CN108743918A - The new application and preparation method thereof of people's beta-alexin 2 - Google Patents
The new application and preparation method thereof of people's beta-alexin 2 Download PDFInfo
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- CN108743918A CN108743918A CN201810641326.0A CN201810641326A CN108743918A CN 108743918 A CN108743918 A CN 108743918A CN 201810641326 A CN201810641326 A CN 201810641326A CN 108743918 A CN108743918 A CN 108743918A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Abstract
The invention discloses new application of people's beta-alexin 2 and preparation method thereof, the present invention proposes application of the people's beta-alexin 2 in preparing treatment fungi especially filamentous fungi infectious disease medicament.The present invention provides the preparation methods of 2 polypeptide of recombined human beta-alexin, HBD2 maturation peptide genes are inserted into pET32a (+) plasmid, correct recombinant plasmid will be built and be transferred to Escherichia coli acquisition expression engineering bacteria, Fiber differentiation expresses engineering bacterium expression HBD2 fusion proteins, finally obtains 2 polypeptide of recombined human beta-alexin by nickel affinity chromatography, fibrin ferment digestion, purifying, concentration again.The new application of people's beta-alexin 2 is proposed in the present invention.Method in the present invention has the characteristics that cheap, yield is more and is easily guaranteed that the biological activity of polypeptide.
Description
Technical field
The present invention relates to a kind of new applications and preparation method thereof of active small molecular protein, especially people's beta-alexin 2
New application and preparation method thereof.
Background technology
In recent years, as broad-spectrum antibiotic, hormone, the extensive use of immunosuppressor and the sprawling of AIDS, organ move
The popularization and application with interventional technique are planted, make the incidence of fungal infection that ascendant trend be presented, fungal infection rate in 2014 is 20
The several times of the nineties in century, Mycotoruloides and aspergillus are the pathogens of current most common fungal infection, and certain fungies cause
Systemic infection lethality be up to 90%.For AIDS patient (AIDS), fungal infection is most common opportunistic infections,
Its morbidity and mortality is higher.Although the antifungal drug of different role mechanism has played important in treating fungal infection
Effect, but as infection bacteria species gradually change, the report in relation to antibody-resistant bacterium gradually increases so that the treatment of fungal infection
It is faced with stern challenge.People's beta-alexin be distributed widely in people, mouse, ox, sheep, pig a variety of organ epithelial cells in, including
Skin, respiratory tract, urethra, enteron aisle and oral mucosa epithelial cell etc..About the antimicrobial acivity of people's beta-alexin, have proven to
Artificial synthesized people's beta-alexin has anti-Staphylococcus aureus, escherichia coli, pseudomonas aeruginosa and Candida albicans
Effect.But the effect about 2 anti-filamentous fungi of people's beta-alexin has no relevant report.Artificial synthesized people's beta-alexin, makes
High price is expensive, yield is few and is not easy to ensure the biological activity of polypeptide.
Currently, being found that about 30 kinds of people's beta-alexin genes with bioinformatics method.The base of beta-alexin
The binding site of NF- κ B, IL-6 and STAT etc. before having because of upstream promoter, when pathogen associative mode molecule (PAMP) and epithelium
After the pattern recognition receptors (including CD14, TLRs etc.) of cell surface combine, participated at a variety of proinflammatory factors (TNF-α, IL-1)
Under, the transcription factors such as activation NF- κ B, then combined, make its gene activation and turned with NF- κ B of promoter region, IL-6 binding sites
Record and translation, belong to inducible expression.
Therefore, the antimycotic relevant application of non-finder's beta-alexin 2 in the prior art, especially anti-filamentous fungi are related
Application.The synthetic method of existing people's beta-alexin 2, exist involve great expense, yield is few and is not easy to ensure the biology of polypeptide
The problem of activity.
Invention content
The purpose of the present invention is to provide the new application and preparation method thereof of people's beta-alexin 2.It is anti-that the present invention proposes people β-
Imperial element 2 is preparing the application in treating fungal infectious disease drug, especially treats filamentous fungi infectious diseases in preparation
Application in drug.The present invention provides a kind of 2 polypeptide of recombined human beta-alexin (recombinant human beta-
Defensin 2, rHBD2) preparation method.The preparation method of 2 polypeptide of recombined human beta-alexin has low cost in the present invention
The characteristics of more than honest and clean, yield and being easily guaranteed that the biological activity of polypeptide.
