CN112552389A - Active peptide fusion protein and preparation method thereof - Google Patents
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Abstract
The invention provides an active peptide fusion protein with an antibacterial function and a preparation method thereof, wherein the fusion protein comprises a human Fc part and a bioactive peptide part which are directly connected or connected through a linking element. The active peptide part of the fusion protein provided by the invention is a novel antibacterial peptide, and the amino acid sequence of the active peptide part is shown as SEQ ID NO: 1-5. The fusion protein provided herein has better antibacterial activity, lower hemolysis and cytotoxicity, and longer half-life. In addition, the preparation method provided by the invention has high yield, and the prepared active peptide has high purity, thereby laying a technical foundation for realizing industrialization.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an active peptide fusion protein with an antibacterial function and a preparation method thereof.
Background
Antimicrobial peptides (AMPs) are a class of biologically short peptides with Antimicrobial activity, generally consisting of 10-60 amino acid residues. The antibacterial peptide is widely distributed and various in nature, is found in organisms from mammals, birds, amphibians, fishes, insects to plants and the like, and is an important component of the innate immune system of an organism. Hundreds of polypeptides having antibacterial activity have been isolated and identified.
The antibacterial peptide has the action mechanism of interacting with the surface of a cell membrane to change the permeability of the cell membrane, break acid-base balance, change osmotic pressure balance and form an ion channel, so that bacteria are cracked and killed, and the antibacterial peptide has a wide inhibiting effect on bacteria, fungi, chlamydia, mycoplasma, viruses, tumor cells and the like. The general combination mode of the antibacterial peptide is non-target combination, is not easy to generate drug resistance, and is effective to antibiotic resistant strains, so that the antibacterial peptide is expected to become a new generation of peptide antibiotics and has wide application value in the directions of medicines, livestock breeding additives, food additives and the like.
The immunoglobulins of the IgG class are the most abundant proteins in human blood and have half-lives of up to 21 days, and the Fc fragment is the main reason for maintaining the longer half-life of IgG immunoglobulins. The Fc fragment binds to the neonatal Fc receptor (FcRn), avoiding degradation into lysosomes. Therefore, the Fc fragment of IgG is often used to connect with active protein or peptide fragment to form fusion protein, so as to improve the half-life of active protein or peptide fragment in vivo and achieve the purpose of long-lasting effect.
At present, because the traditional antibacterial peptide on the market has small fragments, is easy to be degraded by enzyme after entering blood, has short half-life period and poor drug-forming property, the traditional antibacterial peptide molecules need to be modified properly so as to improve the antibacterial potency and reduce or eliminate the side effect.
Chinese patent 201510486586.1 discloses a method for preparing an antibacterial peptide-lysozyme fusion protein, which sequentially comprises an antibacterial peptide thanatin protein, a 3GSA flexible peptide and a T4 lysozyme protein from an N end to a C end, and the protein is proved to have remarkable effects on the aspects of improving the survival rate of piglets, reducing the diarrhea rate of nursery pigs and reducing the incidence rate of respiratory diseases of the nursery pigs, and can be used for preparing related medicinal preparations. However, the fusion protein is mainly applied to livestock breeding, particularly pig breeding, and the application range is small.
Chinese patent 201711091432.8 discloses an antibacterial fusion protein and its use, wherein the fusion protein contains antibacterial protein fragment rBPI21 and antibacterial peptide LL-37, the molecular weight is large, the in vivo action time of the fusion protein can be prolonged, and the fusion protein also has a wider antibacterial spectrum, greatly enhances the antibacterial effect, and can be widely applied to the technical fields of antibiosis, endotoxin resistance and the like. However, the effect of extending the duration of action of the fusion protein by virtue of the molecular weight needs to be further optimized.
