CN106478792A - A kind of three PEPC ec Md3js, preparation method and application - Google Patents

Abstract

The invention discloses a kind of three PEPC ec Md3js, for encoding the nucleotide sequence of described three PEPC ec Md3js as shown in SEQ ID NO.1, the aminoacid sequence of described three PEPC ec Md3js is as shown in SEQ ID NO.2.Using the antibacterial peptide Cec Md of this three PEPC ec Md3js preparation, there is to various bacteria antibacterial activity, heat stability good, and isolate and purify simple, easy to operate, it is suitable to large-scale industrial production, this three PEPC ec Md3js has a good application prospect in fields such as antibacterials, food antibacterial, feed additives.In addition, a kind of present invention also offers preparation method of three PEPC ec Md3js.

Description

A kind of three PEPC ec Md3js, preparation method and application

Technical field

The invention belongs to gene engineering technology field and in particular to a kind of three PEPC ec Md3js, preparation method and should With.

Background technology

Antibacterial peptide is the effective barrier that organism resists extraneous pathogenic microorganism invasion, has broad-spectrum antibacterial action, and Have the characteristics that to be not likely to produce drug resistance, in fields such as agricultural, medical treatment, food, animal feeds, there is preferable application prospect.Mesh Front it has been found that thousands of kinds of antibacterial peptides, but the antibacterial peptide species that can be applied to clinic is still limited.Accordingly it is desirable to Activity preferably novel antimicrobial peptide is obtained by MOLECULE DESIGN and again transformation.

Because antibacterial peptide belongs to small-molecular peptides, its clone, expression, separation, purification and detection operation are all relatively cumbersome, in order to Improve the organic efficiency of destination protein, effectively keep antibacterial peptide activity, reduce host cell toxic, increase antiseptic peptide stability And expression, to express antibacterial peptide generally by the way of expressing in series.

Cecropin antimicrobial peptides (cecropins) are the natural antibacterial peptides that current research is the clearest, effect is best；Housefly giant silkworm The homology of plain antibacterial peptide (Cec Md) and cecropin antimicrobial peptides reaches 81%, but housefly cecropin antibacterial peptide still have certain molten Blood activity, and purification procedures are loaded down with trivial details.

Content of the invention

One kind three PEPC ec Md3js of present invention offer, preparation method and application, using this three PEPC ec Md3js The antibacterial peptide Cec Md hemolytic of preparation is low, antibacterial activity is high, and this preparation method purification procedures is simple.

The invention provides a kind of three PEPC ec Md3js, for encoding the nucleotides sequence of described three PEPC ec Md3js , as shown in SEQ ID NO.1, the aminoacid sequence of described three PEPC ec Md3js is as shown in SEQ ID NO.2 for row.

Present invention also offers a kind of preparation method of three PEPC ec Md3js, comprise the following steps：

Step 1, genes of interest as shown in SEQ ID NO.3 for the synthesizing ribonucleotide sequence；

Step 2, with restricted enzyme Kpn I and Xba I double digestion carrier pGAPZ α A, and reclaims the load obtaining 3147bp Body fragment；Described genes of interest and described carrier segments are connected, obtains the company containing recombinant vector pGAPZ α A/Cec Md3js Connect liquid；

Step 3, the connection liquid of step 2 is converted to competent escherichia coli cell DH5 α, and extracts recombinant vector PGAPZ α A/Cec Md3js, obtains recombinant vector pGAPZ α A/Cec Md3js；

Step 4, recombinant vector pGAPZ α A/Cec Md3js is transformed in host cell, obtains expressing engineering bacteria；

Step 5, the expression engineering bacteria of incubation step 4, carry out the expression of three PEPC ec Md3js, and extract three PEPC ec Md3js, obtains three PEPC ec Md3js.

Preferably, in the preparation method of above-mentioned three PEPC ec Md3js, the host cell of step 4 is Pichia sp. GS115.

Present invention also offers a kind of three PEPC ec Md3js, the application in preparing antibacterial peptide Cec Md.

Present invention also offers a kind of three PEPC ec Md3js, in preparation treatment Gram negative bacterial disease medicine or Application in treatment gram positive bacteria disease medicament.

Present invention also offers a kind of three PEPC ec Md3js, the application in preparing animal feed additive.

