CN104387465A - Antimicrobial peptide AP-57 as well as preparation method and application method thereof - Google Patents

Abstract

The invention discloses an antimicrobial peptide AP-57 and its preparation method and application method. The antimicrobial peptide AP-57 is a polypeptide containing 57 amino acids; the preparation method comprises the following steps: the human source AP-57 eukaryotic expression nucleic acid sequence Optimize the nucleic acid sequence suitable for expression in Escherichia coli; transform the recombinant expression plasmid into engineered Escherichia coli BL21 competent cells; pick a single clone to expand culture and induce expression; affinity chromatography; cation exchange chromatography; final mass spectrometry identification. The present invention has broad-spectrum anti-pathogenic microorganism functions, including anti-bacteria, anti-fungal, anti-virus, anti-mycoplasma, and is a novel human antimicrobial peptide with multiple biological functions; AP-57 antibacterial peptide has significant anti-bacterial and The antifungal effect has a very significant anti-enveloped virus and mycoplasma effect, and has great potential application value in inhibiting the growth, reproduction and dissemination of microorganisms, and preventing or treating pathogenic microorganism infections.

Description

A kind of antibacterial peptide AP-57 and its preparation method and application method

technical field

The invention belongs to the technical field of biochemistry and biomedicine, and in particular relates to an antimicrobial peptide AP-57 and its preparation method and application method.

Background technique

Microbial infections, including bacteria, fungi, viruses, etc., can cause many very serious human diseases including hepatitis, pneumonia, AIDS, tuberculosis, etc. Infectious epidemics caused by pathogenic microorganisms are always one of the important causes of human death.

Since the invention of antibiotics, the lives of countless patients with bacterial infections have been saved. However, the use of a large number of antibiotics not only brings many side effects to patients, but also causes the breeding of a large number of drug-resistant strains. And antibiotics are often ineffective against viral infections.

Antimicrobial peptides are a general term for short peptides with antibacterial activity. In 1975, Swedish scientist G.Boman et al induced and isolated a cecropin from the chrysalis chrysalis, and named it cecropin. Since then, people have successively discovered and isolated a variety of polypeptides with antibacterial activity from bacteria, fungi, amphibians, insects, higher plants, mammals and even humans, such as magainins, melittins, defensins (defensins) and so on. Because this type of active peptide has broad-spectrum and high-efficiency bactericidal activity against bacteria, it is named "antibacterial peptides, ABP", which is translated as antibacterial peptide in Chinese, and its original meaning is antibacterial peptide. With the in-depth development of people's research work, it is found that some antibacterial peptides have a strong killing effect on some fungi, protozoa, viruses and cancer cells, so many scholars also call them peptide antibiotics (Peptide Antibiotics), And antimicrobial peptides (Antimicrobial peptides, AMP). In addition, it has also been reported that some antimicrobial peptides have the functions of promoting blood coagulation, promoting wound healing, anti-inflammation, and regulating immunity. These endogenous antimicrobial peptides play an important role in the body's resistance to pathogenic invasion.

Defensins (defensins) are small in molecular weight (generally less than 60 amino acids, a few have more than 100 amino acids) rich in cysteine (generally contain 6 to 8 conservative cysteine residues, forming 3 to 4 pairs of disulfide bonds) cationic proteins, which belong to the class of antimicrobial peptides. Widely present in biological groups including plants, lower animals and mammals. So far, hundreds of defensins have been discovered in humans, and each defensin contains 6 cysteines, and each defensin has unique expression and functional characteristics. Human defensins are divided into α-type and β-type according to the structure and distribution characteristics. In addition, the cyclic defensin θ-Defensin has been discovered in rhesus monkeys in recent years. Defensins are evolved from the long-term struggle of organisms against diseases, and are also an important part of their own defense system. They also have functions such as anti-microbial, anti-tumor, promotion of blood coagulation and wound healing, anti-inflammation, and immune regulation.

At present, when food safety incidents occur frequently and thorny problems such as drug resistance, drug residues, and antibiotic abuse are unresolved, many scientists and entrepreneurs regard antimicrobial peptides as the most promising alternatives to antibiotics. Most of the antimicrobial peptides have the ability to penetrate the cell membrane, resulting in the incomplete cell membrane and the extravasation of the cytoplasm, thereby killing the target microorganisms and cells. Its mechanism of direct membrane destruction makes it difficult for target microorganisms to develop drug resistance. Some antimicrobial peptides, such as nisin, have long been approved as antibacterial food additives. Therefore, the discovery of a new antimicrobial peptide has great potential application development value.

