CN107641150A - A kind of preparation method of the antibacterial peptide with anti Helicobacter pylori activity - Google Patents
A kind of preparation method of the antibacterial peptide with anti Helicobacter pylori activity Download PDFInfo
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- CN107641150A CN107641150A CN201710995599.0A CN201710995599A CN107641150A CN 107641150 A CN107641150 A CN 107641150A CN 201710995599 A CN201710995599 A CN 201710995599A CN 107641150 A CN107641150 A CN 107641150A
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- antibacterial peptide
- helicobacter pylori
- fusion protein
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Abstract
The invention provides a kind of preparation method of the antibacterial peptide with anti Helicobacter pylori activity, belong to pharmaceutical field.This method includes：Structure coding includes SEQ ID NO:The nucleotide sequence of the fusion protein of antibacterial peptide shown in 1；Structure includes the recombinant vector of nucleotide sequence；Recombinant vector is used for conversion or transfection host cell, and nucleotides sequence is listed in host cell and expresses.This method includes the fusion protein of antibacterial peptide using gene engineering method expression, so as to reduce the isoelectric point of antibacterial peptide, reaches high-caliber expression, reduces production cost.Obtained this antibacterial peptide has anti Helicobacter pylori activity, and has stronger stability and antibacterial activity in gastric environment.
Description
Technical field
The present invention relates to pharmaceutical field, in particular to a kind of antibacterial peptide with anti Helicobacter pylori activity
Preparation method.
Background technology
Helicobacter pylori is to be found by two scientists of Australia the eighties in last century and obtained promise shellfish in 2005
You encourage at physiology.At present, there is more than 50% population in the world infected with this pathogen, wherein 20% the infected will draw
Play the clinical symptoms of chronic gastritis, digestibility and duodenal ulcer, stomach cancer and mucosa associated lymphoid tissue knurl.World health group
Knit and it is classified as I class procarcinogen.More it is widely current in developing country, some regional population's infection rates of China are up to 60%
More than.
Antibiotic is the effective ways for treating Helicobacter pylori infection, but helicobacter pylori produces to Multiple Classes of Antibiotics
Drug resistance, or even same infected patient is to 7 kinds of antibiotic resistances.At present, to there is this obvious disease symptomses individual treatment method
The conjoint therapy of two kinds or three kinds antibiotic is mainly combined with a kind of proton pump, it has 80% eradication rate and subsequent clinic
The improvement of symptom, have become the treatment method of standard.But with the increase of antibiotics resistance bacterial strain, re-infection, concurrent
Disease and the factor such as high medical expense and curative compliance difference limit radical cure of the antibiotic to Helicobacter pylori infection, wherein most
Chief reason is exactly the drug resistance of bacterium.In consideration of it, being badly in need of at present to Helicobacter pylori infection disease, propose that one kind is disobeyed
Rely the therapeutic scheme in antibiotic.
The content of the invention
The first object of the present invention is to provide a kind of antibacterial peptide with anti Helicobacter pylori activity and comprising this
The fusion protein of antibacterial peptide.This antibacterial peptide has anti Helicobacter pylori activity, and has stronger stabilization in gastric environment
Property and antibacterial activity, it causes the perforation of cell membrane to reach the effect of helicobacter pylori resistant by physical action, used
During be not easy to make helicobacter pylori to produce drug resistance.
The second object of the present invention is that providing a kind of above-mentioned antibacterial peptide and fusion protein is preparing treatment pylorus spiral
Applied in the medicine of bacillosis, because the antibacterial peptide will not be destroyed in gastric environment by pepsin, and under gastric acid environment according to
Stronger antibacterial activity so can be kept, the medicine for preparing treatment helicobacter pylori disease can be used for as active component.
The third object of the present invention is to provide a kind of gene for being used to encode above-mentioned antibacterial peptide and above-mentioned for encoding
The gene of fusion protein.
The fourth object of the present invention is that the gene for the gene and encoding fusion protein for providing a kind of encoding antimicrobial peptide exists
Prepare the application in the medicine for the treatment of helicobacter pylori disease.
The fifth object of the present invention is to provide a kind of preparation method of the antibacterial peptide with anti Helicobacter pylori activity,
This method includes the fusion protein of antibacterial peptide using gene engineering method expression, so as to reduce the isoelectric point of antibacterial peptide, makes it
Reach high-caliber expression, reduce production cost.
The sixth object of the present invention is to provide a kind of with the silkworm hemolymph of anti Helicobacter pylori activity and its preparation
Method and purposes.Contain fusion protein and enterokinase, specificity of the fusion protein through enterokinase in this silkworm hemolymph simultaneously
After digestion, obtain with natural structure antibacterial peptide, therefore, the silkworm hemolymph be provided with the activity of helicobacter pylori resistant and
The silkworm hemolymph can be directly oral or is freeze-dried just available for Helicobacter pylori infection is treated, and greatly reduces antibacterial peptide
Production cost.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
In a first aspect, the present invention provides a kind of antibacterial peptide with anti Helicobacter pylori activity, fusion protein, specifically
For：
A kind of antibacterial peptide with anti Helicobacter pylori activity, the amino acid sequence of antibacterial peptide is (1) or (2)：
(1)SEQ ID NO:Sequence shown in 1；
(2) by SEQ ID NO:Sequence shown in 1 by the substitution of one or several amino acid residues and/or missing and/
Or addition obtain and with SEQ ID NO:1 has the derived sequence of identical bioactivity.
A kind of antibacterial peptide with anti Helicobacter pylori activity, the amino acid sequence of antibacterial peptide is (1) or (2)：
(1)SEQ ID NO:Sequence shown in 1；
(2) by SEQ ID NO:Sequence shown in 1 by the substitution of one or several amino acid residues and/or missing and/
Or addition obtain and with SEQ ID NO:1 has the derived sequence of identical bioactivity.
A kind of fusion protein for including above-mentioned antibacterial peptide, fusion protein have one level below amino acid sequence structure：
Carrier protein-connection peptide-protease processing site peptide fragment-antibacterial peptide；
Antibacterial peptide has SEQ ID NO:Amino acid sequence shown in 1.
