CN110128548A - One kind, which has both, adjusts immune, anti-oxidant, anti-inflammatory and multi-functional hybrid peptide of removing toxic substances and the preparation method and application thereof - Google Patents
One kind, which has both, adjusts immune, anti-oxidant, anti-inflammatory and multi-functional hybrid peptide of removing toxic substances and the preparation method and application thereof Download PDFInfo
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- CN110128548A CN110128548A CN201910435730.7A CN201910435730A CN110128548A CN 110128548 A CN110128548 A CN 110128548A CN 201910435730 A CN201910435730 A CN 201910435730A CN 110128548 A CN110128548 A CN 110128548A
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- hybrid peptide
- inflammatory
- peptide
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Abstract
The present invention relates to a kind of have both to adjust immune, anti-oxidant, anti-inflammatory and function of detoxification hybrid peptide and the preparation method and application thereof.Hybrid peptide provided by the invention is named as LTP, obtains for antimicrobial peptide LL-37 and thymopeptide-5 TP5 through protein engineering Computer Design, heterozygosis, optimization, inside and outside secondary screening, amino acid sequence is as shown in SEQ ID NO.1.Hybrid peptide LTP two-way immunoregulatory activity with higher, can enhance normal or immunosuppressive condition lower body immune function, and protection immunosupress is damaged caused by body;Under inflammatory conditions, the oxidation reaction and inflammatory reaction that LTP can also neutralize resolution endotoxin, inhibit body, alleviate damage of the inflammatory reaction to tissue, and have cytotoxicity low, highly-safe, preparation method is simple, the advantages such as low in cost, it can be used as ideal immune, anti-oxidant, anti-inflammatory regulator or anti-oxidant solution phallotoxins be immunized, there is good application potential and value.
Description
Technical field
The present invention relates to genetic engineering and field of biological, and in particular to one kind has immunological regulation, anti-oxidant, anti-inflammatory
Anti-oxidant removing toxic substances hybrid peptide and the preparation method and application thereof is immunized with resolution function.
Background technique
Animals or humans young age, weak, disease, stress, pathogen infection when, frequently result in immunity reduction,
And then secondary infection (bacterium or virus mixed infection) occurs, cause inflammatory reaction (red, hot, swollen, pain etc.).Currently, for sense
Dye and inflammation treatment, tradition again the control strategy that generally uses be use antibiotic and hormone anti-inflammatory drugs (such as hydrocortisone,
Dexamethasone etc.), though can be effectively controlled infective inflammation and non-infectious inflammation, a variety of side effects can be caused by continuing use:
As water-electrolyte metabolism and sugar, fat, protein metabolism serious diseases, and cause adrenal cortex function decline, digestive system simultaneously
Send out disease or aggravate infection etc..In addition, antibiotic health care growth accelerator is widely used in animal husbandry.In infection and inflammation treatment
It is middle there is a problem of using antibiotic or hormone anti-inflammatory drugs it is obvious:, cannot although antibiotic can reduce or kill pathogen
Improve the immune function of body, on the contrary, being also possible to generate endotoxin or exotoxin by the germ that antibiotic kills, and then aggravates scorching
Disease reaction even results in systemic inflammatory response syndrome (Botwinski, 2001), gently then causes animal fever, anorexia, internal
Energy consumes excessively, body tissue is decomposed, immunity and production performance decline, it is heavy then may cause livestock and poultry death (Botwinski,
2001;Zinner,1999).A large number of studies show that, many antibiotic can also promote endogenous toxic material while killing bacterium in recent years
Element be lipopolysaccharides (lipopolysaccharide, LPS) released from bacterial cell membrane (Holzheimer, 2001;
Hurley, 1995), and then LPS is caused largely to assemble, cause inflammatory reaction.Endotoxin LPS usually by enteropathogenic E. Coli,
The cell disruption of the Gram-negative bacterias such as Salmonella, Brucella, proteus, swine flu and haemophilus parasuis produces
It is raw, the release of a variety of body proinflammatory cytokines, such as TNF-α, interleukin 6 (IL-6) and IL-1 β can be induced;Can also simultaneously
Induction, which generates a large amount of free radicals, leads to oxidative damage, and then reduces immunity.Therefore, endotoxin LPS is reduced or eliminated, and is passed through
Anti-oxidation function reduces body oxidative damage, reduces or eliminates infected animal or the inflammatory reaction of people.In conclusion current is anti-
All there is obvious shortcoming in the anti-infective and glucocorticoid anti-inflammatory drugs of raw element, should not continue to use.Therefore, develop it is a kind of it is novel,
It is safe, without side-effects, environmentally friendly and be provided simultaneously with immunological regulation, anti-oxidant, anti-inflammatory properties and the endotoxic active peptide of resolution, to people
Class or animal farming industry have important practical significance and huge application prospect, are new breakthrough and the Xin Li of anti-infective strategy
It reads.
Immune system can protect body from the invasion of external microorganism by immune defense function, at the same can and
When remove the cell of internal aging and canceration.On the one hand, when immune function is lower than normal level, body is easily infected and lures
The generation for sending out malignant tumour etc., to keep the aggravation of patient difficult to treat;But on the other hand, when being immunoreacted excessive
It can cause animal or the strong inflammatory reaction of human body, lead to body injury and each physiological system dysfunction, and then influence body
Normal metabolic process, serious person's even threat to life.It can be seen that it is anti-inflammatory with it is immune be function of immune system two sides
Face, it is closely related, it is indivisible, it is sometimes even overlapped.Therefore, it in traditional medicine field, by anti-inflammatory agent and is immunized
It is not meet body actual needs or anti-inflammatory and immune interaction relationship, and considerably increase that it is well-separated, which to enhance medicine,
Clinic selects the complexity and Antagonism of medicine.In view of the foregoing, preparation safe and efficient, with two-way immunoloregulation function is developed
Or drug is of great significance to improve the immunity function of animal and human body.
Antimicrobial peptide LL-37 is the polypeptide of relative molecular mass about 5000Da (dalton), finds in human body so far
Uniquely with the antibacterial of amphiphilic αhelix in unique member and human body in antibacterial peptide (cathelicidin) family
Peptide.LL-37 is widely distributed in the blood cell and epithelial cell of human body, is had and is neutralized endotoxic effect, can with LPS and
CD14 is combined, and neutralizes the bio-toxicity of LPS;Chemotaxis can be mediated, immunocyte is recruited and reaches infection site, remove cause of disease
Object;And the effects of promoting angiogenesis, it is the anti-inflammatory peptides of a kind of research comparative maturity.