Technical solution of prior art one

There are many types of antimicrobial peptides in various organisms in nature. Hundreds of human antimicrobial peptides have been discovered so far, and most of the antimicrobial peptides containing disulfide bonds are classified as defensins. In the past ten years, non-human antimicrobial peptides have been continuously discovered and identified, but the discovery of new human antimicrobial peptides has been very rare.

Regarding the AP-57 polypeptide, previous research reports have revealed some functions that may be related to immune regulation and colon cancer tumor cell proliferation regulation. These reports cannot speculate and predict that AP-57 has the effect of antibacterial peptides.

The shortcoming of prior art one

Although people have isolated and identified many types of antimicrobial peptides from various organisms in nature, each antimicrobial peptide has the basic characteristics of anti-microbial, but different antimicrobial peptides have different antibacterial spectrum and antibacterial effect. It can be said that each antimicrobial peptide has its own unique efficacy. In particular, the types of human antimicrobial peptides discovered and reported are relatively small. We know that the application of antimicrobial peptides from other species to the human body is likely to cause immune rejection, so the development of antimicrobial peptides from different species is very limited in the development of human drugs.

Searching the literature of the past ten years, we know that the discovery and reporting of new human antimicrobial peptides has been very rare. The AP-57 antimicrobial peptide sequence of the present invention is significantly different from the existing reported antimicrobial peptides in terms of sequence homology. Therefore, the AP-57 antimicrobial peptide of the present invention has great potential application and development value, especially in the development of human antimicrobial infection drugs.

Technical scheme of prior art 2

Currently, antimicrobial peptides containing disulfide bonds are mostly produced by solid-phase synthesis or yeast expression. It is still difficult to use the convenient, fast and economical E. coli cytoplasmic expression, and there are few successful reports. There are also some reports on the successful expression of antimicrobial peptides using Escherichia coli, but the production and preparation process is not widely applicable to the preparation of other antimicrobial peptides. It can be said that different antimicrobial peptides have different molecular weight, isoelectric point, hydrophobicity, spatial structure and other characteristics, so they should have different production and preparation processes.

The shortcoming of prior art two

The solid-phase synthesis of peptides is suitable for target peptides with a small number of amino acids and few disulfide bonds. For proteins with a large number of amino acids and disulfide bonds, their production and quality control are more difficult, and the production cost is also higher.

The yeast expression system is more suitable for the target protein with a large number of amino acids and disulfide bonds. However, compared with the Escherichia coli expression system, there is still an obvious problem of high production cost.

Contents of the invention

The purpose of the embodiments of the present invention is to provide an antimicrobial peptide AP-57 and its preparation method and application method, aiming at solving the problem that there are relatively few types of human antimicrobial peptides in the existing technology, and the solid-phase synthesis of peptides is suitable for the number of amino acids. For target polypeptides with fewer and fewer disulfide bonds, production and quality control are more difficult for proteins with more amino acid numbers and more disulfide bonds, and the production cost is relatively high.

The present invention is achieved in this way, an antibacterial peptide AP-57, the antibacterial peptide AP-57, is a polypeptide, corresponding to the mature protein sequence after the signal peptide is removed from the C10orf99 gene expression product, contains 57 amino acids, and the sequence is:

KRRPAKAWSGRRTRLLCCHRVPSPNSTNLKGHHVRLCKPCKLEPEPRLWVVPGALPQV;

The theoretical molecular weight is: 6.52D; the theoretical isoelectric point is 11.28; it is a strong cationic amphipathic antibacterial peptide.

Another object of the present invention is to provide a kind of preparation method of antibacterial peptide AP-57, the preparation method of this antibacterial peptide AP-57 comprises the following steps:

Step 1, sequence optimization and vector construction: firstly, the human AP-57 eukaryotic expression nucleic acid sequence was optimized to a nucleic acid sequence suitable for expression in Escherichia coli, and constructed on the genetically engineered pET28a vector.

