Preparation and purification method of antibacterial peptide of gangrene protozoa
阅读说明：本技术 抗细菌和疽原虫抗菌肽的制备及纯化方法 (Preparation and purification method of antibacterial peptide of gangrene protozoa ) 是由 邓兴朝 陈欢 于 2019-09-17 设计创作，主要内容包括：本发明实施例公开了抗细菌和疽原虫抗菌肽的制备及纯化方法。该方法包括：通过聚合酶链式反应扩增目标抗菌肽基因；根据所述目标抗菌肽基因,构建对应的表达载体；将所述表达载体转化到宿主细菌,培养得到重组细菌；诱导所述重组细菌表达包含设定标签的重组抗菌肽；对所述包含设定标签的重组抗菌肽进行酶切,获得无标签的重组抗菌肽作为抗菌肽原料对待纯化的抗菌肽原料进行预处理,获得粗提取抗菌肽；使用设定孔径的微孔滤膜过滤所述粗提取抗菌肽,收集滤液；通过二次超滤的方式,对所述滤液进行过滤,获得目标产物；浓缩所述目标产物,制备获得纯化的抗菌肽。整个方法流程简单,耗时缩短,费用降低,可很好的应用于抗菌肽的工业化制备。(The embodiment of the invention discloses a preparation and purification method of antibacterial and gangrene protozoan antibacterial peptide. The method comprises the following steps: amplifying target antibacterial peptide genes by polymerase chain reaction; constructing a corresponding expression vector according to the target antibacterial peptide gene; transforming the expression vector into host bacteria, and culturing to obtain recombinant bacteria; inducing the recombinant bacteria to express a recombinant antibacterial peptide containing a set label; carrying out enzyme digestion on the recombinant antibacterial peptide containing the set label to obtain non-label recombinant antibacterial peptide serving as an antibacterial peptide raw material for preprocessing the antibacterial peptide raw material to be purified to obtain a crude extracted antibacterial peptide; filtering the crude extract antibacterial peptide by using a microporous filter membrane with a set pore diameter, and collecting filtrate; filtering the filtrate in a secondary ultrafiltration mode to obtain a target product; and concentrating the target product to prepare the purified antibacterial peptide. The whole method has simple flow, short time consumption and low cost, and can be well applied to the industrial preparation of the antibacterial peptide.)
1. A method for preparing an antimicrobial peptide, comprising:
amplifying target antibacterial peptide genes by polymerase chain reaction;
constructing a corresponding expression vector according to the target antibacterial peptide gene;
transforming the expression vector into host bacteria, and culturing to obtain recombinant bacteria;
inducing the recombinant bacteria to express a recombinant antibacterial peptide containing a set label;
and carrying out enzyme digestion on the recombinant antibacterial peptide containing the set label to obtain the non-label recombinant antibacterial peptide as the antibacterial peptide raw material.
2. A method for purifying an antimicrobial peptide, comprising:
pretreating an antibacterial peptide raw material to be purified to obtain a crude extracted antibacterial peptide;
filtering the crude extract antibacterial peptide by using a microporous filter membrane with a set pore diameter, and collecting filtrate;
filtering the filtrate in a secondary ultrafiltration mode to obtain a target product;
and concentrating the target product to prepare the purified antibacterial peptide.
3. The purification method according to claim 2, wherein the filtering of the filtrate by means of secondary ultrafiltration to obtain the target product comprises:
performing primary ultrafiltration on the filtrate by using an ultrafiltration membrane with the pore diameter larger than the molecular weight of the antibacterial peptide;
performing secondary ultrafiltration on the filtrate obtained after the primary ultrafiltration by using an ultrafiltration membrane with the pore diameter smaller than the molecular weight of the antibacterial peptide, taking trapped fluid to obtain the target product or,
performing primary ultrafiltration on the filtrate by using an ultrafiltration membrane with the pore diameter smaller than the molecular weight of the antibacterial peptide;
and (3) carrying out secondary ultrafiltration on the filtrate of the trapped fluid after the primary ultrafiltration by using an ultrafiltration membrane with the pore diameter larger than the molecular weight of the antibacterial peptide, and taking the filtrate to obtain the target product.