The mankind start from the research of immune-active peptides 1981, and Jolles etc. is separated from people lactoprotein's hydrolysate for the first time
It obtains a kind of with immunocompetent peptide fragment.Hereafter, scientists have carried out the research in terms of a large amount of immune-active peptides,
In, it is more with thymic peptide research report, it is also more deep.Thymopeptide-5 (TP5), the i.e. activated centre of thymopoietin II, tool
There is biological sexology activity similar with thymopoietin, it can the unbalance immune function of bidirectional modulation, Cell differentiation inducing activity, promotion leaching
Bar cell subsets is developed and is activated, and is a kind of important immunomodulator.
As structure, function and the Study on mechanism to anti-inflammatory peptides and mucin peptide deepen continuously, scientific research personnel
Begin trying, adjusting activity stronger two-way immunomodulatory peptides higher using protein engineering method design safety.Have and grinds
Report is studied carefully by being to obtain Multifucntional polypeptide or by different immune-active peptides and anti-by different types of polypeptide progress heterozygosis
Scorching peptide heterozygosis obtains the anti-inflammatory adjusting drug of novel immune.
Summary of the invention
In order to solve the problems in the existing technology, it is had both the object of the present invention is to provide one kind and adjusts immune, antioxygen
Change, the anti-inflammatory and multi-functional hybrid peptide and the preparation method and application thereof that detoxifies
To achieve the above object, technical scheme is as follows: the present invention is to polypeptide LL-37's and thymic peptide TP5
On the basis of the relationship of sequence, structure and sequential structure and function carries out numerous studies, with Protein Molecular Design technology
Carry out polypeptide LL-37 (amino acid sequence is as shown in SEQ ID NO.3) and thymopeptide-5 TP5 (amino acid sequence such as SEQ ID
Shown in NO.4) heterozygosis optimization it is miscellaneous finally to be obtained by the screening and sequence optimisation to hybrid peptide for a kind of novel immune detoxification
Peptide is closed, is named as LTP, amino acid sequence is as shown in SEQ ID NO.1.Hybrid peptide LTP has the function there are two source of parents peptide simultaneously
Can, that is, there is two-way immunoloregulation function: under normal or immunosuppressive condition, can be improved the immune function of body;In inflammation
Under disease or endotoxin existence, LTP can also anti-oxidant, resolution endotoxin and body inflammatory is inhibited to react.
Firstly, the amino acid sequence of immune detoxification hybrid peptide LTP provided by the invention as shown in SEQ ID NO.1 or is
What replacement, missing or insertion of the amino acid sequence as shown in SEQ ID NO.1 through one or more amino acid obtained has phase
The amino acid sequence of congenerous polypeptide.
It is above-mentioned be transformed on the basis of the amino acid sequence as shown in SEQ ID NO.1 acquisition have identical function
The derived peptides of hybrid peptide LTP include but is not limited to following polypeptide:
(1) it is obtained in C-terminal or N-terminal addition the protein tag sequence of the amino acid sequence as shown in SEQ ID NO.1 more
Peptide, such as: the His label containing 6 His residues is added in the C-terminal or N-terminal of the amino acid sequence as shown in SEQ ID NO.1
Obtained polypeptide;Or it is obtained in C-terminal or N-terminal the addition GST or C-Myc label of the amino acid sequence as shown in SEQ ID NO.1
Polypeptide;
It should be appreciated by those skilled in the art that being added to realize the purpose of being easy to purifying, polypeptide marker at the both ends of polypeptide
Sequence label is ordinary skill in the art means, can't be impacted to the function of polypeptide inherently and activity, therefore,
The above-mentioned LTP derivative obtained in the both ends of the hybrid peptide LTP as shown in SEQ ID NO.1 addition sequence label is also in the present invention
Protection scope in.
(2) the conservative ammonia of one or more amino acid sequences is carried out in the amino acid sequence as shown in SEQ ID NO.1
The polypeptide that base acid is replaced, such as: the 15th Leu, which is replaced with Ile, to cause material change to the function of albumen.
The present invention also provides have immunological regulation, anti-oxidant, resolution endotoxin and anti-inflammatory properties hybrid peptide described in coding
Gene.
In the case where the amino acid sequence of known hybrid peptide LTP, those skilled in the art can be according to for polypeptide table
The needs reached, for the Preference that uses of codon, design has different for degeneracy principle and different plant species based on codon
The encoding gene of the hybrid peptide LTP of nucleotide sequence.
As one embodiment of the present invention, the nucleotide sequence of the gene is as shown in SEQ ID NO.2.Such as SEQ
Gene shown in ID NO.2 is the hybrid peptide LTP encoding gene designed according to the codon preference of Pichia pastoris.With SEQ ID
NO.2 has the gene order of the coding identical function albumen of at least 80%, 85%, 90%, 95%, 98% or 99% homology
Also belong to protection scope of the present invention.
Further, the present invention also provides the biomaterial for containing the heterozygosis DNA encoding peptide, the biomaterial packets
Include recombinant DNA, expression cassette, transposons, plasmid vector, phage vector, viral vectors or host cell.
The host cell includes animal and plant cells or cell line, microbial cell.
Further, the present invention provides the preparation method of the hybrid peptide, comprising: leads the gene for encoding the hybrid peptide
Enter in host cell, expresses the hybrid peptide.
Preferably, the preparation method includes: to connect the encoding gene of the hybrid peptide LTP with expression vector, structure
Recombinant expression carrier is built, by transgenic method, recombinant expression carrier is imported into host cell, obtains importing LTP encoding gene
Host cell.
The transgenic method includes heat stress conversion, electrotransformation, transfection etc..
The host cell includes but is not limited to animal and plant cells, microbial cell.
Preferably, the host cell is yeast, more preferably Pichia pastoris.
When using Pichia pastoris as host cell, such as using the sequence optimized through Pichia pastoris codon preference
Encoding gene shown in SEQ ID NO.2 expresses the hybrid peptide, has more preferably expression quantity.
As one embodiment of the present invention, the preparation of the hybrid peptide is host with Pichia pastoris, with expression vector
PPICZ α A is carrier, expresses hybrid peptide by methanol induction, specifically comprises the following steps:
(1) encoding gene of hybrid peptide LTP is connected to expression vector pPICZ α A, constructs recombinant expression carrier;
(2) above-mentioned recombinant expression carrier is converted into Pichia pastoris GS115, building imports hybrid peptide LTP encoding gene
Recombination engineering;
(3) above-mentioned recombination engineering is cultivated, the expression of methanol induction hybrid peptide LTP is added;
(4) culture solution supernatant is collected, purifying obtains hybrid peptide LTP.