Step 2 Plasmid transformation and small-scale culture: Thaw the engineered Escherichia coli BL21 competent cells on ice, pipette 20-100 μl and place in a 1.5ml EP tube. Add 1-2μl plasmid, mix gently, and let stand on ice for 20-30min. Then heat-shock in a water bath at 42°C for 60-90s, and then immediately ice-bath for 2min. Add 900 μl of LB medium without antibiotics, and incubate at 37°C for 1 hour on a constant temperature shaker. Centrifuge at 6000rpm for 3min, remove the supernatant, 100-200μl LB resuspended bacteria solution, pipette and mix well, spread on the LB solid culture dish added with antibiotics, and incubate overnight at 37℃. Pick a positive single colony and inoculate it into a test tube containing 4ml of LB liquid medium, shake overnight at 37°C and 220rpm, transfer the above bacterial solution to a shaker flask containing 100ml of LB liquid medium at a ratio of 1:1000 , 37°C, shaking at 220 rpm until the OD600 reached 0.6.

Step 3, expanding culture and inducing expression: transfer the above-mentioned bacterial solution to a shaker flask containing 1000-2000ml LB liquid medium at a ratio of 1:1000, culture at 37°C and shake at 220rpm until the OD600 reaches 1.0, add 0.5-1mmol/L IPTG, induced at 37°C for 6 hours, the collected bacteria were resuspended in loading buffer (50mM NaH ₂ PO ₄ .2H ₂ O, 0.5M NaCl, pH 8.0), and crushed in a high-pressure homogenizer. Then centrifuge at 4°C and 13000rpm for 30-40min, take the supernatant and filter it with a 0.22μm filter for subsequent purification;

Step 4, affinity chromatography: load the lysed supernatant in step 3 to the chromatographic column with the affinity filler installed, rinse with 3 times the column volume of loading buffer, wash with elution buffer (50mM NaH ₂ PO ₄ .2H ₂ O, 0.5M NaCl, 500mM imidazole, pH 8.0) were eluted, and the peak protein samples were collected.

Step 5, cation exchange chromatography: put the protein sample on the chromatographic column installed with cationic filler after standard enzyme digestion, rinse with equilibration buffer (20mM Tris, pH8.5), and wash with elution buffer (2M NaCl, 20mM Tris, pH8.5) to elute, collect the effluent peak protein, and retain the sample for SDS-PAGE electrophoresis analysis and mass spectrometry verification. High-purity human AP-57 can be obtained after ion exchange chromatography.

Another object of the present invention is to provide an application method of antibacterial peptide AP-57, which has broad-spectrum anti-pathogenic microorganism functions, including anti-bacteria, anti-fungal, anti-virus, anti-mycoplasma; containing 57 amino acid polypeptide, named AP-57.

The antibacterial peptide AP-57 provided by the present invention and its preparation method and application method, through large-scale expression and activity detection analysis, found that a polypeptide recombined and expressed through E. coli genetic engineering has a function similar to defensins; the present invention also provides The preparation method of the polypeptide is simple and efficient, and at the same time, the polypeptide is a human polypeptide, which is conveniently applied to the development of human medicines, can be used to develop medicines against pathogenic microorganisms, and has great development potential. The novel human antimicrobial peptide with multiple biological functions has great potential application value. The AP-57 antibacterial peptide has significant antibacterial and antifungal effects, and has very obvious anti-enveloped virus and mycoplasma effects. It has great potential application value in inhibiting the growth, reproduction and dissemination of microorganisms, and preventing or treating pathogenic microorganism infections. In addition, the invention provides a simple and efficient production and preparation technical scheme of AP-57, which is convenient for the application of AP-57 antimicrobial peptide in the development of antimicrobial infection drugs, and has great application value.

Description of drawings

Fig. 1 is the flow chart of the preparation method of antimicrobial peptide AP-57 provided by the embodiment of the present invention;

Fig. 2 is a schematic diagram of AP-57 homology comparative analysis provided by the embodiment of the present invention;

Figure 3 is a schematic diagram of the soluble induced expression of AP-57 in Escherichia coli provided by the embodiment of the present invention;

Figure 4 is a schematic diagram of the induced expression analysis of AP-57 in Escherichia coli with different IPTG concentrations provided by the embodiment of the present invention;

Figure 5 is a schematic diagram of AP-57 expression analysis of AP-57 induced by different induction times in Escherichia coli provided by the embodiment of the present invention;

Figure 6 is a schematic diagram of the SDS-PAGE electrophoresis results of AP-57 purified by nickel column affinity chromatography provided in the embodiment of the present invention;

Fig. 7 is a schematic diagram of the SDS-PAGE electrophoresis results of cation chromatography purified AP-57 provided by the embodiment of the present invention;

