CN1966709A - Process for preparing recombinant Sinkiang domestic silkworm antibiotic peptide, Sinkiang domestic silkworm antibiotic peptide therefrom, and use thereof - Google Patents
Process for preparing recombinant Sinkiang domestic silkworm antibiotic peptide, Sinkiang domestic silkworm antibiotic peptide therefrom, and use thereof Download PDFInfo
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Abstract
本发明公开一种重组新疆家蚕抗菌肽的纯化制备方法、重组新疆家蚕抗菌肽及其在抑菌、抗肿瘤方面的应用。重组新疆家蚕抗菌肽是从新疆家蚕中克隆得到的，并在真核表达系统中进行了表达。其制备方法为，将发酵液离心去除沉淀，冷冻干燥后经离子交换层析、疏水作用层析和凝胶过滤层析而分离纯化得到。重组新疆家蚕抗菌肽对细菌和肿瘤细胞具有强烈的抑杀作用，而且还具有无溶血活性和对正常组织细胞没有毒性作用，在制备治疗病原微生物感染性疾病和肿瘤的药物中的应用。
The invention discloses a method for purifying and preparing the recombinant Xinjiang silkworm antibacterial peptide, the recombinant Xinjiang silkworm antibacterial peptide and its application in antibacterial and antitumor aspects. The recombinant Xinjiang silkworm antimicrobial peptide was cloned from Xinjiang silkworm and expressed in a eukaryotic expression system. The preparation method is that the fermented liquid is centrifuged to remove the precipitate, freeze-dried, separated and purified by ion exchange chromatography, hydrophobic interaction chromatography and gel filtration chromatography. The recombinant Xinjiang silkworm antibacterial peptide has a strong inhibitory effect on bacteria and tumor cells, and also has no hemolytic activity and no toxic effect on normal tissue cells, and is used in the preparation of drugs for treating pathogenic microbial infectious diseases and tumors.
Description
Technical field
The invention belongs to biological technical field.Specifically, relate to a kind of method for preparing purified, recombinant Sinkiang domestic silkworm antibiotic peptide of recombinant Sinkiang domestic silkworm antibiotic peptide and in application antibacterial, anti-tumor aspect.
Background technology
The discovery of penicillin makes people break away from fear to the pathogenic micro-organism associated diseases, has caused the appearance of a large amount of antibiotic thus, for the protection human beings'health has been established solid basis.But the continuous generation of the abuse of traditional antibiotic and Resistant strain has had a strong impact on the clinical therapeutic efficacy of infectious diseases.Therefore, the development of antibiotic of new generation becomes the emphasis of 21 century new drug development.Over nearly more than 20 years, the antibacterial peptide of different sources has demonstrated the characteristics of broad spectrum antibiotic activity, and has the incomparable advantage of traditional antibiotic, promptly can not cause the generation of Resistant strain, thereby is expected to become the antibiotic preparation of a new generation.
Silkworm Chang Zuowei auxiliary material in Chinese materia medica is used for treatment of diseases.Natural antibacterial peptide is the useful as drug raw material also.The tussah antibacterial peptide was used for the treatment of in 1996 the new drug " kidney proheparin " of ephritis, hepatitis.1997, the tussah cellulose capsule entered clinical observation with Western medicine two classes treatment hepatitis B.But, because the natural antibacterial peptide content is few, extracting yield low (0.005%), the cost height is failed suitability for industrialized production.
There is long sericulture history in Xinjiang.In secular breed, cultivate the kind that strong resistance against diseases is arranged in a large number.Be cloned into the cDNA molecule of cultivated silkworm antimicrobial peptide 3 instar larvaes that the new silkworm that Zhang Fuchun etc. cultivate from Aksu Prefecture silkworm egg place, Xinjiang is No. three, coded protein amino acid sequence reaches 98% with the homology of the silkworm cecropin B sequence of having reported.And Sinkiang domestic silkworm antibiotic peptide obtained expression in prokaryotic expression system and eukaryotic expression system, for a large amount of preparations of Sinkiang domestic silkworm antibiotic peptide provide solid platform.
The reorganization insect antimicrobial peptide is to utilize biotechnology, antibacterial peptide gene is cloned in the genetic engineering bacterium, thus the sterilization polypeptide that produces.The preparation of recombinant Sinkiang domestic silkworm antibiotic peptide is mainly adopted high-efficient liquid phase chromatogram technology (HPLC) or is adopted prokaryotic expression system to express the antibacterial peptide that merges, after enzyme is cut, obtain required antibacterial peptide with the ion-exchange chromatography separation, the yield of antibacterial peptides that this method produces is low, the cost height.Recombinant Sinkiang domestic silkworm antibiotic peptide is the antibacterial peptide gene that obtains from Aksu, Xinjiang silkworm clone, is transformed in the antibacterial peptide gene engineering bacteria, obtains by fermentation.The purity of the recombinant antibacterial peptide of fermentation gained does not reach biomedical needs, has limited its application.This just need carry out the purifying of the degree of depth.At present, the purification process of recombinant antibacterial peptide is comparatively single, mostly be to use a kind of or two kinds of chromatography methods, but purity is undesirable.Or label combined with antibacterial peptide, use the affinity chromatography technology and carry out purifying.Need behind this method purifying further to handle, label is removed, and cost is higher relatively.Therefore, the optimization of recombinant antibacterial peptide downstream technology becomes the bottleneck of its widespread use.
