CN105238809A - Expressing method of antimicrobial peptide CC31 in bacillus subtilis - Google Patents
Expressing method of antimicrobial peptide CC31 in bacillus subtilis Download PDFInfo
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Abstract
The invention discloses an expressing method of antimicrobial peptide CC31 in bacillus subtilis. Through the molecular biological technique, an expression vector pHT43 is selected to establish a recombinant expression vector, the recombinant expression vector pHT43/CC31 is successfully established and is converted into bacillus subtilis WB800N, and accordingly expression engineering bacteria pHT43/CC31/WB800N are obtained. In LB culture media, by means of IPTG inducible expression, an expression product is purified through the high performance liquid chromatography (HPLC) so that CC31 protein can be obtained, and the correctness is detected through the mass-spectrography (MS). According to the method, the antimicrobial peptide CC31 is expressed in the bacillus subtilis WB800N, separation and purification are easy, and operating is also easy. The expression product has antimicrobial activity to both gram positive bacteria and gram negative bacteria and does not have bacteriostatic activity to beneficial bacteria, the heat stability is good, and the method is suitable for large-scale industrial production and can be applied to the fields of medicine, food, feed additives and cultivation.
Description
Technical field
The present invention relates to biological field, be specifically related to the expression method of a kind of antibacterial peptide CC31 in subtilis.
Background technology
Antibacterial peptide is the integral part of animal and plant and human congenital's immunologic mechanism, is subject to being induced to produce when virus, bacterium etc. are attacked at body.Antibacterial peptide has broad-spectrum antibacterial action, especially more causes the interest of people to it to the killing action that some produce the pathogenic bacteria of antibiotic resistance.Antibacterial peptide is huge at the application potential in the fields such as agricultural, medical treatment, food.Up to now, thousands of kinds of natural antibacterial peptides are had been found that, but the kind with better medical selectivity index is fewer, therefore, people wish by the redesign on molecule, such as, the mode such as design of T1249 obtains desirable novel artificial antibacterial peptide from abundant natural antibacterial peptide resource.
The design feature of heterozygous antibacterial peptide be integrate two (or more) advantage of native peptides, through suitable manually modified after, can maximize favourable factors and minimize unfavourable ones, play anti-microbial activity to the full extent, not cause haemolysis.Cecropin antimicrobial peptides (cecropins) is the natural antibacterial peptide that research is the clearest, effect is best at present; Housefly cecropin antibacterial peptide (CecMd) is this laboratory through induction Musca domestica larva, then clones (homology 81%) that obtain.And Chensirin is the natural antibacterial peptide that the Northeast China wood frog skin of discovered in recent years is secreted, its activity is very high, but has higher hemolytic activity.By carrying out hybrid design to both, obtain low haemolysis, the novel heterozygous antibacterial peptide CC31 of high antibacterial activity, has very large advantage compared with native peptides.This research team by antibacterial peptide CC31 in intestinal bacteria, Pichia anomala expression system successful expression, not yet carry out the research in other bacterial classification expression systems, for this reason, antibacterial peptide CC31 is carried out expression study at subtilis expression system by us.
Subtilis (B.Subtilis) is as the expressive host of foreign gene, tool has the following advantages: (1) B.Subtilis is that aerobic bacteria is not containing thermal source lipopolysaccharides (lipopolysaccharides, LPSs), probiotics no pathogenicity; B.Subtilis is security bacterial strain by U.S. FDA accreditation.(2) B.Subtilis cytolemma is individual layer, and colibacillary be double-deck, the albumen of secretion is than recovery easier under Escherichia coli system, purifying under B.Subtilis system, and easy and simple to handle, many enzymes are all the albumen be secreted into outside born of the same parents that B.Subtilis produces.(3) long-term research shows, B.Subtilis does not have obvious codon-bias.(4) requirement of B.Subtilis to substratum is fairly simple, definitely occupy economic advantages aborning, phage, plasmid all can be used as cloning vector, and B.Subtilis has good Transcription/Translation System, can avoid the formation of the protein of false folding, inclusion body.(5) single-gene of B.Subtilis, the formation etc. of gemma all regulate and control by polygene; The sequence that 16SrRNA3 ' holds also is different from intestinal bacteria.
