CN104961834A - Antimicrobial protein and preparation method thereof - Google Patents
Antimicrobial protein and preparation method thereof Download PDFInfo
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- CN104961834A CN104961834A CN201510462882.8A CN201510462882A CN104961834A CN 104961834 A CN104961834 A CN 104961834A CN 201510462882 A CN201510462882 A CN 201510462882A CN 104961834 A CN104961834 A CN 104961834A
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Abstract
The invention provides an antimicrobial protein and a preparation method, belonging to the field of biochemistry. The antimicrobial protein is composed of a lysozyme, an antimicrobial peptide and a chaperonin, wherein the antimicrobial peptide and chaperonin are sequentially connected to the C terminal of the lysozyme. The invention also provides a preparation method of the antimicrobial protein LYS-ABP-MBP. The antimicrobial protein LYS-ABP-MBP has the advantages of no irritation to the human body, favorable antibacterial effect and favorable bioactivity stability, and is suitable for wound disinfection. The antibacterial effect is obviously higher than that of the single lysozyme or antimicrobial peptide protein.
Description
Technical field
The invention belongs to biochemical field, particularly a kind of antibacterial protein and preparation method thereof.
Background technology
Sterilization refers to kill pathogenic micro-organism but differ kills the method for bacterial spore surely.Material for sterilizing is called sterilizing agent.And thimerosal is exactly the sterilizing agent of liquid as its name suggests.The features such as desirable sterilizing agent should possess that fungicidal spectrum is wide, sterilizing ability is strong, speed of action is fast, good stability, nontoxic, noresidue, non-corrosiveness, nonirritant, soluble in water, safe and cheap and easy to get to humans and animals, environmental pollution degree is low.
Current thimerosal product is mainly divided into environment disinfected class, conventional trick thimerosal and wound thimerosal.Environment disinfected class thimerosal refers to the product of the public place such as routine disinfection sterilization, swimming pool, hotel, restaurant, family, school, the hospital sterilization being mainly used in health and epidemic prevention and disaster area, comprises 84 thimerosals, Peracetic Acid, lysol etc.Conventional trick thimerosal refers to the product being mainly used in trick skin, comprises chlorhexidine acetate, ethanol is.And wound thimerosal class to be mainly used in hospital operation or wound, the wound sterilization of burn and various ulcer, and the conventional wound of individual or ulcer are disinfected, and comprise gentian voiet, hydrogen peroxide, alcohol etc.
At present, traditional wound thimerosal has many shortcomings in actual applications, such as often has pungent taste, has hormesis to wound or affected part.As gentian voiet, i.e. the gentian violet solution of 2%, this solution has the effect accelerating wound incrustation and healing, is usually used in shallow epidermis skin, mucosa infection, but stimulates comparatively large because of this solution, should not be used in mucous membrane or the open surface of a wound, the especially large surface of a wound, and can bring out skin carcinoma; Hydrogen peroxide, i.e. hydrogen peroxide, have sterilization, but illing skin of easily burning when concentration is excessive, and character is unstable, the shelf-time is not long, easily loses efficacy; 75% alcohol effectively can remove bacterium, prevent wound infection, but alcohol has destruction cell and stronger pungency, therefore can not be used in wound, the sterilization of the position mucous membrane such as ulceration position and eye, nose, mouth, vagina, in addition, the container of alcohol storage should be tight, answers jam-pack with rear bottleneck, and preventing volatilization from causing content to decline affects fungicidal effectiveness.
In addition, chemical sterilizing agent also also exists the problem of chemical residual.And for the thimerosal of plant extract, also exist that effect is low, the shortcoming of raw material poor stability, and in leaching process, apply chemical solvents, also there is the problem of certain chemical residual.
Summary of the invention
The object of the invention is the shortcoming for prior art, provide a kind of without chemical residual, to human body nonirritant and the antibacterial protein of good antimicrobial effect, sterilize for wound.
For realizing object of the present invention, the present invention adopts following technical scheme:
A kind of antibacterial protein, be made up of N,O-Diacetylmuramidase, antibacterial peptide and chaperone, described antibacterial peptide and chaperone are connected to the C-terminal of N,O-Diacetylmuramidase successively.