Disclosure of Invention
In order to solve the problems, the invention provides an active peptide fusion protein and a preparation method thereof, aiming at the problems of short half-life and poor pharmaceutical property of the traditional antibacterial peptide. The active peptide provided by the invention is a novel antibacterial bioactive peptide, and the fusion protein of the active peptide has better antibacterial activity, lower hemolytic property and cytotoxicity and longer half-life. In addition, the preparation method provided by the invention has high yield, and the prepared active peptide has high purity, thereby laying a technical foundation for realizing industrialization.
In one aspect, the present invention provides a biologically active peptide, which is an antimicrobial peptide. The amino acid sequence of the bioactive peptide is SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5; preferably SEQ ID NO: 2. the bioactive peptide does not belong to any traditional classified antibacterial peptide, belongs to novel antibacterial peptide, and is subjected to amino acid sequence optimization modification on the basis of the traditional mammal-derived antibacterial peptide.
It should be clear that the bioactive peptides provided by the present invention may exert an antibacterial effect when present alone or when present in a fusion with other proteins.
In another aspect, the present invention provides a fusion protein with antibacterial function, wherein the fusion protein comprises: biologically active peptides (antimicrobial peptides) and other proteins with antimicrobial action.
The fusion protein is a monomer or a homodimer; the interaction of the two monomers of the homodimer is either non-covalent or covalent binding.
Further, the fusion protein comprises: a biologically active peptide having an antibacterial effect, an Fc fragment or a mutant thereof, and a linking element.
Specifically, the fusion protein can be represented as REPT @ Fc, wherein REPT represents a bioactive peptide; @ represents a linking element, binding REPT and Fc together; fc represents human Fc fragment or its mutant.
Preferably, the fusion protein is a monomer.
Preferably, the fusion protein is a homodimer; the homodimer comprises two identical peptide chains (or monomers), and the interaction of the two monomers is non-covalent or covalent.
Further, the amino acid sequence of the bioactive peptide is SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5; preferably SEQ ID NO: 2.
furthermore, the bioactive peptide does not belong to any traditional classified antibacterial peptide, belongs to a novel antibacterial peptide, and is subjected to amino acid sequence optimization modification on the basis of the traditional mammal-derived antibacterial peptide.
In particular, the Fc fragment is a constant region fragment of an IgG immunoglobulin.
Further, the Fc fragment comprises immunoglobulin CH2 and CH3 regions; the immunoglobulin is from human or other mammals.
Preferably, the Fc fragment is derived from a human, further preferably a human IgG4 immunoglobulin.
Specifically, the fusion protein is formed by connecting REPT with an Fc fragment directly or indirectly through a linking element, wherein the linking element is composed of 2-20 flexible amino acids, and the amino acids are selected from at least one of Gly, Ser, Ala and Thr.
Further, the ground linking element is (Ala-Ala-Ala-Ala-Ser)n(ii) a Wherein n is an integer between 1 and 5, preferably 2.
In some embodiments, the active peptide fusion protein is SEQ ID NO: 6 or an amino acid sequence substantially identical to SEQ ID NO: 6 has more than 90% of homology and derivative proteins with the same functions.
In still another aspect, the present invention provides a biomaterial comprising the aforementioned gene encoding a fusion protein.
Specifically, the biological material includes, but is not limited to, a plasmid vector containing the gene encoding the fusion protein and a transgenic host cell.
In yet another aspect, the present invention provides a method for preparing the aforementioned fusion protein.
Specifically, the method comprises the following steps:
(1) gene synthesis: artificially synthesizing a coding gene of a target protein, and performing codon optimization on the coding gene;
(2) transformation and expression: constructing the optimized coding gene on a eukaryotic expression vector, transfecting the eukaryotic expression vector into eukaryotic cells through electrotransfer or a chemical reagent, and expressing the eukaryotic expression vector in the eukaryotic cells;
(3) and separating and purifying the target fusion protein.
The transformation expression method of the step (2) comprises transient transfection expression and stable transfection expression.
Preferably, the transient transfection expression eukaryotic expression vector is pTT 5.
Preferably, the transient transfection expressing eukaryotic cell is an Expi CHO-S cell.