Summary of the invention
Purification process at present recombinant antibacterial peptide is comparatively single, and purity is not high, and the higher relatively present situation of purifying cost.The invention provides a kind of preparation method of recombinant Sinkiang domestic silkworm antibiotic peptide, and the application of antibacterial peptide in preparing anti-infective and antitumor drug is provided.
Simultaneously, the present invention also provides a kind of highly purified recombinant Sinkiang domestic silkworm antibiotic peptide.
The invention provides the concrete preparation method of a kind of recombinant Sinkiang domestic silkworm antibiotic peptide and obtain highly purified recombinant Sinkiang domestic silkworm antibiotic peptide.The present invention precipitates the centrifugal removal of fermented liquid, 100 ℃ of water-bath 20-40min, and centrifugal removal precipitation through after the lyophilize, is carried out separation and purification by ion exchange chromatography, hydrophobic interaction chromatography and gel permeation chromatography and is obtained.
Concrete ion exchange chromatography is that collected fermentation dry powder is dissolved in 10-100mMNa
2HPO
4-NaH
2PO
4Go up sample after the pH6.0-9.0 damping fluid to same damping fluid counter-balanced anion-exchange column, exchange column length 10-160mm, diameter 5-20mm is with the 10-100mM Na of the NaCl that contains 1.0-2.0M
2HPO
4-NaH
2PO
4The pH6.0-9.0 buffer solution for gradient elution is collected II, III peak.
Concrete hydrophobic interaction chromatography is, active peak II, the III that ion exchange chromatography obtains is splined on through with containing 1.5-2.5M NH
4(SO
4)
210-100mM Na
2HPO
4-NaH
2PO
4PH6.0-9.0 damping fluid equilibrated hydrophobic chromatography post, column length 10-100mm, diameter 5-20mm uses 10-100mM Na
2HPO
4-NaH
2PO
4The pH6.0-9.0 buffer solution for gradient elution is collected II, III peak.
Concrete gel permeation chromatography is, active peak II, III that hydrophobic interaction chromatography obtained are splined on Sephadex G-75 gel-filtration column, gel-filtration column length 1000mm, and diameter 20mm carries out wash-out with redistilled water, collects the III peak, is the antibacterial peptide peak.
The present invention also specifically provides a kind of highly purified recombinant Sinkiang domestic silkworm antibiotic peptide, with the gene clone of its described antibacterial peptide of encoding to carrier, enter then in the host cell express after, with the centrifugal removal precipitation of fermented liquid, through after the lyophilize, carry out separation and purification by ion exchange chromatography, hydrophobic interaction chromatography and gel permeation chromatography and obtain.
Concrete, the recombinant Sinkiang domestic silkworm antibiotic peptide that obtains belongs to the strongest cecropin category-B of sterilizing power (cecropin B), and its coded antibacterial peptide has 63 amino acid, and wherein mature peptide is 37 amino acid, leading peptide is 26 amino acid, does not contain halfcystine, does not have a disulphide bridges; The 8th be phenylalanine, be different from other antibacterial peptide again, produce and to be different from the characteristic of other antibacterial peptide, even higher activity is arranged.
Its DNA base sequence:
ATG AAT TTC GCA AAG ATC CTA TTT TTC GTC TTC GCT CTG
GTG CTG GCT TTG AGC ATG ACC AGC GCT GCT CCC GAG CCC
AAG TGG AAG ATC TTC AAG AAA ATT GAA AAA ATG GGC AGG
AAC ATT CGT GAC GGC ATC GTC AAA GCG GGC CCG GCG ATC
GAG GTC CTT GGT TCG GCT AAA GCT ATA GGA AAA TGA
Its aminoacid sequence:
MNFAKILFFV FALVLALSMT SAAPEPKWKI FKKIEKMGRN
IRDGIVKAGP AIEVLGSAKA IGK
The present invention also provides the recombinant Sinkiang domestic silkworm antibiotic peptide that above-mentioned purifying process obtained relevant biological characteristics.Be specially, antibacterial peptide is a small-molecular peptides, has thermostability, can 100 ℃ of water-bath 10min to 10h, and recombinant Sinkiang domestic silkworm antibiotic peptide is behind heated and boiled 8h, and anti-microbial activity does not change yet, shows stronger anti-microbial activity and the thermostability of recombinant Sinkiang domestic silkworm antibiotic peptide performance; The acid-and base-resisting characteristic: in the scope of pH at 3-11, the antibacterial ability of recombinant Sinkiang domestic silkworm antibiotic peptide remains unchanged; Simultaneously, recombinant Sinkiang domestic silkworm antibiotic peptide still has stronger anti-microbial activity in concentration is the NaCl of 0-5M.