In some embodiments, chaperone described in described antibacterial protein is maltose binding protein.
In some embodiments, described antibacterial protein, its aminoacid sequence is as shown in SEQ IDNo.1.
Present invention also offers the DNA molecular of the antibacterial protein described in coding.
Present invention also offers the preparation method of antibacterial protein of the present invention, N,O-Diacetylmuramidase, antibacterial peptide are connected with expression vector orientation with the gene fragment of chaperone, expression after transformed host cell, extraction, purifying.
In some embodiments, described expression vector is pBV220.
In some embodiments, described host cell is intestinal bacteria or yeast.
In some embodiments, described extraction comprises bacterium collection, fragmentation, centrifuging and taking supernatant.
In some embodiments, described purifying is affinitive layer purification and ion-exchange purification.
Present invention also offers the application of antibacterial protein of the present invention in preparation wound class thimerosal.
Therefore the invention provides the application of described antibacterial protein LYS-ABP-MBP in preparation wound class thimerosal.
As shown from the above technical solution, the invention provides a kind of antibacterial protein, be made up of N,O-Diacetylmuramidase, antibacterial peptide and chaperone, described antibacterial peptide and chaperone are connected to the C-terminal of N,O-Diacetylmuramidase successively.Provide the preparation method of described antibacterial protein LYS-ABP-MBP simultaneously.Antibacterial protein LYS-ABP-MBP of the present invention is to human body nonirritant and good antimicrobial effect, and fungistatic effect is obviously better than independent N,O-Diacetylmuramidase and antimicrobial peptide protein, and biological activity stability is good, is suitable for wound sterilization.
Embodiment
The invention discloses a kind of antibacterial protein, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Product of the present invention is described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope product as herein described is changed or suitably change with combination, realize and apply the technology of the present invention.
For realizing object of the present invention, the present invention adopts following technical scheme:
The invention provides a kind of antibacterial protein, be made up of N,O-Diacetylmuramidase, antibacterial peptide and chaperone, described antibacterial peptide and chaperone are connected to the C-terminal of N,O-Diacetylmuramidase successively.
N,O-Diacetylmuramidase (lysozyme, LYS) also known as muramidase (muramidase) or N-acetylmuramide lycanohydrlase (N-acetylmuramide glycanohydrlase), be a kind of alkaline enzyme that can be hydrolyzed mucopolysaccharide in pathogenic bacterium.N,O-Diacetylmuramidase is mainly through the β-1 between the-acetylmuramic acid in destruction cell walls and NAG, 4 glycosidic links, make the insoluble mucopolysaccharide of cell walls resolve into solubility glycopeptide, cause cell wall rupture content overflow and make bacterolysis, thus reach the object of killing bacterium.N,O-Diacetylmuramidase also directly can be combined with electronegative viral protein, forms double salt, make virally inactivated with DNA, RNA, apoprotein.Therefore, this enzyme has antibacterial, anti-inflammatory, the effect such as antiviral, and nontoxic, have no side effect.
Antibacterial peptide (antimicrobial peptide, ABP) be that the one produced through induction in organism has bioactive micromolecule polypeptide, molecular weight is about 2000 ~ 7000, be made up of 20 ~ 60 amino-acid residues, the Antibacterial mechanism of antibacterial peptide is that antibacterial peptide molecule can be bored a hole and form ion duct on bacterial cell plasma membrane, cause bacterial cell membrane structure deteriorate, cause water-soluble substances in born of the same parents to ooze out in a large number, and finally cause bacterial death.Antibacterial peptide has the features such as strong basicity, thermostability and broad-spectrum antimicrobial.Antibacterial peptide, to bacterium, especially has very strong lethal effect to some resistance pathogenic bacteria.In addition, some antibacterial peptide has killing action to fractionated viral, fungi, protozoon and cancer cells etc., even can improve immunizing power, accelerating wound healing process.
N,O-Diacetylmuramidase can extract and obtain in a large number from egg white, but this method to there is raw material quality wayward, have the danger of potential pathogen contamination.Antibacterial peptide can extract and obtain from fruit bat, but the raw-material source of this method is wayward, and extract the yield of antibacterial peptides obtained low, poor activity, quality instability is also wayward.In addition, N,O-Diacetylmuramidase and antibacterial peptide biologically stable very poor, room temperature is placed and active is just dropped to original less than 50% in several days, and therefore, N,O-Diacetylmuramidase or antibacterial peptide cannot use as wound sterilizing agent separately.