Fig. 8 is the result figure of inhibition experiment of AP-57 provided by the embodiment of the present invention to Escherichia coli (ATCC 25922) motility;

Fig. 9 is the bacteriostatic experiment result figure of AP-57 provided by the embodiment of the present invention to Staphylococcus aureus (ATCC 25923);

Fig. 10 is the bacteriostatic experiment result figure of AP-57 provided by the embodiment of the present invention to actinomycetes (clinical patient isolate);

Fig. 11 is the bacteriostatic experiment result figure of AP-57 provided by the embodiment of the present invention to Aspergillus niger (ATCC16404);

Fig. 12 is the bacteriostasis experiment result figure of AP-57 provided by the embodiment of the present invention to mycoplasma (clinical patient isolate);

Fig. 13 is a graph showing the results of the inhibition experiment of AP-57 on lentivirus (HEK293 cell packaging) infection ability provided by the embodiment of the present invention.

Detailed ways

In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

The application principle of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

The antibacterial peptide AP-57 of the embodiment of the present invention is a polypeptide, corresponding to the mature protein sequence of the C10orf99 gene expression product after removing the signal peptide, containing 57 amino acids, and the sequence is:

KRRPAKAWSGRRTRLLCCHRVPSPNSTNLKGHHVRLCKPCKLEPEPRLWVVPGALPQV;

Theoretical molecular weight (MW) is: 6.52D; theoretical isoelectric point (pI) is 11.28; it is a strong cationic amphipathic antibacterial peptide, which has broad-spectrum anti-pathogenic microorganism functions, including anti-bacteria, anti-fungal, anti-virus, Anti-mycoplasma.

The novel antimicrobial peptide has broad-spectrum antimicrobial efficacy, and the antimicrobial peptide has a sequence shown in SEQ ID NO: 1 and contains 57 amino acids. Name it AP-57 (antimicrobial peptide-57, Antimicrobial peptide57). The AP-57 polypeptide has broad-spectrum antimicrobial efficacy, and the invention also provides a preparation method of the AP-57 antimicrobial peptide. At the same time, the polypeptide is a human polypeptide, which is conveniently applied to the development of human medicines, can be used to develop medicines against pathogenic microorganisms, and has great development potential.

As shown in Figure 1, the preparation method of the antimicrobial peptide AP-57 of the embodiment of the present invention comprises the following steps:

S101, sequence optimization and vector construction: firstly, the human AP-57 eukaryotic expression nucleic acid sequence was optimized as a nucleic acid sequence suitable for expression in Escherichia coli, and constructed on the genetically engineered pET28a vector.

S102, plasmid transformation and small-scale culture: the plasmid was transformed into engineered Escherichia coli BL21 competent cells, and cultured upside down at 37°C overnight. Pick a positive single colony and inoculate it into a test tube containing 4ml of LB liquid medium, shake overnight at 37°C and 220rpm, transfer the above bacterial solution to a shaker flask containing 100ml of LB liquid medium at a ratio of 1:1000 , 37°C, shaking at 220 rpm until the OD600 reached 0.6.

S103, expanded culture and induced expression: transfer the above bacterial solution to a shaker flask containing 1000-2000ml LB liquid medium at a ratio of 1:1000, culture at 37°C with shaking at 220rpm until the OD600 reaches 1.0, add 0.5 -1mmol/L IPTG, induced at 37°C for 6h, the collected bacteria were resuspended in loading buffer (50mM NaH ₂ PO ₄ .2H ₂ O, 0.5M NaCl, pH 8.0), and crushed in a high-pressure homogenizer. Then centrifuge at 4°C and 13000rpm for 30-40min, take the supernatant and filter it with a 0.22μm filter for subsequent purification;

S104, Affinity chromatography: Load the lysed supernatant from step 3 to the chromatographic column installed with the affinity filler, rinse with 3 times the column volume of loading buffer, and wash with elution buffer (50mM NaH ₂ PO ₄ .2H ₂ O, 0.5M NaCl, 500mM imidazole, pH 8.0) was eluted, and the peak protein sample was collected.

S105, cation exchange chromatography: put the protein sample on the chromatographic column equipped with cation filler after standard digestion, rinse with equilibration buffer (20mM Tris, pH8.5), wash with elution buffer ( 2M NaCl, 20mM Tris, pH8.5) to elute, collect the eluted peak protein, and retain the sample for SDS-PAGE electrophoresis analysis and mass spectrometry verification. High-purity human AP-57 can be obtained after ion exchange chromatography.