Simultaneously, the present invention also specifically provide antibacterial peptide anti-infective and antitumor in application, concrete with the application of recombinant Sinkiang domestic silkworm antibiotic peptide in the medicine of preparation treatment cause pathogeny imcrobe infection disease and tumour that is obtained.
The recombinant Sinkiang domestic silkworm antibiotic peptide that the present invention obtains can significantly suppress other microbial growth, all microbial bacterias comprise: intestinal bacteria, streptococcus aureus, bacillus megaterium, Corynebacterium glutamicum, Bacillus thuringiensis, proteus vulgaris, Actinomyces pyogenes, suis, Brucella etc. show that recombinant Sinkiang domestic silkworm antibiotic peptide has the function of broad-spectrum antimicrobial.
Simultaneously, the recombinant Sinkiang domestic silkworm antibiotic peptide of the present invention's acquisition can significantly suppress the growth of tumour cell.Hela, SiHa, the isocellular growth of MGC, MFC there is significant inhibitory effect.Compare with the undressed control group of cell, the tumour that does not generate tumour or generation is significantly less than control group.Confirm that further recombinant Sinkiang domestic silkworm antibiotic peptide can suppress growth of tumor in the body.
Applied clinically at present most antibiotic do not press down tumoricidal function, but Sinkiang domestic silkworm antibiotic peptide of the present invention has significant tumoricidal effect external, can significantly suppress the growth of tumour cell, the antibacterial peptide that suppresses tumor promotion that has in other source of reporting with document is compared, and the tumors inhibition activity of recombinant silkworm antibacterial peptide is high 50 times.Simultaneously, Sinkiang domestic silkworm antibiotic peptide of the present invention can significantly suppress the growth of mouse interior tumor.
In addition, from moderate and severe cervical and cervical cancer patient sampling, separate obtaining 4 kinds of microorganisms.16s rRNA and form and biochemical cultural characters, preliminary evaluation are bacteriums such as enterococcus faecalis, staphylococcus, rod bacterium, coryneform bacteria; Use pcr analysis, from these 4 kinds of bacteriums, amplify the HPV16 specific fragment.Further, the recombinant Sinkiang antibacterial peptide of gained of the present invention can preferably suppress this 4 kinds of microbial growths, and its MIC is respectively 0.023 μ g/ml, 0.014 μ g/ml, 0.008 μ g/ml, 0.030 μ g/ml.
By implementing the concrete technical scheme of the present invention, realizes content of the present invention, can realize the recombinant Sinkiang domestic silkworm antibiotic peptide pathogenic micro-organism that obtained and the interaction in vitro result of tumour cell, specify the drug effect and the potential effect of the present invention's realization:
1, the antimicrobial spectrum of recombinant Sinkiang domestic silkworm antibiotic peptide: recombinant Sinkiang domestic silkworm antibiotic peptide has the function of broad-spectrum antimicrobial, microorganism is had significant inhibitory effect, and all microorganisms comprise intestinal bacteria, streptococcus aureus, bacillus megaterium, Corynebacterium glutamicum, Bacillus thuringiensis, proteus vulgaris, Actinomyces pyogenes, suis, Brucella, enterococcus faecalis, staphylococcus, rod bacterium, coryneform bacteria etc.
2, the minimal bactericidal concentration of recombinant Sinkiang domestic silkworm antibiotic peptide (MBC): the recombinant Sinkiang antibacterial peptide is starkly lower than penbritin to the minimal bactericidal concentration of microorganism, shows stronger antibacterial ability.Recombinant Sinkiang domestic silkworm antibiotic peptide comprises intestinal bacteria 0.030 μ g/ml to the minimal bactericidal concentration of microorganism, streptococcus aureus 0.007-0.011 μ g/ml, bacillus megaterium 0.010-0.014 μ g/ml, Corynebacterium glutamicum 0.018-0.021 μ g/ml, Bacillus thuringiensis 0.012-0.016 μ g/ml, proteus vulgaris 0.013-0.017 μ g/ml, Actinomyces pyogenes 0.028-0.033 μ g/ml, suis 0.009-0.011 μ g/ml, Brucella 0.005-0.006 μ g/ml, enterococcus faecalis 0.020-0.025 μ g/ml, staphylococcus 0.013-0.015 μ g/ml, rod bacterium 0.005-0.010 μ g/ml, coryneform bacteria 0.028-0.032 μ g/ml etc.