Preferably, the stable transfection expression eukaryotic expression vector is pcDNA3.1 (+).
Preferably, the stably transfected expression eukaryotic cell is a CHO cell.
The purification method of step (3) includes but is not limited to affinity chromatography, ion exchange method, size exclusion method and ultrafiltration method.
In some embodiments, the optimized REPT @ Fc nucleotide sequence is as set forth in SEQ ID NO: shown at 7.
In still another aspect, the present invention provides the use of the above fusion protein in the preparation of an antibacterial agent.
Specifically, the bacteria on which the antibacterial agent acts include, but are not limited to, gram-negative bacteria, gram-positive bacteria or fungi; further, the bacteria acting on the antibacterial agent include, but are not limited to, Escherichia coli, Bacillus subtilis, Staphylococcus aureus or Candida albicans.
Specifically, the application includes, but is not limited to, preparing an antibacterial preparation by using the fusion protein as an additive.
The antibacterial preparation includes but is not limited to injection, solution, gel, emulsion, inhalant, eye drop and spray.
The antimicrobial preparation may also contain other acceptable carriers or additives, including but not limited to ethanol, microcrystalline cellulose, sodium hydroxymethyl cellulose, carbomer, polysorbates, poloxamer, tween-80, and glycerol.
In yet another aspect, the present invention provides an antimicrobial formulation.
The antibacterial preparation comprises the fusion protein.
The addition amount of the fusion protein in the antibacterial preparation is 0-1000mg/g, preferably 100-200 mg/g.
The antibacterial preparation includes but is not limited to injection, solution, gel, emulsion, inhalant, eye drop and spray.
The antimicrobial preparation may also contain other acceptable carriers or additives, including but not limited to ethanol, microcrystalline cellulose, sodium hydroxymethyl cellulose, carbomer, polysorbates, poloxamer, tween-80, and glycerol.
The research of the invention shows that the active peptide fusion protein has broad spectrum and wide application range on gram-negative bacteria, gram-positive bacteria or fungi, which indicates that the active peptide provided by the invention can be used for preparing antibacterial preparations and provides a new choice for the current sterilization and disinfection field.
Drawings
FIG. 1 is a schematic representation of a REPT @ Fc dimer.
FIG. 2 is a vector map of pTT5-REPT @ Fc and pCDNA3.1(+) -REPT @ Fc.
FIG. 3 shows the SDS-PAGE results of the fusion proteins.
FIG. 4 shows the HPLC-SEC results for the fusion protein.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
EXAMPLE 1 antimicrobial susceptibility testing of bioactive peptides
The 5 bioactive peptides tested in this example were obtained by chemical synthesis or by clonal expression methods conventional in the art, and in either case, were peptide products identified for sequence correctness. In this example, the chemical synthesis was performed.
1) The experimental strains are 18 standard strains and clinical isolated strains preserved in laboratories, and comprise drug-resistant bacteria.
2) Culture medium: MH agar culture medium is purchased from China pharmaceutical biological product verification, and MH broth culture medium and brain heart infusion culture medium are products of DIFCO company.
3) The test drugs are: the bioactive peptide to be detected is synthesized by a outsourcing company by adopting a chemical solid phase, and the purity is detected to be more than 95%.
4) The test steps are as follows: performing drug sensitivity test by two-fold dilution method, and respectively amplifying 18 test bacteria with nutritional brain heart infusion solution or MH broth culture medium;
after the sample to be tested is dissolved, diluting the sample to various required concentrations by using two times of corresponding culture medium, and respectively adding a proper amount of the diluted sample into the pore plate;
adding the amplified test bacteria into corresponding pore plates in equal volume, culturing at 37 deg.C for 24 hr, and determining OD600And (4) judging the MIC (minimum inhibitory concentration).
SEQ ID NO: 1-5 MIC (mu g/mL) statistical table of minimum inhibitory concentration of bioactive peptide:
note: the bacteria marked in the table above are drug-resistant bacteria.