The present invention, which passes through test in vivo and in vitro, to be proved, it is normal and immune scarce that hybrid peptide LTP can not only improve body
Immunocompetence under the state of falling into, improves the expression quantity of cell factor, promotes mouse growth, alleviate immune deficiency to mouse spleen with
And it is injured caused by thymus gland;And the inflammatory reaction of LPS induction can be inhibited during inflammatory reaction, enhance the anti-of body
Oxidability reduces cytokine-expressing amount, alleviates inflammatory conditions and injures caused by mouse weight and enteron aisle, has good
Anti-inflammatory dual regulation is immunized.
Based on above-mentioned function, the present invention hybrid peptide is provided or the hybrid peptide that is prepared using the preparation method or
The encoding gene of the hybrid peptide or biomaterial containing the heterozygosis DNA encoding peptide are in preparing immunomodulator
Using.
Preferably, the immunomodulator is immunopotentiator.
The present invention also provides the hybrid peptide or the hybrid peptides or the hybrid peptide that are prepared using the preparation method
Encoding gene or biomaterial containing the heterozygosis DNA encoding peptide preparing anti-inflammatory preparation or resolution/antiendotoxin preparation
In application.
Preparation of the present invention includes drug, health care product and food or feed addictive.
Above-mentioned immunopotentiator can be used for a variety of including the immunity of organism caused by cyclophosphamide (CY) inhibits reaction
The immunosuppressive prevention and treatment of type.
Above-mentioned anti-inflammatory preparation or resolution endotoxin preparation can be used for the inflammation including the inflammatory reaction that LPS is induced
Or the prevention and treatment of endotoxemia.
The present invention also provides a kind of product, the product is drug, health care product and food or feed addictive, the production
Product include the hybrid peptide TP5 or the hybrid peptide comprising using the preparation method of above-mentioned hybrid peptide to be prepared.
In the product, other active constituent groups can be compounded using the hybrid peptide as effective component or by the hybrid peptide
At the effective component of drug, health care product and food or feed addictive.
Preferably, described pharmaceutical composition also includes the receivable carrier of pharmaceutical field or auxiliary material.
The beneficial effects of the present invention are: first passage of the present invention obtains LL-37 and TP5 heterozygosis, optimized and screening
Anti-inflammatory hybrid peptide LTP is immunized, polypeptide LTP has the function there are two source of parents peptide simultaneously, that is, there is two-way immunoloregulation function, and
And compared with the corresponding activity of source of parents peptide LL-37 and TP5, immunoregulatory activity, antioxidant activity, anti-inflammatory activity and resolution
Activity of endotoxin is stronger: under normal or immunosuppressive condition, can significantly improve the immune function of body, protect immunosupress
It is damaged caused by body;Under inflammatory conditions, LTP may also suppress body inflammatory reaction, alleviate inflammatory reaction to the damage of tissue
Wound;LTP has cytotoxicity low, highly-safe simultaneously, prepares convenient, low-cost advantage, can be used as preferably immune adjust
Agent, antioxidant, anti-inflammatory agent, endotoxin antidote are saved, medicine, food, health care, the feed, nutrition of humans and animals are widely used in
Equal fields, have huge application value.
The preparation method of hybrid peptide provided by the invention, it can be achieved that hybrid peptide LTP a large amount of, efficient preparation, it is obtained miscellaneous
Closing peptide LTP has two-way immunological regulation and resolution endotoxin and antioxidant activity, and without obvious toxic-side effects.
Detailed description of the invention
Fig. 1 is the molecular docking figure of candidate hybrid peptide in the embodiment of the present invention 1, wherein A figure is hybrid peptide molecular docking 3D
Simulation drawing, B figure are energy (positive value representative absorption energy, the negative value representative that hybrid peptide is absorbed or discharged during molecular docking
It releases energy).
Fig. 2 is the building flow chart of recombinant expression carrier pPICZ α A-LTP in the embodiment of the present invention 1.
The PCR that Fig. 3 is recombinant expression carrier pPICZ α A-LTP in the embodiment of the present invention 1 identifies gel electrophoresis result,
In, M is DNA molecular amount standard;Swimming lane 1-3 is the target fragment comprising polypeptide LTP encoding gene.
Electrophoresis and the mass spectrum inspection that Fig. 4 is the hybrid peptide LTP of the Pichia yeast engineering expression purified in the embodiment of the present invention 1
It surveys as a result, A is SDS-PAGE electrophoresis detection result, wherein M is protein molecular weight standard;Swimming lane 1-2 is methanol induction 144h
The band of the purified destination protein LTP of fermented supernatant fluid;B is the Mass Spectrometer Method result figure of purification of hybrid peptide LTP.
Fig. 5 is hybrid peptide LTP and its source of parents peptide LL-37, TP5 in the embodiment of the present invention 2 to the neutralization activity of LPS, wherein
LTP, LL-37 and TP5 respectively represent polypeptide LTP, LL-37 and TP5, and PMB represents polymyxin B.
Fig. 6 is hybrid peptide LTP and its source of parents peptide LL-37, TP5 in the embodiment of the present invention 3 to the cell of mouse macrophage
The influence of survival rate.
Fig. 7 be in the embodiment of the present invention 4 hybrid peptide LTP to the cytokine-expressing amount of mouse macrophage RAW264.7
It influences;Wherein, A figure is the expression quantity of TNF-α;B figure is the expression quantity of IFN-γ;Control represents normal group, and LPS is represented
LPS induces the model group of inflammation, and LL-37 represents test group 1 (LL-37 processing is added under normal condition), and TP5 represents test group 2
(TP5 processing is added under normal condition), LTP represent test group 3 (LTP processing is added under normal condition), and LL-37+LPS is represented
Test group 4 (is added after LL-37 processing and induces inflammatory model with LPS), and TP5+LPS represents test group 5 and (uses after TP5 processing is added
LPS induces inflammatory model), LTP+LPS represents test group 2 (LPS, which is induced, is added LTP processing under inflammatory conditions);Both * represent
Comparing has significant difference (p < 0.5), and * *, which both represents to compare, extremely significant sex differernce (p < 0.01).
Fig. 8 A- Fig. 8 F is respectively the immunosuppressed mice that hybrid peptide LTP induces cyclophosphamide in the embodiment of the present invention 5
Immunoregulation effect;Wherein, Fig. 8 A is influence of the LTP to immunosuppressed mice weight;Fig. 8 B is LTP immunosuppressed mice spleen
Influence;Fig. 8 C is influence of the LTP to immunosuppressed mice thymus gland;Fig. 8 D is shadow of the LTP to mouse macrophage phagocytic activity
It rings;Fig. 8 E is influence of the LTP to mouse cytokine IFN-γ burst size;Fig. 8 F is that LTP discharges mouse cytokine IL-6
The influence of amount;Control represents blank group, CY representative model group, and LTP+CY represents test group;* representing has with blank control group
Significant difference (p < 0.5), #, which is represented, has significant difference (p < 0.5) with model group.