Technical scheme of the present invention：The new application of people's beta-alexin 2, people's beta-alexin 2 are preparing treatment fungal infectious
Application in disease medicament.
In the new application of people's beta-alexin 2 above-mentioned, the fungal infectious disease is filamentous fungi infectious diseases.
In the new application of people's beta-alexin 2 above-mentioned, people's beta-alexin 2 is 2 polypeptide of recombined human beta-alexin.
In the new application of people's beta-alexin 2 above-mentioned, people's beta-alexin 2 is taken, is added one or more pharmaceutically acceptable
Carrier or excipient, be prepared into it is various treatment fungal infections pharmaceutical preparations.
In the new application of people's beta-alexin 2 above-mentioned, the pharmaceutical preparation of the treatment fungal infection is oral preparation：Piece
Agent, capsule, oral solution, suspension；Or ejection preparation：Solution, suspension, the powder-injection of injectable；Or external preparation：
Cream or solution.
In the new application of people's beta-alexin 2 above-mentioned, the preparation method of 2 polypeptide of recombined human beta-alexin, according to
The HBD2 gene orders that genbank is provided synthesize HBD2 maturation peptide genes；HBD2 maturation peptide genes are inserted into pET32a (+) matter
Grain；Correct recombinant plasmid will be built and be transferred to Escherichia coli Rossetta-gami (2), obtain expression engineering bacteria Rosseta-
gami(2)-pET32a(+)/HBD2；Fiber differentiation expresses engineering bacteria Rosseta-gami (2)-pET32a (+)/HBD2 expression
HBD2 fusion proteins collect bacterium cellular content；By nickel affinity chromatography by the HBD2 fusion protein purifications in cellular content,
Using the label protein assigned by pET32a (+) plasmid in fibrin ferment digestion HBD2 fusion proteins, then it is pure through nickel affinity chromatography
Change, desalination, concentration obtain 2 polypeptide of recombined human beta-alexin.
In the preparation method of 2 polypeptide of recombined human beta-alexin above-mentioned, the HBD2 maturations peptide gene is inserted into pET32a (+)
Plasmid, be HBD2 maturations peptide gene and pET32a (+) plasmid are first subjected to endonuclease reaction respectively with BamH I and Xhol I, then
The HBD2 maturations peptide gene after digestion and the pET32a (+) after digestion are attached reaction under the action of T4 DNA ligases.
In the preparation method of 2 polypeptide of recombined human beta-alexin above-mentioned, in the connection reaction, the HBD2 after digestion is ripe
Peptide gene and the molecule mole ratio of pET32a (+) plasmid after digestion are (3~10):1.
In the preparation method of 2 polypeptide of recombined human beta-alexin above-mentioned, the expression engineering bacteria Rosseta-gami (2)-
PET32a (+)/HBD2 is through IPTG induced expression HBD2 fusion proteins.
In the preparation method of 2 polypeptide of recombined human beta-alexin above-mentioned, the engineering bacteria Rosseta-gami (2)-
PET32a (+)/HBD2 expresses HBD2 fusion proteins, and optimal expression condition is best a concentration of 0.5mM of IPTG, when most preferably inducing
Between be 6h, optimum temperature be 34 DEG C.
Compared with the prior art, the present invention show that people's beta-alexin 2 can destroy bacterium eucaryotic cell structure, to unicellular fungi and more
Cell fungi all has inhibition or killing effect.The present invention proposes people's beta-alexin 2 and is preparing treatment fungal infectious disease
Application in drug is especially preparing the application in treating filamentous fungi infectious disease medicament, is expanding people's beta-alexin 2
Application range.The present invention provides a kind of preparation methods of people's beta-alexin 2 (2 polypeptide of recombined human beta-alexin).The preparation
HBD2 maturation peptide genes are inserted into pET32a (+) plasmid by method, will be built correct recombinant plasmid and are transferred to Escherichia coli
Rossetta-gami (2) obtains expression engineering bacteria Rosseta-gami (2)-pET32a (+)/HBD2, and Fiber differentiation expresses engineering
Bacterium Rosseta-gami (2)-pET32a (+)/HBD2 expresses HBD2 fusion proteins, finally passes through nickel affinity chromatography, fibrin ferment again
Digestion, purifying, concentration obtain 2 polypeptide of recombined human beta-alexin (recombinant human beta-defensin 2,
rHBD2).The present invention provides new way for the preparation of 2 polypeptide of people's beta-alexin.The preparation method has cheap, yield
The characteristics of mostly and being easily guaranteed that the biological activity of polypeptide.