4. The purification method according to claim 2, wherein the concentrating the target product to obtain the purified antimicrobial peptide comprises:
and concentrating the target product through a nanofiltration membrane to prepare the purified antibacterial peptide.
5. The purification method according to claim 2, wherein the pore size of the microfiltration membrane is set to 0.1 to 1 μm.
6. The purification method according to claim 2, wherein when the antibacterial peptide raw material to be purified is a secretory expression genetically engineered antibacterial peptide, the pretreatment specifically comprises:
collecting fermentation liquor and centrifuging;
and taking the centrifuged supernatant to obtain the crude extract antibacterial peptide.
7. The purification method according to claim 2, wherein when the antibacterial peptide raw material to be purified is a genetically engineered antibacterial peptide expressed in cells, the pretreatment specifically comprises:
lysing cells and centrifuging the cell lysate;
and taking the centrifuged supernatant to obtain the crude extract antibacterial peptide.
8. The purification method according to claim 2, wherein when the antibacterial peptide raw material to be purified is a bacterial antibacterial peptide, the pretreatment specifically comprises:
collecting viable bacteria from the culture medium;
cracking live bacteria and centrifuging live bacteria lysate;
and taking the centrifuged supernatant to obtain the crude extract antibacterial peptide.
9. The purification method according to claim 2, wherein the activity of the purified antimicrobial peptide is detected by an agar diffusion method.
10. An antibacterial peptide against bacteria and cellulitis, which is obtained by the production method and the purification method according to any one of claims 1 to 9.
Technical Field
The invention relates to the technical field of biology, in particular to a preparation and purification method of antibacterial peptide resisting bacteria and gangrene protozoa.
Background
Antimicrobial peptides, which may also be referred to as antimicrobial peptides, are small molecule polypeptides that are ubiquitous in organisms in nature. It is capable of resisting the invasion of external pathogens, is an important component of the innate immune system, and provides the first line of defense for most organisms.
The antibacterial peptide has the activities of broad-spectrum sterilization, virus inhibition, tumor cell inhibition and the like, is not easy to generate drug resistance, has high thermal stability, has great killing capability to multiple drug-resistant bacteria, does not damage normal cells of human bodies, has small toxicity, can enhance the immune function and accelerate wound healing, and has great application prospect in the aspects of medicine, food processing, daily chemicals, agriculture, livestock and poultry industry and the like.
Although the antibacterial peptide has wide application prospect and huge development potential, the industrialization process of the antibacterial peptide is slow. At present, the main method for producing the antibacterial peptide is to produce the antibacterial peptide by a genetic engineering technology, the production cost is high, the antibacterial peptide is easy to carry out enzymolysis, and the industrial scale needs to be broken through. Moreover, the activity of the produced antibacterial peptide is not ideal enough, and has a large gap compared with the traditional antibiotics.
In the process of implementing the invention, the inventor finds that the following problems exist in the related art: the traditional gene engineering method for producing the antibacterial peptide has a series of defects of easy enzymolysis, insufficient active yield, certain toxic action on host cells and the like, and is difficult to realize large-scale industrial production.
Disclosure of Invention
Aiming at the technical problems, the embodiment of the invention provides a preparation and purification method of antibacterial peptide for resisting bacteria and gangrene protozoa, so as to solve the problems that the existing antibacterial peptide production and purification method has various defects and can not well realize large-scale industrial production.
In a first aspect of the embodiments of the present invention, there is provided a method for preparing an antimicrobial peptide. The method comprises the following steps:
amplifying target antibacterial peptide genes by polymerase chain reaction; constructing a corresponding expression vector according to the target antibacterial peptide gene; transforming the expression vector into host bacteria, and culturing to obtain recombinant bacteria; inducing the recombinant bacteria to express a recombinant antibacterial peptide containing a set label; and carrying out enzyme digestion on the recombinant antibacterial peptide containing the set label to obtain the non-label recombinant antibacterial peptide as the antibacterial peptide raw material.