Summary of the invention
For solving the problem, the invention provides that a kind of separation and purification is simple, purity of protein is high, be suitable for the expression method of antibacterial peptide CC31 in subtilis of large-scale industrial production.
For achieving the above object, the technical scheme that the present invention takes is:
The expression method of antibacterial peptide CC31 in subtilis, comprises the steps:
The synthesis of S1, antibacterial peptide gene and the structure of cloning vector
S11, design following two to four primers, often pair all has complementary sequence;
F1：5′-GC
TCTAGAGGTTGGCTGAAAAAAATCGGCAAGAAGATTGA-3′
R1：5′-ACGGGTATGTTGGCCAACACGTTCAATCTTCTTGCCGATTTTTTTC-3′
F2：5′-ACGTGTTGGCCAACATACCCGTATTATTCCGCTGCCGCTGGGTTATTTTGCGAA-3′
R2：5′-TCC
CCCGGGTTAGGTTTTTTTCGCAAAATAACCCAGCGGCAGCG-3′
S12, primers F 1 to be extended respectively with R1, F2 and R2 in following PCR reaction system, obtain extension liquid P1, P2.Concrete steps following (the PCR reaction system for P1): open PCR instrument preheating in advance, 2 × TaqMasterMix25 μ L, upstream primer F1 (10 μMs) 2 μ L, downstream primer R1 (10 μMs) 2 μ L, RNase-Freewater21 μ L are joined in PCR reaction tubes, mix, after brief centrifugation, be placed in PCR instrument.Reaction conditions is: 94 DEG C of denaturation 2min; 30 circulations (72 DEG C extend 30s for 94 DEG C of sex change 30s, 63 DEG C of annealing 30s); 72 DEG C of ends extend 2min, must extend liquid P1.In like manner, extension liquid P2 can be obtained;
S13, corresponding extension liquid P1 and the P2 obtained using first time PCR carry out PCR as template each other and primer mutually, concrete steps are as follows: open PCR instrument preheating in advance, 2 × TaqMasterMix25 μ L, P10.5 μ L, P20.5 μ L, RNase-Freewater24 μ L are joined in PCR reaction tubes, mix, after brief centrifugation, be placed in PCR instrument.Reaction conditions is: 94 DEG C of denaturation 2min; 30 circulations (72 DEG C extend 30s for 94 DEG C of sex change 30s, 53 DEG C of annealing 30s); 72 DEG C of ends extend 2min, obtain antibacterial peptide gene;
S14, the antibacterial peptide gene of gained to be connected with cloning vector pMD18-T carrier, in 0.2mL Eppendorf tube, to add pMD18-TVector1 μ L, the antibacterial peptide gene 2 μ L after glue reclaims, ddH
2o2 μ L, Solution1 (thawed on ice) 5 μ L, full dose is 10 μ L; Mix, after brief centrifugation; Be placed in and connect instrument, 4 DEG C, 12h, obtains pMD18-T/CC31;
S15, the intestinal bacteria containing expression vector pHT43 are inoculated into 3mL contain in the LB substratum of 10 μ g/mLCm, 37 DEG C, 200rpm shaken overnight is cultivated, extract plasmid, cloned plasmids correct to itself and-20 DEG C of discriminatings of preserving is carried out double digestion respectively, and it is as follows that enzyme cuts system: the distilled water 6 μ L of XbaI1 μ L, SamI1 μ L, plasmid DNA 10 μ L, CutSmartBuffer2 μ L, sterilizing; Endonuclease reaction condition is: 37 DEG C of water-baths, 3h, and wherein plasmid DNA is respectively: pHT43, pMD-18T/CC31 plasmid;
S16, get mentioned reagent in PCR reaction tubes, brief centrifugation after mixing, make solution aggregation bottom test tube, after 37 DEG C of water-bath 3h, endonuclease reaction is complete;
S17, the CC31 gene fragment that enzyme is cut cut with enzyme after plasmid pHT43 be connected, can be built into plasmid vector pHT43/CC31, ligation system is as follows: CC31 gene 6 μ L, 10 × T4DNAligaseBuffer1 μ L, plasmid pHT432.5 μ L, T4DNAligase0.5 μ L; Ligation condition is: 16 DEG C of water-baths, spends the night;
S2, recombinant expression vector pHT43/CC31 are transformed in host cell, build three kinds and express engineering bacterial strain;
S3, be that inductor is expressed by the expression engineering bacteria of step S2 gained with IPTG, obtain expression product;
The expression product of S4, separation and purification gained, obtains antimicrobial peptide protein CC31, and detects its exactness.