Further, above-mentioned carrier protein include silkworm baculovirus core polyhedrin, glutathione sulfydryl transferase,
Green fluorescent protein or ubiquitin sample little albumen.
Further, the amino acid sequence of above-mentioned silkworm baculovirus core polyhedrin is SEQ ID NO:2.
Further, above-mentioned connection peptide includes (GGGGS)n, wherein n=1~4.
Further, above-mentioned protease processing site peptide fragment be specific proteins restriction endonuclease action site, specific protein
White restriction endonuclease includes enterokinase, fibrin ferment, factor Xa, PreScission protease, Tobacco mosaic virus enzyme, people
Rhinoviral protease, ubiquitin sample little albumen enzyme.
Further, above-mentioned protease processing site peptide fragment include LVPRGS, IEGR, IDGR, LEVLFQ, ENLYFQG,
LEVLFQG or ubiquitin-like protein tertiary structure.
Further, above-mentioned protease processing site peptide fragment is DDDDK, and DDDDK is the action site of enterokinase.
Further, above-mentioned enterokinase is ox intestine kinase, and the amino acid sequence of ox intestine kinase is SEQ ID NO:3.
Further, the amino acid sequence of above-mentioned fusion protein is SEQ ID NO:4.
Second aspect, the present invention provides a kind of antibacterial peptide, fusion protein is preparing the medicine for the treatment of helicobacter pylori disease
In application, be specially：
A kind of application of antibacterial peptide in the medicine for preparing treatment helicobacter pylori disease, the amino acid sequence of antibacterial peptide is such as
SEQ ID NO:Shown in 1.
A kind of medicine or health products for being used to treat helicobacter pylori, it includes such as SEQ ID NO:Antibacterial shown in 1
Peptide and pharmaceutically acceptable carrier or auxiliary material.
A kind of application of fusion protein in the medicine for preparing treatment helicobacter pylori disease, fusion protein have with next
Grade amino acid sequential structure：
Carrier protein-connection peptide-protease processing site peptide fragment-antibacterial peptide；
Antibacterial peptide has SEQ ID NO:Amino acid sequence shown in 1.
Further, above-mentioned carrier protein include silkworm baculovirus core polyhedrin, glutathione sulfydryl transferase,
Green fluorescent protein or ubiquitin sample little albumen.
Further, above-mentioned protease processing site peptide fragment be specific proteins restriction endonuclease action site, specific protein
White restriction endonuclease includes enterokinase, fibrin ferment, factor Xa, PreScission protease, Tobacco mosaic virus enzyme, people
Rhinoviral protease, ubiquitin sample little albumen enzyme.
Further, above-mentioned protease processing site peptide fragment include DDDDK, LVPRGS, IEGR, IDGR, LEVLFQ,
ENLYFQG, LEVLFQG or ubiquitin-like protein tertiary structure.
Further, the amino acid sequence of above-mentioned fusion protein is SEQ ID NO:4.
Further, above-mentioned helicobacter pylori disease includes the gastritis as caused by Helicobacter pylori infection, alimentary canal is burst
Ulcer, lymphoproliferative gastric lymphoma, stomach cancer.
A kind of medicine or health products for being used to treat helicobacter pylori, it includes fusion protein and pharmaceutically may be used
The carrier or auxiliary material of receiving, fusion protein have one level below amino acid sequence structure：
Carrier protein-connection peptide-protease processing site peptide fragment-antibacterial peptide；
Antibacterial peptide has SEQ ID NO:Amino acid sequence shown in 1.
Further, in addition to enterokinase, the amino acid sequence of fusion protein is SEQ ID NO:4.
The third aspect, a kind of gene for being used for antibacterial peptide of the coding with anti Helicobacter pylori activity of present invention offer,
The gene of fusion protein, it is specially：
A kind of gene for being used to encode the antibacterial peptide with anti Helicobacter pylori activity, the amino acid sequence of antibacterial peptide is such as
SEQ ID NO:Shown in 1.
A kind of gene for being used to encode the fusion protein comprising above-mentioned antibacterial peptide, fusion protein have one level below amino acid
Sequential structure：
Carrier protein-connection peptide-protease processing site peptide fragment-antibacterial peptide；
Antibacterial peptide has SEQ ID NO:Amino acid sequence shown in 1.
Further, above-mentioned carrier protein include silkworm baculovirus core polyhedrin, glutathione sulfydryl transferase,
Green fluorescent protein or ubiquitin sample little albumen.
Further, above-mentioned protease processing site peptide fragment be specific proteins restriction endonuclease action site, specific protein
White restriction endonuclease includes enterokinase, fibrin ferment, factor Xa, PreScission protease, Tobacco mosaic virus enzyme, people
Rhinoviral protease, ubiquitin sample little albumen enzyme.
Further, above-mentioned protease processing site peptide fragment include DDDDK, LVPRGS, IEGR, IDGR, LEVLFQ,
ENLYFQG, LEVLFQG or ubiquitin-like protein tertiary structure.
Further, the amino acid sequence of above-mentioned fusion protein is SEQ ID NO:4.
A kind of recombinant vector for including said gene.
A kind of host cell for including above-mentioned recombinant vector.
Further, above-mentioned host cell includes bacterium, zooblast or plant cell.
A kind of recombinant virus for including said gene.
Fourth aspect, the present invention provides a kind of gene of encoding antimicrobial peptide, the gene of fusion protein is preparing treatment pylorus
Application in the medicine of pylori disease, it is specially：
A kind of application of gene of encoding antimicrobial peptide in the medicine for preparing treatment helicobacter pylori disease, the ammonia of antibacterial peptide
Base acid sequence such as SEQ ID NO:Shown in 1.
A kind of application of gene of encoding fusion protein in the medicine for preparing treatment helicobacter pylori disease, fusion protein
With one level below amino acid sequence structure：
Carrier protein-connection peptide-protease processing site peptide fragment-antibacterial peptide；
Antibacterial peptide has SEQ ID NO:Amino acid sequence shown in 1.