Also a kind of method preparing antibacterial peptide Cec Md using described three PEPC ec Md3js of the present invention, specifically include with Lower step：

Step a, with pH 8.0, the PB buffer of 0.01mol/L is solvent, and compound concentration is the three PEPC ec of 1mg/mL Md3js solution；Then dose volume fraction is 50% formic acid solution, and three PEPC ec Md3js solution are pressed with formic acid solution According to 1000:12 volume ratio mixing, 50 DEG C of water-bath 36h, obtain cutting solution；

Step b, will cut solution with distilled water and dilute 3 times, and vacuum lyophilization obtains solid antimicrobial peptide；

Solid antimicrobial peptide is redissolved with distilled water, then through vacuum lyophilization, obtains antibacterial peptide Cec Md crude product by step c；

Step d, the purification of antibacterial peptide Cec Md

By antibacterial peptide Cec Md crude product through 5000kDa membrance separation, the separating liquid obtaining, through G50 column chromatography, is resisted Bacterium PEPC ec Md.

One kind three PEPC ec Md3js that the present invention provides, and apply technique for gene engineering efficient table in Pichia sp. Reached this three PEPC ec Md3js, empirical tests, using the antibacterial peptide Cec Md hemolytic of this three PEPC ec Md3js preparation low, There is antibacterial activity, heat stability to various bacteria good, expression is high, and isolates and purifies simple, easy to operate, is suitable to extensive work Industry metaplasia is produced, and this three PEPC ec Md3js has in the field such as medical (antibacterials), food (antibacterial), feed additive Good application prospect.

Brief description

Fig. 1 is that the PCR primer of recombinant vector pGAPZ α A/Cec Md3js identifies electrophoretogram；

Fig. 2 is that the double digestion product of recombinant vector pGAPZ α A/Cec Md3js identifies electrophoretogram；

Fig. 3 is that the linearisation product of recombinant vector pGAPZ α A/Cec Md3js identifies electrophoretogram；

Fig. 4 is that the bacterium colony PCR of yeast transformant identifies electrophoretogram；

Fig. 5 is the Tricine-SDS-PAGE analysis collection of illustrative plates of three PEPC ec Md3js；

Fig. 6 is the efficient liquid phase chromatographic analysis result of three PEPC ec Md3js；

Fig. 7 is the mass spectrometry results of three PEPC ec Md3js.

Wherein, in Fig. 1, M swimming lane is DNA standard molecular weight (Maker), and 1, No. 2 swimming lanes are recombinant vector pGAPZ α A/ The PCR primer (704bp) of Cec Md3js；In Fig. 2, M swimming lane is DNA standard molecular weight (Maker), and 1, No. 2 swimming lanes are restructuring The double digestion product (396bp) of carrier pGAPZ α A/Cec Md3js；In Fig. 3, M swimming lane is DNA standard molecular weight (Maker), No. 1 Swimming lane is the linearisation product (3543bp) of recombinant vector pGAPZ α A/Cec Md3js；In Fig. 4, M swimming lane is DNA standard molecular weight (Maker), No. 1 swimming lane is the bacterium colony PCR primer (783bp) of yeast transformant；In Fig. 5, M swimming lane is standard protein Marker, 1 Number swimming lane is three PEPCs ec Md3cs (13.91kDa), and No. 2 swimming lanes are three PEPCs ec Md3js (13.36kDa), No. 3 swimming lanes For Cec Md2cs (9.08kDa), No. 4 swimming lanes are Cec Md2js (8.79kDa), and No. 5 swimming lanes are bigeminy PEPC ec Md2ys (8.77kDa), No. 6 swimming lanes are antibacterial peptide Cec Md (4.26kDa), and No. 7 swimming lanes are Cec Md3jn sample supernatant, No. 8 swimming lanes For Cec Md3cn sample supernatant, No. 9 swimming lanes are blank.

Specific embodiment

The present invention is described in detail with reference to the accompanying drawings and detailed description, it is to be understood that the protection of the present invention Scope is not limited by specific embodiment.

The invention provides a kind of three PEPC ec Md3js, for encoding the nucleotides sequence of described three PEPC ec Md3js , as shown in SEQ ID NO.1, the aminoacid sequence of described three PEPC ec Md3js is as shown in SEQ ID NO.2 for row.

Based on same inventive concept, present invention also offers a kind of preparation method of three PEPC ec Md3js, including with Lower step：

Step 1, genes of interest as shown in SEQ ID NO.3 for the synthesizing ribonucleotide sequence.

It should be noted that for the expression improving antibacterial peptide, using series design, it then follows Pichia sp. preferences are former Then, the nucleotide sequence for encoding three PEPC ec Md3js as shown in SEQ ID NO.1 for the design, and 5 ' in this sequence The restriction enzyme site GGTACC and start codon ATG of Kpn I is added at end；And the restriction adding Xba I is held in this sequence 3 ' Property restriction enzyme site TCTAGA and termination codon TAA, obtain the genes of interest as shown in SEQ ID NO.3.