Fig. 9 A- Fig. 9 E is hybrid peptide LTP in the embodiment of the present invention 6 to the inhibiting effect of mouse inflammatory reaction;Wherein, Fig. 9 A
Influence for LTP to mouse weight;Fig. 9 B is the influence of LTP mouse intestinal length;Fig. 9 C is that LTP is complete to mouse intestinal tissue
Property influence;Fig. 9 D is influence of the LTP to mouse cytokine IFN-γ burst size;Fig. 9 E is LTP to mouse cytokine IL-6
The influence of burst size;Control represents blank group, LPS representative model group, and LTP+LPS represents test group;* it represents and blank pair
Have significant difference (p < 0.5) according to group, #, which is represented, has significant difference (p < 0.5) with model group.
Figure 10 A- Figure 10 C is hybrid peptide LTP in the embodiment of the present invention 7 to the antioxidation of mouse;Wherein, Figure 10 A is
Influence of the LTP to mice serum MDA level;Figure 10 B is LTP on the active influence of mice serum SOD;Figure 10 B is LTP to small
The active influence Control of mouse change of serum C AT represents blank group, LPS representative model group, and LTP+LPS represents test group;* represent with
Blank control group has significant difference (p < 0.5), and #, which is represented, has significant difference (p < 0.5) with model group.
Specific embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real
Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field
Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified, wherein big
Enterobacteria competent cell Top 10, Pichia pastoris GS115 and expression vector pPICZ α A are purchased from Invitrogen company.
The preparation of anti-inflammatory hybrid peptide LTP is immunized in embodiment 1
1, the acquisition of heterozygosis peptide sequence
It is ground by the relationship of sequence, structure and sequential structure to polypeptide LL-37 and thymopeptide-5 TP5 and function
Study carefully, using Protein Molecular Design technology carry out polypeptide LL-37 and thymopeptide-5 TP5 heterozygosis, obtain candidate hybrid peptide LTP,
TP5-LL-37 and LL-37-TP5 ', amino acid sequence is respectively such as SEQ ID NO.1, SEQ ID NO.5, SEQ ID NO.6
It is shown.Myeloid differentiation protein-2 (MD-2) assigns TLR4 to the reactivity of various ligands (including LPS) in conjunction with TLR4;Secondly,
MD-2 can promote the expression of TLR4 and TLR2, and closely related with the distribution of TLR4 in the cell.Thus MD-2 is not only
The accessory molecule of TLR4, but also be the regulatory molecule in the innate immunity, it may be in the pathologic, physiologics mistake such as infection, inflammation, immune
There is wider biological function in journey.The marking situation of this research and utilization itself and MD-2 molecular docking, to its anti inflammatory immunity
Effect is predicted that as a result as shown in Figure 1, three kinds of candidate hybrid peptides can release energy in conjunction with MD-2, but LTA discharges energy
Amount is more, and it is more stable to illustrate that it is combined, and has better anti inflammatory immunity activity, so finally obtaining hybrid peptide LTP, amino
Acid sequence is as shown in SEQ ID NO.1.
2, recombinant expression carrier constructs
According to hybrid peptide LTP (abbreviation polypeptide LTP) amino acid sequence and Pichia pastoris codon preference, design and synthesize more
It connect by the encoding gene (sequence is as shown in SEQ ID NO.2) of peptide LTP with expression vector pPICZ α A, conversion to large intestine bar
10 competent cell of bacterium Top, constructs recombinant expression carrier pPICZ α A-LTP, and vector construction process is as shown in Figure 2.
Plasmid is extracted according to the operating method of the small extraction reagent kit of Tiangeng biochemical technology (immune) Co., Ltd's plasmid, to extract
Plasmid as template carry out PCR amplification LTP gene,
Upstream primer: 5 '-GGTACCATTGGTAAGGAATT-3 ';Downstream primer: 5 '-
GATGATGATGAGTAACATCC-3′.Amplified production is subjected to agarose gel electrophoresis, is observed under ultraviolet lamp, identification conversion knot
Fruit, as shown in figure 3, there are LTP gene purpose bands (120bp) in display amplified production.Recombinant plasmid is sequenced into one
Whether the insertion of step identification target fragment is correct, and examines the fidelity of Insert Fragment.Recombinant expression carrier is confirmed through sequencing
PPICZ α A-LTP is constructed successfully.
3, the preparation of Pichia pastoris competent cell
The single colonie of Pichia pastoris GS115 is inoculated in 3-5ml YPD (2% tryptone, 2% glucose, 1% yeast
Powder, pH 7.0) in fluid nutrient medium, in 30 DEG C shaken cultivation 12 hours or so, be inoculated in by the volume ratio of 1:100-1:50
In 100ml YPD fluid nutrient medium, in 30 DEG C of shaken cultivations to OD600For 1.3-1.5 or so, stop culture.Culture solution is transferred to
In centrifuge tube, places on ice 10 minutes, be then centrifuged 5 minutes collection thallus in 4 DEG C, 1500 × g.Pre-cooling is added into thallus
Sterile water gently suspension cell, after placing 15-30 minute on ice, in 4 DEG C, 1500 × g centrifugation 5 minutes.It discards supernatant, is added
The sterile sorbitol solution (1M) of 10ml pre-cooling, is resuspended thallus, obtains competent cell suspension.Every 200 μ l of pipe is distributed into standby
Pichia pastoris GS115 competent cell, immediately using or be stored in -70 DEG C.
4, the building of the Pichia yeast engineering of hybrid peptide LTP is expressed
Above-mentioned steps 3 are constructed to obtained recombinant expression carrier pPICZ α A-LTP and convert Pichia pastoris GS115 bacterial strain, tool
Body method is as follows:
Linearized recombinant plasmid pPICZ alpha A-LTP 5-10 μ g and 200 μ l has activated the impression of Pichia pastoris GS115
State mixing with cells, moves into electric revolving cup, ice bath 5-30min, and electricity turns several seconds (electricity never vibrates during turning) after ice bath, then
Mixed liquor is placed in rapidly the 1M sorbitol solution for being rapidly added 1ml ice bath on ice, so that thallus is suspended and is mixed and goes to 1.5ml
It in EP pipe, is placed on ice, 2ml YPD fluid nutrient medium is then added, 30 DEG C of culture 3h make bacterial strain recover and form resistance.To
Recombinant bacterium takes 200 μ l culture solutions to be coated on the YPDS plate containing 100 μ g/ml kanamycins after forming resistance, is then inverted plate
It is incubated overnight in 30 DEG C.It selects the single colonie grown on above-mentioned YPDS plate and is inoculated in the YPD liquid containing 100 μ g/ml kanamycins
It is incubated overnight in body culture medium, mentions middle amount kit extraction plasmid using plasmid is small, then pass through PCR and verify identification positive transformants
Son obtains the Pichia yeast engineering of expression hybrid peptide LTP.