Wherein, the host cell in described step S2 is subtilis WB800N.
Wherein, the expression product in described step S3 is centrifuged supernatant.
Wherein, separation and purification described in described step S4 adopts high performance liquid chromatography (HPLC) purifying, utilizes mass spectroscopy (MS) to detect its exactness.
The antibacterial peptide CC31 of the above-mentioned gained application in preparation treatment gram-negative and/or gram-positive microorganism disease medicament, animal feedstuff additive.
The present invention selects pHT43 as expression vector, and insert a multiple clone site (BamHI, XbaI, AatII, SmaI) and efficient SD sequence in pHT43 carrier, selected restriction enzyme site is XbaI and SmaI.And Host Strains has multiple, but natural B.Subtilis can secrete some extracellular proteases in exocytosis process, the fundamental property of target protein may be changed, so occurred much knocking out the B.Subtilis that can secrete extracellular enzyme gene, as WB600, WB700, WB800N etc., the present invention selects the WB800N having knocked out 8 genes to be Host Strains.
The present invention has following beneficial effect:
Using gene engineering technique is high expression antibacterial peptide CC31 in subtilis, confirm that kind of an antibacterial peptide has germicidal action to various bacteria through experiment, expression product stability is better, separation and purification is simple, easy to operate, easy amplification, is suitable for large-scale industrial production, has important value to the efficient CC31 antiseptic feed additive of development of new, antibacterials etc.
Accompanying drawing explanation
Fig. 1 be in the embodiment of the present invention first time PCR primer electrophorogram.Wherein, 1 is DNA standard molecular weight; 5:P2 fragment 73bp; 6 is P1 fragment 62bp.
Fig. 2 is second time PCR primer electrophorogram in the embodiment of the present invention.Wherein, 1 is DNA standard molecular weight; 2 is heterozygous antibacterial peptide CC31113bp.
Fig. 3 is that in the embodiment of the present invention, recombinant clone plasmid is template PCR primer agarose gel electrophoresis figure.Wherein, 1 is heterozygous antibacterial peptide CC31113bp; 4 is DNA standard molecular weight; 5 is negative control.
Fig. 4 is the agarose gel electrophoresis figure of recombinant clone plasmid double digestion product in the embodiment of the present invention.Wherein, 1,5 is DNA standard molecular weight; 3 is heterozygous antibacterial peptide CC31113bp; 4: negative control.
Fig. 5 is the PCR qualification of recombinant expression vector in the embodiment of the present invention.Wherein, 3 is restructuring support C C31PCR product 283bp.Primer is respectively:
F31：5’-TTGTCAGTTTGCCGATTACA-3’
R31：5’-CTATTCCTAATAAGCCGATA-3’
Fig. 6 is that the enzyme of recombinant plasmid in the embodiment of the present invention cuts qualification.Wherein, 1 is DNA standard molecular weight; 3 is restructuring support C C31 digestion products 113bp.