Description of the drawings
Fig. 1 is pET32a (+) plasmid construct schematic diagram；
Fig. 2 is pET32a (+)/HBD2 Prokaryotic Recombinant Plasmids structural schematic diagrams；
Fig. 3 is the double digestion qualification result of pET32a (+)/HBD2 recombinant plasmids, the label in figure be：1-pET32a(+)/
HBD2 recombinant plasmids (non-digestion), 2-pET32a (+)/HBD2 recombinant plasmid Xhol and Apal double digestion products, 3-DNA standard scores
Son amount Marker；
Fig. 4 is the sequencing result of pET32a (+)/HBD2 recombinant plasmids；
Fig. 5 is HBD2 fusion protein induced expression PAGE gel electrophoresis results, the label in figure be：1- protein marks
Quasi-molecule amount Marker, 2-Rossetta-gami (2)-pET32a (+)/HBD2 induced products, 3-Rossetta-gami (2)-
The non-induced products of pET32a (+)/HBD2；
Fig. 6 is the Western Blot qualification results that HBD2 merges HBD2 albumen, the label in figure be：1- protein standards
Molecular weight Marker, 2-Rossetta-gami (2)-pET32a (+) is induced without IPTG, 3-Rossetta-gami (2)-
PET32a (+)/HBD2 is induced through IPTG；
Fig. 7 is the SDS-PAGE electrophoresis results for recombinating HBD2 polypeptides, the label in figure be：M- protein standard markers
Marker, 1- digestion products penetrate liquid, and 2-Binding Buffer wash the liquid that penetrates of column, 3-Elution buffer eluents
Penetrate liquid, 4- digestion products；
Fig. 8 is the Western Blot qualification results for recombinating HBD2 polypeptides, the label in figure be：1- recombinates HBD2 and merges egg
In vain, the rHBD2 of 2- purifying, the pyrolysis product after the induction of 3-Rossetta-gami (2)-pET32a (+) bacterium.
Specific implementation mode
The present invention will be further described below with reference to the drawings, but is not intended as the foundation limited the present invention.
It is the experimental example of the present invention below：
Experimental example 1：Express the structure experiment of engineering bacteria Rosseta-gami (2)-pET32a (+)/HBD2
The structure of 1.pET32a (+)/HBD2 recombinant plasmids
(1) the extracting of plasmid pET32a (+)：Culture e. coli jm109, which is shaken, with LB culture mediums (contains pET32a (+) matter
Grain), bacterium cell is collected, is operated with plasmid extraction kit, pET32a (+) plasmid (structure of pET32a (+) plasmid is obtained
Schematic diagram is shown in Fig. 1).
(2) restriction enzyme reaction：By the HBD2 maturations peptide gene of synthesis and pET32a (+) plasmid with I Hes of BamH
Xhol I carries out endonuclease reaction respectively under optimum conditions, and reaction product row glue recycling after agarose electrophoresis obtains digestion production
Object.
(3) connection reaction：Above-mentioned digestion products are attached reaction again after glue recovery purifying.It is anti-in T4 DNA ligases
Lower progress should be acted on, the molecule mole ratio of Insert Fragment and carrier in connecting reaction is by 6:1 principle carries out (pET32a
(+)/HBD2 Prokaryotic Recombinant Plasmids structural schematic diagrams are shown in Fig. 2).
(4) transformed competence colibacillus cell：Use CaCl2E. coli jm109 is prepared into competent cell by method, and conventional method will
Connection product converts escherichia coli jm109 competent cell, and converted product is inoculated on the LB tablets containing ampicillin
PCR and digestion with restriction enzyme identification are carried out after overnight incubation picking positive bacterium colony culture in second day.
2. structure expression engineering bacteria Rosseta-gami (2)-pET32a (+)/HBD2
(1) competent cell is prepared：Use CaCl2Escherichia coli Rosseta-gami (2) is prepared into competent cell by method.