5, the inducing expression of hybrid peptide LTP
The Pichia yeast engineering for selecting expression hybrid peptide LTP is inoculated in the BMGY culture containing 100 μ g/ml kanamycins
In base, 30 DEG C, 180-200rpm shaking table culture to OD600When for 2-6, low-speed centrifugal collects thallus;Bacterium is resuspended with BMMY culture medium
Body is to OD600It is 1,30 DEG C, 200rpm shaken cultivation.During shaken cultivation, every addition methanol for 24 hours to final concentration of 5%
Carry out the inducing expression of polypeptide LTP.2ml bacterium solution is taken in methanol induction 144h, 12000rpm is centrifuged 5min, after collecting supernatant
It is placed in -20 DEG C of preservations.The culture supernatant of collection is subjected to Tricine-SDS-PAGE electrophoresis detection, electrophoresis result is shown, is trained
It supports and contains polypeptide LTP in supernatant, polypeptide LTP successful expression, the expression quantity of polypeptide LTP can reach 46mg/L or so after measured.
6, the purifying of hybrid peptide LTP
When synthesizing the encoding gene of LTP, it is added to histidine tag (6 × His) in its C-terminal, therefore, polypeptide LTP can
With the Ni in Ni-NTA Sepharose chromatographic column2+In conjunction with being washed using various concentration imidazole elution to fermented supernatant fluid
It takes off and collects eluting peak sample, it can be achieved that polypeptide LTP's isolates and purifies.The Ni-NTA Sepharose chromatographic column of polypeptide LTP is pure
Change method collects each eluting peak sample referring to chromatographic column product operation instruction, pure by Tricine-SDS-PAGE electrophoresis detection
Change effect, the molecular size range of LTP is 3.6kDa, as a result as shown in the A of Fig. 4, the results showed that, it is purified obtain purity compared with
High polypeptide LTP;The Mass Spectrometer Method result of the LTP of purifying is as shown in the B of Fig. 4, the results showed that, its molecular weight of purified polypeptide with
The theoretical molecular weight of LTP is consistent.The present invention contains the LTP nucleotide sequence that restriction enzyme site terminator and N-terminal contain His label
As shown in SEQ ID NO.7.
Neutralization of the 2 hybrid peptide LTP of embodiment to LPS
It with water is different by hybrid peptide LTP and its source of parents peptide LL-37, TP5 dissolved dilution using no heat source tiny electrolytic cell
The solution (0-64 μ g/mL) of concentration, takes the LTP polypeptide solution of above-mentioned each concentration of 100 μ L to mix with LPS (1EU/mL) respectively.
After 37 DEG C of incubation 30min, using colour developing mechanism tachypleus amebocyte lysate box detection polypeptide LTP, LL-37, TP5 to the neutralization ratio of LPS, with mostly viscous
Rhzomorph B (PMB) is as control.As a result as shown in figure 5, polypeptide LTP LPS neutralization activity with higher, LPS neutralization activity
It is suitable with polymyxin B, concentration be 8 μ g/mL when, hybrid peptide LTP to the neutralization ratio of LPS close to 100%, and its neutralize live
Property is significantly higher than its source of parents peptide LL-37 and TP5.
Influence of the 3 hybrid peptide LTP of embodiment to mouse macrophage cell survival rate
The macrophage RAW264.7 of logarithmic growth phase is inoculated in 96 orifice plates, and initial cell culture density is 1 × 104
A/mL, every 100 μ L of hole, at 37 DEG C, 5%CO2Under conditions of after overnight incubation, be separately added into a series of concentration gradients LTP,
LL-37 and TP5 (0-100 μ g/mL) solution after culture for 24 hours, survive to mouse macrophage using CCK8 method detection polypeptide LTP
The influence of rate.As a result as shown in fig. 6, the cytotoxicity of hybrid peptide LTP is significantly reduced compared with its source of parents peptide LL-37, and in 0-100 μ
The survival rate of macrophage is greater than 80% in g/mL concentration range, shows that the cytotoxicity of LTP is lower, safety with higher
Property.
Immunoregulatory activity of the 4 hybrid peptide LTP of embodiment in mouse macrophage
Hybrid peptide LTP and its source of parents peptide LL-37, TP5 DMEM culture medium is diluted, compound concentration is the more of 10 μ g/mL
Peptide solution, detection hybrid peptide LTP and its source of parents peptide LL-37, TP5 is under normal condition and under the inflammatory conditions of LPS induction
The influence of the cytokine secretions such as TNF-α, the IFN-γ of mouse macrophage RAW264.7.It is respectively set normal group
(Control), model group (LPS), test group 1 (LL-37), 4 (LL-37 of test group 2 (TP5), test group 3 (LTP) and test group
+ LPS), test group 5 (TP5+LPS), test group 6 (LTP+LPS), wherein normally organize without any processing;1,2,3 point of test group
Final concentration of 10 μ g/mL LL-37, TP5 or LTP solution is not added after cell pellet overnight culture;Test group 4,5,6 is respectively thin
Final concentration of 10 μ g/mL LL-37, TP5 or LTP solution is added in born of the same parents after being incubated overnight, after one hour, model group and test group 4,
5, the LPS solution of the 6 final concentration of 100ng/mL of addition.Cytokine TNF-α and IFN-γ are detected using Elisa method, as a result
As shown in fig. 7, hybrid peptide LTP be remarkably improved the mouse macrophage under normal condition cytokine TNF-α (A of Fig. 7),
The expression quantity of IFN-γ (B of Fig. 7), and the expression quantity of LTP group Macrophage Cell factor TNF-α, IFN-γ is significantly higher than LL-
37 groups and TP5 group.But at the same time, when cell is in the inflammatory conditions of LPS induction, LTP can significantly inhibit TNF-α again
The expression of (A of Fig. 7), IFN-γ (B of Fig. 7) cell factor, and inhibitory effect is better than its source of parents peptide LL-37 and TP5.Thus it says
Bright, hybrid peptide LTP has two-way immunoregulation effect, can both enhance the immunocompetence under cell normal condition, can also inhibit scorching
The inflammatory reaction of cell under symptom state, and its immune antiphlogistic effects is superior to its source of parents peptide LL-37 and TP5.