3, recombinant Sinkiang domestic silkworm antibiotic peptide suppresses the effect of growth of tumour cell: recombinant Sinkiang domestic silkworm antibiotic peptide can significantly suppress tumour cell Hela, SiHa, the isocellular growth of MGC, MFC.The dosage of inhibition fully to tumour cell is respectively, Hela:0.054-0.084 μ g/ml; SiHa:0.058-0.079 μ g/ml; MGC:0.055-0.081 μ g/ml; MFC:0.062-0.078 μ g/ml.Recombinant Sinkiang domestic silkworm antibiotic peptide has stronger tumor inhibition effect.
4, reagent required for the present invention is analytical pure, and the preparation method is conventional purified technology of protein, and is easy and simple to handle, and required time is short, and cost is lower, can directly amplify production.
Description of drawings:
Fig. 1 is the ion-exchange chromatography figure of recombinant Sinkiang domestic silkworm antibiotic peptide;
Fig. 2 is the hydrophobic interaction chromatography figure of recombinant Sinkiang domestic silkworm antibiotic peptide;
Fig. 3 is recombinant Sinkiang domestic silkworm antibiotic peptide Sephadex G-75 gel permeation chromatography figure.
Embodiment:
Below, for embodiment the present invention is described, still, the present invention is not limited to following embodiment.In addition, in following explanation, if no special instructions, then % all refers to weight percent.
Embodiment 1: the preparation method one of recombinant Sinkiang domestic silkworm antibiotic peptide
Fermented liquid 12000rpm, the centrifugal removal precipitation of 5min, 100 ℃ of water-bath 30min, 12000rpm, the centrifugal removal precipitation of 10min ,-20 ℃ of preservations are standby after the lyophilize.
The first step, ion exchange chromatography: above-mentioned collected fermentation dry powder 40mg is dissolved in 10mMNa
2HPO
4-NaH
2PO
4The pH6.0 damping fluid, 12000rpm, 4 ℃ of centrifugal 10min get supernatant, and the filter membrane with 0.45 μ m filters again.Be splined on 10mM Na
2HPO
4-NaH
2PO
4PH7.0 damping fluid counter-balanced Q Sepharose Fast Flow anion-exchange column, exchange column length 10mm, diameter 5mm is washed till the complete wash-out in percolation peak through 2.5 identical damping fluids of column volume.10mM Na with the NaCl that contains 2.0M
2HPO
4-NaH
2PO
4The pH6.0 buffer solution for gradient elution is collected II, III peak.
In second step, hydrophobic interaction chromatography: active peak II, the III that ion exchange chromatography obtains is splined on through with containing 1.5M NH
4(SO
4)
210mM Na
2HPO
4-NaH
2PO
4The abundant counter-balanced Phehyl of pH6.0 damping fluid Sepharose HP hydrophobic chromatography post, column length 10mm, diameter 5mm is washed till the complete wash-out in percolation peak through 3 identical damping fluids of column volume.Use 10mMNa
2HPO
4-NaH
2PO
4The pH6.0 buffer solution for gradient elution is collected II, III peak;
The 3rd step, gel permeation chromatography: with the redistilled water balance Sepadex G-75 gel-filtration column of 2 column volumes, gel-filtration column length 1000mm, diameter 20mm.Active peak II, III that hydrophobic interaction chromatography obtained are splined on the Sephadex G-75 gel-filtration column that balance has been got well, carry out wash-out, collect the III peak, be the antibacterial peptide peak, can obtain highly purified recombinant Sinkiang domestic silkworm antibiotic peptide with redistilled water.
Embodiment 2: the preparation method two of recombinant Sinkiang domestic silkworm antibiotic peptide
Fermented liquid 12000rpm, the centrifugal removal precipitation of 5min, 100 ℃ of water-bath 30min, 12000rpm, the centrifugal removal precipitation of 10min ,-20 ℃ of preservations are standby after the lyophilize.
The first step, ion exchange chromatography: above-mentioned collected fermentation dry powder 40mg is dissolved in 100mMNa
2HPO
4-NaH
2PO
4The pH9.0 damping fluid, 12000rpm, 4 ℃ of centrifugal 10min get supernatant, and the filter membrane with 0.45 μ m filters again.Be splined on 10mM Na
2HPO
4-NaH
2PO
4PH7.0 damping fluid counter-balanced Q Sepharose Fast Flow anion-exchange column, exchange column length 160mm, diameter 20mm is washed till the complete wash-out in percolation peak through 2.5 identical damping fluids of column volume.100mM Na with the NaCl that contains 2.0M
2HPO
4-NaH
2PO
4The pH9.0 buffer solution for gradient elution is collected II, III peak.