From MIC analysis, SEQ ID NO: 1-5, wherein the peptide has high candida inhibitory effect, and the peptide has the amino acid sequence shown in SEQ ID NO: 2, and thus is further developed as a candidate sequence, but this is not intended to limit the scope of the claims of the present invention, within which the sequence shown in SEQ ID NO: 1-5 can achieve corresponding technical effects.
Example 2 construction of a recombinant cell line expressing a fusion REPT @ Fc protein
Construction of the REPT @ Fc plasmid: the nucleotide sequence of REPT @ Fc (SEQ ID NO: 7) is obtained by artificial synthesis after codon optimization according to the gene sequence of human IgG4 searched in a gene library, the REPT @ Fc gene is synthesized by a gene synthesis company and constructed into pTT5 and pcDNA3.1(+) by TA cloning, and the accuracy of constructed plasmids is verified by sequencing to obtain recombinant plasmid DNA.
The REPT @ Fc amino acid sequence is SEQ ID NO: 6, the nucleotide encoding the REPT @ Fc amino acid is SEQ ID NO: 7.
the method for constructing the expression vector containing the active peptide fusion protein comprises the following steps:
(1) synthesizing a REPT @ Fc gene fragment by adopting a whole gene synthesis technology: SEQ ID NO: 7. SEQ ID NO: 7 comprises the sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10. wherein, SEQ ID NO: 8 is a bioactive peptide REPT gene sequence which is designed according to CHO cell preference and the coded amino acid sequence is SEQ ID NO: 2; SEQ ID NO: 9 is the gene sequence of the linking element AAAAT, functioning to link REPT and Fc; SEQ ID NO: 10 is a gene sequence of human Fc, and the amino acid sequence is as follows: SEQ ID NO: 11, the function of the peptide is to enhance the bacteriostatic ability of the peptide and prolong the half-life period of the peptide; the 5 'and 3' ends have restriction enzymes EcoRI and BamHI, respectively.
The REPT @ Fc amino acid sequence expressed by the sequence is shown as SEQ ID NO: and 6.
(2) Vector assembly and cloning:
in this example, an expression vector was constructed by a Gibson assembly method; specifically, the REPT @ Fc gene fragment synthesized in the step (1) is used as a template, a primer is designed by a conventional method for PCR amplification, and a 681bp fragment is recovered, namely the active peptide fusion protein gene. The active peptide fusion protein gene is digested by NcoI and XhoI in 50 times amount in CutSmart buffer at 37 deg.c, digested for 1 hr, and the pTT5 and pCDNA3.1 vector are digested with EcoRI and BamHI to recover 5.3K linearized vector. Linear vector DNA100ng, PCR fragment DNA500ng, was added to 10. mu.L of 2 XGibson Mix, sterile water was added to 20. mu.L and incubated at 50 ℃ for half an hour. 10 mu L of the assembly product is taken to transform a chemically competent cell DH10B of escherichia coli, the transformed bacterium liquid is coated on an LB plate containing 100 mu g/mL of ampicillin to be cultured overnight at 37 ℃, a monoclonal extracted plasmid is picked the next day to be sequenced, and an expression vector containing REPT @ Fc is constructed and obtained, wherein the vectors are named as pTT5-REPT @ Fc and pCDNA3.1(+) -REPT @ Fc. The map of the constructed vector is shown in FIG. 2.
The method for constructing the recombinant CHO cell strain containing the pCDNA3.1(+) -REPT @ Fc expression vector comprises the following steps:
the recombinant vector pcDNA3.1(+) -REPT @ Fc with correct sequencing is transformed into a CHO cell strain by a conventional method, and the obtained recombinant CHO cell is named CHO-pcDNA3.1(+) -REPT @ Fc.