Chaperone (chaperone) refers to that those assist other macromolecular structures to fold/move back the protein of pleat, assembling/disintegration, quits work after these macromolecular structures have possessed normal biological function.Chaperone can prevent the formation of incorrect folding intermediate and not have the incorrect gathering of the protein protomer assembled, and assists assembling and the disintegration of polypeptide chain transmembrane transport and large oligomeric protein.
N,O-Diacetylmuramidase, antibacterial peptide and chaperone are undertaken merging the antibacterial protein obtaining a kind of N,O-Diacetylmuramidase, antibacterial peptide and chaperone and merge by genetic engineering technique by the present invention, and described antibacterial peptide and chaperone are connected to the C-terminal of N,O-Diacetylmuramidase successively.
N,O-Diacetylmuramidase and antibacterial peptide carry out merging and not only have N,O-Diacetylmuramidase and destroy cell walls function but also have the function that antibacterial peptide destroys bacterial cell membrane, and N,O-Diacetylmuramidase and antibacterial peptide produce the sterilization effect that synergistic effect greatly strengthen antibacterial protein simultaneously.Chaperone is connected to the C-terminal of antibacterial peptide simultaneously, the expression amount of macromole in intestinal bacteria that N,O-Diacetylmuramidase and antibacterial peptide merge can be improved, macromole correctly the folding in intestinal bacteria body simultaneously helping N,O-Diacetylmuramidase and antibacterial peptide to merge, the macromole that N,O-Diacetylmuramidase and antibacterial peptide are merged is solvable, avoids the formation of inclusion body.
In some embodiments, described chaperone is maltose binding protein (MBP), and the antibacterial protein that namely described N,O-Diacetylmuramidase, antibacterial peptide and chaperone merge is the antibacterial protein LYS-ABP-MBP of N,O-Diacetylmuramidase, antibacterial peptide and maltose binding protein fusion.
In some specific embodiments, described antibacterial protein LYS-ABP-MBP, its aminoacid sequence is as shown in SEQ ID No.1.
Present invention provides the DNA molecular of antibacterial protein of the present invention of encoding.Due to the degeneracy of codon, the nucleotide sequence of a variety of antibacterial protein of the present invention of can encoding can be there is.For the DNA molecular of coding antibacterial protein of the present invention, those skilled in the art can utilize existing known method manufacture synthesis easily.Such as, by selecting the codon of the amino-acid residue corresponding to the aminoacid sequence designed by forming, can determine and provide the DNA molecular of the aminoacid sequence corresponding to antibacterial protein easily.
Present invention also offers the preparation method of described antibacterial protein LYS-ABP-MBP, N,O-Diacetylmuramidase, antibacterial peptide are connected with expression vector orientation with the gene fragment of chaperone, expression after transformed host cell, extraction, purifying.
The method utilizing genetic engineering technique to obtain DNA molecular at present has a variety of, comprises full genome synthesis, digestion with restriction enzyme and pcr amplification.The preferred full genome of preparation method of the present invention synthesizes described DNA molecular.
In some embodiments, the nucleotide sequence being reassembled as full genome composite coding lysozyme-antibacterial peptide of above-mentioned preparation method.
PMAL-p2x fusion protein expression vector self contains maltose binding protein gene order, selects pMAL-p2x plasmid to be template, design primer, uses round pcr to amplify MBP gene fragment.
Expression vector described in preparation method of the present invention is pBV220.
Host cell described in preparation method of the present invention is E. coli expression strains or yeast.Wherein E. coli expression strains comprises Rosetta series and BL21 series bacterial strain.
In some embodiments, host cell is e. coli bl21 series bacterial strain.E. coli bl21 series bacterial strain comprises e. coli strain bl21, e. coli bl21 (DE3) bacterial strain, e. coli bl21 (DE3) pLysS bacterial strain.