Fig. 7 is that the Tricine-SDS-PAGE of 3 antibacterial peptides in the embodiment of the present invention analyzes.Wherein, 1 is empty plasmid supernatant liquor; 3 is CC31 sample supernatant 3.6kDa; 5 is standard protein Marker.
Embodiment
In order to make objects and advantages of the present invention clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment 1
Construction of expression vector
The synthesis of a antibacterial peptide gene and the structure of cloning vector
SOEingPCR technology is adopted to complete heterozygous antibacterial peptide CC31 gene chemical synthesis in vitro.Antibacterial peptide design following two is to four primers, and often pair all has complementary sequence, through two-step pcr synthetic antimicrobial peptide gene, and is connected with pMD18-T cloning vector, forms cloning recombinant plasmids pMD18-T/CC31.
CC31 aminoacid sequence: GWLKKIGKKIERVGQHTRIIPLPLGYFAKKT
CC31：113bp
GC
TCTAGAGGTTGGCTGAAAAAAATCGGCAAGAAGATTGAACGTGTTGGCCAACATACCCGTATTATTCCGCTGCCGCTGGGTTATTTTGCGAAAAAAACC
CCCGGGGGA
F1：5′-GC
TCTAGAGGTTGGCTGAAAAAAATCGGCAAGAAGATTGA-3′
R1：5′-ACGGGTATGTTGGCCAACACGTTCAATCTTOTTGCCGATTTTTTTC-3′
F2：5′-ACGTGTTGGCCAACATACCCGTATTATTCCGCTGCCGCTGGGTTATTTTGCGAA-3′
R2：5′-TCC
CCCGGGTTAGGTTTTTTTCGCAAAATAACCCAGCGGCAGCG-3。
Adopt SOEingPGR method synthetic antibacterial peptide genetic results as Fig. 1,2, method is as follows:
During first time PCR, F1 with just, F2 and R2 extends respectively, obtain extension liquid P1, P2.Reaction system following (for F1 and R1):
Open PCR instrument preheating in advance, mentioned reagent is joined in PCR reaction tubes, mix rear brief centrifugation, put into PCR instrument.Reaction conditions is: 94 DEG C denaturation 2min:30 circulation (72 DEG C extend 30s for 94 DEG C of sex change 30s, 63 DEG C of annealing 30s): 72.C extends 2min eventually.Question response terminates, and therefrom gets 5 μ L and carries out electrophoresis detection (1.5% sepharose containing EB), and temporary-4 DEG C of remaining part is deposited.
During second time PCR, mutually carry out PCR as template each other and primer using the corresponding extension liquid (P1 and P2) that first time PCR obtains, reaction system is as follows:
Open PCR instrument preheating in advance, mentioned reagent is joined in PCR reaction tubes, mix rear brief centrifugation, put into PCR instrument.Reaction conditions is: 94 DEG C of denaturation 2min; 30 circulations (72 DEG C extend 30s for 94 DEG C of sex change 30s, 66 DEG C of annealing 30s); 72 DEG C of ends extend 5min.Treat that PCR terminates to become CC31 gene, detect with the gene of 1.5% agarose gel electrophoresis to synthesis.
The connection of goal gene and cloning vector:
Antibacterial peptide gene is connected with cloning vector pMD18-T carrier, in 0.2mL Eppendorf tube, adds pMD18-TVector1 μ L, the antibacterial peptide gene 2 μ L after glue reclaims, ddH
2o2 μ L, Solution1 (thawed on ice) 5 μ L, full dose is 10 μ L; Mix rear brief centrifugation; Put into and connect instrument, 4 DEG C, 12h, obtains pMD18-T/CC31 and carries out the qualification result such as PCR, double digestion as Fig. 3,4.