In step S101:

In the eukaryotic expression nucleic acid sequence (SEQ ID NO: 3) of 57 amino acids encoding AP-57, there are 9 rare codons in Escherichia coli, all of which are replaced with codons preferred by Escherichia coli. At the same time, the stop codon in SEQ ID NO: 3 was replaced with the stop codon TGA preferred by Escherichia coli. The final optimized sequence is SEQ ID NO: 5; the detailed replacement is shown in Table 1:

Table 1: Codon optimization of the nucleic acid sequence encoding AP-57

*Number of codons: the corresponding number of encoded amino acids in the nucleic acid sequence encoding AP-57

1. Sequence composition:

The human AP-57 precursor encodes a small molecular weight protein of 81 amino acids, and the mature human AP-57 only has 57 amino acids (theoretical MW: 6.52; theoretical pi: 11.28). The sequence includes 16 basic amino acids, 2 acidic amino acids and 4 cysteines, so under physiological conditions, the protein is positively charged.

2. Homology comparison:

As shown in Figure 2, homology comparison analysis (DNAMAN software) shows that C10orf99 has homology with common model animals Gorilla (Gorilla), cynomolgus monkey (Machin), rat (Rat) and mouse (Mouse) About 96%, 82% and 47%. In terms of conservation, the N-terminal and C-terminal parts of the sequence, the positions of most basic amino acids, and the 4 cysteine positions are highly conserved. In addition, it can be clearly seen from the sequence that the protein presents a remarkable amphipathic characteristic in which the N-terminus is hydrophilic and the C-terminus is sparse in multiple species.

Three, AP-57 expression and preparation:

Step 1: First, optimize the human AP-57 eukaryotic expression nucleic acid sequence (sequence 3) into a nucleic acid sequence (sequence 5) suitable for expression in Escherichia coli. The sequence was commissioned to be synthesized by Beijing Jinweizhi Company and constructed on genetically engineered pET28a carrier.

Step 2: Transform the recombinant expression plasmid into the engineered Escherichia coli BL21 competent cells, screen the positive strains on the LB plate, pick a single colony and inoculate it in a test tube containing 4ml LB liquid medium, shake overnight at 37°C and 220rpm , and then transfer the above bacterial solution to a shaker flask containing 100ml LB liquid medium at a ratio of 1:1000, shake and cultivate at 37°C at 220rpm until the OD600 reaches 0.6, add 1mmol/L IPTG, and induce 4 After 1 hour, the bacterial liquid was collected and analyzed by SDS-PAGE, the results are shown in Figure 3;

Swimming lane 1: precipitation of cell lysate induced by IPTG; lane 2: precipitation of cell lysate cultured without IPTG; lane 3: supernatant of cell lysate cultured without IPTG; lane 4: supernatant of cell lysate cultured without IPTG clear. It indicated that AP-57 recombinant protein was expressed in soluble form.

Step 3, the effect of different IPTG concentrations on the induced expression of the target protein: divide the bacterial solution into 4 test tubes, 4ml in each tube, add IPTG to the final concentrations of 0mmol/L, 0.2mmol/L, 0.5mmol/L, 0.8mmol/L, 1mmol/L, 37°C, 220rpm shaking induction for 6 hours, collect the bacteria, break the bacteria and centrifuge to get the supernatant for SDS-PAGE analysis, as shown in Figure 4, the concentration of IPTG above 0.5mmol/L can activate AP- High-level expression of 57 recombinant proteins;

Swimming lane 1 is not induced by IPTG; Swimming lanes 2-5 respectively correspond to the expression induced by the bacteria under the conditions of 0.2mmol/L, 0.5mmol/L, 0.8mmol/L, 1mmol/L final concentration of IPTG;

Step 4, the effect of different induction times on the induced expression of the target protein: divide the bacterial solution into 5 tubes, 4ml in each tube, add IPTG to a final concentration of 1mmol/L, shake at 37°C and 220rpm, and set the induction interval as 0h, Take samples at 2h, 4h, 6h, and 20h, collect the bacteria, and centrifuge the bacteria to get the supernatant for SDS-PAGE analysis, as shown in Figure 5, which shows that the effect of induction for 6h is better;

Swimming lanes 1-5 are induction culture 0h, 2h, 4h, 6h and 20h respectively;