Example 3 expression of the biologically active peptide fusion protein REPT @ Fc
The recombinant CHO cell obtained in example 2 CHO-pcDNA3.1(+) -REPT @ Fc was expressed in terms of 3-5X 105cells/mL subculture under the conditions of 37 ℃ and CO 25%, rotation speed of 120rpm, humidity of 70-80%, culturing for 3-4 days, and cell density of 3-4 × 106cells/mL. Sampling and counting on the day of transfection, and controlling the cell density to be not higher than 4X 106cells/mL, cell viability greater than 95%. The amount of DNA used was 1. mu.g/mL and the amount of PEI was 3. mu.g/mL, the DNA and PEI were added to opti-MEM medium 10% of the cell culture volume and incubated at 37 ℃ for 15min, then they were slowly added to the cell culture broth using a pipette, which should be completed within 3min, and the transfected cells were then placed on a shaker for further culture. After 24h of transfection, 10% Supplement feeding medium is added, the temperature is reduced to 32 ℃ for continuous culture, and the content of glucose is controlled to be 2-8g/L in the culture process. When the viable cell density is lower than 70%, taking out the cellsThe suspension was centrifuged at 10000rpm for 30min to collect the REPT @ Fc supernatant.
Example 4 purification of a biologically active peptide fusion protein REPT @ Fc
Protein a affinity media (available from GE, usa) was equilibrated with PBS (pH 7.0) buffer, and then the supernatant from example 4 was loaded onto a Protein a affinity column, washed with greater than 3 column volumes of PBS (pH 7.0) buffer, eluted with 100mM citrate buffer (pH 3.0), and the eluate was collected and neutralized with 100mM Tris-HCl (pH 9.0) buffer.
Example 5 identification of a biologically active peptide fusion protein REPT @ Fc
The SDS-PAGE results of the fusion proteins collected in example 4 are shown in FIG. 3, and the HPLC-SEC results are shown in FIG. 4.
Example 6 antimicrobial susceptibility assay of the biologically active peptide fusion protein REPT @ Fc
The purified bioactive peptide fusion protein REPT @ Fc of example 4 was subjected to an antimicrobial susceptibility test according to the method of example 1, and the results obtained were as follows:
the result shows that the bioactive peptide fusion protein REPT @ Fc has good antibacterial effect compared with the pure bioactive peptide, and the minimum inhibitory concentration is lower, namely, the usage amount is less when the same inhibitory effect is achieved.
Example 6 SD rats acute toxicity test by intravenous administration of REPT @ Fc
REPT @ Fc was administered by a single intravenous injection in SD rats to evaluate toxicity responses and animal mortality caused by REPT @ Fc. A total of 2 groups of 10 animals, male and female halves, were designed for this experiment. The test was administered at a dose of 0 (negative control) and 200mg/kg body weight, respectively. Animals were closely observed for clinical symptoms 6 hours after dosing, and thereafter for 14 consecutive days. Animals were weighed 1 time before dosing and 1 time per week thereafter. At the end of the observation period, all surviving animals were dissected.
No animal death was observed throughout the test period. No obvious abnormality was observed in clinical observation of each group of animals during the test period. The average body weight of the female and male animals before, 7 and 14 days after the administration at 200mg/kg body weight groups was not statistically different from that of the 0mg/kg group animals. The gross anatomy during the observation period had no post-dose related abnormalities.
As can be seen, the Maximum Tolerated Dose (MTD) of the REPT @ Fc fusion protein is more than 200mg/kg, and the safety is good.
Example 7 preparation and detection of other fusion proteins
Reference examples 2-6 were made to the preparation and detection methods of the nucleic acid sequences utilizing SEQ ID NOs: 1. 3, 4 and 5 to prepare and detect. The amino acid sequence of the fusion protein 1 is shown as SEQ ID NO: 12 is shown in the specification; the amino acid sequence of the fusion protein 3 is shown as SEQ ID NO: 13 is shown in the figure; the amino acid sequence of the fusion protein 4 is shown as SEQ ID NO: 14 is shown in the figure; the amino acid sequence of the fusion protein 5 is shown as SEQ ID NO: shown at 15. The results are as follows:
the results show that the bioactive peptide fusion proteins 1, 3, 4 and 5 have better antibacterial effect and lower minimum inhibitory concentration compared with the simple bioactive peptides (SEQ ID NO: 1, 3, 4 and 5), namely, the usage amount is less when the same inhibitory effect is achieved. And the bioactive peptide protein has good safety.