B builds the recombinant expression vector of antibacterial peptide
The cloning vector of antibacterial peptide gene and the double digestion of expression vector:
Intestinal bacteria containing expression vector pHT43 being inoculated into 3mL contains in the LB substratum of 10 μ g/mLCm, 37 DEG C of 200rpm shaken overnight are cultivated, extract plasmid, cloned plasmids correct to itself and-20 DEG C of discriminatings of preserving is carried out double digestion respectively, it is as follows that enzyme cuts system:
Endonuclease reaction condition is: 37 DEG C of water-baths, 3h, and wherein plasmid DNA is respectively: pHT43, pMD-18T/CC31 plasmid.Get mentioned reagent in PCR reaction tubes, brief centrifugation after mixing, make solution aggregation bottom test tube, after 37 DEG C of water-bath 3h, endonuclease reaction is complete.Get 5 μ L reaction solutions to mix with 1 μ L6 × LoadingBuffer, in the 1%Agarosegel electrophoresis detection containing EB.After electrophoresis detection is correct, the double digestion liquid of remaining 15 μ L is carried out glue recovery.
The connection of antibacterial peptide gene and expression vector pHT43:
The CC31 gene fragment that enzyme is cut cut with enzyme after plasmid pHT43 be connected, can be built into plasmid vector pHT43/CC31, ligation system is as follows:
Ligation condition is: 16 DEG C of water-baths, spends the night.Recombinant expression vector carries out the qualification result such as PCR, double digestion as Fig. 5,6.
(1) genetic engineering bacterium is built:
Single colony inoculation of picking Host Strains WB800N, in 2mLSPI substratum, is placed in 50mL centrifuge tube, incubated overnight in 200r/min, 37 DEG C of shaking tables, therefrom gets 100 μ L and transfers in the SPI substratum of 5mL, and in shaking table, the same terms is cultivated, and starts to survey OD after 3h
600value, every 20 minutes once, OD
600when value is between 0.6 ~ 0.8, therefrom gets 200 μ L bacterium liquid and transfer and be placed in 50mL centrifuge tube in 2mLSPII substratum, 90min (37 DEG C, 200r/min) cultivated by shaking table.20 μ L100 × EGTA solution are added rapidly, at cultivation 10min after 90min.Last packing, every 0.5mL is a, is placed in 1.5mL centrifuge tube ,-80 DEG C of preservations.Get subtilis WB800N competent cell 0.5mL and be placed in an aseptic 1.5mLEP pipe, add 10 μ L plasmids simultaneously, mix gently, 37 DEG C, in 200r/min shaking table, after cultivating 90min, 8000g is centrifugal, discards 350 μ L supernatant liquors, resuspended thalline.Get on LB flat board that appropriate bacterium liquid coats containing 10 μ g/mLCm, 37 DEG C of constant incubator incubated overnight.
(2) abduction delivering product purification qualification:
Picking contrast bacterium (the subtilis WB800N containing empty plasmid pHT43) and each single bacterium colony of recombinant bacterium pHT43/CC31/WB800N, be inoculated in 5.0mLLB liquid nutrient medium (containing Cm5 μ g/mL) respectively.37 DEG C, 200rpm air surge incubator overnight incubation, this bacterium liquid is as seed liquor.Get the LB substratum (containing Cm) that seed liquor 500 μ L is placed in 50mL, inject 250mL triangular flask, 37 DEG C, 200rpm air surge incubator is cultivated, as bacterium liquid OD
600nm starts when being 0.85 to induce (t=0), adds IPTG (volumetric concentration is 0.8mmol/L).Sample after induction 24h, 10000g, 4 DEG C of centrifugal 10min, from supernatant liquor, get 20 μ L mix with isopyknic 2 × SDS sample-loading buffer, ultrasonic disruption 1min, then boiling water bath 10min, be prepared into albumen sample, for Tricine-SDS-PAGE detected result as Fig. 7.PHT43/CC31/WB800N recombinant bacterium is expressed supernatant liquor by Mini ultrafiltration system 5000kda membrane sepn, parting liquid is upper liquid phase separation after crossing G50 chromatography column.Get 3 μ L AG HPLC and analyze crude product, moving phase is water and acetonitrile, carries out the gradient elution of 30min.First by the start gradient balance 10min of HPLC containing the water of 95% and the acetonitrile of 5%.Then inject ready sample, the reaction times is 40min, collects sample out from detector.Finally detect the appearance time of CC31 between 10 ~ 12min with the water of 25% and acetonitrile mixture cleaning 30min, the HPLC of 75%, purity is 89.27%.