In second step, hydrophobic interaction chromatography: active peak II, the III that ion exchange chromatography obtains is splined on through with containing 2.5M NH
4(SO
4)
2100mM Na
2HPO
4-NaH
2PO
4The abundant counter-balanced Phehyl of pH9.0 damping fluid Sepharose HP hydrophobic chromatography post, column length 100mm, diameter 20mm is washed till the complete wash-out in percolation peak through 3 identical damping fluids of column volume.Use 100mMNa
2HPO
4-NaH
2PO
4The pH9.0 buffer solution for gradient elution is collected II, III peak;
The 3rd step, gel permeation chromatography: as above-mentioned technological step, will collect the III peak, and be the antibacterial peptide peak, and can obtain same highly purified recombinant Sinkiang domestic silkworm antibiotic peptide.
Embodiment 3: the preparation method three of recombinant Sinkiang domestic silkworm antibiotic peptide
Preparation as above-mentioned embodiment raw material.
The first step, ion exchange chromatography: above-mentioned collected fermentation dry powder 40mg is dissolved in 50mMNa
2HPO
4-NaH
2PO
4The pH8.0 damping fluid, 12000rpm, 4 ℃ of centrifugal 10min get supernatant, and the filter membrane with 0.45 μ m filters again.Be splined on 10mM Na
2HPO
4-NaH
2PO
4PH7.0 damping fluid counter-balanced Q Sepharose Fast Flow anion-exchange column, exchange column length 100mm, diameter 10mm is washed till the complete wash-out in percolation peak through 2.5 identical damping fluids of column volume.50mM Na with the NaCl that contains 1.5M
2HPO
4-NaH
2PO
4The pH8.5 buffer solution for gradient elution is collected II, III peak.
In second step, hydrophobic interaction chromatography: active peak II, the III that ion exchange chromatography obtains is splined on through with containing 2.0M NH
4(SO
4)
210mM Na
2HPO
4-NaH
2PO
4The abundant counter-balanced Phehyl of pH8.5 damping fluid Sepharose HP hydrophobic chromatography post, column length 50mm, diameter 15mm is washed till the complete wash-out in percolation peak through 3 identical damping fluids of column volume.Use 80mMNa
2HPO
4-NaH
2PO
4The pH6.5 buffer solution for gradient elution is collected II, III peak;
The 3rd step, gel permeation chromatography: as above-mentioned technological step, will collect the III peak, and be the antibacterial peptide peak, and can obtain same highly purified recombinant Sinkiang domestic silkworm antibiotic peptide.
Embodiment 4: the preparation method four of recombinant Sinkiang domestic silkworm antibiotic peptide
Preparation as above-mentioned embodiment raw material.
The first step, ion exchange chromatography: concrete experimental procedure as described above, will collect fermentation dry powder and be dissolved in 10-100mM Na
2HPO
4-NaH
2PO
4The pH6.0-9.0 damping fluid, 12000rpm through centrifugal, gets supernatant, filters with filter membrane again.Go up sample after the damping fluid to same damping fluid counter-balanced anion-exchange column, exchange column length 10-160mm, diameter 5-20mm is with the 10-100mM Na of the NaCl that contains 1.0-2.0M
2HPO
4-NaH
2PO
4The pH6.0-9.0 buffer solution for gradient elution is collected II, III peak.
In second step, hydrophobic interaction chromatography: active peak II, the III that concrete experimental procedure as described above, ion exchange chromatography obtain is splined on through with containing 1.5-2.5M NH
4(SO
4)
210-100mMNa
2HPO
4-NaH
2PO
4PH6.0-9.0 damping fluid equilibrated hydrophobic chromatography post, column length 10-100mm, diameter 5-20mm uses 10-100mM Na
2HPO
4-NaH
2PO
4The pH6.0-9.0 buffer solution for gradient elution is collected II, III peak.
The 3rd step, gel permeation chromatography: as above-mentioned technological step, will collect the III peak, and be the antibacterial peptide peak, and can obtain same highly purified recombinant Sinkiang domestic silkworm antibiotic peptide.
Embodiment 5: recombinant Sinkiang domestic silkworm antibiotic peptide suppresses the effect of growth of tumour cell
With aseptic redistilled water purified recombinant Sinkiang domestic silkworm antibiotic peptide is carried out doubling dilution by 10 times, have 8 titres, each titre is provided with 3 repetitions, every hole application of sample amount 100 μ l, and setting up normal liver cell HL-7702 simultaneously is control group.With 5 * 10
4The MGC cell of/ml or normal liver cell 200 μ l are added in the 96 porocyte culture plates, in 37 ℃, and 5%CO
2Cultivate in the incubator.Behind the 24h, remove old DMEM substratum, every hole adds new DMEM substratum 100 μ l again, and the sample with above-mentioned different titers adds with every hole 100 μ l application of sample amounts simultaneously, behind the mixing, inserts CO again gently
2Cultivate 24h in the incubator.Supernatant is removed in suction, and PBS washs gently, removes supernatant.Every hole adds the fresh DMEM substratum of 80 μ l, adds the MTT solution (5mg/ml) of 20 μ l again, and vibration gently continues to cultivate 4h; Stop cultivating, the careful substratum that goes in the hole of inhaling, every hole adds the dimethyl sulfoxide (DMSO) (DMSO) of 100 μ l, and vibration is gently cultivated 30min for 37 ℃, measures the light absorption value in each hole at the 490nm place of enzyme-linked immunosorbent assay instrument.EC
50It is the drug level when killing and wounding 50% cell.The results are shown in Table 1.