Further, above-mentioned carrier protein include silkworm baculovirus core polyhedrin, glutathione sulfydryl transferase,
Green fluorescent protein or ubiquitin sample little albumen.
Further, above-mentioned protease processing site peptide fragment be specific proteins restriction endonuclease action site, specific protein
White restriction endonuclease includes enterokinase, fibrin ferment, factor Xa, PreScission protease, Tobacco mosaic virus enzyme, people
Rhinoviral protease, ubiquitin sample little albumen enzyme.
Further, above-mentioned protease processing site peptide fragment include DDDDK, LVPRGS, IEGR, IDGR, LEVLFQ,
ENLYFQG, LEVLFQG or ubiquitin-like protein tertiary structure.
Further, the amino acid sequence of above-mentioned fusion protein is SEQ ID NO:4.
A kind of application of recombinant vector in the medicine for preparing treatment helicobacter pylori disease, recombinant vector include coding
SEQ ID NO:The gene order of antibacterial peptide shown in 1；Or the gene order comprising fusion protein, fusion protein have following
Primary amino acid sequences structure：
Carrier protein-connection peptide-protease processing site peptide fragment-antibacterial peptide；
Antibacterial peptide has SEQ ID NO:Amino acid sequence shown in 1.
A kind of application of recombinant virus in the medicine for preparing treatment helicobacter pylori disease, recombinant vector include coding
SEQ ID NO:The gene order of antibacterial peptide shown in 1；Or the gene order comprising fusion protein, fusion protein have following
Primary amino acid sequences structure：
Carrier protein-connection peptide-protease processing site peptide fragment-antibacterial peptide；
Antibacterial peptide has SEQ ID NO:Amino acid sequence shown in 1.
A kind of application of host cell in the medicine for preparing treatment helicobacter pylori disease, recombinant vector include coding
SEQ ID NO:The gene order of antibacterial peptide shown in 1；Or the gene order comprising fusion protein, fusion protein have following
Primary amino acid sequences structure：
Carrier protein-connection peptide-protease processing site peptide fragment-antibacterial peptide；
Antibacterial peptide has SEQ ID NO:Amino acid sequence shown in 1.
Further, above-mentioned helicobacter pylori disease includes the gastritis as caused by Helicobacter pylori infection, alimentary canal is burst
Ulcer, lymphoproliferative gastric lymphoma, stomach cancer.
5th aspect, the present invention provide a kind of preparation method of the antibacterial peptide with anti Helicobacter pylori activity, specifically
For：
A kind of preparation method of the antibacterial peptide with anti Helicobacter pylori activity, it includes：
Structure coding includes SEQ ID NO:The nucleotide sequence of the fusion protein of antibacterial peptide shown in 1；
Structure includes the recombinant vector of nucleotide sequence；
Recombinant vector is used for conversion or transfection host cell, and nucleotides sequence is listed in host cell and expresses.
Further, above-mentioned fusion protein has one level below amino acid sequence structure：
Carrier protein-connection peptide-protease processing site peptide fragment-antibacterial peptide；
Antibacterial peptide has SEQ ID NO:Amino acid sequence shown in 1.
Further, above-mentioned carrier protein include silkworm baculovirus core polyhedrin, glutathione sulfydryl transferase,
Green fluorescent protein or ubiquitin sample little albumen.
Further, above-mentioned protease processing site peptide fragment be specific proteins restriction endonuclease action site, specific protein
White restriction endonuclease includes enterokinase, fibrin ferment, factor Xa, PreScission protease, Tobacco mosaic virus enzyme, people
Rhinoviral protease, ubiquitin sample little albumen enzyme.
Further, above-mentioned protease processing site peptide fragment include DDDDK, LVPRGS, IEGR, IDGR, LEVLFQ,
ENLYFQG, LEVLFQG or ubiquitin-like protein tertiary structure.
Further, above-mentioned recombinant vector includes recombinant plasmid, and the expression vector of recombinant plasmid is pFastDual carriers.
Further, above-mentioned recombinant vector also includes recombinant shuttle vector, and recombinant shuttle vector is by recombinant plasmid transformed
Into competent escherichia coli cell, and the obtained restructuring Bacmid of restructuring occurs in the presence of recombinase.
Further, above-mentioned recombinant vector also includes recombinant virus, and recombinant virus is by the way that recombinant shuttle vector is transfected
It is made into silkworm BmN cells.
Further, above-mentioned host cell is silkworm chrysalis cell.
Further, express making nucleotides sequence be listed in host cell, after obtaining fusion protein, in addition to：With special
Property protein incision enzyme to fusion protein carry out digestion.
6th aspect, the present invention provide a kind of silkworm hemolymph with anti Helicobacter pylori activity and preparation method thereof with
Purposes, it is specially：
A kind of preparation method of the silkworm hemolymph with anti Helicobacter pylori activity, it includes：Utilize the shaft-like disease of silkworm
Malicious double-promoter expression system co-expresses fusion protein and enterokinase, and such as SEQ ID NO are included in fusion protein:It is anti-shown in 1
The specific proteins action site of bacterium peptide and enterokinase.
Further, the amino acid sequence of above-mentioned fusion protein such as SEQ ID NO:Shown in 4.
Further, it is above-mentioned to co-express fusion protein and enterokinase using silkworm baculovirus double-promoter expression system
Method includes：
Structure coding such as SEQ ID NO:The amino acid sequence of fusion protein shown in 4；
Structure such as SEQ ID NO:Amino acid sequence and coding such as SEQ ID NO shown in 4:Ox intestine kinase shown in 3
Amino acid sequence recombinant vector；
Recombinant vector is used to transfect silkworm BmN cells, after obtaining recombinant virus, then recombinant virus is expressed in silkworm chrysalis,
Obtain the silkworm hemolymph for including fusion protein and enterokinase.
Further, above-mentioned recombinant vector includes recombinant plasmid, and the expression vector of recombinant plasmid is pFastDual carriers.