Sequence listing
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<211> 196
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 11
Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Ser
1 5 10 15
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
20 25 30
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
35 40 45
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
50 55 60
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
65 70 75 80
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
85 90 95
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
100 105 110
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
115 120 125
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
130 135 140
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
145 150 155 160
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
165 170 175
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
180 185 190
Ser Arg Trp Gln
195
<210> 12
<211> 220
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 12
Met Gly Arg Phe Lys Arg Phe Arg Lys Lys Phe Lys Lys Leu Phe Lys
1 5 10 15
Lys Leu Ser Ala Ala Ala Ala Ser Thr His Thr Ser Pro Pro Ser Pro
20 25 30
Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys
35 40 45
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
50 55 60
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
65 70 75 80
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
85 90 95
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
100 105 110
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
115 120 125
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
130 135 140
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
145 150 155 160
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
165 170 175
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
180 185 190
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
195 200 205
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
210 215 220
<210> 13
<211> 219
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 13
Gly Arg Lys Lys Arg Lys Arg Lys Lys Lys Lys Lys Leu Lys Lys Lys
1 5 10 15
Leu Ser Ala Ala Ala Ala Ser Thr His Thr Ser Pro Pro Ser Pro Ala
20 25 30
Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro
35 40 45
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
50 55 60
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
65 70 75 80
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
85 90 95
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
100 105 110
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
115 120 125
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
130 135 140
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
145 150 155 160
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
165 170 175
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
180 185 190
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
195 200 205
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
210 215
<210> 14
<211> 219
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 14
Ser Leu Lys Lys Lys Leu Lys Lys Lys Lys Lys Arg Lys Arg Lys Lys
1 5 10 15
Arg Gly Ala Ala Ala Ala Ser Thr His Thr Ser Pro Pro Ser Pro Ala
20 25 30
Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro
35 40 45
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
50 55 60
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
65 70 75 80
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
85 90 95
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
100 105 110
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
115 120 125
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
130 135 140
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
145 150 155 160
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
165 170 175
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
180 185 190
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
195 200 205
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
210 215
<210> 15
<211> 220
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 15
Met Ser Leu Lys Lys Lys Leu Lys Lys Lys Lys Lys Arg Lys Arg Lys
1 5 10 15
Lys Arg Gly Ala Ala Ala Ala Ser Thr His Thr Ser Pro Pro Ser Pro
20 25 30
Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys
35 40 45
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
50 55 60
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
65 70 75 80
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
85 90 95
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
100 105 110
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
115 120 125
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
130 135 140
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
145 150 155 160
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
165 170 175
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
180 185 190
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
195 200 205
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
210 215 220
Claims (10)
1. The bioactive peptide with the antibacterial function is characterized in that the amino acid sequence of the bioactive peptide is SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5, or a pharmaceutically acceptable salt thereof.
2. A fusion protein with antibacterial function is characterized in that the fusion protein comprises bioactive peptide and other protein; the amino acid sequence of the bioactive peptide is selected from SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5, or a pharmaceutically acceptable salt thereof.
3. The fusion protein of claim 2, wherein the fusion protein is a monomer or homodimer; the interaction of the two monomers of the homodimer is either non-covalent or covalent binding.
4. The fusion protein of claim 2, wherein the other protein comprises a human Fc fragment; the bioactive peptide is connected with the human Fc fragment directly or through a linking element.
5. The fusion protein of claim 4, wherein the human Fc fragment is selected from the group consisting of the CH2-CH3 constant regions of human IgG1 or IgG4 or a variant thereof obtained by mutating an amino acid fragment thereof.
