination lysozyme activity
The bacteriostatic experiment of 2.6 recombination lysozymes
As shown in figure 8, to gram-positive bacteria bacillus subtilis (B.subiilis) and Gram-negative bacteria Escherichia coli
(E.coli) when carrying out bacteriostatic experiment, it can produce apparent raw inhibition zone, the results showed that the recombination lysozyme is to both having
Effect.As shown in figure 9, selecting Escherichia coli (E.coli), staphylococcus glucose coccus (S.aureus), bacillus subtilis
(B.subiilis), the antibacterial model of the observations such as vibrio parahemolyticus (V.Parahemolyticus), Pseudomonas aeruginosa recombination lysozyme
When enclosing, the recombination lysozyme has the above bacterium as the result is shown inhibits to grow to some extent, wherein the growth to S.aureus
Inhibition reaches 83.57%, reaches 36.34% to the growth inhibition of E.coli.
3 discuss and look forward to
By the property of research recombination lysozyme, reaction optimal pH is 6.0, and shows wider pH value and be resistant to (pH for this experiment
Value is 5-9), 50% enzyme activity is still kept after tolerance 50 minutes.In terms of temperature, optimum temperature is 30 DEG C, and at 40 DEG C
Hereinafter, enzyme activity is always 90% or more.In ions Na+、Fe3+、Li+There is a facilitation to enzymatic activity, and K+、Mg2+To enzyme
Activity has inhibiting effect.SDS, EDTA, TEMD, Trition X-100 and Tween-20 have facilitation to enzymatic activity, and
DTT and PMSF has inhibiting effect to enzymatic activity, and several cysteines that the prefabricated effect of DTT shows to recombinate in lysozyme form two
Sulfide linkage, and disulfide bond plays an important role in the stability for maintaining albumen.At antibacterial aspect, lysozyme is recombinated to Escherichia coli
(E.coli), staphylococcus glucose coccus (S.aureus), bacillus subtilis (B.subiilis), vibrio parahemolyticus
(V.Parahemolyticus), Pseudomonas aeruginosa (P.aeruginosa) has inhibitory effect, wherein the effect of gram-positive bacteria
It becomes apparent from.In conclusion the wider pH value tolerance of the recombination lysozyme, and to the difference of Gram-positive and Gram-negative bacteria
Fungistatic effect all presents the recombination lysozyme and has a wide range of applications future.
Communicable disease causes tremendous influence to shrimp culture industry, seriously restricts its sustainable development.Make for a long time
With antibiotic there are the hidden danger of safety and drug resistance, lysozyme is a kind of native protein as the nospecific immunity factor,
It is added in prawn feed, can solve in breeding production thus lead to problems such as immunity of prawn reduction and medicament residue,
Therefore the present invention can provide theoretical foundation for the development and utilization of lysozyme.
Above-mentioned embodiment is only a preferred solution of the present invention, not the present invention is made in any form
Limitation, there are also other variations and modifications on the premise of not exceeding the technical scheme recorded in the claims.
Sequence table
<110>Institutes Of Technology Of Zhejiang
<120>a kind of recombination lysozyme from hydriopsis cumingii gene
<130> 2018.11
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 492
<212> DNA
<213>hydriopsis cumingii (Hyriopsis cumingii)
<400> 1
atggctgcat cttcaaactc acagtttctg acaaaactat gcgctgaact tgagaaggac 60
gaaggagttg tacacaaaat atacacagac cacctgggct atttgcactt tggtattggt 120
caccttatca ccgagaatga tccagaatac ggcaagtcag ttggcagccc ggtctgcaag 180
gagagagtgg atgaagtctt caagaaagac gttcaaaccg ctttggaggg atgctcccga 240
ctgtttccag actttcagga actccctgag gaggctcagc tcgttatcgc caatatgatg 300
tttaatctgg gagagaccaa actcgcaaag tttgtgaagt ttcgagctgc cttggaggct 360
agggattggt ccaaagctgc agacgagatg gtggacagca ggtggtacaa acaagtgaca 420
acgcgagcca acaggcttgt cgcgagaata agaaacttgg ctgatctcga gcaccaccac 480
caccaccact ga 492
<210> 2
<211> 215
<212> PRT
<213>lysozyme (lysozyme)
<400> 2
Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser
1 5 10 15
Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp
20 25 30
Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Asp Ile
35 40 45
Gly Ser Glu Phe Met Ala Ala Ser Ser Asn Ser Gln Phe Leu Thr Lys
50 55 60
Leu Cys Ala Glu Leu Glu Lys Asp Glu Gly Val Val His Lys Ile Tyr
65 70 75 80
Thr Asp His Leu Gly Tyr Leu His Phe Gly Ile Gly His Leu Ile Thr
85 90 95
Glu Asn Asp Pro Glu Tyr Gly Lys Ser Val Gly Ser Pro Val Cys Lys
100 105 110
Glu Arg Val Asp Glu Val Phe Lys Lys Asp Val Gln Thr Ala Leu Glu
115 120 125
Gly Cys Ser Arg Leu Phe Pro Asp Phe Gln Glu Leu Pro Glu Glu Ala
130 135 140
Gln Leu Val Ile Ala Asn Met Met Phe Asn Leu Gly Glu Thr Lys Leu
145 150 155 160
Ala Lys Phe Val Lys Phe Arg Ala Ala Leu Glu Ala Arg Asp Trp Ser
165 170 175
Lys Ala Ala Asp Glu Met Val Asp Ser Arg Trp Tyr Lys Gln Val Thr
180 185 190
Thr Arg Ala Asn Arg Leu Val Ala Arg Ile Arg Asn Leu Ala Asp Leu
195 200 205
Glu His His His His His His
210 215
<210> 3
<211> 26
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 3
aggaattcat ggctgcatct tcaaac 26
<210> 4
<211> 31
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 4
accctcgaga tcagccaagt ttcttattct c 31
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