In a second aspect of the embodiments of the present invention, there is provided a method for purifying an antimicrobial peptide. The purification method comprises the following steps:
pretreating an antibacterial peptide raw material to be purified to obtain a crude extracted antibacterial peptide; filtering the crude extract antibacterial peptide by using a microporous filter membrane with a set pore diameter, and collecting filtrate; filtering the filtrate in a secondary ultrafiltration mode to obtain a target product; and concentrating the target product to prepare the purified antibacterial peptide.
Optionally, filtering the filtrate by means of secondary ultrafiltration to obtain a target product, specifically including:
performing primary ultrafiltration on the filtrate by using an ultrafiltration membrane with the pore diameter larger than the molecular weight of the antibacterial peptide; performing secondary ultrafiltration on the filtrate obtained after the primary ultrafiltration by using an ultrafiltration membrane with the pore diameter smaller than the molecular weight of the antibacterial peptide, and taking trapped fluid to obtain the target product, or
Performing primary ultrafiltration on the filtrate by using an ultrafiltration membrane with the pore diameter smaller than the molecular weight of the antibacterial peptide; and (3) carrying out secondary ultrafiltration on the filtrate of the trapped fluid after the primary ultrafiltration by using an ultrafiltration membrane with the pore diameter larger than the molecular weight of the antibacterial peptide, and taking the filtrate to obtain the target product.
Optionally, the concentrating the target product to prepare a purified antimicrobial peptide specifically includes: and concentrating the target product through a nanofiltration membrane to prepare the purified antibacterial peptide.
Alternatively, the pore size of the microfiltration membrane is set to 0.1 to 1 μm.
Optionally, when the antibacterial peptide raw material to be purified is a secretory-expressed genetically engineered antibacterial peptide, the pretreatment specifically includes: collecting fermentation liquor and centrifuging; and taking the centrifuged supernatant to obtain the crude extract antibacterial peptide.
Optionally, when the antibacterial peptide raw material to be purified is a genetically engineered antibacterial peptide expressed in cells, the pretreatment specifically includes: lysing cells and centrifuging the cell lysate; and taking the centrifuged supernatant to obtain the crude extract antibacterial peptide.
Optionally, when the antibacterial peptide raw material to be purified is a bacterial antibacterial peptide, the pretreatment specifically includes:
collecting viable bacteria from the culture medium; cracking live bacteria and centrifuging live bacteria lysate; and taking the centrifuged supernatant to obtain the crude extract antibacterial peptide.
Alternatively, the activity of the purified antimicrobial peptide is assayed by an agar diffusion method.
In a third aspect of the embodiments of the present invention, there is provided an antimicrobial peptide against bacteria and gangrene protozoa. The antibacterial peptide for resisting bacteria and cellulitis protozoides is prepared by the preparation method and the purification method.
According to the technical scheme provided by the embodiment of the invention, the preparation and purification method is simple in process, short in time consumption and low in cost, and can be well applied to industrial preparation of the antibacterial peptide. The purification method of the antibacterial peptide can keep the activity and purity of the antibacterial peptide to the maximum extent, and has good application prospect.
In addition, in the process of producing and preparing the antibacterial peptide, the heterologous host is used for expressing the antibacterial peptide containing the fusion protein, and a special label column is used or the traditional purification steps are simplified, so that the defect of low target gene expression is overcome, the toxicity to host cells is reduced, the high-purity antibacterial peptide can be obtained, and the steps of separating and purifying the antibacterial peptide are reduced.