Table 1: recombinant Sinkiang domestic silkworm antibiotic peptide is in external effect of press down killing tumor growth
| Sample concentration (μ g/ml) | A490 | |
| MGC | HL-7702 | |
| 2.74 2.74×10-1 2.74×10-2 2.74×10-3 2.74×10-4 2.74×10-5 2.74×10-6 2.74×10-7 | 0.095 0.284 0.296 0.429 0.592 0.763 0.915 1.057 | 0.083 0.085 0.088 0.082 0.085 0.083 0.081 0.086 |
Applied clinically at present most antibiotic press down tumoricidal function, and as can be seen, but Sinkiang domestic silkworm antibiotic peptide has significant tumoricidal effect external from above-mentioned table.
Embodiment 6: recombinant Sinkiang domestic silkworm antibiotic peptide is to the interaction in vitro of pathogenic micro-organism and tumour cell
1, the antimicrobial spectrum of recombinant Sinkiang domestic silkworm antibiotic peptide: the confession sample basis that recombinant silkworm antibacterial peptide is made into 27.4 μ g/ml.Make flat board with following listed pathogenic bacteria in the LB solid medium, with the punch tool punching of diameter 3mm, every hole application of sample amount is 20 μ l, is equivalent to 0.548 μ g/ml.Every kind of pathogenic bacteria is repeated 3 times.Cultivate 18-24h for 37 ℃, measure bacteriostatic diameter, the results are shown in Table 2:
Table 2: the antimicrobial spectrum of recombinant Sinkiang domestic silkworm antibiotic peptide
| Strains tested | Antibacterial circle diameter (mm) |
| Escherichia coli and staphylococcus aureus bacillus megaterium Corynebacterium glutamicum Dipel proteus vulgaris Actinomyces pyogenes streptococcus Brucella enterococcus faecalis staphylococcus | 5 18 13 10 15 13 5 16 20 7 15 19 5 |
2, the minimal bactericidal concentration of recombinant Sinkiang domestic silkworm antibiotic peptide (MBC)
The minimal bactericidal concentration of recombinant Sinkiang domestic silkworm antibiotic peptide of the present invention adopts the positive contrast of common drug penbritin, the negative contrast of sterile distilled water.Each 3 of sterile test tube of test, and indicate drug level and action time, referring to table 3.
Table 3: the comparison of the minimal bactericidal concentration of recombinant Sinkiang domestic silkworm antibiotic peptide and penbritin
| Strains tested | Minimal bactericidal concentration | |
| Antibacterial peptide (μ g/ml) | Penbritin (μ g/ml) | |
| Escherichia coli and staphylococcus aureus bacillus megaterium Corynebacterium glutamicum Dipel proteus vulgaris Actinomyces pyogenes streptococcus Brucella enterococcus faecalis staphylococcus rod bacterium corynebacteria | 0.030 0.008 0.012 0.020 0.014 0.015 0.030 0.010 0.006 0.023 0.014 0.008 0.030 | 0.8 5.0 3.3 0.5 11.0 0.2 13.9 9.6 16.7 10.0 11.3 17.4 12.6 |
3, recombinant Sinkiang domestic silkworm antibiotic peptide suppresses the effect of growth of tumour cell
Use aseptic redistilled water the recombinant Sinkiang domestic silkworm antibiotic peptide of purified acquisition is carried out 10 times of doubling dilutions, have 8 titres, each titre is provided with 3 repetitions, every hole application of sample amount 100 μ l, and setting up normal liver cell HL-7702 simultaneously is control group.MGC cell or the normal liver cell 200 μ l of 5 * 104/ml are added in the 96 porocyte culture plates, in 37 ℃, 5%CO
2Cultivate in the incubator.Use the lethal effect of mtt assay working sample pair cell behind the 24h.EC50 is the drug level when killing and wounding 50% cell.The results are shown in Table 4.