Step 5, expansion culture: the target protein is amplified and cultured in a 2L shake flask and induced to express the target protein according to the optimized conditions (37°C, 220rpm shaking, 0.5mmol/LIPTG induction for 6h), collect the bacterial liquid, centrifuge, and remove the supernatant , bacteria were resuspended in loading buffer (50mM NaH ₂ PO ₄ .2H ₂ O, 0.5M NaCl, pH8.0), crushed in a high-pressure homogenizer, centrifuged at 4°C, 13000rpm for 40min, and taken Clear 0.22μm filter for subsequent purification;

Step 6, affinity chromatography: load the sample from the previous step to the chromatographic column installed with affinity filler, rinse with 3 times the column volume of loading buffer, wash with elution buffer (50mM NaH ₂ PO _{4.2H 2} O, 0.5M NaCl, 500mM imidazole, pH 8.0) to elute, collect the effluent peak protein, and perform SDS-PAGE electrophoresis analysis, as shown in Figure 6;

Lane 1 is the supernatant before sample loading; Lane 2 is the elution peak 1 of affinity chromatography; Swimming lane 3 is the elution peak 2 of affinity chromatography;

Step 7, cation exchange chromatography: put the protein sample on the chromatographic column equipped with cation packing after standard enzyme digestion, rinse with equilibration buffer (20mM Tris, pH8.5), and wash with elution buffer (2M NaCl, 20mM Tris, pH8.5) to elute, collect the effluent peak protein, and retain the sample for SDS-PAGE electrophoresis analysis, as shown in Figure 7, after ion exchange chromatography, high-purity human AP-57 can be obtained;

M: Marker; lane 1 is the elution peak 1 of cationic chromatography; lane 2 is the elution peak 2 of cationic chromatography;

Step 8, mass spectrometry analysis and identification: Beijing Huada Protein Research and Development Center Co., Ltd. used Q-TOF tandem mass spectrometry for mass spectrometry analysis. The identification results showed that the expressed and purified protein corresponded to the protein encoded by C10orf99 in the database.

AP-57 is named C10orf99 (open reading frame 99 of chromosome 10) in the database.

The application effect of the present invention is further described in conjunction with the following experiments:

AP-57 function analysis:

Experimental materials: Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 19115, Aspergillus niger ATCC16404, Candida albicans ATCC10231, Saccharomyces cerevisiae ATCC 2601 were purchased from ATCC; Pseudomonas aeruginosa (PA01), typhoid fever Bacillus (RE88), Candida albicans CMCC (B) 98001 were preserved in this laboratory; actinomycetes and mycoplasma were isolated from clinical patients. The Ad5 type adenovirus carrying EGFP protein is preserved in our laboratory. The lentivirus carrying EGFP protein was purchased from Guangzhou Saiye Biotechnology Co., Ltd. Modified 1640 medium was purchased from sigma. Bovine serum albumin was purchased from AID SCIENCE; antimicrobial peptide LL-37 was purchased from Shanghai Aobo Biotechnology Co., Ltd.; PBS buffer was purchased from sigma;

1 Antibacterial Analysis

Resuspend the seed-preserving bacteria with modified serum-free 1640 to make a bacterial suspension, and resuscitate for 1-3 days. Bacteria were measured by the McFarland standard turbidimetric method, and adjusted to a bacterial suspension with a concentration of 1×10 ⁸ /mL. The modified serum-free 1640 medium was used for the dilution of the concentration gradient of the bacterial solution. Spread the bacterial solution on a 24-well or 96-well cell culture plate, let it stand for 30 minutes, and set up a blank control group, a PBS control group, a bovine serum albumin (BSA) negative control group, a LL-37 positive control group, and an AP-57 experimental group. The negative control group, the positive control group and the experimental group were added bovine serum albumin, LL-37 and AP-57 with different concentration gradients respectively, and three replicate wells were set up. Incubate at 37°C, observe the growth of bacteria at regular intervals, record, take pictures, and count the minimum effective antibacterial concentration (MES) and inhibition rate (inhibition rate=(1-experimental group living body number or mycelia length or movement speed/control group living body number or hyphal length or movement speed) × 100%, the control is the blank control, that is, the control without any treatment).

The results show that AP-57 treatment can significantly reduce the movement speed of Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi, resulting in the aggregation of flora, see Figure 8 and Table 2; and can significantly inhibit the growth of Staphylococcus aureus (ATCC 25923) , see Figure 9 and Table 2; also can significantly inhibit the growth of actinomycetes (clinical patient isolates), see Figure 10 and Table 2.