In some preferred embodiments, described host cell is e. coli bl21 (DE3) pLysS.E. coli bl21 (DE3) pLysS is with pLysS plasmid, and this plasmid is with the gene of coding T7 N,O-Diacetylmuramidase.T7 N,O-Diacetylmuramidase can reduce the expression background of T7 promotor goal gene below, but does not affect the target protein expression level that induction produces, can the expression of more rigorous control T7 polysaccharase.
Need after prokaryotic cell prokaryocyte protein expression to extract from host cell, then carry out separation and purification, obtain target protein.Extract described in preparation method of the present invention and comprise bacterium collection, fragmentation, centrifuging and taking supernatant.
Further, the present invention carries out separation and purification by the method for affinity chromatography to the target protein given expression to.
Affinity chromatography utilizes special between molecule with its part, reversible affine keying action and carries out a kind of chromatographic technique of being separated.It the affinity molecule with special construction is made solid-phase adsorbent be placed in chromatography column, and when wanting separated mixed liquid of protein to pass through chromatography column, the protein with sorbent material with affinity will be trapped in chromatography column by adsorbing.Those do not have the protein of avidity owing to not adsorbed, and directly flow out, thus separate with separated protein, then select suitable elutriant, change conjugation condition and are got off by combined Protein elution, reach separation object.
In some embodiments, the chromatography media of described affinity chromatography is Q Sepharose FastFlow.
The present invention utilizes engineered method, prepare (1) lysozyme-antibacterial peptide-MBP (maltose binding protein) fusion rotein (hereinafter referred to as LYS-ABP-MBP), (2) antibacterial peptide-N,O-Diacetylmuramidase-MBP fusion rotein (hereinafter referred to as ABP-LYS-MBP), (3) lysozyme-antibacterial peptide-GST (Glutathione S transferase) fusion rotein (hereinafter referred to as LYS-ABP-GST), (4) antibacterial peptide-N,O-Diacetylmuramidase-gst fusion protein (hereinafter referred to as ABP-LYS-GST), (5) lysozyme-antibacterial peptide fusion rotein (hereinafter referred to as LYS-ABP), (6) antibacterial peptide-lysozyme fusion protein (hereinafter referred to as ABP-LYS).Adopt the antibacterial effect of the more above-mentioned fusion rotein of antibacterial spot method.The fungistatic effect that result shows antibacterial protein LYS-ABP-MBP of the present invention and lysozyme-antibacterial peptide fusion rotein LYS-ABP is best, and fungistatic effect is better than the fungistatic effect of independent N,O-Diacetylmuramidase and antimicrobial peptide protein and ABP-LYS-MBP.
Further, the present invention, by LYS-ABP-MBP and LYS-ABP, after room temperature lucifuge places 1 month, investigates the biological activity of 2 kinds of albumen by antibacterial spot method.After result is presented at room temperature placement, LYS-ABP deactivation rate is very fast, and LYS-ABP-MBP can keep 1 month activity.Show that antibacterial protein LYS-ABP-MBP of the present invention has good biological activity stability.
Therefore the invention provides the application of described antibacterial protein LYS-ABP-MBP in preparation wound class thimerosal.
As shown from the above technical solution, the invention provides a kind of antibacterial protein, be made up of N,O-Diacetylmuramidase, antibacterial peptide and chaperone, described antibacterial peptide and chaperone are connected to the C-terminal of N,O-Diacetylmuramidase successively.Provide the preparation method of described antibacterial protein LYS-ABP-MBP simultaneously.Antibacterial protein LYS-ABP-MBP of the present invention is to human body nonirritant and good antimicrobial effect, and fungistatic effect is obviously better than independent N,O-Diacetylmuramidase and antimicrobial peptide protein, and biological activity stability is good, is suitable for wound sterilization.
In order to make those skilled in the art understand technical scheme of the present invention better, below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1:
1, plasmid construction
1.1, the gene order of full genome synthesis lysozyme-antibacterial peptide (LYS-ABP) and antibacterial peptide-N,O-Diacetylmuramidase (ABP-LYS), and at two ends design restriction enzyme site, use EcoRI/BamHI double digestion, reclaim small segment.
1.2, with PMal-P2x plasmid for template, design primer, uses round pcr to amplify maltose binding protein (MBP) gene fragment, introduces restriction enzyme site BamHI/HindIII simultaneously, PCR fragment, after BamHI/HindIII double digestion, reclaims fragment.