Drawings
FIG. 1 is a schematic diagram of an embodiment of a method for preparing and purifying an antimicrobial peptide according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of an embodiment of secondary ultrafiltration according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of one embodiment of an expression vector according to an embodiment of the present invention;
FIG. 4 is a schematic diagram of an embodiment of electrophoresis results according to an embodiment of the present invention;
fig. 5 is a schematic diagram of an embodiment of a bacteriostatic experiment according to an embodiment of the invention.
SEQ ID No.1 is an upstream primer sequence;
SEQ ID No.2 is a downstream primer sequence.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It will be understood that when an element is referred to as being "secured to" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may be present. As used in this specification, the terms "vertical," "horizontal," "left," "right," "up," "down," "inner," "outer," "bottom," and the like are used in the orientation or positional relationship indicated in the drawings for convenience in describing the invention and for simplicity in description, and do not indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and are therefore not to be considered limiting. Furthermore, the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
FIG. 1 is a process for preparing an antimicrobial peptide according to the present invention. The preparation method can be shown in fig. 1, and the preparation method can comprise the following steps:
and S110, amplifying the target antibacterial peptide gene by using a polymerase chain reaction.
S120, constructing a corresponding expression vector according to the target antibacterial peptide gene.
S130, transforming the expression vector into host bacteria, and culturing to obtain the recombinant bacteria.
S140, inducing the recombinant bacteria to express the recombinant antibacterial peptide containing the set label.
S150, carrying out enzyme digestion on the recombinant antibacterial peptide containing the set label to obtain the non-label recombinant antibacterial peptide as an antibacterial peptide raw material.
In the method, heterologous host expression is used for expressing the fusion protein-containing antibacterial peptide, and a special label column is combined. The preparation method improves the defect of low expression of target genes, reduces the poison to host cells, can obtain high-purity antibacterial peptide, and reduces the steps of separation and purification of the antibacterial peptide.
With continued reference to fig. 1, the present invention provides a flow chart of a method for purifying an antimicrobial peptide. Is used in combination with the preparation method to realize the large-scale production of the antibacterial peptide. The purification method may comprise the steps of:
s210, pretreating the antibacterial peptide raw material to be purified to obtain the crude extracted antibacterial peptide.
Specifically, according to different genetic engineering methods for obtaining the antibacterial peptide raw material, a corresponding pretreatment mode can be adopted to obtain crude extract antibacterial extract. In some embodiments, when the antimicrobial peptide starting material to be purified is a secretionally expressed genetically engineered antimicrobial peptide, the pretreatment may be: the fermentation broth was first collected and centrifuged. And then, taking the supernatant after centrifugation to obtain the crude extract antibacterial peptide.
And when the antibacterial peptide raw material to be purified is a genetically engineered antibacterial peptide expressed in cells, the pretreatment step may include: first, cells are lysed and the cell lysate is centrifuged. And then, taking the centrifuged supernatant to obtain the crude extracted antibacterial peptide.
If the antibacterial peptide raw material to be purified is a bacterial antibacterial peptide, the step of pretreating may include: first, live bacteria are collected from the culture medium. Then, live bacteria are lysed and the live bacteria lysate is centrifuged. And finally, taking the centrifuged supernatant to obtain the crude extracted antibacterial peptide.
S220, filtering the crude extract antibacterial peptide by using a microporous filter membrane with a set pore diameter, and collecting filtrate.
In step 220, the crude antimicrobial peptide is further purified by microfiltration. In particular, the microfiltration step may be carried out using a microfiltration membrane of any suitable pore size. Preferably, a microfiltration membrane having a pore size of 0.1 to 1 μm can be optionally used.
And S230, filtering the filtrate in a secondary ultrafiltration mode to obtain a target product.
"Secondary ultrafiltration" means that a filtration space having an upper molecular weight limit and a lower molecular weight limit is formed by two times of ultrafiltration with different pore sizes to obtain a final target product.
In some embodiments, as shown in fig. 2, the process of secondary ultrafiltration may include the steps of:
and S231, performing primary ultrafiltration on the filtrate by using an ultrafiltration membrane with the pore diameter smaller than the molecular weight of the antibacterial peptide.