2 Antifungal analysis

Aspergillus niger ATCC16404, Candida albicans ATCC 10231, Saccharomyces cerevisiae ATCC 2601, and Candida albicans CMCC (B) 98001 were resuscitated on the slant, and the fungi were picked on the serum-free modified 1640 medium with an inoculation loop, mixed by pipetting, and cultured overnight at 37°C. After growing to a suitable concentration, dilute to different concentration gradients, spread 24-well cell culture plates or 96-well cell culture plates, and let stand for 30 minutes. Set blank control group, PBS control group, bovine serum albumin (BSA) negative control group, LL-37 positive control group, AP-57 experimental group. The negative control group, the positive control group and the experimental group were added bovine serum albumin, LL-37 and AP-57 with different concentration gradients respectively, and three replicate wells were set up. Incubate at 37°C, observe the growth of fungi at regular intervals, record, take pictures, and count the minimum effective antibacterial concentration (MES) and inhibition rate (inhibition rate=(1-experimental group living body number or mycelium length/control group living body number or Mycelium length) × 100%, the control is the blank control, that is, the control without any treatment). The results showed that AP-57 had obvious inhibitory effect on the growth of Aspergillus niger, as shown in Figure 11 and Table 2.

3 Anti-mycoplasma analysis

Mycoplasma hominis was cultured in modified 1640 medium with 10% fetal bovine serum. Dilute it to different concentration gradients with improved 1640 medium, add it to a 24-well plate covered with slides, set up a blank control group, a PBS control group, a bovine serum albumin (BSA) negative control group, and a LL-37 positive control group , AP-57 experimental group. The negative control group, the positive control group and the experimental group were added bovine serum albumin, LL-37 and AP-57 with different concentration gradients respectively, and three replicate wells were set up. Incubate at 37°C for 1 to 6 hours, stain with Hoechst, record, take pictures, and count the minimum effective antibacterial concentration (MES) and inhibition rate (inhibition rate=(1-experimental group living body number/control group living body number)×100%, the control is Blank control, that is, the control without any treatment). The results showed that AP-57 had a significant inhibitory effect on the growth of Mycoplasma (clinical patient isolate), as shown in FIG. 12 and Table 2.

4 Antiviral Analysis

Lentiviral particles carrying EGFP were packaged in HEK293 cells. Dilute it with improved 1640 medium and add AP-57 antimicrobial peptides with different concentration gradients, as well as PBS control, equal concentration of BSA control, and equal concentration of LL-37 positive control, treat for 2-4 hours, and then add cultured with 24-well plate of HEK293 cells, 10% serum, DMEM medium, put into cell incubator and incubate for 24-48h, observe infection efficiency under fluorescent microscope, take pictures, record, statistics (inhibition rate=(1-experimental group infection rate/ Infection rate of the control group) × 100%, the control is the control of the blank group, that is, the control without adding any treatment). The results showed that AP-57 had a significant inhibitory effect on the infection of lentivirus (packaged in HEK293 cells), as shown in Figure 13 and Table 2.

Table 2.AP-57 inhibits microorganism experiment result

The AP-57 antimicrobial peptide of the present invention has significant antibacterial and antifungal effects, although the effect is slightly lower than the positive control LL-37 antibacterial peptide in terms of inhibition of some bacteria, but for the used actinomycetes and black Aspergillus, the minimum effective concentration reached 12.33 μg/ml and 28.55 μg/ml, which was significantly better than the effect of the control LL-37 antimicrobial peptide. For the mycoplasma and lentivirus used in this example, the minimum effective concentration reaches 4.77 μg/ml and 16.50 μg/ml, which is also better than the effect of the control LL-37 antimicrobial peptide. Generally speaking, compared with the positive control antimicrobial peptide LL-37 that many research institutes and enterprises are competing to develop, AP-57 obviously has its unique advantages. In terms of market prospects, antimicrobial peptides can be used not only for clinical patients, but also for food additives. Feed additives are used for sterilization, such as clinically used polymyxin, and milk that has long been approved as a green food additive by many countries. streptavidin (nisin) and so on. Therefore, the AP-57 provided by the present invention has great potential application value in inhibiting the growth, reproduction and dissemination of microorganisms, and preventing or treating pathogenic microorganism infections.