1.3, by ready commercialization plasmid pBV220 EcoRI/HindII double digestion, and large fragment is reclaimed.
1.4, three sections of fragments that above-mentioned steps reclaims are carried out orientation in T4 ligase enzyme system to connect, maltose binding protein (MBP) is connected to the C-terminal of gene order fragment, and be transformed in DH5a engineering bacteria and carry out evaluation and screening, finally filter out the recombinant plasmid of lysozyme-antibacterial peptide-maltose binding protein fragment (pBVLYS-ABP-MBP) and antibacterial peptide-N,O-Diacetylmuramidase-maltose binding protein fragment (pBVABP-LYS-MBP).
1.5, by above-mentioned two recombinant plasmid transformed to e. coli bl21 Host Strains, obtain and can express engineering bacteria pBVLYS-ABP-MBP/BL21 and pBVABP-LYS-MBP/BL21 of LYS-ABP-MBP and ABP-LYS-MBP.
Continue according to the method described above to build other two kinds of recombinant bacterial strains, first the gene order of full genome synthesis lysozyme-antibacterial peptide and antibacterial peptide-N,O-Diacetylmuramidase, and at two ends design restriction enzyme site, use EcoRI/BamHI double digestion, reclaim small segment.Next is with pGEX-4-T-1 plasmid for template, and design primer, uses round pcr to amplify GEX gene fragment, introduce restriction enzyme site BamHI/HindIII simultaneously, and PCR fragment, after BamHI/HindIII double digestion, reclaims fragment.Then by ready commercialization plasmid pBV220 EcoRI/HindII double digestion, and large fragment is reclaimed.Finally the three sections of fragments reclaimed are carried out orientation in T4 ligase enzyme system to connect, GEX gene fragment is connected to the C-terminal of gene order fragment, and be transformed in DH5a engineering bacteria and carry out evaluation and screening, finally filter out the recombinant plasmid of lysozyme-antibacterial peptide-GEX fragment (pBVLYS-ABP-GEX) and antibacterial peptide-N,O-Diacetylmuramidase GEX fragment (pBVABP-LYS-GEX).By above-mentioned two recombinant plasmid transformed to e. coli bl21 Host Strains, obtain engineering bacteria pBVLYS-ABP-GEX/BL21 and pBVABP-LYS-GEX/BL21 that can express LYS-ABP-GEX and ABP-LYS-GEX.
The aminoacid sequence of LYS-ABP-MBP fusion rotein is shown in SEQ ID No:1;
The aminoacid sequence of ABP-LYS-MBP fusion rotein is shown in SEQ ID No:2;
The aminoacid sequence of LYS-ABP-GST fusion rotein is shown in SEQ ID No:3;
The aminoacid sequence of ABP-LYS-GST fusion rotein is shown in SEQ ID No:4;
The aminoacid sequence of LYS-ABP fusion rotein is shown in SEQ ID No:5;
The aminoacid sequence of ABP-LYS fusion rotein is shown in SEQ ID No:6.
2, the mass propgation of engineering bacteria
1. bacterial classification: pMAL-LYS-ABP/BL21, pMAL-ABP-LYS/BL21, pGEX-LYS-ABP/BL21, pGEX-ABP-LYS/BL21.
2. LB substratum (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L pH7.2) is distributed into 250mL, 2000mL triangle shaking flask, 121 DEG C of autoclaving 15min, for subsequent use.
3. engineering bacteria spawn culture: have on the LB flat board of engineering bacteria bacterium colony long, picking list bacterium colony is inoculated in 5mL LB substratum respectively, 37 DEG C when being cultured to OD600=0.6, in 250mL LB substratum of transferring, about 15 little the stoppings when OD600=0.6 of amplification culture are cultivated.
4. transfer: be transferred in the 2000mL Erlenmeyer flask containing LB by the engineering bacteria that 250mL LB cultivates, 37 DEG C, 200rpm, cultivates 20hr and continues to cultivate.When being cultured to OD600=0.6, add IPTG to final concentration 1mM in 37 DEG C of inducing culture 3hr.