Further, above-mentioned expression vector also includes recombinant shuttle vector, and recombinant shuttle vector is by recombinant plasmid transformed
Into competent escherichia coli cell, and the obtained restructuring Bacmid of restructuring occurs in the presence of recombinase.
Silkworm hemolymph made from method is prepared as above in one kind.
A kind of medicine or health products for being used to treat helicobacter pylori disease, it includes silkworm hemolymph and medicine and pharmacology can
The auxiliary material or carrier of receiving.
A kind of preparation method for the medicine or health products for being used to treat helicobacter pylori disease, it includes freezing silkworm hemolymph
Do into powder.
A kind of application of silkworm hemolymph in the medicine for preparing treatment helicobacter pylori disease.
Further, above-mentioned helicobacter pylori disease includes the gastritis as caused by Helicobacter pylori infection, alimentary canal is burst
Ulcer, lymphoproliferative gastric lymphoma, stomach cancer.
Compared with prior art, the beneficial effect of present disclosure for example including：
At present, using in the method for antibiosis extract for treating Helicobacter pylori infection, bacterium is easily by variation to antibiotic
Resistance is produced, that is, drug-fast bacteria occurs.With the increase of antibiotics resistance bacterial strain, re-infection, complication and high medical expense and
The factors such as curative compliance difference so that antibiotic can not effect a radical cure to Helicobacter pylori infection.
And present disclosure provide such as SEQ ID NO:Antibacterial peptide shown in 1, it has stronger to helicobacter pylori
Lethal effect.This antibacterial peptide will not be such that the resistance to the action of a drug of helicobacter pylori has with the use of antibacterial peptide as antibiotic
Increased, this antibacterial action for being primarily due to antibiotic is usually to act on special acceptor or enzyme, and bacterium is easily by change
It is different that resistance is produced to antibiotic；And antibacterial peptide typically no special acceptor in antibacterial, it is mainly made by physical action
Reach antibacterial effect into the perforation of cell membrane, so the use of the antibacterial peptide is not allowed to be also easy to produce resistance bacterium and cross tolerance.
Meanwhile in Helicobacter pylori infection, harmful side effect can be produced to body with antibiosis extract for treating, this is main
It is because antibiotic can stimulate endotoxic release, can also causes septic shock to even result in death, but use present disclosure
The antibacterial peptide provided is then without this phenomenon, and the antibacterial peptide also has suppression helicobacter pylori induction generation harmful
The effect of cell factor.
Present disclosure provide this antibacterial peptide be filtered out from antibacterial peptide storehouse there is strong helicobacter pylori resistant
The antibacterial peptide of activity.Experiment shows that the antibacterial peptide not only has stability in gastric environment, is not destroyed by pepsin, together
When under the sour environment of gastric juice (pH be 1.3~1.8) can still keep stronger antibacterial activity, it is to helicobacter pylori
Disease caused by infection has the effect of preferable, advantageously accounts for the antibiotics resistance occurred in Helicobacter pylori infection treatment
And its huge difficult medical problem of side effect.
However, due to the particular surroundings of stomach, when treating helicobacter pylori with the antibacterial peptide, due to gastric mucosal barrier be present
(i.e. helicobacter pylori is colonized in the epidermal surface under stomach lining rete malpighii, and antibacterial peptide needs to arrive at pylorus spiral for effect
The field planting position competence exertion antibacterial activity of bacillus.), orally substantial amounts of antibacterial peptide medicament, utilization are existing for need when causing to treat
Chemical synthesising technology production antibacterial peptide it is with high costs, the financial burden of patient can be increased in long therapeutic procedure.In view of
This, present disclosure also provides a kind of preparation method of the antibacterial peptide with anti Helicobacter pylori activity, and this method uses base
Because engineering method expression includes the fusion protein of antibacterial peptide, so as to reduce the isoelectric point of antibacterial peptide, reach high-caliber
Expression, reduce production cost.
In addition, being expressed using technique for gene engineering in obtained fusion protein, contain such as SEQ ID NO:Antibacterial shown in 1
Peptide.The fusion protein is after protease specificity digestion, you can obtains the above-mentioned antibacterial peptide with natural structure, plays anti-pylorus
The activity of helicobacter infection.
In silkworm hemolymph with anti Helicobacter pylori activity that present disclosure provides and preparation method thereof, using family
Silkworm utilizes baculoviral while expressed fusion protein (containing antibacterial peptide) and enterokinase as expression vector.Due in fusion protein
Specific cleavage site containing enterokinase, the fusion protein is after the specific digestion of enterokinase, you can obtaining has naturally
The antibacterial peptide of structure, so that the silkworm hemolymph collected has anti Helicobacter pylori activity.Because enterokinase is mankind's intestines
Enzyme present in road, there is oral administration safety, therefore the silkworm hemolymph can be directly oral without purifying, it is deep and remote so as to solve treatment
N of high dose oral antibacterial peptide, this costly great difficult problem are needed during door pylori.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described.
Fig. 1 is the PGQ containing antibacterial peptide provided in the embodiment of the present invention 3 fusion protein F AMP amino acid sequence；
Fig. 2 is the fusion protein F AMP provided in the embodiment of the present invention 3 protein steric structural figure；
Fig. 3 is the pFastDual plasmid maps containing double-promoter provided in the embodiment of the present invention 4；
Fig. 4 is the anti Helicobacter pylori activity inhibition zone of three kinds of silkworm hemolymphs in the embodiment of the present invention 5；
The electrophoretogram of the polypeptide of acquisition is purified in Fig. 5 Tricine-SDS-PAGE analysis silkworm hemolymphs.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition suggested according to normal condition or manufacturer are carried out.Agents useful for same or the unreceipted production firm person of instrument, it is
The conventional products that can be obtained by commercially available purchase.
The feature and performance of the present invention are described in further detail with reference to embodiments：
Embodiment 1
The present embodiment provides a kind of antibacterial peptide with anti Helicobacter pylori activity, its amino acid sequence such as SEQ ID
NO:Shown in 1.