6. The fusion protein of claim 4, wherein the linking element is 2-20 flexible amino acids selected from one or more of Gly, Ser, Ala, and Thr.
7. The fusion protein of claim 6, wherein the linking element is (Ala-Ala-Ala-Ala-Ser)n(ii) a Wherein n is an integer between 1 and 5, preferably 2.
8. The fusion protein of any one of claims 1-7, wherein the fusion protein is prepared by:
(1) gene synthesis: artificially synthesizing the encoding gene of the active peptide fusion protein, and carrying out codon optimization;
(2) transformation and expression: inserting the optimized coding gene into a eukaryotic expression vector, transferring the optimized coding gene into a eukaryotic host cell through electrotransfer or a chemical reagent, and expressing the optimized coding gene in the eukaryotic host cell;
(3) separating and purifying the active peptide fusion protein.
9. The method according to claim 8, wherein the transformation expression in step (2) is transient expression, the eukaryotic expression vector is pTT5, and the eukaryotic host cell is Expi CHO-S cell; the transformation expression in the step (2) is stable transformation expression, the eukaryotic expression vector is pcDNA3.1, and the eukaryotic cell is a CHO cell; the purification method in the step (3) comprises one or more of affinity chromatography, ion exchange method, size exclusion method and ultrafiltration method.
10. Use of a biologically active peptide according to claim 1 and a fusion protein according to claims 2 to 7 for the preparation of an antibacterial agent, wherein said bacterium is a gram-negative bacterium, a gram-positive bacterium or a fungus; the bacteria are escherichia coli, bacillus subtilis, staphylococcus aureus or candida albicans; the antibacterial preparation is injection, solution, gel, emulsion, inhalant, eye drop or spray.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2022205822A1 (en) * | 2021-03-31 | 2022-10-06 | 杭州先端生物科技有限公司 | Cathelicidin for inhibiting novel coronavirus infections and use thereof |
Citations (5)
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|---|---|---|---|---|
| CN109021109A (en) * | 2018-07-20 | 2018-12-18 | 上海交通大学 | A kind of ox source property anti-Staphylococcus aureus fusion antibody scFv-Fc and preparation method thereof |
| CN110637035A (en) * | 2017-05-25 | 2019-12-31 | 百时美施贵宝公司 | Fusion of modified IgG1Fc domain to anti-CD 40 domain antibody |
| CN110719915A (en) * | 2017-05-25 | 2020-01-21 | 百时美施贵宝公司 | Antibodies comprising modified heavy chain constant regions |
| CN111214644A (en) * | 2020-03-16 | 2020-06-02 | 中国人民解放军第四军医大学 | Application of Fenmu polypeptide in preparation of medicine for inhibiting II type herpes simplex virus infection |
| CN113444163A (en) * | 2020-03-26 | 2021-09-28 | 潘彩秀 | Active peptide and preparation method thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110637035A (en) * | 2017-05-25 | 2019-12-31 | 百时美施贵宝公司 | Fusion of modified IgG1Fc domain to anti-CD 40 domain antibody |
| CN110719915A (en) * | 2017-05-25 | 2020-01-21 | 百时美施贵宝公司 | Antibodies comprising modified heavy chain constant regions |
| CN109021109A (en) * | 2018-07-20 | 2018-12-18 | 上海交通大学 | A kind of ox source property anti-Staphylococcus aureus fusion antibody scFv-Fc and preparation method thereof |
| CN111214644A (en) * | 2020-03-16 | 2020-06-02 | 中国人民解放军第四军医大学 | Application of Fenmu polypeptide in preparation of medicine for inhibiting II type herpes simplex virus infection |
| CN113444163A (en) * | 2020-03-26 | 2021-09-28 | 潘彩秀 | Active peptide and preparation method thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022205822A1 (en) * | 2021-03-31 | 2022-10-06 | 杭州先端生物科技有限公司 | Cathelicidin for inhibiting novel coronavirus infections and use thereof |
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