Immunoregulation effect of the 5 hybrid peptide LTP of embodiment to immunosuppressed mice
Using C57BL/6 system male mice, (20~22g of weight, purchase tie up tonneau China experimental animal from Beijing to the present embodiment
Technology Co., Ltd.) carry out zoopery, guideline of the whole experiment process all referring to European experimental animal Ethics Committee
(86/609/EEC), and obtain the license of experimental animal Ethics Committee, China Agricultural University.Animal feeding environment is cleaning
Grade, 22 ± 2 DEG C of environment temperature, humidity 50%~55%, 8:00~20:00 illumination.6~8, the every cage of mouse raisings, can be free
It ingests drinking-water.
1, influence of the hybrid peptide LTP to the weight and immune organ weight of immunosuppressed mice
36 healthy male mices are taken to be randomly divided into 3 groups, every group 12.It is divided into blank group (Control): physiological saline;
Model group (CY): injection cyclophosphamide CY (100mg/kg);Test group (LTP+CY): injection LTP (10mg/kg) and cyclophosphamide
CY(100mg/kg)。
Intraperitoneal injection hybrid peptide LTP (dosage 10mg/kg) is carried out to test group mouse, continuous 14 days, often
It is primary, and blank group and model group give the physiological saline of respective volume.The 8th day of self administration of medication starts, to model group and test
Group mouse carries out intraperitoneal injection of cyclophosphamide 100mg/kg, and blank group is given same amount of normal saline, injected every other day, and co-injection 4 times,
Prepare the animal model of immunologic hypofunction.After the last administration, mouse cervical dislocation is put to death, records mouse weight, takes spleen
And thymus gland and claim its weight in wet base respectively, calculate mouse spleen index and thymus index (index and spleen index=mouse spleen weight/weight.Thymus gland
Index=mouse thymus weight/weight).As a result as shown in Fig. 8 A, Fig. 8 B and Fig. 8 C, the results showed that, the body of model group mouse
Weight, index and spleen index and thymus index are significantly reduced than blank group, illustrate that cyclophosphamide can inhibit mouse growth, reduce immune device
Official's index inhibits immune;And weight, index and spleen index and the thymus index of test group mouse are restored to the normal water of blank group
It is flat, illustrate that hybrid peptide LTP can promote the growth of immunologic hypofunction mouse, and have to the immune organ of immunologic hypofunction mouse
There is protective effect.
2, influence of the hybrid peptide LTP to the macrophage phagocytic activity of immunosuppressed mice
36 healthy male mices are taken to be randomly divided into 3 groups, every group 12.It is divided into blank group: physiological saline;Model group: note
Penetrate cyclophosphamide CY (100mg/kg);Test group: injection LTP (10mg/kg) and cyclophosphamide CY (100mg/kg).
The administration mode of blank group, model group and test group with described in above-mentioned steps 1, last dose for 24 hours after, will be small
After mouse blood sampling is put to death, impregnate 5-10s in 75% ethyl alcohol respectively, the RPMI1640 of injection 4mL addition heparin in intraperitoneal,
It is centrifuged eluate, abandons supernatant, isolated peritoneal macrophage.0.5mL is added into precipitating containing 10% fetal calf serum
RPMI1640 is resuspended, and cell concentration is adjusted 5 × 106A/mL is inoculated in 96 orifice plates, in 37 DEG C, 5%CO2It is cultivated under environment
After 3h, supernatant is abandoned, dimethyl diaminophenazine chloride normal saline solution is added, lytic cell after 10min detects its extinction under 540nm wavelength
Degree.Experimental result is as in fig. 8d, the results showed that, compared to the blank group, the neutral red blood cell phagocytic rate of model group significantly reduces,
Cyclophosphamide can inhibit the phagocytic activity of mouse macrophage, and be added LTP can restore immunologic hypofunction mouse macrophage it is thin
The phagocytic activity of born of the same parents, and then the body inherency of the mouse of booster immunization hypofunction is immune.
(3) influence of the hybrid peptide LTP to the cytokine release amount of immunosuppressed mice
36 healthy male mices are taken to be randomly divided into 3 groups, every group 12.It is divided into blank group: physiological saline;Model group: note
Penetrate cyclophosphamide CY (100mg/kg);Test group: injection LTP (10mg/kg) and cyclophosphamide CY (100mg/kg).
The administration mode of blank group, model group and test group with described in above-mentioned steps 1, last dose for 24 hours after, eyeball
Blood is taken, serum is separated, using the content of cell factor in ELISA method detection mice serum (IFN-γ, IL-6).Experimental result
As shown in Fig. 8 E, Fig. 8 F, the results showed that, compared to the blank group, the content of the cell factor (IFN-γ, IL-6) of model group mouse
It significantly reduces;And the cell factor (IFN-γ, IL-6) that hybrid peptide LTP is remarkably improved in immunosuppressed mice serum is administered
Content, and then improve immunologic hypofunction mouse immunocompetence.
Anti-inflammatory effect of the 6 hybrid peptide LTP of embodiment to inflammatory conditions mouse
Using C57BL/6 system male mice, (20~22g of weight, purchase tie up tonneau China experimental animal from Beijing to the present embodiment
Technology Co., Ltd.) zoopery is carried out, entire process of testing is all referring to the guideline of European experimental animal Ethics Committee
(86/609/EEC), and obtain the license of experimental animal Ethics Committee, China Agricultural University.Animal feeding environment is cleaning
Grade, 22 ± 2 DEG C of environment temperature, humidity 50%~55%, 8:00~20:00 illumination.6~8, the every cage of mouse raisings, can use by oneself
It ingests drinking-water.
1, hybrid peptide LTP is to the weight of inflammatory conditions mouse and the influence of enteron aisle
36 healthy male mices are taken to be randomly divided into 3 groups, every group 12.It is divided into blank group (Control): physiological saline;
Model group (LPS): LPS (10mg/kg);Test group (LTP+LPS): LTP (10mg/kg), LPS (10mg/kg).
Intraperitoneal injection hybrid peptide LTP (dosage 10mg/kg) is carried out to test group mouse, continuous 7 days, daily
Once, the physiological saline of respective volume is given to blank group and model group mouse.After last dose 1h, to model group and test
Group mouse carries out intraperitoneal injection LPS (10mg/kg), and blank group gives same amount of normal saline, and mouse cervical dislocation is put to death after 6h, note
The weight and enteron aisle length for recording mouse take mouse jejunum to carry out the observation of H&E stained slice.Experimental result such as Fig. 9 A, Fig. 9 B and figure
Shown in 9C, the results showed that, the weight and enteron aisle length of model group mouse are significantly reduced than blank group, and pass through sections observation
It was found that the intestinal villus form of model group is impaired, while there is apparent oedema phenomenon in submucosa, illustrates that the inflammation of LPS induction is anti-
Mouse weight decline, gut atrophy, damage should be will lead to;And the weight and enteron aisle length of test group mouse are restored to blank group
Normal level, intestinal villus form and oedema phenomenon have clear improvement, and illustrate the inflammation that hybrid peptide LTP can protect LPS to induce
Reaction is damaged caused by mouse weight and enteron aisle.