The screening of the antibacterial peptide and experimentation are as follows：
A. the screening of antibacterial peptide：Antibacterial peptide has oneself unique antimicrobial spectrum, not every antibacterial peptide such as antibiotic
All there is antibacterial activity to helicobacter pylori, inventor screens tool by experimental study from ten hundreds of antibacterial peptide storehouses
There is 25 kinds of the antibacterial peptide of anti-Gram-negative bacteria, and by being chemically synthesized to obtain.
B. the preparation of antibacterial peptide test sample：According to minimal inhibitory concentration experimental method the antibacterial peptide of above-mentioned chemical synthesis with
Doubling dilution is 64 μ g/mL, 32 μ g/mL, 16 μ g/mL, 8 μ g/mL, 4 μ g/mL, 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL.
C. minimal inhibitory concentration is detected：The helicobacter pylori ATCC34504 standards of logarithmic phase are added in every test tube
Bacterial strain reaches 106CFU/mL, 37 DEG C of culture 36h, the antibacterial peptide for not seeing obvious bacterial growth are dense under the conditions of microaerophilic
Spend the minimal inhibitory concentration for the antibacterial peptide.
Experimental result is shown, in all synthetic antibacterial peptides, the antibacterial peptide minimal inhibitory concentration (MIC) finally obtained is
1 μ g/mL antibacterial peptide sequence is：GVLSNVIGYLKKLGTGALNAVLKQ, i.e. SEQ ID NO:Antibacterial peptide shown in 1, is designated as
PGQ, pI/Mw:10.00/2.5KDa.
Embodiment 2
The antibacterial peptide PGQ that the present embodiment simulation gastric environment is provided embodiment 1 carries out bacteriostasis experiment：
A. simulated gastric fluid (containing pepsin and stomach acidity material, pH is 1.3~1.8) is prepared；
B. the culture of different strains helicobacter pylori is carried out, bacterial strain includes：ATCC34504 reference cultures and it is located away from stomach
Clinical strains in the stomach of ulcer and patients with gastric cancer.
C. the helicobacter pylori 10 of logarithmic phase is added in simulated gastric fluid6CFU/mL, then plus antibacterial peptide PGQ reaches dense
Spend be 4 μ g/mL (4 × MIC) and without plus antibacterial peptide as compareing each three groups, respectively in 15min, 30min, 60min and
120min takes 10 μ L mixed liquor to be diluted to 50 μ L, is coated in solid medium, and the bacterium colony on per flat board is counted after culture 36h.Press
Formula calculates sterilizing rate：
Sterilizing rate=(1- add the bacterium solution flat-plate bacterial colony number of antibacterial peptide/without plus antibacterial peptide bacterium solution flat-plate bacterial colony number) ×
100%
Experimental result：Antibacterial peptide PGQ is not less than 50% in 15min, to the sterilizing rate of helicobacter pylori；
During 30min, 80% is not less than to the sterilizing rate of helicobacter pylori；In 60min, helicobacter pylori sterilizing rate is not less than
90%；In 120min, 100% is nearly reached to the sterilizing rate of helicobacter pylori.Thus illustrate, antibacterial peptide PGQ not only exists
In gastric environment there is stability not destroyed by pepsin, while the work of helicobacter pylori resistant is kept under gastric acid environment
Property.
Embodiment 3
The antibacterial peptide PGQ with anti Helicobacter pylori activity that embodiment 1 provides is with 3 positive net charges and is
The small peptide of small-molecular-weight, antibacterial peptide PGQ isoelectric point (PI) are：10.0, if the antibacterial peptide is directly a large amount of in host cell
Expression, there is considerable influence to the physiological pH of host cell, and then influence the survival of host cell, be unfavorable for antibacterial peptide PGQ height
Horizontal expression.In fact, inventor in experiments it is found that：Do not merge and directly express antibacterial peptide, antibacterial peptide is in host cell
It can not be expressed at all.
In consideration of it, the present embodiment provides a kind of fusion protein for being used to express antibacterial peptide PGQ, the fusion protein has following
Primary amino acid sequences structure：
Carrier protein-connection peptide-protease processing site peptide fragment-antibacterial peptide；
Wherein, carrier protein includes silkworm baculovirus core polyhedrin, glutathione sulfydryl transferase, green fluorescence
Albumen or ubiquitin sample little albumen.Optionally, carrier protein is silkworm baculovirus core polyhedrin, and its amino acid sequence is
SEQ ID NO:2(GenBank:AFJ06818.1).This silkworm core polyhedrin can form crystal structure, be easy to exposure and melt
The restriction enzyme site of specific proteins restriction endonuclease in hop protein, so as to avoid influence of the carrier protein to antibacterial peptide PGQ.
Wherein, connection peptide includes (GGGGS)n, wherein n=1~4.Optionally, n=2~3, or n=3.(GGGGS)nIt is logical
Often it is the jointing of connection peptide, GGGGS number very little, otherwise can not can not can influence the synthesis effect of fusion protein too much
Rate.With 2~3 more preferably, this connection peptide causes protease enzyme site to expose relatively to GGGGS number, is advantageous to digestion.
Wherein, protease processing site peptide fragment, corresponding specific proteins restriction endonuclease are any one in table 1 1~8
Group.Thus, this fusion protein is after specific proteins endonuclease digestion, you can obtains the antibacterial peptide PGQ of natural structure, plays
Anti Helicobacter pylori activity.
Protease processing site peptide fragment in the fusion protein of table 1.
| Label | Protease processing site peptide fragment | Specific proteins restriction endonuclease |
| 1 | DDDDK | Enterokinase |
| 2 | LVPRGS | Fibrin ferment |
| 3 | IEGR | Factor Xa |
| 4 | IDGR | Factor Xa |
| 5 | LEVLFQ | PreScission protease |
| 6 | ENLYFQG | Tobacco mosaic virus enzyme |
| 7 | LEVLFQG | ERC group virus's protease |
| 8 | Identify the three-level knot of ubiquitin-like protein | Structure ubiquitin sample little albumen enzyme |
Specifically, constructed in the present embodiment such as SEQ ID NO:Fusion protein shown in 4, fusion protein F AMP is designated as,
pI/Mw:6.27/34.5Kda its amino acid sequence is as shown in Figure 1.