Step 2, with restricted enzyme Kpn I and Xba I double digestion carrier pGAPZ α A, and reclaims the load obtaining 3147bp Body fragment；Described genes of interest and described carrier segments are connected, obtains the company containing recombinant vector pGAPZ α A/Cec Md3js Connect liquid.Specifically include following steps：

Step 2.1, with restricted enzyme Kpn I and Xba I double digestion carrier pGAPZ α A, the double digestion of carrier pGAPZ α A System as shown in table 1, obtains carrier double digestion solution；

5 μ L carrier double digestion solution are taken to mix homogeneously with 6 × Loading Buffer of 1 μ L, in 1% agar containing EB Sugared electrophoresis detection, after electrophoresis detection is correct, by the carrier double digestion solution of remaining 15 μ L according to DNA gel reclaim reagent cassette method Carry out glue reclaim, obtain the carrier segments of 3147bp.

The double digestion system of table 1 carrier pGAPZ α A

Wherein, the condition of double digestion is：Each material in double digestion system shown in table 1 is added in PCR pipe and mixes, in 37 DEG C water-bath 3h.

Step 2.2, described genes of interest and described carrier segments is connected, its linked system is as shown in table 2, the bar of connection Part is 16 DEG C of water-bath 2h, obtains the connection liquid containing recombinant vector pGAPZ α A/Cec Md3js.

Table 2 genes of interest and the linked system of carrier segments

Step 3, the connection liquid of step 2.2 is converted to competent escherichia coli cell DH5 α (this step is mainly Recombinant vector pGAPZ α A/Cec Md2ys in connection liquid converted to competent escherichia coli cell DH5 α), 37 DEG C are shaken Swing culture 1h, be coated on containing 25 μ g/mL Zeocin^TMOn the less salt LB plating medium of (bleomycin), it is upside down in 37 DEG C of perseverances In warm incubator, until forming single bacterium colony, picking single bacterium colony carries out PCR primer (704bp) identification, sieve to incubated overnight (12-16h) Select of the positive colony containing recombinant vector pGAPZ α A/Cec Md3js, and extract the recombinant vector in positive colony PGAPZ α A/Cec Md3js, obtains recombinant vector pGAPZ α A/Cec Md3js.The formula of described less salt LB plating medium is： 10g/L tryptone, 5g/L yeast extract, 15g/L agar, 5g/L sodium chloride, balance of water, pH 7.5.Described PCR primer identification System is as shown in table 3.

The PCR primer identification system of table 3 recombinant vector pGAPZ α A/Cec Md3js

Wherein, the primer sequence of PCR primer (704bp) the identification employing of step 3 is：

Forward Primer：5’-GTCCCTATTTCAATCAATTGAA-3’

3’AOX1 Primer：5’-GCAAATGGCATTCTGACATCC-3’.

PCR primer qualification result such as Fig. 1 of recombinant vector pGAPZ α A/Cec Md3js, has band at 704bp, and weight is described Group carrier pGAPZ α A/Cec Md3js successfully constructs；Kpn I and Xba I double digestion of recombinant vector pGAPZ α A/Cec Md3js produces Thing qualification result, as shown in Fig. 2 having band at 396bp, illustrates that recombinant vector pGAPZ α A/Cec Md3js successfully constructs.

Step 4, recombinant vector pGAPZ α A/Cec Md3js is transformed in host cell (Pichia pastoris GS115), obtains Expression engineering bacteria.

Step 4.1, the preparation of Pichia pastoris GS115 competent cell

Step 4.1.1, activation：Take -80 DEG C of frozen Pichia pastoris GS115s, using YPD plate loop method, culture temperature Spend for 28-30 DEG C (about 2 days), obtain the single bacterium colony of the first monoclonal cell；

The single bacterium colony of picking first monoclonal cell, using YPD plate loop method, cultivation temperature is 28-30 DEG C (about 2 My god), obtain the single bacterium colony of the second monoclonal cell；

The single bacterium colony of picking second monoclonal cell adopts YPD plate loop method, and cultivation temperature is 28-30 DEG C (about 2 My god), obtain the single bacterium colony of Pichia pastoris GS115.

Wherein, adopt described in step 4.1.1 and can wrap up YPD flat board with preservative film during YPD plate loop method.