(2) the extracting of recombinant plasmid pET32a (+)/HBD2：Culture e. coli jm109 is shaken with LB culture mediums (to contain
PET32a (+)/HBD2 plasmids), bacterium cell is collected, is operated with plasmid extraction kit, pET32a (+)/HBD2 matter is obtained
Grain.
(3) transformation experiment：Conventional method is by pET32a (+)/HBD2 recombinant plasmid transformed Escherichia coli Rosseta-gami
(2) converted product is inoculated in second day picking positive of overnight incubation on the LB tablets containing ampicillin by competent cell
PCR and digestion with restriction enzyme identification are carried out after bacterium colony culture.
3. experimental result
(1) the double digestion of pET32a (+)/HBD2 recombinant plasmids and sequencing qualification result
PET32a (+)/HBD2 recombinant plasmids are through nucleic acid restriction endonuclease Xhol and Apal double digestion, digestion products fine jade
The band (see Fig. 3) of visible 4000+bp and 2000 ± bp sizes after sepharose electrophoresis are consistent with expected primer size.It will
The sequencing result (see Fig. 4) of pET32a (+)/HBD2 recombinant plasmids carries out blast comparisons, the HBD2 mature peptide sequences being inserted into plasmid
Row are correct, no mutation, and reading frame is correct.
Experimental example 2：Induced expression, identification and the optimum induction of HBD2 fusion proteins
The induced expression of 1.HBD2 fusion proteins and identification
(1) the induced expression of HBD2 fusion proteins：Expression bacterial strain Rossetta-gami (2)-pET32a that glycerine is preserved
(+)/HBD2 and Rossetta-gami (2)-pET32a (+) is inoculated in respectively in 5mL LB/AMP fluid nutrient mediums, 37 DEG C of oscillations
Overnight incubation takes 50 μ L bacterium solutions with 1:100 inoculum concentration is reinoculated in 5mL LB/AMP fluid nutrient mediums, 37 DEG C,
200rpm shaken cultivation 4h are not added with IPTG until thalline reaches exponential phase (A600 ≈ 0.6) as positive control, remaining
Addition IPTG to final concentration of 0.5mM, then continue to cultivate 4h for 37 DEG C, centrifugation goes to use 0.01mol/LPBS buffer solutions after supernatant
After 1mL washs bacterium cell twice, 0.5mLPBS is added, bacterium cell is resuspended.
(2) sds polyacrylamide gel electrophoresis (SDS-PAGE) is analyzed：It is slow that 5 × SDS albumen loadings are added in above-mentioned sample
Fliud flushing, 100 DEG C, 10min is boiled in boiling, and cooling is spare.Prepare 5% concentration glue and 14% separation gel, the 15 μ L albumen samples that will be prepared
Product and 3 μ L protein moleculars Marker are splined in well.Deposition condition is：Sample in concentrating glue voltage be 80V, sample into
Voltage is 120V after entering separation gel, and electrophoresis time is about 2h.After electrophoresis, gel is peeled, coomassie brilliant blue staining liquid is added,
Dye 1h or so.ddH2Eluent (methanol 30ml+ glacial acetic acid 10mL+ddH is used in O rinsings afterwards for several times2O 60mL) it is carried out on shaking table
2 decolorations, until protein band is clear.Electrophoresis result is recorded using gel imager.
(3) immune protein trace (Western blot) is identified：By Rossetta-gami (2)-pET32a (+) bacterium of collection
The bacteria suspension that suspension and addition IPTG are induced respectively takes 50 μ L that the boiling of 10 μ L 5 × SDS protein electrophoresis sample-loading buffers is added and boils
Protein band on gel is transferred on pvdf membrane after electrophoresis by 10min, row SDS-PAGE electrophoresis, successively respectively with
Goat polyclonal antibodies HBD2 (1:2000), rabbit-anti sheep IgG antibody (1:4000) it is incubated, is developed with ECL luminescent solutions, retain figure
Picture.