S232, carrying out secondary ultrafiltration on the filtrate of the trapped fluid after the primary ultrafiltration by using an ultrafiltration membrane with the pore diameter larger than the molecular weight of the antibacterial peptide.
S233, filtering liquid is taken to obtain the target product.
Of course, in other embodiments, the order of the two ultrafiltration steps may be changed, and is not limited to the use of the steps shown in fig. 2. For example, the filtrate is first subjected to primary ultrafiltration using an ultrafiltration membrane having a pore size greater than the molecular weight of the antimicrobial peptide. Then, carrying out secondary ultrafiltration on the filtrate obtained after the primary ultrafiltration by using an ultrafiltration membrane with the pore diameter smaller than the molecular weight of the antibacterial peptide, and taking the trapped fluid to obtain the target product.
The two modes can achieve the same effect, and impurities exceeding the upper limit of the molecular weight and the lower limit of the molecular weight are removed, and the target product is reserved so as to achieve the purification effect.
S240, concentrating the target product to prepare and obtain the purified antibacterial peptide.
The antibacterial peptide with higher concentration can be obtained after further concentration and enrichment of the target product. In particular, the concentration and enrichment process may be carried out in any suitable manner. In some embodiments, the target product may be concentrated by nanofiltration membrane to obtain purified antimicrobial peptide.
The activity of the purified antimicrobial peptide can be detected specifically by the agar diffusion method. The agar diffusion method specifically comprises the following steps:
firstly, when the solid detection culture medium is cooled to about 50 ℃, adding 2% of indicator bacterium suspension, fully shaking up, adding 10ml of the culture medium into a sterile culture dish, punching a plurality of holes on each flat plate by using a 2.7mm hollow puncher after solidification, adding 6 mu l of purified antibacterial peptide to be detected into the holes, standing for 20min, putting the plates into a 37 ℃ constant temperature incubator for incubation for 18h, and measuring the diameter of a bacteriostatic ring. If a remarkable inhibition circle (the diameter is larger than a set threshold value) appears, the purified antibacterial peptide has activity.
The preparation and purification method disclosed in the embodiment of the invention can be used for preparing various types of antibacterial peptides. In some embodiments, the present embodiments also provide an antimicrobial peptide having antibacterial and anti-cellulite activity. The antibacterial peptide for resisting bacteria and gangrene protozoa is prepared by the preparation method and the purification method.
The following describes the specific procedures of the preparation and purification methods of the above antibacterial peptides in detail with reference to specific examples. It should be noted that the specific examples are only for illustration and are not intended to limit the scope of the present application. The numerical values or ranges in this particular example are necessarily absolute values and may be subject to variation within tolerances.
1) Amplification of target genes by Polymerase Chain Reaction (PCR):
extracting total RNA from African emperor scorpion venom according to the kit specification. Reverse transcription was performed using a reverse transcription kit to obtain total cDNA. And (3) amplifying the antibacterial peptide Scorpine gene by using an upstream primer and a downstream primer by using the total cDNA as a template.
The sequence of the upstream primer is shown as SEQ ID No. 1:
5’-CTGCGATCCGGCTGGATTAACGAGGAGAAG-3’
the sequence of the downstream primer is shown as SEQ ID No. 2:
5’-ATTACTCGAGTTAGTAGGAGAGAGGGGTGCC-3’
wherein, the PCR amplification process comprises the following steps: 94 ℃ for 4 min; 94 ℃ for 30 s; 50 ℃ for 30 s; at 72 deg.C for 1min for 30 cycles; 72 ℃ for 7 min; 4 ℃ for 12 min. And after the reaction is finished, sending the PCR amplification product to a sequencing company for sequencing, and identifying the sequencing to correctly amplify the antibacterial peptide Scorpine gene.