Embodiment 2
Purified product CC31 embodiment 1 obtained carries out Activity determination.
Minimal bactericidal concentration adopts 96 well plate method to measure.Choose intestinal bacteria in flat board, stop newborn chain coccobacillus, streptococcus aureus, Salmonellas, short lactobacillus, lactobacillus crispatus, lactobacillus delbruckii, bifidus bacillus, pichia spp SMD1168 and Pichia pastoris GS115 single bacterium colony in appropriate liquid substratum, 37 DEG C, four kinds of pathogenic bacterium, 200rpm air concussion overnight incubation, four kinds of milk-acid bacteria anaerobism 37 DEG C, 200rpm shakes overnight incubation, two kinds of pichia spp 30 DEG C, 200rpm air concussion cultivates 48h.By antibacterial peptide 1 × PBS classification dilution be: 2000,1500,750,500,375,250,187.5,125,93.75,62.5,46.875,31.25,23.475 and 15.625 μMs amount to 16 gradients.Bacterium liquid after cultivating is diluted to 2 × 10
5~ 7 × 10
5cFU/mL, is added drop-wise in 3 96 well culture plates by the test bacterium liquid diluted respectively, often 1 ~ 12 hole of row, every hole 60 μ L, and then every hole drips the antibacterial peptide of 20 μ L, and every hole adds 120 μ L substratum.Each sample sets 3 repetitions, simultaneously using not containing test bacterium as negative control, four kinds of pathogenic bacterium in 37 DEG C, constant temperature culture, rotating speed is 100rpm, cultivates 24h; 37 DEG C of four kinds of milk-acid bacterias, anaerobism constant temperature culture, cultivates 24h; 30 DEG C of two kinds of pichia spp, constant temperature culture, rotating speed is 100rpm, cultivates 48h.The nothing that detects by an unaided eye muddiness, be the minimal inhibitory concentration (MIC) of tested bacterium.Minimal bactericidal concentration adopts 96 well plate method to measure.Mixed solution porose before MIC being got 5 μ L moves in corresponding fresh culture 96 orifice plate, then cultivation 48h is carried out, observations, the bacterium not having muddiness can enter as the original species of 99.5% is killed, and is the minimum bactericidal concentration (MBC) of this bacterium.The results are shown in Table 1.
The minimal inhibitory concentration (MIC) of table 1 antibacterial peptide CC31 and minimal bactericidal concentration (MBC)
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (6)
1. the expression method of antibacterial peptide CC31 in subtilis, is characterized in that, comprise the steps:
The synthesis of S1, antibacterial peptide gene and the structure of cloning vector
S11, design following two to four primers, often pair all has complementary sequence;
F1：5′-GC
TCTAGAGGTTGGCTGAAAAAAATCGGCAAGAAGATTGA-3′
R1：5′-ACGGGTATGTTGGCCAACACGTTCAATCTTCTTGCCGATTTTTTTC-3′
F2：5′-ACGTGTTGGCCAACATACCCGTATTATTCCGCTGCCGCTGGGTTATTTTGCGAA-3′
R2：5′-TCC
CCCGGGTTAGGTTTTTTTCGCAAAATAACCCAGCGGCAGCG-3′
S12, primers F 1 to be extended respectively with R1, F2 and R2, obtain extending liquid P1, P2, concrete steps are as follows: open PCR instrument preheating in advance, 2 × TaqMasterMix25 μ L, upstream primer F1 (10 μMs) 2 μ L, downstream primer R1 (10 μMs) 2 μ L, RNase-Freewater21 μ L are joined in PCR reaction tubes, mix, after brief centrifugation, be placed in PCR instrument, reaction conditions is: 94 DEG C of denaturation 2min; 30 circulations; 72 DEG C of ends extend 2min, must extend liquid P1, repeat above technique, must extend liquid P2;