2, influence of the hybrid peptide LTP to the cytokine-expressing amount of inflammatory conditions mouse
36 healthy male mices are taken to be randomly divided into 3 groups, every group 12.It is divided into blank group: physiological saline;Model group: LPS
(10mg/kg);Test group: LTP (10mg/kg), LPS (10mg/kg).
The administration mode of blank group, model group and test group is with described in above-mentioned 1, after last dose 1h, to model group
It is carried out intraperitoneal injection LPS (10mg/kg) with test group mouse, blank group gives same amount of normal saline, and eyeball takes blood after 6h, separation
Serum, using the content of cell factor in ELISA method detection mice serum (IFN-γ, IL-6).Experimental result such as Fig. 9 D, Fig. 9 E
It is shown, the results showed that, compared to the blank group, cell factor (IFN-γ, IL-6) content of model group mouse significantly increases;And it tries
The expression quantity of cell factor (IFN-γ, IL-6) of LPS induction can be significantly inhibited by testing group administration hybrid peptide LTP, and then inhibit scorching
Disease reaction.
Antioxidation of the 6 hybrid peptide LTP of embodiment to inflammatory conditions mouse
1, Scavenging activity of the hybrid peptide LTP to DPPH (1,1- diphenyl -2- trinitrophenyl-hydrazine) free radical
1.5mL sample liquid is mixed with the 1mmol/L DPPH ethanol solution of equivalent, and 25 DEG C are protected from light 30min,
517nm measures its light absorption value Asample;DPPH ethanol solution is substituted with the dehydrated alcohol of equivalent, is mixed instead with 1.5mL sample
It answers, surveys its light absorption value Asample blank in 517nm wavelength;Analyte sample fluid is not added, measures 1.5mL under 517nm wavelength
The light absorption value Acontrol of DPPH ethyl alcohol and 1.5mL water mixed liquid.Test result shows that hybrid peptide LTP has stronger DPPH clear
Removing solid capacity, the clearance rate of hybrid peptide LTP is 63.26 ± 5.69% when 10 mg/mL.
2, Scavenging activity of the hybrid peptide LTP to superoxide anion
2.8ml Tris-HCl-EDTA (pH 8.2) buffer is added in 0.1ml sample to be tested, and 25 DEG C of water-baths keep the temperature 10min
0.1ml 3.0mM pyrogallol solution is added afterwards, mixes rapidly, the every 30s of 325nm surveys light absorption value, and 5min terminates.Make light absorption value with
The regression equation of time change seeks its slope.Superoxide anion Scavenging activity calculation formula are as follows:
Scavenging activity (%)=(VControl-VSample)/VControl× 100%
In formula, VControlIt is control group mouse thymus cells rate (Δ A/min);VSampleIt is sample sets pyrogallol oxidation speed
Rate (Δ A/min).
Test result shows that hybrid peptide LTP has a stronger superoxide anion Scavenging activity, hybrid peptide LTP when 10mg/mL
Clearance rate be 44.57 ± 3.14%.
3, the reducing power of hybrid peptide LTP
1.0ml sample is mixed with 1.0ml sodium radio-phosphate,P-32 solution (pH6.6) and 1% potassium ferricyanide of 1.0ml, 50 DEG C of hatchings
20min.1.0ml 10%TCA solution is added, 5000 × g is centrifuged 10min.2.0ml supernatant and 2.0ml deionized water, 0.4ml
The mixing of 0.1% ferric trichloride, is stored at room temperature 10min, and 700nm surveys light absorption value.Test result shows that hybrid peptide LTP has preferably
Reducing power, the reducing power of hybrid peptide LTP is 0.54 ± 0.07 when 10mg/mL.
4, hybrid peptide LTP is on inflammatory conditions mice serum MDA level and the active influence of SOD and CAT
36 healthy male mices are taken to be randomly divided into 3 groups, every group 12.It is divided into blank group: physiological saline;Model group: LPS
(10mg/kg);Test group: LTP (10mg/kg), LPS (10mg/kg).
The administration mode of blank group, model group and test group is with described in above-mentioned 1, after last dose 1h, to model group
It is carried out intraperitoneal injection LPS (10mg/kg) with test group mouse, blank group gives same amount of normal saline, and eyeball takes blood after 6h, separation
Serum measures mice serum malonaldehyde (MDA) concentration and superoxide dismutase (SOD), catalase (CAT) activity,
Measuring method is operated according to the specification of kit (Nanjing is built up Bioisystech Co., Ltd and provided).Experimental result is as schemed
10A, Figure 10 B, shown in Figure 10 C, the results showed that, compared to the blank group, model group mice serum MDA it is horizontal it is significant increase, and SOD
And CAT activity significantly reduces;And MDA level significantly reduces in test group mice serum, SOD and the significant raising of CAT activity.
In conclusion hybrid peptide LTP has two-way immunological regulation and the anti-oxidant and endotoxic effect of resolution.One side
Face, LTP can enhance the immunocompetence of normal condition or immunological function repression state lower body, in terms of promoting body inherent immunity
Macrophage improves the expression quantity of cell factor to the phagocytic activity of foreign matter, and protection immunosupress is damaged caused by body.Cause
This, polypeptide LTP can be used for preventing and treating due to immune defense and immune surveillance function it is low caused by infection.
On the other hand, hybrid peptide LTP can clear up endotoxin, inhibit body inflammatory reaction, reduce under inflammatory conditions cell because
The burst size of son alleviates damage of the inflammatory reaction to tissues such as the enteron aisles of animal and people.Therefore, hybrid peptide LTP can be used for inflammation again
The treatment of disease property disease (such as enteritis).
Oxidative stress is alleviated by the activity of the intracellular antioxidase of modulate host again, finally makes antioxygen in host cell
Change enzymatic activity and tends to normal level.
In addition, hybrid peptide LTP has good DPPH clearance rate, superoxide anion Scavenging activity and reducing power, explanation
Hybrid peptide can removing machine interior free yl body is no longer influenced by caused by free radical further damage.At the same time, hybrid peptide
LTP can also enhance the activity of antioxidase SOD and CAT in Mice Body, enhance the ability of body Scavenger of ROS and free radical, right
Active oxygen in environment has a degree of tolerance, then plays its antioxidation.Hybrid peptide LTP can reduce Mice Body
The content of interior MDA reduces membrane damage caused by oxidative stress.Therefore, hybrid peptide LTP can also be used for the preparation of anti-oxidation medicine.