5. the collection of bacterium and cracking: the engineering bacteria containing abduction delivering is placed in 4 DEG C of refrigerators, place and make thalline natural sedimentation in 24 hours, supernatant is removed after thalline natural sedimentation, add the damping fluid (50mM Tris-HCl, 2mM EDTA, 0.1%Triton X-100, PH 8.0) of equivalent, forward in other container, put into-20 DEG C of refrigerators, treat completely freezing after, take out after dissolving completely, then repeat freeze thawing twice.The centrifugal 20min of 12000g, gets supernatant.
3, purifying
1. DNA digestion: in the liquid after freeze thawing, adds DNA enzymatic with the DNA of thalline release of degrading, normal temperature about 5 hours, DNA digestion.
2. column chromatography consummateization:
Chromatography media: Q Sepharose Fast Flow;
Chromatography column specification: internal diameter 50cm, high 40cm, medium height about 10cm, medium volume is about 200mL;
Monitoring: 280nm
Balance liquid is 25mM Tris-HCl damping fluid, pH8.
First with more than balance liquid balance chromatography column 5 column volumes, flow velocity 20mL/min, after then sample balance liquid dilutes 3 times, carry out loading, flow velocity is 20mL/min, continues balance about 5 column volumes after upper complete sample, use 25mM Tris-HCl damping fluid, pH8,0.15M NaCl washes foreign protein and is returned to baseline to uv-absorbing, uses elutriant 25mM Tris-HCl damping fluid, pH8,0.3M NaCl wash-out target protein, and collect, be stoste.Finally use regenerated liquid 25mM Tris-HCl damping fluid, pH8,2M NaCl regenerates medium.The ethanol that can add 20% after medium washing is put into 4 DEG C and is preserved.
By purifying, obtain 4 kinds of fusion roteins, be respectively LYS-ABP-MBP, ABP-LYS-MBP, LYS-ABP-GST and ABP-LYS-GST.Use zymoplasm to LYS-ABP-MBP by the restriction enzyme site designed between albumen, ABP-LYS-MBP 2 kinds of fusion roteins cut, condition is 5IU/mL, room temperature, 12 hours, obtain enzyme and cut liquid, then by the method for the flat ultrafiltration of 10kD, results permeate, the method re-using the flat ultrafiltration of 1kD molecular weight cut-off obtains LYS-ABP and ABP-LYS two kinds of target proteins.
So far, obtain 6 kinds of fusion roteins altogether, through inspection, electrophoresis purity and HPLC purity are all greater than 95%.
Embodiment 2: fungistatic effect is studied
Adopt the antibacterial effect of antibacterial spot method antibacterial protein more of the present invention and antibacterial peptide, N,O-Diacetylmuramidase.
1, laboratory apparatus and reagent:
Water isolation type electro-heating standing-temperature cultivator, LB substratum, penbritin, intestinal bacteria, streptococcus aureus, P. aeruginosa.
2, experimental technique:
According to " Chinese Pharmacopoeia " three (2005 editions) annex IX A inhibition zone method, in 8 cm dishes, pour LB substratum about the 20mL containing 1.5% agar into, for subsequent use after cooled and solidified; Get and cultivate intestinal bacteria, streptococcus aureus and each 50 μ L of Pseudomonas aeruginosa liquid that OD600 is about 0.6, about 50 DEG C are cooled to 5mL, pour into above in ready culture dish after cultivating mixing containing the LB of 0.5% agar, cooled and solidified is as assay plate.Evenly punch with punch tool in assay plate, add respectively in hole: 100 μ g/mL penbritins (positive control), 2 μ g/mL6 kind fusion protein sample, 2 μ g/mL N,O-Diacetylmuramidases sample (ovum gallinaceum extracts clearly), 2 μ g/mL antibacterial peptide samples (fruit bat extraction), physiological saline (negative control), each 150 μ L, around the hole that detects by an unaided eye after cultivating 24 hours at 37 DEG C, whether occur inhibition zone.There is inhibition zone to appear as bacteriostatic action, do not have inhibition zone to appear as and there is no bacteriostatic action.Measure the diameter of inhibition zone while having bacteriostatic action, result is as table 1.