Fusion protein F AMP, using silkworm baculovirus core polyhedrin as carrier protein, its isoelectric point is 6.27, is connect
The physiological pH of nearly bombyx mori cell, is advantageous to the high level expression in silkworm.Meanwhile fusion protein F AMP, in antibacterial peptide PEG
Before added sequence D DDDK, it is that (amino acid sequence is SEQ ID NO to ox intestine kinase:3, pI/Mw:5.97/27.3Kda
GenBank:ABB82940.1 specific cleavage site amino terminal sequence).Thus, the fusion protein F AMP for expressing to obtain is through ox
After enterokinase specificity digestion, you can obtain the antibacterial peptide PGQ of natural structure, play anti Helicobacter pylori activity.
Protease processing site peptide fragment in fusion protein F AMP, from the specific cleavage site aminoterminal of ox intestine kinase
Sequence, it the advantage is that：Enterokinase is the normal enzyme in digestion, beneficial to health, need not after recombination expression
Purifying, can be directly oral.
Fusion protein F AMP protein steric structural figure is as shown in Fig. 2 from Figure 2 it can be seen that digestion in fusion protein F AMP
Outside silkworm baculovirus core polyhedrin, antibacterial peptide PGQ is easy to carry out enzyme-specific to it outside extending in site
Cut, obtain the antibacterial peptide PGQ of natural structure.
Embodiment 4
The method that the present embodiment provides the fusion protein F AMP provided in a kind of specific expressed embodiment 3：
It is capable of the antibacterial peptide of low cost production using technique for gene engineering, is to solve antibacterial peptide to treat high this problem of cost
Effective way.What the report about antibacterial peptide gene engineering was more both at home and abroad is expressed in Escherichia coli and Pichia pastoris,
Although these documents provide technical method and directive function to probing into for antibacterial peptide, this kind of method has following defect：1. its
The product of expression is the product obtained after nonspecific protease digestion, and the antibacterial peptide of gained can not retain original natural knot
Structure.This will cause antibacterial peptide to be difficult to keep original antibacterial activity.2. the albumen using escherichia expression system expression is most
It is to exist in the form of inclusion body, it is necessary to which strict Purification just has bioactivity.In addition, Escherichia coli contain endogenous toxic material
Element, contain culture medium simultaneously, this allow for expressed albumen have to pass through it is strict after purification could be medicinal.It is 3. red using finishing
Carbon source during Yeast expression using methanol as culture medium, methanol are to have severe toxicity to human body, and synthetic product must enter tight to culture medium
The purifying of lattice can just take.These all considerably increase the production cost of antibacterial peptide, in helicobacter pylori n of high dose oral
Still reach to the effect less than low cost.
It is (including anti-come expressed fusion protein FAMP using baculoviral using silkworm as expression vector in the present embodiment
Bacterium peptide PEG).Strict host's parasitics of virus, invertebrate virus is safe to vertebrate people.Silkworm, it is a kind of
Medicine-food two-purpose insect (see《Chinese Pharmacopoeia》, 2015 editions).It is as follows using the advantage of this expression system：
1. silkworm individual is larger, it is of moderate size, easily operated, control, and growth period is short, albumen synthesis capability is strong.Silk
Composition is exactly mainly protein.In silkworm larva and pupa, the protein content of recombination expression is higher by the 10-100 of culture cell expression quantity
Times, the amount that pharmaceutical protein produces can greatly be improved.
2. the fat-body tissue of silkworm larva or pupa has the function of synthesizing and secrete lipoprotein and glycoprotein, to external source egg
It can be glycosylated in vain, phosphorylation, accurate protein folding and the correct disulfide bond of formation, expressed albumen are shown well
Biological activity, play inherent immunity effect alexin (being exactly that a kind of antibacterial peptide wherein contains more disulfide bond).
3. silkworm is with a history of thousands of years in raising and train for China, raising is experienced.Cost of material is low, is fed with mulberry leaf
Silkworm only 0.05 yuan/bar or so.To the direction that man-made feeds carry out sterileization, inexpensive, extensive whole year continuously raises
Development.
4. silkworm chrysalis itself is edible, ancient Chinese, which is bred silkworms, not takes silk but in order to edible.Contain egg in silkworm hemolymph
White enzyme inhibitor, there is stabilization to expression product.The protein of expression can be not required to purify, and be ground after silkworm is dried or is freeze-dried
Powder is broken into, can directly add food, be particularly suitable as the production of oral drugs.
5. baculovirus expression vector system marking protein, the technological process of production is uncomplicated, is adapted to several genes expression,
Replacement of products is easily most completed in 15 days cans soon, while the strong security of product.
Double-promoter expression system is used in the present embodiment, while expresses such as SEQ ID NO:Fusion protein shown in 4
FAMP and such as SEQ ID NO:Ox intestine kinase (Erk) shown in 3.Wherein, limited in fusion protein F AMP containing EcoR I and Xba I
Restriction enzyme site, contain Xho I and the restriction sites of Kpn I in ox intestine kinase.By encoding fusion protein FAMP gene and coding ox
After the Gene Exchange of enterokinase, then the two is cloned into pFastDual carriers simultaneously, as shown in Figure 3, you can obtain one kind
The recombinant plasmid of gene simultaneously comprising fusion protein F AMP and ox intestine kinase, is designated as pFastDual- (FAMP) (Erk).Simultaneously
The gene of encoding fusion protein FAMP gene and coding ox intestine kinase is cloned into pFastDual carriers respectively respectively, obtained
For the recombinant plasmid of control, pFastDual- (FAMP), pFastDual- (Erk) are designated as respectively.