Step 4.1.2, cultivates in a small amount：The single bacterium colony of picking Pichia pastoris GS115 is inoculated into 5mL YPD fluid medium In, 28-30 DEG C, 200r/min shake-flask culture 16-18h (OD₆₀₀Value 2.0 about), obtain Pichia pastoris GS115 bacteria suspension.

It should be noted that shake-flask culture typically overnight, is not required to survey OD in step 4.1.2₆₀₀Value.

Step 4.1.3, amplification culture：By 5mL Pichia pastoris GS115 bacterial suspension inoculation to 500mL YPD fluid medium In, and press 1:100～200 volume ratio inoculation, culture bottle adopts the culture bottle of 2L, wherein every built-in 250mL of 1L culture bottle YPD liquid, in order to allow Pichia pastoris GS115 fully absorb inhaling of oxygen, 28-30 DEG C, 200r/min shake-flask culture 16-18h (OD₆₀₀Value 2.0 about), obtain Pichia pastoris GS115 culture bacterium solution.

It should be noted that shake-flask culture typically overnight, is not required to survey OD in step 4.1.3₆₀₀Value.

Step 4.1.4, Pichia pastoris GS115 is cultivated bacterium solution ice bath 10min, moves into the centrifuge tube of 4 DEG C of pre-coolings, and then 4 DEG C 1500 × g centrifugation 5min, removes supernatant, collects precipitation；And the resuspended precipitation of sterilized water with 4 DEG C of pre-coolings of 500mL, obtain first Yeast re-suspension liquid.

Step 4.1.5, the first yeast re-suspension liquid is centrifuged 5min in 4 DEG C of 1500 × g, removes supernatant, collects the first yeast Bacterium is precipitated；And the sterilized water resuspended yeast precipitation with 4 DEG C of pre-coolings of 250mL, obtain the second yeast re-suspension liquid.

Step 4.1.6, the second yeast re-suspension liquid is centrifuged 5min in 4 DEG C of 1500 × g, removes supernatant, collects the second yeast Bacterium is precipitated；And the resuspended second yeast precipitation of aseptic 1mol/L Sorbitol with 4 DEG C of pre-coolings of 20mL, obtain the first Sorbitol weight Suspension.

Step 4.1.7, the first Sorbitol re-suspension liquid is centrifuged 5min in 4 DEG C of 1500 × g, removes supernatant, collects the 3rd yeast Bacterium is precipitated；And the resuspended 3rd yeast precipitation of aseptic 1mol/L Sorbitol with 4 DEG C of pre-coolings of 1mL, obtain Pichia pastoris GS115 Cell.

Step 4.1.8, prepares the competence of Pichia pastoris GS115 cell, obtains Pichia pastoris GS115 competent cell, Ice bath preserves, and the preparation same day on the same day uses, can not be frozen.

Step 4.2, the linearisation of recombinant vector pGAPZ α A/Cec Md3js

The recombinant vector pGAPZ α A/Cec Md3js of step 3 is carried out linearized enzyme digestion with restriction enzyme A vr II, Obtain linearisation reaction mixture, the wherein reaction system of linearized enzyme digestion as shown in table 4, reaction condition is 37 DEG C of water-bath 3h；

5 μ L linearisation reaction mixture are taken to mix homogeneously with 6 × Loading Buffer of 1 μ L, in 1% agar containing EB Sugared electrophoresis detection, after electrophoresis detection is correct, by the linearisation reaction mixture of remaining 15 μ L according to DNA gel reclaim reagent cassette method Carry out glue reclaim, obtain linearisation recombinant vector pGAPZ α A/Cec Md3js.

Wherein, kit method carry out glue reclaim step as follows：Add in linearisation reaction mixture and be equivalent to linearly Change the NaAC (3mol/L) of 1/10 times of volume of reaction mixture, be equivalent to 2.5 times of volume dehydrated alcohol of linearisation reaction mixture, -80 DEG C place more than 0.5h；Supernatant is abandoned after 12 000r/min centrifugation 10min；70% absolute ethanol washing precipitation, vacuum drying, obtain To linearisation recombinant vector pGAPZ α A/Cec Md3js, -80 DEG C save backup.

Wherein, the identification knot of the linearisation product of recombinant vector pGAPZ α A/Cec Md3js in linearisation reaction mixture As shown in figure 3, there being band at 3543bp, then the linearisation of explanation recombinant vector pGAPZ α A/Cec Md3js is successful for fruit.