2) Construction of pSUMO/scorpine expression vector:
2.1) purifying the PCR amplification product by using a PCR product purification Kit, then carrying out enzyme digestion and purification on the PCR amplification product by using restriction enzymes BamH I and Xho I, carrying out electrophoresis on the product by using 1% agarose Gel Extraction Kit, and purifying the product by using an agarose Gel Extraction Kit to obtain the antibacterial peptide Scorpine gene DNA.
2.2) cutting the pSUM0 plasmid by BamH I and Xho I, and purifying to obtain a linearized pSUMO plasmid vector.
2.3) the linearized pSUM0 plasmid vector and the antibacterial peptide Scorpine gene DNA were ligated with a ligation kit overnight at 16 ℃ to obtain a ligation product.
3) Verification of recombinant genes:
the ligation products were transformed into DH5 α -competent bacteria by heat shock (42 ℃ C., 90s) in a volume ratio of ligation product to DH5 α -competent bacteria of 1: 100. then, the bacterial colonies were spread on a solid LB medium (containing 50mg/ml kanamycin), cultured overnight in a 37 ℃ incubator, picked up, and sent to a sequencer for sequencing and identification, which confirmed that the insertion site of the target gene was correctly inserted.
A schematic diagram of the pSUM0/scorpine expression vector constructed is shown in FIG. 3. Wherein f1 origin refers to a gene replicon, kanr refers to a kanamycin resistance gene, ori refers to a gene replication origin, T7 promoter refers to a prokaryotic gene promoter T7, lac refers to a lactose gene, 6XHis refers to six amino acid tags, and SUM0 is a small ubiquitin-like tag.
4) Expression and purification of recombinant antibacterial peptide Scorpine protein:
the pSUM0/scorpine expression vector (i.e., ligation product) was transformed into BL-21(DH3) pLys competent bacteria using heat shock (42 ℃, 90s) with a volume ratio of pSUM0/scorpine expression vector to BL-21(DH3) pLys competent bacteria of 1: 100. Then, the cells were plated on a solid LB medium (containing 50mg/ml kanamycin) and cultured overnight in a 37 ℃ incubator.
Single bacteria were picked, dropped into liquid LB medium (containing 50mg/ml kanamycin), and cultured on a shaker at 37 ℃ and 220rpm, and then transferred to a new liquid LB medium (containing 50mg/ml kanamycin) at a ratio of 1:1000, and cultured on a shaker at 37 ℃ and 220 rpm.
When the culture was carried out to a 0D600 value of about 0.4, 0.5mM isopropylthio-. beta. -D-galactoside (IPTG) was added and subjected to shaking induction at 28 ℃ for 6 hours. Then, the mixture was centrifuged at 10000rpm at 4 ℃ for 10min, and the supernatant was discarded to collect bacterial precipitates.
Then, a part of the bacterial pellet was sonicated (power 400W, sonication for 10 seconds each,
Finally, respectively storing the bacterial sediment before the ultrasonic treatment, the bacterial supernatant after the ultrasonic treatment and the bacterial sediment after the ultrasonic treatment at the temperature of minus 20 ℃ for standby or immediately purifying.
The three samples were examined by 12% polyacrylamide gel electrophoresis (SDS-PAGE). FIG. 4 is a schematic diagram showing the result of electrophoresis of the SDS-PAGE. As shown in FIG. 4, the recombinant Scorpine protein has a molecular weight of approximately 18kDa (including the tag His-SUM0 molecular weight (10kDa), and exists in soluble form in bacteria.
5) Pretreating an antibacterial peptide raw material to be purified:
collecting the live bacteria which successfully express the target antibacterial peptide in the step 4 from a liquid or solid culture medium, cracking the cells, and then centrifuging and taking the supernatant to obtain the crude antibacterial peptide.
6) Microfiltration of crude extract antimicrobial peptides:
and (3) carrying out microfiltration on the supernatant obtained in the step (5) by adopting an inorganic ceramic microfiltration experimental device with the pore diameter of 0.2 mu m. The operating parameters of the microfiltration are 40 ℃, 0.3MPa and pH7.5.