The present invention also carried out hybrid peptide LTP for example terminus amidated LTP of derivative and to the 16th amino acids into
The functional experiment of the corresponding replaced LTP of row, the results showed that, the said derivative of hybrid peptide LTP similarly has and hybrid peptide
LTP is similar to be immunized anti-inflammatory two-way regulating function.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
Sequence table
<110>China Agricultural University
<120>one kind, which has both, adjusts immune, anti-oxidant, anti-inflammatory and multi-functional hybrid peptide of removing toxic substances and the preparation method and application thereof
<130> KHP191110425.4
<160> 7
<170> SIPOSequenceListing 1.0
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<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Ile Gly Lys Glu Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu
1 5 10 15
Arg Asn Leu Val Pro Arg Thr Glu Arg Lys Asp Val Thr
20 25
<210> 2
<211> 87
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
attggtaagg aatttaagag aatcgttcaa agaattaagg atttcttgag aaacttggtt 60
cctagaactg aaagaaagga tgttact 87
<210> 3
<211> 37
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<213>artificial sequence (Artificial Sequence)
<400> 3
Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu
1 5 10 15
Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val
20 25 30
Pro Arg Thr Glu Ser
35
<210> 4
<211> 5
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Arg Lys Asp Val Thr
1 5
<210> 5
<211> 29
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<213>artificial sequence (Artificial Sequence)
<400> 5
Arg Lys Asp Val Thr Ile Gly Lys Glu Phe Lys Arg Ile Val Gln Arg
1 5 10 15
Ile Lys Asp Phe Leu Arg Asn Leu Val Pro Arg Thr Glu
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<213>artificial sequence (Artificial Sequence)
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1 5 10 15
Arg Asn Leu Val Pro Arg Thr Glu Thr Val Asp Lys Arg
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<213>artificial sequence (Artificial Sequence)
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ggtaccatcg gtaaggaatt caagagaatc gttcaaagaa tcaaggactt cttgagaaac 60
ttggttccaa gaactgaaag aaaggacgtt actcatcatc atcatcatca ttgatctaga 120
Claims (10)
1. anti-oxidant removing toxic substances hybrid peptide is immunized in one kind, which is characterized in that the amino acid sequence of the hybrid peptide such as SEQ ID NO.1
Shown or replacement, missing or insertion for the amino acid sequence as shown in SEQ ID NO.1 through one or more amino acid obtains
The amino acid sequence with identical function polypeptide.
2. encoding the gene of hybrid peptide described in claim 1.
3. gene according to claim 2, which is characterized in that the nucleotide sequence of the gene such as SEQ ID NO.2 institute
Show, or there is the coding identical function of at least 80%, 85%, 90%, 95%, 98% or 99% homology with SEQ ID NO.2
The gene order of albumen.
4. the biomaterial containing gene described in Claims 2 or 3, which is characterized in that the biomaterial include recombinant DNA,
Expression cassette, transposons, plasmid vector, phage vector, viral vectors or host cell.
5. the method for preparing hybrid peptide described in claim 1 characterized by comprising by gene described in claim 2 or 3
It imports in host cell, expresses the hybrid peptide.
6. according to the method described in claim 5, it is characterized in that, the host cell is yeast, preferably Pichia pastoris.
7. hybrid peptide described in claim 1 uses gene or power described in claim 5 or 6 the methods or Claims 2 or 3
Benefit requires 4 biomaterials preparing the application in immunomodulator.
8. hybrid peptide described in claim 1 or the hybrid peptide being prepared using claim 5 or 6 the methods or right are wanted
Biomaterial described in 2 or 3 genes or claim 4 is asked to prepare answering in anti-inflammatory preparation or resolution/antiendotoxin preparation
With.
9. application according to claim 7 or 8, which is characterized in that the preparation include drug, health care product and food or
Feed addictive.
10. a kind of product, the product includes drug, health care product and food or feed addictive, which is characterized in that includes power
Benefit requires 1 hybrid peptide or the hybrid peptide comprising being prepared using claim 5 or 6 the methods.
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Cited By (3)
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CN111848817A (en) * | 2020-07-28 | 2020-10-30 | 中国农业大学 | Multifunctional hybrid peptide with antibacterial, antiviral, immunoregulatory and anti-inflammatory activities, and preparation method and application thereof |
CN111944058A (en) * | 2020-07-28 | 2020-11-17 | 中国农业大学 | Multifunctional hybrid peptide with antibacterial, anti-inflammatory, endotoxin neutralization and immunoregulation activities, and preparation method and application thereof |
CN111944059A (en) * | 2020-07-28 | 2020-11-17 | 中国农业大学 | Multifunctional hybrid peptide with antibacterial, immunoregulatory, anti-infection and anti-inflammatory activities, and preparation method and application thereof |
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CN104017084A (en) * | 2014-05-07 | 2014-09-03 | 中国农业大学 | Heterozygous antibacterial and antiviral polypeptide as well as preparation method and application thereof |
CN105968214A (en) * | 2016-06-21 | 2016-09-28 | 中国农业大学 | Antibacterial and antiviral hybrid peptide as well as preparation method and application thereof |
CN107312094A (en) * | 2017-07-06 | 2017-11-03 | 上海海洋大学 | A kind of heterozygous antibacterial peptide and its preparation method and application |
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2019
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CN104017084A (en) * | 2014-05-07 | 2014-09-03 | 中国农业大学 | Heterozygous antibacterial and antiviral polypeptide as well as preparation method and application thereof |
CN105968214A (en) * | 2016-06-21 | 2016-09-28 | 中国农业大学 | Antibacterial and antiviral hybrid peptide as well as preparation method and application thereof |
CN107312094A (en) * | 2017-07-06 | 2017-11-03 | 上海海洋大学 | A kind of heterozygous antibacterial peptide and its preparation method and application |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN111848817A (en) * | 2020-07-28 | 2020-10-30 | 中国农业大学 | Multifunctional hybrid peptide with antibacterial, antiviral, immunoregulatory and anti-inflammatory activities, and preparation method and application thereof |
CN111944058A (en) * | 2020-07-28 | 2020-11-17 | 中国农业大学 | Multifunctional hybrid peptide with antibacterial, anti-inflammatory, endotoxin neutralization and immunoregulation activities, and preparation method and application thereof |
CN111944059A (en) * | 2020-07-28 | 2020-11-17 | 中国农业大学 | Multifunctional hybrid peptide with antibacterial, immunoregulatory, anti-infection and anti-inflammatory activities, and preparation method and application thereof |
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