Table 4: recombinant Sinkiang domestic silkworm antibiotic peptide suppresses the effect of growth of tumour cell
| For test agent | EC50(μg/ml) | |
| MGC | HL-7702 | |
| Fermented liquid recombinant Sinkiang domestic silkworm antibiotic peptide (lot number 1) recombinant Sinkiang domestic silkworm antibiotic peptide (lot number 2) recombinant Sinkiang domestic silkworm antibiotic peptide (lot number 3) | 8000 35.2 1.38 0.072 | - - 437.5 285.2 |
Applied clinically at present most antibiotic press down tumoricidal function, and as can be seen, but Sinkiang domestic silkworm antibiotic peptide has significant tumoricidal effect external from above-mentioned table.
Recombinant Sinkiang domestic silkworm antibiotic peptide can significantly suppress microbial growth, and all microbial bacterias comprise: intestinal bacteria, streptococcus aureus, bacillus megaterium, Corynebacterium glutamicum, Bacillus thuringiensis, proteus vulgaris, Actinomyces pyogenes, suis, Brucella, enterococcus faecalis, staphylococcus, rod bacterium, coryneform bacteria etc.The result shows that recombinant Sinkiang domestic silkworm antibiotic peptide has the function of broad-spectrum antimicrobial.In addition, recombinant Sinkiang domestic silkworm antibiotic peptide can significantly suppress the growth of tumour cell, recombinant Sinkiang domestic silkworm antibiotic peptide is 0.072 μ g/ml to MGC cell inhibiting dosage, the antibacterial peptide that suppresses tumor promotion that has in other source of reporting with document is compared, and the tumors inhibition activity of recombinant silkworm antibacterial peptide is high 50 times.
Claims (10)
1, a kind of preparation method of recombinant Sinkiang domestic silkworm antibiotic peptide is characterized in that, with the centrifugal removal precipitation of fermented liquid, 100 ℃ of water-bath 20-40min, centrifugal removal precipitation through after the lyophilize, is carried out separation and purification by ion exchange chromatography, hydrophobic interaction chromatography and gel permeation chromatography.
2, the preparation method of recombinant Sinkiang domestic silkworm antibiotic peptide as claimed in claim 1 specifically is characterised in that:
(1), ion exchange chromatography: collected recombinant Sinkiang domestic silkworm antibiotic peptide fermentation dry powder is dissolved in 10-100mM Na
2HPO
4-NaH
2PO
4Go up sample after the pH6.0-9.0 damping fluid to same damping fluid counter-balanced Q Sepharose Fast Flow anion-exchange column, exchange column length 10-160mm, diameter 5-20mm is with the 10-100mMNa of the NaCl that contains 1.0-2.0M
2HPO
4-NaH
2PO
4The pH6.0-9.0 buffer solution for gradient elution is collected II, III peak;
(2), hydrophobic interaction chromatography: active peak II, the III that step (1) is obtained is splined on through with containing 1.5-2.5M NH
4(SO
4)
210-100mM Na
2HPO
4-NaH
2PO
4PH6.0-9.0 damping fluid equilibrated hydrophobic chromatography post, column length 10-100mm, diameter 5-20mm uses 10-100mMNa
2HPO
4-NaH
2PO
4The pH6.0-9.0 buffer solution for gradient elution is collected II, III peak;
(3), gel permeation chromatography: active peak II, III that step (2) is obtained are splined on Sepadex G-75 gel-filtration column, gel-filtration column length 1000mm, diameter 20mm carries out wash-out with redistilled water, collects the III peak.
3, a kind of recombinant Sinkiang domestic silkworm antibiotic peptide is characterized in that, with the gene clone of the described antibacterial peptide of coding to carrier, enter then in the host cell express after, utilize the described recombinant Sinkiang domestic silkworm antibiotic peptide of claim 1 to carry out separation and purification and obtain.
4, as recombinant Sinkiang domestic silkworm antibiotic peptide as described in the claim 3, its biological characteristics is that behind the heated and boiled 8h, anti-microbial activity does not change yet, and recombinant Sinkiang domestic silkworm antibiotic peptide has anti-microbial activity and thermostability; In the scope of pH at 3-11, the antibacterial ability of recombinant Sinkiang domestic silkworm antibiotic peptide remains unchanged; Recombinant Sinkiang domestic silkworm antibiotic peptide has anti-microbial activity in concentration is the NaCl of 0-5M.
5, a kind of sequence as recombinant Sinkiang domestic silkworm antibiotic peptide as described in the claim 3, it is characterized in that, 1 to 63 amino acid in this recombinant Sinkiang domestic silkworm antibiotic peptide sequence, this antibacterial peptide precursor has 63 amino acid, wherein mature peptide is 37 amino acid, leading peptide is 26 amino acid, do not contain halfcystine, do not have a disulphide bridges, the 8th be phenylalanine.
6, a kind of application as recombinant Sinkiang domestic silkworm antibiotic peptide as described in the claim 3 is characterized in that, the application of described recombinant Sinkiang domestic silkworm antibiotic peptide in preparation treatment cause pathogeny imcrobe infection disease and tumour medicine.