The optimum induction of 2.HBD2 fusion proteins
(1) the optimization of induction time and IPTG concentration：By Rosetta-gami (2)-pET32a (+)/HBD2 fresh mediums
According to 1: 100 ratio transferred species in the LB culture mediums of ampicillin (100 μ g/mL of final concentration) amplification cultivation to A600 ≈
0.6, be separately added into final concentration of 0.5,1.0,1.5, the expression of 2.0mM IPTG induction HBD2 fusion proteins；37 DEG C of induction trainings
It supports, the culture of each IPTG concentration takes 1mL sample row 14%SDS-PAGE electrophoresis after culture 4,6,8,12h respectively；Using
Gel imager records electrophoresis result and analyzes HBD2 fusion protein expressions using 4.3 softwares of BandScan.
(2) the optimization of cultivation temperature：By Rosetta-gami (2)-pET32a (+)/HBD2 fresh mediums according to 1:100
Ratio transferred species in the LB culture mediums of ampicillin (100 μ g/mL of final concentration) amplification cultivation be added eventually to A600 ≈ 0.6
The expression of a concentration of 0.5mM IPTG inductions HBD2 fusion proteins, takes 1mL samples after 38,34,30,26 DEG C of Fiber differentiation 6h respectively
Conduct 14%SDS-PAGE electrophoresis；Electrophoresis result is recorded using gel imager and HBD2 is analyzed using 4.3 softwares of BandScan
Fusion protein expression.
3. experimental result
(1) the induced expression result of HBD2 fusion proteins
PAGE gel electrophoresis result shows (see Fig. 5), Rosetta-gami (2)-pET32a (+)/HBD2 protokaryon tables
Expression engineered bacteria induces without IPTG and through occurring a HBD2 expressing fusion protein item after IPTG induced expressions at about 25KDa
Band, and the expression quantity of the destination protein after IPTG induced expressions and expressing quantity dramatically increase.
(2) the Western Blot qualification results of HBD2 fusion proteins：Immune protein Blot experiment result shows (see Fig. 6),
There are about the albumen of 25KDa prints by Rossetta-gami (2)-pET32a (+)/HBD2 when using goat polyclonal antibodies HBD2 as primary antibody
Mark band, Rossetta-gami (2)-pET32a (+) bacterium cell induction after pyrolysis product in have not seen western blot band.
(3) the optimal conditions of induced expression HBD2 fusion proteins：Production is detected under different IPTG concentration and corresponding induction time
Raw HBD2 fusion proteins account for the ratio of mycoprotein, and HBD2 fusion proteins account for the percentage knot of bacterial protein in each culture
Fruit is shown in Table 1, wherein inducing 6 hours HBD2 fusion proteins generated with 0.5mM IPTG, at most (it is total that HBD2 fusion proteins account for thalline
The 54.1% of albumen), therefore select 0.5mM as the best induced concentrations of IPTG, 6 hours are best induction time.Different
The HBD2 fusion proteins that generation is detected under cultivation temperature account for the ratio of mycoprotein, the HBD2 fusion eggs generated when being cultivated at 34 DEG C
It is white at most (HBD2 fusion proteins account for the 55.9% of bacterial protein), therefore select 34 DEG C as inducing temperature.That is induced expression
The optimal conditions of HBD2 fusion proteins is induced 6 hours with 0.5mM IPTG at 34 DEG C.
1 HBD2 fusion protein optimum induction results of table
Experimental example 3：The purifying for recombinating HBD2 polypeptides obtains
The affinitive layer purification of 1.HBD2 fusion proteins
(1) expression bacterial strain Rossetta-gami (2)-pET32a (+)/HBD2 that glycerine preserves is inoculated in 5mL LB/AMP
In fluid nutrient medium, 37 DEG C of shaken cultivations are stayed overnight, and take 50 μ L bacterium solutions with 1:100 inoculum concentration is reinoculated on 1L LB/AMP liquid
In body culture medium, 37 DEG C, 200rpm shaken cultivation 4h, until thalline reaches exponential phase (A600 ≈ 0.6), addition IPTG to end
Then a concentration of 0.5mM continues to cultivate 6h for 34 DEG C, centrifugation goes after supernatant to wash bacterium cell with 0.01mol/LPBS buffer solutions 1mL
After twice, Binding Buffer bufferings are added in the ratio that 1mL Binding Buffer are added in 10mL cultures for bacterium cell
Bacterium cell is resuspended in liquid.
By supernatant be placed in ultrasonication on ice (No. 6 probe, 100 power, ultrasonic 3S, be spaced 3S, act on 30min), 4
DEG C, 10000rpm centrifuge 20min, collect supernatant.