The reaction system that table 4 linearized enzyme digestion is processed

Step 4.3, the linearisation recombinant vector pGAPZ α A/Cec Md3js electricity of step 4.2 is transformed into step 4.1.8 In Pichia pastoris GS115 competent cell, obtain expressing engineering bacteria, specifically include:

By 80 μ L Pichia pastoris GS115 competent cells and 5-10 μ g or 5-10 μ L linearisation recombinant vector pGAPZ α A/ Cec Md3js ice bath mixes, and proceeds in the 2mm electricity revolving cup of ice bath pre-cooling；Continue ice bath 5min；Electroporated, wherein electric shock turns Change condition is identical with the condition of electroporated saccharomyces cerevisiae (Saccharomyces cerevisiae), is：Voltage be 1500V or 2000V；Electric capacity is 25 μ F；Resistance is 200 Europe, and temperature is 0 DEG C.

After electroporated, add the cold 1mol/L Sorbitol of 1mL ice bath toward in 2mm electricity revolving cup immediately, and the product that will shock by electricity It is transferred in a 15mL Conical sterile tube, then the electric shock product in 15mL Conical sterile tube is stood in 30 DEG C Culture 1-2h, obtains electric transformed yeast.

Take 10 μ L, 25 μ L, 50 μ L, 100 μ L, the electric transformed yeast of 200 μ L, be respectively coated in 5 different YPDS cultures Base flat board is (containing 100 μ g/mL Zeocin^TM) upper (the electric transformed yeast coating of little density is more beneficial for Zeocin^TMResistance selects)； Cultivate 3-10 days in 30 DEG C, until yeast monoclonal bacterium colony occurs；10-20 yeast monoclonal bacterium colony of picking is in YPDS culture medium Flat board (the bleomycin Zeocin containing 100 μ g/mL^TM) on carry out streak culture, obtain the single bacterium colony of yeast transformant.

Step 4.4, the positive transformant in the single bacterium colony of yeast transformant of screening step 4.3.

Step 4.4.1, PCR identifies：

The single bacterium colony of the yeast transformant of picking step 4.3 is inoculated in 5mL and contains 25 μ g/mL Zeocin^TMYPD liquid training In foster base, in 30 DEG C of 200r/min shaking table cultures 15-16h (overnight), obtain yeast transformant bacteria suspension.

Take 500 μ L yeast transformant bacteria suspension centrifugations, abandon supernatant, uncap, in 500W microwave heating to boiling, in liquid nitrogen Place 5min, be subsequently adding 50 μ L sterilized water, in 500W microwave heating to boiling, centrifugation, collect supernatant, and using supernatant as The pastoris genomic dna template of PCR.

Using primer pair Forward Primer：5 '-GTCCCTATTTCAATCAATTGAA-3 ' and 3 ' AOX1Primer： 5 '-GCAAATGGCATTCTGACATCC-3 ' carry out bacterium colony PCR, and whether identification recombinant vector pGAPZ α A/Cec Md3js integrates Enter in Pichia sp. genome, wherein as shown in table 5, the reaction condition of bacterium colony PCR is the reaction system of bacterium colony PCR：94 DEG C pre- Degeneration 5min；94 DEG C of degeneration 30s；54.8 DEG C of annealing 30s；72 DEG C of extension 1min, totally 30 circulations；72 DEG C extend 7min eventually.

After bacterium colony PCR reaction terminates, 3 μ L PCR primer are taken to be examined in 2% agarose gel electrophoresiies containing EB Survey, result, as shown in figure 4, having band at 783bp, illustrates that this yeast transformant is positive colony, the bacterium solution of this yeast transformant For positive colony bacterium solution.

The reaction system of table 5 bacterium colony PCR

Step 4.4.2, sequencing identification

By the positive colony bacterium solution of step 4.4.1 and glycerol according to 5:1 volume ratio mix homogeneously, send Harbin to win bodyguard Bioisystech Co., Ltd is sequenced, and sequencing result is contrasted with designed genes of interest with DNAMAN, if positive gram Really contain designed genes of interest in grand, then this positive colony is expression engineering bacteria, and this expression engineering bacteria is used for expressing The genes of interest fragment containing in recombinant vector pGAPZ α A/Cec Md3js.

Step 5, the expression engineering bacteria of incubation step 4.4.2, carry out the expression of three PEPC ec Md3js, and extract three PEPC ec Md3js, obtains three PEPC ec Md3js.

Step 5.1, the expression of three PEPC ec Md3js

In 100 μ g/mL Zeocin^TMScreening stability inferior preferably expresses engineering bacteria, -80 DEG C of preservations.