To slow down the contamination level of the membrane, in this example, every 20min of operation the connection was changed by the pipeline valve and back-flushed with permeate for 2 min. And (4) supplementing deionized water into the feed liquid every 20min until the volume concentration factor reaches 5, and stopping the operation.
7) Secondary ultrafiltration:
wherein, when the millipore ultrafiltration membrane is used for the first time, the smooth surface is downwards placed in a beaker, and the beaker is rinsed in sterilized water for more than 1 hour and water is changed for three times to complete the preparation of the ultrafiltration membrane.
In addition, the smooth surface of the ultrafiltration membrane is upwards arranged on a small-sized ultrafilter, 3ml of micro-filtered raw material liquid is filled in the ultrafiltration membrane, a rubber tube is connected, nitrogen is filled in the ultrafiltration membrane, the pressure is within the range of 2.5-4.0 atmospheric pressure, and the ultrafiltration membrane is placed on a magnetic stirrer and is carried out at the temperature of 4 ℃.
In this embodiment, firstly, an ultrafiltration membrane with 10000 cut-off molecular weight is used to obtain a filtrate; ultrafiltering with ultrafiltration membrane with molecular weight cutoff of 1000, and collecting the retentate.
8) And (4) nanofiltration:
and (3) adopting small-sized laboratory nanofiltration equipment, selecting a polyether sulfone roll-type membrane (the effective membrane area is 0.25m) with the molecular weight cutoff of 160D, and concentrating the ultrafiltered raw material liquid through the nanofiltration equipment to ensure that the volume concentration multiple is 5 to obtain the purified antibacterial peptide. Wherein the nanofiltration pressure is 2bar, and the feed liquid temperature is room temperature.
9) And (3) detecting the activity of the purified antibacterial peptide:
and identifying the biological activity of the purified antibacterial peptide by using an agar diffusion method. The method comprises the following specific steps:
uniformly mixing standard strain E.coli K12D31 into 10ml LB agar medium of 45-50 deg.C in Petri dish with inner diameter of 8.65cm to make viable count reach 106Per ml, and after coagulation, a 2.7mm diameter hole was punched.
Then, the purified antimicrobial peptide stock solution and 5 concentrations of antimicrobial peptide solution formed by diluting the stock solution by four times are added into holes according to 6 mul/hole, and the result is observed after culturing for 18h, and the existence of inhibition zones is observed.
As shown in FIG. 5, significant inhibition zones appeared in the stock solutions of antimicrobial peptides, 1/2 and 1/4 dilutions of the stock solutions, indicating that the purified antimicrobial peptides were active and successfully purified.
In conclusion, the preparation and purification method of the antibacterial peptide provided by the embodiment of the invention solves the problem of toxicity of the antibacterial peptide to a host after expression, and can effectively separate and purify the antibacterial peptide. And the micro-filtration, ultra-filtration, nano-filtration and the like are used for realizing industrial production, and the technology is widely applied in the pharmaceutical industry, thereby being beneficial to large-scale production and popularization.
In the purification method provided by the embodiment of the invention, the cost of the separation and purification method is obviously reduced, the method can be used for preparing medicines, can also keep the activity and purity of the antibacterial peptide to the maximum extent, and has good application prospect.
It should be understood that the technical solutions and concepts of the present invention may be equally replaced or changed by those skilled in the art, and all such changes or substitutions should fall within the protection scope of the appended claims.
Sequence listing
<110> Shenzhen Shenjin technology Limited
<120> preparation and purification method of antibacterial and cellulitis antibacterial peptide
<141> 2019-09-17
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213> Homo sapiens
<400> 1
ctgcgatccg gctggattaa cgaggagaag 30
<210> 2
<211> 31
<212> DNA
<213> Homo sapiens
<400> 2
attactcgag ttagtaggag agaggggtgc c 31