(3) the HBD2 fusion proteins of expression are purified with reference to GE companies HisTrapTMFF nickel purification column specifications, received
Collect eluent.
2. fibrin ferment endonuclease reaction
(1) the eluent being collected into carries out concentration desalination with ultra-filtration centrifuge tube (10KDa)：By be collected into after purification
HBD2 fusion proteins centrifuge 20-40min through 4000 × g of ultra-filtration centrifuge tube, set liquid in pipe are abandoned, with sterile ddH2O supplies inner tube
Liquid, repeated centrifugation 2 times are finally collected inner tube liquid, are quantified with BCA protein quantification kits to remove salinity.
(2) fibrin ferment endonuclease reaction：HBD2 fusion proteins are diluted to 1mg/mL with 0.1mol/L PBS buffer solution, are added
Fibrin ferment (final concentration 10U/mL) mixing, 25 DEG C of water-bath 14h.
(3) the purifying after recombinant protein digestion：With reference to GE company HisTrapTMFF nickel purification column specifications to expression
HBD2 fusion proteins are purified, and eluent is collected.
3. recombinating the acquisition and identification of HBD2 polypeptides
(1) the purifying after recombinant protein digestion：With reference to GE company HisTrapTMFF nickel purification column specifications to expression
HBD2 fusion proteins are purified, and eluent is collected.
(2) the eluent being collected into carries out concentration desalination with ultra-filtration centrifuge tube (3KDa)：Method is the same, with BCA protein quantifications
Kit is quantified.
(3) by quantitative recombination HBD2 polypeptides row SDS-PAGE electrophoresis and Western Blot identifications.
4. experimental result
(1) the SDS-PAGE electrophoresis results of HBD2 polypeptides are recombinated：As shown in Figure 7, after fibrin ferment digestion, label protein quilt
Excision (about 18KDa) completely.Digestion products are purified and desalination, concentration, obtain rHBD2 (about 6KDa).
(2) the Western Blot qualification results of HBD2 polypeptides are recombinated：The Western of HBD2 fusion proteins and rHBD2
Blot experimental results show the western blot band (see Fig. 8) of the about left and right 6KDa and 25KDa, molecular size range and theoretical value
It is consistent, and western blot band is had not seen in the pyrolysis product after the induction of Rossetta-gami (2)-pET32a (+) bacterium.
Experimental example 4：It recombinates the antifungal activity of HBD2 polypeptides and is detected with the antifungic action of combining of antifungal drug
1. the preparation of fungi
The fungi strain that recovery preserves, including candida albicans bacterium (ATCC24433), candida albicans bacterium (are clinically separated
Strain), neogenesis cryptococcus (clinical separation strain), aspergillus niger (clinical separation strain), microsporum canis (clinical separation strain), red hair tinea
Unicellular fungi bacterium cell is diluted to 1 by bacterium (clinical separation strain), acrothesium floccosum (clinical separation strain) with YPG culture mediums
×105Many cells fungal spore is diluted to 1 × 10 by CFU/mL with PDA culture medium5CFU/mL blows and even is prepared into bacteria suspension.
2.rHBD2 the preparation of polypeptide and antifungal drug
RHBD2 polypeptides are diluted to 2000 with 10mM PBS, 1000,500,250,125,62.6,31.25,15.625,
7.8mg/mL.Positive drug is Itraconazole (ITRAC), amphotericin B (AMPHO), 5-flurocytosine (5-FLU), is used respectively
Water for injection is diluted.
The detection of 3.MIC, MFC
The antifungal activity in vitro that rHBD2 polypeptides are observed using micro-amounts of liquids dilution method is added in 96 well culture plates per hole
Enter 90 μ L of bacteria suspension, adds the rHBD2 polypeptides or different antifungal drugs or corresponding buffer solution of various concentration.It is unicellular
Fungi is cultivated 2 days at 37 DEG C, and many cells fungi is cultivated 5 days at 28 DEG C.RHBD2 polypeptide minimums to have no fungi growth phenomenon are dilute
Degree of releasing is used as minimal inhibitory concentration (MIC).10uL cultures are drawn from the hole for having no bacterium cell growth is inoculated with corresponding blank training
Base is supported, suitable culture conditions are provided, the rHBD2 polypeptide minimum dilutions to have no bacterium cell growth phenomenon are dense as minimum sterilization
It spends (MBC/MFC).