Expression engineering bacteria is seeded in the 50mL conical flask equipped with 20mL YPD fluid medium, in 30 DEG C, 200r/ Min cultivates 72h, obtains expressing engineering bacteria bacteria suspension；

The expression engineering bacteria bacteria suspension taking 1mL in sterilized 100mL YPD fluid medium, with 8 layers of sterilized non-fat Gauze seals, and in 30 DEG C, 200r/min culture 2-4d, obtains bacteria liquid sample；

Step 5.2, the extraction of three PEPC ec Md3js

Take bacteria liquid sample 4mL of step 5.1,12000r/min is centrifuged 5min, collect supernatant, then the supernatant of collection is entered Row concentrates, and obtains three PEPC ec Md3js concentrated solutions, by three PEPC ec Md3js concentrated solutions with 20% ethanol elution remove impurity, so Use the ultracentrifugation pipe ultracentrifugation of 3kD, lyophilization afterwards, obtain three PEPC ec Md3js.

It should be noted that step 5.1 and step 5.2 can also adopt following processing mode filtering and concentrating：Work will be expressed Journey bacterium is seeded in YPD fluid medium, 30 DEG C, 240r/min culture 72h, and 4000r/min is centrifuged 20min, takes supernatant, 0.45 μm of membrane filtration, filtrate adopts TCA method to concentrate, and lyophilization obtains three PEPC ec Md3js；

Step 5.3, prepares the antibacterial peptide Cec Md (nucleotide of described antibacterial peptide Cec Md using three PEPC ec Md3js Sequence is as shown in SEQ IDNO.4).

Step 5.3.1, with pH 8.0, the PB buffer of 0.01mol/L is solvent, and compound concentration is three peptides of 1mg/mL Cec Md3js solution, then dose volume fraction is the formic acid solution of 50% (v/v), by three PEPC ec Md3js solution and first Acid solution is according to 1000:12 volume ratio mixing, 50 DEG C of water-bath 36h, obtain cutting solution.

It should be noted that above-mentioned PB buffer is the common phosphate buffer in this area, NaH can be adopted₂PO₄With Na₂HPO₄Preparation.

Step 5.3.2, will cut solution with distilled water and dilute 3 times, and vacuum lyophilization obtains solid antimicrobial peptide.

Solid antimicrobial peptide is redissolved by step 5.3.3 with distilled water, then through vacuum lyophilization, removes formic acid with thorough, obtain To antibacterial peptide Cec Md crude product.

Step 5.3.4, the purification of antibacterial peptide Cec Md

The hydrolyzate (antibacterial peptide Cec Md crude product) of three PEPC ec Md3js is passed through Mini ultrafiltration system 5000kDa Membrance separation, separating liquid crosses upper liquid phase purification after G50 chromatographic column, obtains the antibacterial peptide Cec Md of purification, the antibacterial peptide Cec of this purification Md can be used for preparing the application in treatment Gram negative bacterial disease medicine or treatment gram positive bacteria disease medicament, or For preparing animal feed additive.

Fig. 5 is that the Tricine-SDS-PAGE of bigeminy PEPC ec Md2ys (8.77kDa) analyzes collection of illustrative plates, and wherein M swimming lane is mark Quasi- albumen Marker, No. 1 swimming lane is three PEPCs ec Md3cs (13.91kDa), and No. 2 swimming lanes are three PEPC ec Md3js (13.36kDa), No. 3 swimming lanes are Cec Md2cs (9.08kDa), and No. 4 swimming lanes are Cec Md2js (8.79kDa), and No. 5 swimming lanes are Bigeminy PEPC ec Md2ys (8.77kDa), No. 6 swimming lanes are antibacterial peptide Cec Md (4.26kDa), and No. 7 swimming lanes are Cec Md3jn sample Product supernatant, No. 8 swimming lanes are Cec Md3cn sample supernatant, and No. 9 swimming lanes are blank, wherein 1,3,4,5,7, No. 8 swimming lanes It is in order that the auxiliary sample that makes a distinction of different sample electrophoresis collection of illustrative plates, be to observe three PEPC ec Md3js and anti-for convenience Bacterium PEPC ec Md pillar location, is not related to content of the invention.

Detect the purity of the antibacterial peptide Cec Md that three PEPC ec Md3js are prepared from, concrete bag below using HPLC method Include：

Take 3 μ L AG HPLC analyses, mobile phase is water and acetonitrile, carries out the gradient elution of 30min.First HPLC is used Water containing 95% and 5% acetonitrile start gradient balance 10min.It is then injected into ready sample, the response time is 40min, collects sample out from detector.Finally clean 30min with 25% water and 75% acetonitrile mixture, detection Result is shown in Fig. 6 and Fig. 7, and result shows, antibacterial peptide Cec Md has higher purity.