S13, corresponding extension liquid P1 and the P2 obtained using first time PCR carry out PCR as template each other and primer mutually, concrete steps are as follows: open PCR instrument preheating in advance, 2 × TaqMasterMix25 μ L, P10.5 μ L, P20.5 μ L, RNase-Freewater24 μ L are joined in PCR reaction tubes, mix, after brief centrifugation, be placed in PCR instrument, reaction conditions is: 94 DEG C of denaturation 2min; 30 circulations; 72 DEG C of ends extend 2min, obtain antibacterial peptide gene;
S14, the antibacterial peptide gene of gained to be connected with cloning vector pMD18-T carrier, in 0.2mL Eppendorf tube, to add pMD18-TVector1 μ L, the antibacterial peptide gene 2 μ L after glue reclaims, ddH
2o2 μ L, Solution1 (thawed on ice) 5 μ L, full dose is 10 μ L; Mix, after brief centrifugation; Be placed in and connect instrument, 4 DEG C, 12h, obtains pMD18-T/CC31;
S15, the intestinal bacteria containing expression vector pHT43 are inoculated into 3mL contain in the LB substratum of 10 μ g/mLCm, 37 DEG C, 200rpm shaken overnight is cultivated, extract plasmid, cloned plasmids correct to itself and-20 DEG C of discriminatings of preserving is carried out double digestion respectively, and it is as follows that enzyme cuts system: the distilled water 6 μ L of XbaI1 μ L, SamI1 μ L, plasmid DNA 10 μ L, CutSmartBuffer2 μ L, sterilizing; Endonuclease reaction condition is: 37 DEG C of water-baths, 3h, and wherein plasmid DNA is respectively: pHT43, pMD-18T/CC31 plasmid;
S16, get mentioned reagent in PCR reaction tubes, brief centrifugation after mixing, make solution aggregation bottom test tube, after 37 DEG C of water-bath 3h, endonuclease reaction is complete;
S17, the CC31 gene fragment that enzyme is cut cut with enzyme after plasmid pHT43 be connected, can be built into plasmid vector pHT43/CC31, ligation system is as follows: CC31 gene 6 μ L, 10 × T4DNAligaseBuffer1 μ L, plasmid pHT432.5 μ L, T4DNAligase0.5 μ L; Ligation condition is: 16 DEG C of water-baths, spends the night;
S2, recombinant expression vector pHT43/CC31 are transformed in host cell, build three kinds and express engineering bacterial strain;
S3, be that inductor is expressed by the expression engineering bacteria of step S2 gained with IPTG, obtain expression product;
The expression product of S4, separation and purification gained, obtains antimicrobial peptide protein CC31, and detects its exactness.
2. the expression method of antibacterial peptide CC31 in subtilis as claimed in claim 1, it is characterized in that, the host cell in described step S2 is subtilis WB800N.
3. the expression method of antibacterial peptide CC31 in subtilis as claimed in claim 1, it is characterized in that, the expression product in described step S3 is centrifuged supernatant.
4. the expression method of antibacterial peptide CC31 in subtilis as claimed in claim 1, it is characterized in that, separation and purification described in described step S4 adopts high performance liquid chromatography (HPLC) purifying, utilizes mass spectroscopy (MS) to detect its exactness.
5. the expression method of antibacterial peptide CC31 in subtilis as claimed in claim 1, it is characterized in that, 30 circulations in described step S12 and step S13 are: 94 DEG C of sex change 30s, and 63 DEG C of annealing 30s, 72 DEG C extend 30s.
6. the expression method of antibacterial peptide CC31 in subtilis as claimed in claim 1, is characterized in that, the antibacterial peptide CC31 of the gained application in preparation treatment gram-negative and/or gram-positive microorganism disease medicament, animal feedstuff additive.
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