4. combining antifungic action detection
80uL fungies bacteria suspension is added per hole in 96 orifice plates and the rHBD2 polypeptides and 10uL of 10uL various concentrations are a kind of
Various concentration antifungal drug measures the MIC of rHBD2 polypeptides and 4 kinds of antimicrobial drug combinations with gridiron pattern method, to calculate
FIC indexes.The function and effect of rHBD2 polypeptides and antifungal drug compatibility are judgment basis by FIC indexes.FIC indexes=first
MIC/ second prescription used time MIC when MIC/ first prescription used time MIC+ second medicines are combined when medicine is combined.Criterion：Index≤0.5 FIC is
Synergistic effect；FIC indexes > 0.5~1 is summation action；FIC indexes > 1~2 is unrelated effect；FIC indexes > 2 makees for antagonism
With.
5. experimental result
(1) the antifungal activity in vitro of rHBD2 polypeptides：RHBD2 polypeptides have unicellular fungi and many cells fungi relatively strong
Bacteriostasis, extremely strong bactericidal effect (being shown in Table 2) is also shown to many cells fungi.
2 rHBD2 polypeptide antifungal activity experimental results of table
(2) rHBD2 polypeptides and antifungal drug combine antifungic action：RHBD2 polypeptides and antifungal drug Itraconazole
(ITRAC), amphotericin B (AMPHO), 5-flurocytosine (5-FLU) synergy, to common disease originality unicellular fungi, packet
It includes candida albicans bacterium, neogenesis cryptococcus and shows as synergistic effect (being shown in Table 3).
3 rHBD2 polypeptides of table combine antifungic action with antifungal drug
The embodiment of the present invention.
Embodiment 1：Injection.
5 grams of 2 polypeptide of recombined human beta-alexin is taken, addition prepares the corresponding auxiliary material of injection, and 1000 injections are made, and uses
It is infected in prevention fungal infection, especially filamentous fungi.Every 5ml, 2 polypeptide 5mg of beta-alexin containing recombined human.Vein of being grown up is noted
It penetrates, is made into 25 μ g/ml and uses, each 1-3 branch, 1-3 times on the one.
Embodiment 2：Cream.
5 grams of 2 polypeptide of recombined human beta-alexin, addition is taken to prepare the corresponding auxiliary material of cream, 100 emulsifiable pastes are made in mixing
Agent, for treating filamentous fungi skin infection.Every 5g, 2 polypeptide 50mg of beta-alexin containing people.Affected part, 3- on the one are smeared in external application
5 times.
Embodiment 3：The preparation method of 2 polypeptide of recombined human beta-alexin.
According to the HBD2 gene orders that genbank is provided, HBD2 maturation peptide genes are synthesized；HBD2 maturation peptide genes are inserted into
PET32a (+) plasmid；Correct recombinant plasmid will be built and be transferred to Escherichia coli Rossetta-gami (2), obtain expression engineering bacteria
Rosseta-gami(2)-pET32a(+)/HBD2；Fiber differentiation expression engineering bacteria Rosseta-gami (2)-pET32a (+)/
HBD2 expresses HBD2 fusion proteins, collects bacterium cellular content；The HBD2 in cellular content is merged by nickel affinity chromatography
Protein purification, using the label protein assigned by pET32a (+) plasmid in fibrin ferment digestion HBD2 fusion proteins, then through nickel parent
2 polypeptide of recombined human beta-alexin is obtained with chromatographic purifying, desalination, concentration.
The HBD2 maturations peptide gene is inserted into pET32a (+) plasmid, is first by HBD2 maturations peptide gene and pET32a (+) matter
Grain carries out endonuclease reaction respectively with BamH I and Xhol I, then by the HBD2 maturations after digestion under the action of T4 DNA ligases
PET32a (+) after peptide gene and digestion is attached reaction.
In the connection reaction, HBD2 maturations peptide gene and the molecule of pET32a (+) plasmid after digestion after digestion are rubbed
Your number ratio is 6:1.
Expression engineering bacteria Rosseta-gami (2)-pET32a (+)/HBD2 merges egg through IPTG induced expressions HBD2
In vain.