The antibacterial peptide Cec Md that three PEPC ec Md3js are prepared from carries out Activity determination, specifically includes：

Minimal bactericidal concentration (MIC) adopts 96 well plate method to measure.By Lactobacillus brevis, Deshi Lactobacilluss, bacillus bifiduss, Bi Chi Yeast GS115 and stop the newborn chain coccobacilluss, staphylococcus aureuses, Salmonella, escherichia coli in plate loop method, and picking Single bacterium colony is cultivated with corresponding fluid medium.Wherein stop the newborn chain coccobacilluss, staphylococcus aureuses, Salmonella, large intestine 37 DEG C of bacillus, 200r/min air concussion overnight incubation；Lactobacillus brevis, Deshi Lactobacilluss, 37 DEG C of bacillus bifiduss anaerobism, 200r/ Min concussion and cultivate is overnight；In 30 DEG C, 200r/min air concussion cultivates 48h to Pichia pastoris GS115.

Antibacterial peptide Cec Md 1 × PBS is diluted to 2000 μm of ol/L, 1500 μm of ol/L, 750 μm of ol/L, 500 μ respectively mol/L、375μmol/L、250μmol/L、187.5μmol/L、125μmol/L、93.75μmol/L、62.5μmol/L、46.875 μm ol/L, 31.25 μm of ol/L, 23.475 μm of ol/L and 15.625 μm of ol/L amount to 16 gradients.By the bacterium solution dilution after culture To 2 × 10⁵-7×10⁵CFU/mL, the test having diluted bacterium solution is added drop-wise in 3 96 well culture plates respectively, the 1-12 often going Hole, every hole 60 μ L, the then antibacterial peptide of every hole Deca 20 μ L, every hole adds 120 μ L culture medium.Each sample sets 3 repetitions, with When cultivate 48h using without test bacterium as negative control.Detect by an unaided eye and have or not muddiness, the minimum of as tested bacterium is antibacterial dense Degree.

Minimum bactericidal concentration is adopted (MBC) and is measured with 96 well plate method.The mixed liquor in holes all before MIC is taken 5 μ L to move into corresponding In fresh culture 96 orifice plate, then carry out cultivating 48h, observed result, do not have the muddy original species that can regard 99.5% to enter Antibacterial be killed, be the minimum bactericidal concentration of this bacterium.Result is as shown in table 6.

MIC and MBC of table 6 antibacterial peptide Cec Md

Note："-" indicates Wu obvious Mlc

By SWISS-MODEL software to series connection peptide hydrolysis products prediction, find that three PEPC ec Md3js contain alpha-helix Structure, catenation sequence used by three PEPC ec Md3js has for Pro in the heart in the connection of enterokinase formic acid (DPP) peptide chain The selective toxicity of preferable cell, its hemolytic activity reduces, and having of the selection of cell largely reduces.

Explore the hemolytic activity of antibacterial peptide by the burst size measuring haemachrome.Take the fresh sheep that concentration is 2% Red blood cell suspension, is mixed homogeneously with 0.1mL test antibacterial peptide.Concentration after test antibacterial peptide mixes is 100 μ g/mL, 50 μ G/mL, 25 μ g/mL, 12.5 μ g/mL, incubate 1h in 37 DEG C of constant incubators after mix homogeneously, 2000r/min is centrifuged 10min. Supernatant is transferred to 96 orifice plates, measures light absorption value under 570nm using microplate reader.Using aseptic normal saline and 0.1% (v/v) Triton-100 is as the standard of percent hemolysis 0% and 100%.Negative control PBS, positive control Use 0.1%TritonX-100.Each sample setting 3 is parallel, and result takes its meansigma methods.Hemolysis rate (%)=(developmental tube extinction Degree-negative control pipe absorbance)/(positive control pipe absorbance-negative control pipe absorbance) × 100%.Homopolypeptide is not to silk floss Sheep red blood cell hemolytic activity impact result is as shown in table 7, when concentration is 100 μ g/mL, 50 μ g/mL and 25 μ g/mL, three PEPC ec The hemolytic activity of Md3js and antibacterial peptide Cec Md is substantially less than housefly cecropin antibacterial peptide；When concentration is 12.5 μ g/mL, three The hemolytic activity of PEPC ec Md3js is 1.22, and the hemolytic activity of antibacterial peptide Cec Md is 1.47, all relatively low.