By recombinant plasmid pFastDual- (FAMP) (Erk) obtained above, pFastDual- (FAMP), pFastDual-
(Erk) after identifying, convert into competent escherichia coli cell, specially DH10Bac E.coli.Synthesized in helper plasmids
Recombinase help it is lower recombinate, screen and obtain recombinant shuttle vector with identification, be respectively：Bacmid-(FAMP)(Erk)、
Bacmid-(FAMP)、Bacmid-(Erk)。
Again by recombinant shuttle vector Bacmid- (FAMP) (Erk) obtained above, Bacmid- (FAMP), Bacmid-
(Erk), transfect Bombyx noriN cell and obtain recombinant virus：BmNPV-(FAMP)(Erk)、BmNPV-(FAMP)、BmNPV-(Erk)
Virus.
Finally make recombinant virus BmNPV- (FAMP) (Erk), BmNPV- (FAMP) and BmNPV- (Erk) respectively in silkworm silkworm
Expressed in pupa, inoculation recombinant virus collects the silkworm hemolymph of above-mentioned three kinds of silkworm chrysalises after 96 hours.
Embodiment 5
The anti Helicobacter pylori activity for three kinds of silkworm hemolymphs that the present embodiment obtains to embodiment 4 is tested：
With the helicobacter pylori bacterium solution (10 of exponential phase8CFU/mL) it is evenly coated in sterile cotton balls containing 5% Sheep Blood
Helicobacter pylori agar medium on, 5mm aperture is beaten above, in each aperture plus 25 μ L pFastDual-
(FAMP) (Erk) silkworm hemolymph, normal silkworm silkworm hemolymph, pFastDual- (FAMP) silkworms hemolymph, pFastDual- (Erk)
Silkworm hemolymph, isolate and purify pFastDual- (FAMP) silkworm hemolymphs and pFastDual- (Erk) silkworms hemolymph obtain FAMP and
Antibacterial peptide PGQ (2 μ g/mL), the antibacterial peptide PGQ (2 μ g/mL) of chemical synthesis that digestion obtains after Erk, micro- lower 37 DEG C of aerobic condition
Cultivate 48h.
The test structure of three kinds of silkworm hemolymph anti Helicobacter pylori activities is as shown in Figure 4：
Wherein, (1) is pFastDual- (FAMP) (Erk) silkworm hemolymph；(2) it is normal silkworm silkworm hemolymph；(3) it is
PFastDual- (FAMP) silkworm hemolymph；(4) it is pFastDual- (Erk) silkworm hemolymph；(5) it is to isolate and purify pFastDual-
(FAMP) silkworm hemolymph and pFastDual- (Erk) silkworms hemolymph obtain antibacterial peptide PGQ (2 μ of digestion acquisition after FAMP and Erk
g/mL)；(6) it is the antibacterial peptide PGQ (2 μ g/mL) of chemical synthesis.
As seen from Figure 4, BmNPV- (FAMP) is expressed, BmNPV- (Erk) the silkworm hemolymph of silkworm pupa does not show
Go out anti Helicobacter pylori activity, it is primarily due to BmNPV- (FAMP) and BmNPV- (Erk) expression product is respectively
FAMP and Erk, it can not form the antibacterial peptide PGQ of natural structure.
And the silkworm hemolymph for expressing BmNPV- (FAMP) (Erk) silkworm pupa has anti Helicobacter pylori activity, this
Be primarily due to expression product existing FAMPs of the BmNPV- (FAMP) (Erk) in silkworm chrysalis has Erk, expressed FAMP to exist again
The antibacterial peptide PGQ of natural structure is obtained under the digestion of Erk protease, so as to play anti Helicobacter pylori activity.
To further determine that Erk digestions FAMP can obtain antibacterial peptide PGQ and have anti Helicobacter pylori activity.It is right
The silkworm hemolymph of BmNPV- (FAMP), BmNPV- (Erk) expression is purified using AKTA protein purifications system through row, is obtained pure
Fusion protein F AMP and ox intestine kinase Erk.To FAMP after purification and Erk in 37 DEG C and suitable buffer solution through row digestion,
The polypeptide of acquisition is purified in Tricine-SDS-PAGE analysis silkworm hemolymphs, as a result as shown in Figure 5.In Fig. 5, chemical synthesis it is more
Peptide M is Marker, and (1) is the FAMP that pFastDual- (FAMP) silkworm hemolymph isolates and purifies acquisition；(2) it is purifying in (1)
FAMP passes through the Erk protease enzyme restriction enzyme mappings obtained from the purifying of pFastDual- (Erk) silkworms hemolymph；(3) after for digestion in (2)
Isolate and purify the antibacterial peptide PGQ of acquisition；(4) it is the antibacterial peptide PGQ of chemical synthesis.As seen from Figure 5, FAMP is true after digestion
It is real to obtain the antibacterial peptide that there is identical molecular weight with the PGQ peptides of chemical synthesis, and the antibacterial peptide is through anti Helicobacter pylori activity
Identification, it has the PGQ peptide identicals anti Helicobacter pylori activity (MIC=1 μ g/mL) with chemical synthesis.
For the anti Helicobacter pylori activity of the obtained silkworm hemolymph of further checking, 100 infection BmNPV- are taken
(FAMP) the silkworm hemolymph of the silkworm of (Erk), determines its anti Helicobacter pylori activity, and with the antibacterial peptide PGQ of chemical synthesis
Compare, the anti Helicobacter pylori activity of silkworm hemolymph is suitable with 2 μ g/mL antibacterial peptide PGQ antibacterial activities, (5) seen in Fig. 4 and
(6) inhibition zone shown in.Thus illustrate that the silkworm hemolymph that present embodiment provides has anti Helicobacter pylori activity, can be used for
The disease caused by preventing and treating Helicobacter pylori infection is prepared, reduces relative gastritis, digestive tract ulcer, lymphoproliferative stomach
Lymthoma, the incidence of stomach cancer.
Stability test is carried out after silkworm hemolymph is freeze-dried, after storage 6 months, the work of its helicobacter pylori resistant
Property does not change.As can be seen here, the antibacterial peptide obtained by silkworm hemolymph can be stored for a long time, in addition, silkworm hemolymph can be straight
Interface takes.Therefore, this method can directly obtain antibacterial peptide PGQ with BmNPV- (FAMP) (Erk) in silkworm and have anti-pylorus
Pylori active, can be directly oral without purifying, can greatly reduce the production cost of antibacterial peptide.