Engineering bacteria Rosseta-gami (the 2)-pET32a (+)/HBD2 expresses HBD2 fusion proteins, optimal expression condition
For best a concentration of 0.5mM of IPTG, best induction time is 6h, and optimum temperature is 34 DEG C.
Certainly, the present invention can also have other various embodiments, without deviating from the spirit and substance of the present invention, ripe
It knows those skilled in the art and makes various corresponding change and deformations, but these corresponding changes and change in accordance with the present invention
Shape should all belong to the protection domain of appended claims of the invention.
Claims (9)
1. the new application of people's beta-alexin 2, it is characterised in that：People's beta-alexin 2 is preparing treatment fungal infectious disease drug
In application.
2. the new application of people's beta-alexin 2 according to claim 1, it is characterised in that：The fungal infectious disease is
Filamentous fungi infectious diseases.
3. the new application of people's beta-alexin 2 according to claim 1 or 2, it is characterised in that：People's beta-alexin 2
For 2 polypeptide of recombined human beta-alexin.
4. the new application of people's beta-alexin 2 according to claim 3, it is characterised in that：People's beta-alexin 2 is taken, is added one
Kind or a variety of pharmaceutically acceptable carriers or excipient, are prepared into various treatment fungal infectious disease pharmaceutical preparations.
5. the new application of people's beta-alexin 2 according to claim 3 or 4, the preparation of 2 polypeptide of recombined human beta-alexin
Method, it is characterised in that：According to the HBD2 gene orders that genbank is provided, HBD2 maturation peptide genes are synthesized；By HBD2 mature peptides
Gene is inserted into pET32a (+) plasmid；Correct recombinant plasmid will be built and be transferred to Escherichia coli Rossetta-gami (2), obtain table
Expression engineered bacteria Rosseta-gami (2)-pET32a (+)/HBD2；Fiber differentiation expresses engineering bacteria Rosseta-gami (2)-
PET32a (+)/HBD2 expresses HBD2 fusion proteins, collects bacterium cellular content；It will be in cellular content by nickel affinity chromatography
HBD2 fusion protein purifications, utilize the label egg assigned by pET32a (+) plasmid in fibrin ferment digestion HBD2 fusion proteins
In vain, then through nickel affinity chromatography purifying, desalination, concentration 2 polypeptide of recombined human beta-alexin is obtained.
6. the preparation method of 2 polypeptide of recombined human beta-alexin according to claim 5, it is characterised in that：The HBD2 at
Ripe peptide gene is inserted into pET32a (+) plasmid, is first to divide HBD2 maturations peptide gene and pET32a (+) plasmid BamH I and Xhol I
Do not carry out endonuclease reaction, then under the action of T4DNA ligases by after digestion HBD2 maturations peptide gene and digestion after
PET32a (+) is attached reaction.
7. the preparation method of 2 polypeptide of recombined human beta-alexin according to claim 6, it is characterised in that：The connection is anti-
The molecule mole ratio of Ying Zhong, the HBD2 maturations peptide gene after digestion and pET32a (+) plasmid after digestion is (3~10):1.
8. the preparation method of 2 polypeptide of recombined human beta-alexin according to claim 7, it is characterised in that：The expression work
Journey bacterium Rosseta-gami (2)-pET32a (+)/HBD2 is through IPTG induced expression HBD2 fusion proteins.
9. the preparation method of 2 polypeptide of recombined human beta-alexin according to claim 8, it is characterised in that：The engineering bacteria
Rosseta-gami (2)-pET32a (+)/HBD2 expresses HBD2 fusion proteins, and optimal expression condition is：Best IPTG is a concentration of
0.5mM, best induction time are 6h, and optimum temperature is 34 DEG C.
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| CN101570760A (en) * | 2009-06-15 | 2009-11-04 | 四川大学 | Recombinant mouse beta-alexin 3 polypeptide, preparation and use thereof |
| WO2013039857A1 (en) * | 2011-09-12 | 2013-03-21 | modeRNA Therapeutics | Engineered nucleic acids and methods of use thereof |
| CN103088050A (en) * | 2013-01-09 | 2013-05-08 | 华南理工大学 | Mature human beta-defensin-2 (HBD-2) and preparation method thereof |
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Application publication date: 20181106 |