The table 7 not impact to sheep red blood cell (SRBC) hemolytic activity for the homopolypeptide

Note：Between different letter representation same concentrations difference antibacterial peptides, there were significant differences (P<0.05)；" " represents and does not examine Measure result.

Pichia sp. can shorten the cultivation cycle of strain during fermenting and producing, simplifies operating procedure, decreases The consuming of data and reduce fermentation costs, possesses eukaryotic cell albumen synthesis path.Alcohol oxidase (Alochol Oxidase, AOX1) gene promoter is Pichia sp. for the powerful of expression that is strict and starting regulation and control source protein, On the basis of this, for the induction of foreign protein, glycosylation modified, disulfide bond modification and processing, all there is certain biology Function.Although the culture medium composition needed for yeast is single, but can quick high density life under simple condition of culture Long, stable hereditary property in Pichia yeast is reconstituted in for recombiant plasmid, less can secrete oneself protein, but whole cell Dry weight is up to 130g/L it can be seen that yeast is the optimal strain of expression foreign protein.

Although preferred embodiments of the present invention have been described, but those skilled in the art once know basic creation Property concept, then can make other change and modification to these embodiments.So, claims are intended to be construed to including excellent Select embodiment and fall into being had altered and changing of the scope of the invention.

Obviously, those skilled in the art can carry out the various changes and modification essence without deviating from the present invention to the present invention God and scope.So, if these modifications of the present invention and modification belong to the scope of the claims in the present invention and its equivalent technologies Within, then the present invention is also intended to comprise these changes and modification.

Claims (7)

1. a kind of three PEPC ec Md3js are it is characterised in that be used for encoding the nucleotide sequence of described three PEPC ec Md3js As shown in SEQ ID NO.1, the aminoacid sequence of described three PEPC ec Md3js is as shown in SEQ ID NO.2.

2. a kind of preparation method of three PEPC ec Md3js described in claim 1 is it is characterised in that comprise the following steps：

Step 1, genes of interest as shown in SEQ ID NO.3 for the synthesizing ribonucleotide sequence；

Step 2, with restricted enzyme Kpn I and Xba I double digestion carrier pGAPZ α A, and reclaims the carrier-pellet obtaining 3147bp Section；Described genes of interest and described carrier segments are connected, obtains the connection containing recombinant vector pGAPZ α A/Cec Md3js Liquid；

Step 3, the connection liquid of step 2 is converted to competent escherichia coli cell DH5 α, and extracts recombinant vector pGAPZ α A/Cec Md3js, obtains recombinant vector pGAPZ α A/Cec Md3js；

Step 4, recombinant vector pGAPZ α A/Cec Md3js is transformed in host cell, obtains expressing engineering bacteria；

Step 5, the expression engineering bacteria of incubation step 4, carry out the expression of three PEPC ec Md3js, and extract three PEPC ec Md3js, obtains three PEPC ec Md3js.

3. the preparation method of three PEPC ec Md3js according to claim 2 is it is characterised in that the host cell of step 4 For Pichia pastoris GS115.

4. three PEPC ec Md3js described in a kind of claim 1, the application in preparing antibacterial peptide Cec Md.

5. three PEPC ec Md3js described in a kind of claim 1, treat Gram negative bacterial disease medicine in preparation or control Treat the application in gram positive bacteria disease medicament.

6. three PEPC ec Md3js described in a kind of claim 1, the application in preparing animal feed additive.

7. a kind of method preparing antibacterial peptide Cec Md using three PEPC ec Md3js described in claim 1, its feature exists In specifically including following steps：

Step a, with pH 8.0, the PB buffer of 0.01mol/L is solvent, and compound concentration is the three PEPC ec of 1mg/mL Md3js solution；Then dose volume fraction is 50% formic acid solution, and three PEPC ec Md3js solution are pressed with formic acid solution According to 1000:12 volume ratio mixing, 50 DEG C of water-bath 36h, obtain cutting solution；

Step b, will cut solution with distilled water and dilute 3 times, and vacuum lyophilization obtains solid antimicrobial peptide；

Solid antimicrobial peptide is redissolved with distilled water, then through vacuum lyophilization, obtains antibacterial peptide Cec Md crude product by step c；

Step d, the purification of antibacterial peptide Cec Md

By antibacterial peptide Cec Md crude product through 5000kDa membrance separation, the separating liquid obtaining, through G50 column chromatography, obtains antibacterial peptide Cec Md.