Although illustrate and describing the present invention with specific embodiment, but will be appreciated that without departing substantially from the present invention's
Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
SEQUENCE LISTING
<110>Anhui Science and Technology College
<120>A kind of preparation method of the antibacterial peptide with anti Helicobacter pylori activity
<160> 4
<170> PatentIn version 3.5
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Gly Val Leu Ser Asn Val Ile Gly Tyr Leu Lys Lys Leu Gly Thr Gly
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Arg Lys Lys His Leu Ile Glu His Glu Lys Glu Glu Lys Gln Trp Asp
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Leu Leu Asp Asn Tyr Met Val Ala Glu Asp Pro Phe Leu Gly Pro Gly
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Lys Asn Gln Lys Leu Thr Leu Phe Lys Glu Val Arg Asn Val Lys Pro
65 70 75 80
Asp Thr Met Lys Leu Ile Val Asn Trp Ser Gly Lys Glu Phe Leu Arg
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Glu Thr Trp Thr Arg Phe Val Glu Asp Ser Phe Pro Ile Val Asn Asp
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Pro Asn Arg Cys Tyr Lys Phe Leu Ala Gln His Ala Leu Arg Trp Asp
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Tyr Ile Gly Thr Asp Ser Ala Glu Glu Glu Glu Ile Leu Ile Glu Val
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Ser Leu Val Phe Lys Ile Lys Glu Phe Ala Pro Asp Ala Pro Leu Phe
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Asn Glu Lys Cys Gln Gln Gln Met Pro Glu Tyr Asn Ile Thr Glu Asn
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Ser Asp Asp Asp Asp Lys Gly Val Leu Ser Asn Val Ile Gly Tyr Leu
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Lys Lys Leu Gly Thr Gly Ala Leu Asn Ala Val Leu Lys Gln
290 295 300
Claims (10)
1. a kind of preparation method of the antibacterial peptide with anti Helicobacter pylori activity, it is characterised in that it includes：
Structure coding includes SEQ ID NO:The nucleotide sequence of the fusion protein of antibacterial peptide shown in 1；
Structure includes the recombinant vector of the nucleotide sequence；
The recombinant vector is used for conversion or transfection host cell, and the nucleotides sequence is listed in table in the host cell
Reach.
2. the preparation method of antibacterial peptide according to claim 1, it is characterised in that the fusion protein has one level below
Amino acid sequence structure：
Carrier protein-connection peptide-protease processing site peptide fragment-antibacterial peptide；
The antibacterial peptide has SEQ ID NO:Amino acid sequence shown in 1.
3. the preparation method of antibacterial peptide according to claim 2, it is characterised in that it is shaft-like that the carrier protein includes silkworm
Viral core polyhedrin, glutathione sulfydryl transferase, green fluorescent protein or ubiquitin sample little albumen.
4. the preparation method of antibacterial peptide according to claim 2, it is characterised in that the protease processing site peptide fragment is
The action site of specific proteins restriction endonuclease, the specific proteins restriction endonuclease include enterokinase, fibrin ferment, factor Xa,
PreScission protease, Tobacco mosaic virus enzyme, ERC group virus's protease or ubiquitin sample little albumen.
5. the preparation method of antibacterial peptide according to claim 2, it is characterised in that the protease processing site peptide fragment bag
Include DDDDK, LVPRGS, IEGR, IDGR, LEVLFQ, ENLYFQG, LEVLFQG or ubiquitin-like protein tertiary structure.
6. the preparation method of antibacterial peptide according to claim 1, it is characterised in that the recombinant vector includes restructuring matter
Grain, the expression vector of the recombinant plasmid is pFastDual carriers.
7. the preparation method of antibacterial peptide according to claim 6, it is characterised in that the recombinant vector is also worn including restructuring
Shuttle carrier, the recombinant shuttle vector are by the recombinant plasmid transformed into competent escherichia coli cell, and in recombinase
In the presence of the obtained restructuring Bacmid of restructuring occurs.
8. the preparation method of antibacterial peptide according to claim 7, it is characterised in that the recombinant vector also includes restructuring disease
Poison, the recombinant virus are made by the way that the recombinant shuttle vector is transfected into silkworm BmN cells.
9. the preparation method of antibacterial peptide according to claim 8, it is characterised in that the host cell is silkworm chrysalis cell.
10. the preparation method of antibacterial peptide according to claim 5, it is characterised in that be listed in institute making the nucleotides sequence
State in host cell and express, after obtaining fusion protein, in addition to：Enzyme is carried out to the fusion protein with specific proteins restriction endonuclease
Cut.
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Cited By (2)
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|---|---|---|---|---|
| CN110256537A (en) * | 2019-06-14 | 2019-09-20 | 华中科技大学同济医学院附属同济医院 | Bifunctional polypeptides and its application with helicobacter pylori resistant and inhibition inflammatory factor |
| CN112079915A (en) * | 2020-09-14 | 2020-12-15 | 四川昕泰科技有限公司 | Polypeptide and preparation method and application thereof |
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| WO2017004591A2 (en) * | 2015-07-02 | 2017-01-05 | Dana-Farber Cancer Institute, Inc. | Stabilized anti-microbial peptides |
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| WO2017004591A2 (en) * | 2015-07-02 | 2017-01-05 | Dana-Farber Cancer Institute, Inc. | Stabilized anti-microbial peptides |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110256537A (en) * | 2019-06-14 | 2019-09-20 | 华中科技大学同济医学院附属同济医院 | Bifunctional polypeptides and its application with helicobacter pylori resistant and inhibition inflammatory factor |
| CN110256537B (en) * | 2019-06-14 | 2022-10-11 | 华中科技大学同济医学院附属同济医院 | Bifunctional polypeptide with helicobacter pylori resistance and inflammation factor inhibition functions and application thereof |
| CN112079915A (en) * | 2020-09-14 | 2020-12-15 | 四川昕泰科技有限公司 | Polypeptide and preparation method and application thereof |
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