The invention provides a convenient and efficient production and preparation technical scheme of AP-57, which is convenient for the application of the AP-57 antimicrobial peptide in the development of antimicrobial infection drugs, and has great application value.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (5)

1. a preparation method of cecropin A P-57, is characterized in that, the preparation method of this cecropin A P-57 comprises the following steps:

Step one, sequence optimisation and vector construction: first people source AP-57 eukaryotic expression nucleotide sequence is optimized for the nucleotide sequence being applicable to escherichia coli expression, and be implemented on genetically engineered pET28a carrier;

Step 2, Plastid transformation and in a small amount cultivation: by Plastid transformation through engineering approaches e. coli bl21 competent cell, be inverted overnight incubation for 37 DEG C.The positive single colony inoculation of picking is in the test tube that 4ml LB liquid nutrient medium is housed, 37 DEG C, 220rpm jolting is spent the night, and the ratio in 1: 1000 is got bacterium liquid and transferred in the shaking flask that 100ml LB liquid nutrient medium is housed, 37 DEG C, 220rpm jolting is cultured to OD600 and reaches 0.6;

Step 3, enlarged culturing and abduction delivering: get bacterium liquid transfer in the shaking flask that 1000-2000ml LB liquid nutrient medium is housed in the ratio of 1: 1000 again, 37 DEG C, 220rpm jolting is cultured to OD600 and reaches 1.0, add 0.5-1mmol/L IPTG, 37 DEG C of induction 6h, collect thalline and are resuspended in sample-loading buffer, 50mMNaH ₂pO ₄2H ₂o, 0.5M NaCl, pH8.0, broken in high pressure homogenizer, then in 4 DEG C, centrifugal 30-40min under 13000rpm condition, gets supernatant after 0.22 μm of frit, for subsequent purification;

Step 4, affinity chromatography: by supernatant loading to the chromatography column installing affine filler, after the sample-loading buffer drip washing of 3 times of column volumes, use elution buffer wash-out, elution buffer 50mM NaH ₂pO ₄2H ₂o, 0.5M NaCl, 500mM imidazoles, pH8.0, collects eluting peak protein sample;

Step 5, cation-exchange chromatography: protein sample is splined on after standard enzyme is cut the chromatography column having installed cationic fillers, with level pad, after 20mM Tris, pH8.5 drip washing, use elution buffer 2MNaCl, 20mM Tris, pH8.5 wash-out, collects eluting peak albumen, keep sample and do SDS-PAGE electrophoretic analysis and mass spectrum checking, highly purified people AP-57 can be obtained after ion exchange chromatography.

2. the preparation method of cecropin A P-57 as claimed in claim 1, is characterized in that, in step one, and the nucleotide sequence 174bp of ripe AP-57:

AAGAGGCGTCCTGCCAAGGCCTGGTCAGGCAGGAGAACCAGGCTCTGCTGCCACCGAGTCCCTAGCCCCAACTCAACAAACCTGAAAGGACATCATGTGAGGCTCTGTAAACCATGCAAGCTTGAGCCAGAGCCCCGCCTTTGGGTGGTGCCTGGGGCACTCCCACAGGTGTAG。

3. the preparation method of cecropin A P-57 as claimed in claim 1, is characterized in that, in step one, be applicable to the nucleotide sequence 174bp of the improved ripe AP-57 of escherichia coli expression:

AAGCGCCGTCCTGCCAAGGCCTGGTCAGGCCGCCGCACCCGCCTCTGCTGCCACCGCGTCCCTAGCCCTAACTCAACAAACCTGAAAGGACATCATGTGCGCCTCTGTAAACCATGCAAGCTGGAGCCAGAGCCTCGCCTTTGGGTGGTGCCTGGGGCACTCCCACAGGTGTGA。

4. a cecropin A P-57, is characterized in that, this cecropin A P-57, is polypeptide, corresponding C10orf99 gene expression product remove signal peptide after maturation protein sequence, containing 57 amino acid, sequence is:

KRRPAKAWSGRRTRLCCHRVPSPNSTNLKGHHVRLCKPCKLEPEPRLWVVPGALPQV；

Theoretical molecular is: 6.52D; Theoretical iso-electric point is 11.28; For the amphiphilic antibacterial peptide of strong cation.

5. an application method of cecropin A P-57, is characterized in that, this cecropin A P-57 has the function of broad-spectrum disease resistance pathogenic microorganism, comprises antibacterium, antimycotic, antiviral, mycoplasma; Containing 57 amino acid whose polypeptide, called after AP-57.