7, as the application of recombinant Sinkiang domestic silkworm antibiotic peptide as described in the claim 6, it is characterized in that, recombinant Sinkiang domestic silkworm antibiotic peptide has the function of broad-spectrum antimicrobial, can suppress microbial growths such as intestinal bacteria, streptococcus aureus, bacillus megaterium, Corynebacterium glutamicum, Bacillus thuringiensis, proteus vulgaris, Actinomyces pyogenes, suis, Brucella.
8, application as recombinant Sinkiang domestic silkworm antibiotic peptide as described in the claim 6, it is characterized in that recombinant Sinkiang domestic silkworm antibiotic peptide comprises the minimal bactericidal concentration MBC of microorganism: intestinal bacteria 0.030 μ g/ml, streptococcus aureus 0.007-0.011 μ g/ml, bacillus megaterium 0.010-0.014 μ g/ml, Corynebacterium glutamicum 0.018-0.021 μ g/ml, Bacillus thuringiensis 0.012-0.016 μ g/ml, proteus vulgaris 0.013-0.017 μ g/ml, Actinomyces pyogenes 0.028-0.033 μ g/ml, suis 0.009-0.011 μ g/ml, Brucella 0.005-0.006 μ g/ml, enterococcus faecalis 0.020-0.025 μ g/ml, staphylococcus 0.013-0.015 μ g/ml, rod bacterium 0.005-0.010 μ g/ml, coryneform bacteria 0.028-0.032 μ g/ml
9, as the application of recombinant Sinkiang domestic silkworm antibiotic peptide as described in the claim 6, it is characterized in that recombinant Sinkiang domestic silkworm antibiotic peptide is respectively the dosage of inhibition fully of tumour cell Hela, SiHa, MGC, MFC growth: Hela 0.054-0.084 μ g/ml, SiHa 0.058-0.079 μ g/ml, MGC 0.055-0.081 μ g/ml, MFC 0.062-0.078 μ g/ml.
10, as the application of recombinant Sinkiang domestic silkworm antibiotic peptide as described in the claim 7, it is characterized in that the recombinant Sinkiang antibacterial peptide preferably suppresses enterococcus faecalis, staphylococcus, rod bacterium, 4 kinds of microbial growths of coryneform bacteria and is respectively: 0.023 μ g/ml, 0.014 μ g/ml, 0.008 μ g/ml, 0.030 μ g/ml.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173517A (en) * | 2013-03-01 | 2013-06-26 | 浙江大学 | Microdetermination method for measuring minimum inhibitory concentration and minimal bactericidal concentration of cationic antimicrobial peptide |
| CN104490720A (en) * | 2015-01-18 | 2015-04-08 | 新疆大学 | Antibacterial peptide hand sanitizer and preparation method thereof |
| CN110129350A (en) * | 2019-05-21 | 2019-08-16 | 四川大学 | A kind of preparation method of bacterial strain inhibiting microbial growth and insole |
| CN110218245A (en) * | 2019-05-30 | 2019-09-10 | 青岛红樱桃生物技术有限公司 | A kind of cecropin A TMP7 with bacteriostatic activity and its in the application being used to prepare in antibacterial agent |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1309824C (en) * | 2004-08-27 | 2007-04-11 | 广州维观生物科技有限公司 | Antibiotic peptide gene converted viscons bacillus engineering bacterium and its preparing method and use |
| CN1294261C (en) * | 2005-01-04 | 2007-01-10 | 南京师范大学 | High efficiency production method for recombinant silkworm antibacterial peptide CM4 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173517A (en) * | 2013-03-01 | 2013-06-26 | 浙江大学 | Microdetermination method for measuring minimum inhibitory concentration and minimal bactericidal concentration of cationic antimicrobial peptide |
| CN103173517B (en) * | 2013-03-01 | 2014-07-30 | 浙江大学 | Microdetermination method for measuring minimum inhibitory concentration and minimal bactericidal concentration of cationic antimicrobial peptide |
| CN104490720A (en) * | 2015-01-18 | 2015-04-08 | 新疆大学 | Antibacterial peptide hand sanitizer and preparation method thereof |
| CN110129350A (en) * | 2019-05-21 | 2019-08-16 | 四川大学 | A kind of preparation method of bacterial strain inhibiting microbial growth and insole |
| CN110218245A (en) * | 2019-05-30 | 2019-09-10 | 青岛红樱桃生物技术有限公司 | A kind of cecropin A TMP7 with bacteriostatic activity and its in the application being used to prepare in antibacterial agent |
| CN110886049A (en) * | 2019-09-19 | 2020-03-17 | 西南大学 | Fabrication of fibrous membranes that can promote wound healing with antibacterial peptides from Bombyx mori |
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| CN1966709B (en) | 2010-06-02 |
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