The mean value of the diameter (mm) of the inhibition zone of table 1 each sample three bacteriostatic tests:
| Classification | Intestinal bacteria | Streptococcus aureus | Pseudomonas aeruginosa |
| LYS-ABP-MBP | 52 | 26 | 18 |
| ABP-LYS-MBP | 37 | 18 | 11 |
| LYS-ABP-GST | 0 | 0 | 0 |
| ABP-LYS-GST | 0 | 0 | 0 |
| LYS-ABP | 50 | 25 | 16 |
| ABP-LYS | 35 | 18 | 10 |
| N,O-Diacetylmuramidase | 24 | 12 | 9 |
| Antibacterial peptide | 12 | 11 | 5 |
| Negative control | 0 | 0 | 0 |
| Positive control | 55 | 46 | 40 |
From table 1 result, the fungistatic effect of LYS-ABP-MBP and LYS-ABP is best, and fungistatic effect is better than the fungistatic effect of independent N,O-Diacetylmuramidase and antimicrobial peptide protein and ABP-LYS-MBP.
Embodiment 3: the biological activity stability of antibacterial protein is investigated
By LYS-ABP-MBP and LYS-ABP, after room temperature lucifuge places 1 month, investigated the biological activity of 2 kinds of albumen by the antibacterial spot method in embodiment 2, result table 2.
Table 2 LYS-ABP-MBP and LYS-ABP room temperature place the mean value of the diameter (mm) of the inhibition zone of three bacteriostatic tests after 1 month:
| Classification | Intestinal bacteria | Streptococcus aureus | Pseudomonas aeruginosa |
| LYS-ABP-MBP | 55 | 23 | 17 |
| LYS-ABP | 21 | 11 | 5 |
From table 2 result, after room temperature is placed, LYS-ABP deactivation rate is very fast, and LYS-ABP-MBP can keep 1 month activity.Show that antibacterial protein LYS-ABP-MBP of the present invention has good biological activity stability.
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 (8)
1. an antibacterial protein, is made up of N,O-Diacetylmuramidase, antibacterial peptide and chaperone, and described antibacterial peptide and chaperone are connected to the C-terminal of N,O-Diacetylmuramidase successively.
2. antibacterial protein according to claim 1, described chaperone is maltose binding protein, and its aminoacid sequence is as shown in SEQ ID No.1.
3. the DNA molecular of the antibacterial protein of coding described in claim 1 or 2.
4. the preparation method of antibacterial protein according to claim 1, is connected N,O-Diacetylmuramidase, antibacterial peptide with expression vector orientation with the gene fragment of chaperone, expression after transformed host cell, extraction, purifying.
5. preparation method according to claim 4, expression vector is pBV220, and described host cell is intestinal bacteria or yeast.
6. preparation method according to claim 4, is characterized in that, described extraction comprises bacterium collection, fragmentation, centrifuging and taking supernatant.
7. preparation method according to claim 4, is characterized in that, described purifying is affinitive layer purification.
8. the application of the antibacterial protein described in claim 1 or 2 in preparation wound class thimerosal.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1873251A1 (en) * | 2006-06-29 | 2008-01-02 | Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus | Expression vector(s) for enhanced expression of a protein of interest in eukaryotic or prokaryotic host cells |
| CN104761621A (en) * | 2014-01-06 | 2015-07-08 | 长春新悦然科技有限公司 | Antibacterial proteins |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1873251A1 (en) * | 2006-06-29 | 2008-01-02 | Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus | Expression vector(s) for enhanced expression of a protein of interest in eukaryotic or prokaryotic host cells |
| CN104761621A (en) * | 2014-01-06 | 2015-07-08 | 长春新悦然科技有限公司 | Antibacterial proteins |
Non-Patent Citations (3)
| Title |
|---|
| YIGONG GE,ET AL.: "In Vitro Antibacterial Properties of Pexiganan, an Analog of Magainin", 《ANTIMICROBIAL AGENTS AND CHEMOTHERAPY》 * |
| 史春林等: "抗菌肽在宿主防御中的作用", 《中国生物工程杂志》 * |
| 赵广荣: "《现代制药工艺学》", 28 February 2015, 清华大学出版社 * |
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Application publication date: 20151007 |