CN110128548B

CN110128548B - Hybrid peptide with functions of regulating immunity, resisting oxidation, resisting inflammation and detoxifying, and preparation method and application thereof

Info

Publication number: CN110128548B
Application number: CN201910435730.7A
Authority: CN
Inventors: 张日俊; 张璐璐; 卫旭彪; 黄燕; 阿曼; 斯大勇; 李仲玄; 程俊豪; 杜孟思
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2019-05-23
Filing date: 2019-05-23
Publication date: 2020-12-01
Anticipated expiration: 2039-05-23
Also published as: CN110128548A

Abstract

The invention relates to a hybrid peptide with functions of regulating immunity, resisting oxidation, resisting inflammation and detoxifying, and a preparation method and application thereof. The hybrid peptide provided by the invention is named LTP, and is obtained by carrying out protein engineering computer design, hybridization, optimization and in-vitro and in-vivo double screening on antibacterial peptide LL-37 and thymopentin TP5, wherein the amino acid sequence is shown as SEQ ID NO. 1. The hybrid peptide LTP has high bidirectional immunoregulation activity, can enhance the immune function of an organism in a normal or immunosuppressive state, and protects the damage of immunosuppression to the organism; under the inflammatory state, the LTP can neutralize and digest endotoxin, inhibit the oxidation reaction and inflammatory reaction of organisms, relieve the damage of the inflammatory reaction to tissues, has the advantages of low cytotoxicity, high safety, simple preparation method, low cost and the like, can be used as an ideal immune, antioxidant and anti-inflammatory regulator or immune antioxidant detoxification peptide, and has good application potential and value.

Description

Hybrid peptide with functions of regulating immunity, resisting oxidation, resisting inflammation and detoxifying, and preparation method and application thereof

Technical Field

The invention relates to the field of genetic engineering and biological preparations, in particular to an immune antioxidant detoxification hybrid peptide with functions of immunoregulation, oxidation resistance, inflammation resistance and digestion, and a preparation method and application thereof.

Background

In the case of young age, weakness, diseases, stress, pathogenic infection, etc., animals or humans often cause a decrease in immunity, and further, secondary infection (mixed infection of bacteria or viruses) occurs, causing inflammatory reaction (red, heat, swelling, pain, etc.). At present, for the treatment of infection and inflammation, the traditional and commonly used prevention and treatment strategy is to use antibiotics and hormone anti-inflammatory drugs (such as hydrocortisone, dexamethasone and the like), although the antibiotics and hormone anti-inflammatory drugs can effectively control infectious inflammation and non-infectious inflammation, the continuous use can cause various side effects: such as serious disorders of water and salt metabolism and sugar, fat and protein metabolism, and cause deterioration of adrenal cortex function, complications of digestive system or aggravated infection. In addition, antibiotic health promoting growth promoter is widely used in animal husbandry. There are significant problems with the use of antibiotic or hormonal anti-inflammatory drugs in the treatment of infection and inflammation: antibiotics can reduce or kill pathogenic bacteria, but cannot improve the immune function of the body, on the contrary, the bacteria killed by the antibiotics can produce endotoxin or exotoxin, further aggravate inflammatory reaction and even cause systemic inflammatory response syndrome (Botwinski,2001), which causes animals to have fever, anorexia, excessive consumption of energy in vivo, decomposition of body tissues, reduction of immunity and productivity, and death of livestock and poultry (Botwinski, 2001; nneZir, 1999). In recent years, a lot of studies have shown that many antibiotics, while killing bacteria, can also promote the release of endotoxin, i.e. Lipopolysaccharide (LPS), from the bacterial cell membrane (Holzheimer, 2001; Hurley,1995), which in turn causes the LPS to accumulate in a large amount and cause inflammatory reaction. Endotoxin LPS is usually produced by cell disintegration of gram-negative bacteria such as pathogenic Escherichia coli, Salmonella, Brucella, Proteus, swine influenza and Haemophilus parasuis, and can induce release of various proinflammatory cytokines such as TNF-alpha, interleukin 6(IL-6) and IL-1 beta; and simultaneously, a large amount of free radicals are induced to be generated to cause oxidative damage, so that the immunity is reduced. Therefore, endotoxin LPS is reduced or eliminated, the oxidative damage of the organism is reduced through the antioxidant function, and the inflammatory reaction of a patient animal or a human is reduced or eliminated. In summary, the existing antibiotic anti-infection and glucocorticoid anti-inflammatory drugs have obvious defects and are not suitable for continuous use. Therefore, the development of a novel, safe, environment-friendly active peptide with no side effect and simultaneously having the functions of immunoregulation, oxidation resistance and anti-inflammation and endotoxin digestion has important practical significance and huge application prospect for human or animal breeding industry, and is a new breakthrough and new concept of anti-infection strategy.

The immune system can protect the body from being invaded by external microorganisms through the immune defense function, and can remove aging and canceration cells in the body in time. On one hand, when the immune function is lower than the normal level, the organism is very easy to be infected and induce the generation of malignant tumor, and the like, thereby causing the aggravation of the disease of the patient to be difficult to treat; on the other hand, however, when the immune response is excessive, the immune response may cause a strong inflammatory response in animals or humans, which may result in body damage and dysfunction of various physiological systems, which may further affect the normal metabolic processes of the body, and even endanger life of the serious people. It follows that anti-inflammatory and immune are two aspects of immune system function, closely related, inseparable, and sometimes even overlapping. Therefore, in the traditional medicine field, the complete separation of anti-inflammatory drugs and immune-enhancing drugs is not in accordance with the actual needs of the body or the interaction relationship between anti-inflammatory and immune, and greatly increases the complexity and antagonism of clinical drug selection. In view of the above, it is important to develop safe and efficient preparations or drugs having a bidirectional immunoregulatory function to improve the immune function of animals and humans.

The antimicrobial peptide LL-37, a polypeptide with a relative molecular mass of about 5000Da (daltons), is the only member of the antimicrobial peptide (cathelicidin) family found in humans so far, and is the only antimicrobial peptide with an amphipathic alpha-helix structure in humans. LL-37 is widely distributed in blood cells and epithelial cells of human body, has the effect of neutralizing endotoxin, can be combined with LPS and CD14, and neutralizes the biotoxicity of LPS; can mediate chemotaxis, recruit immune cells to the infected part and eliminate pathogens; and promoting angiogenesis, and the like, and is a relatively mature anti-inflammatory peptide.

The human studies on immunologically active peptides began in 1981 when Jolles et al first isolated an immunologically active peptide fragment from human milk protein hydrolysates. After that, many studies on immunoactive peptides have been conducted by scientists, and among them, studies on thymosin have been reported and conducted more deeply. Thymopentin (TP5), the active center of thymopoietin II, has biological activity similar to that of thymopoietin, can regulate unbalanced immune function bidirectionally, induce cell differentiation, promote lymphocyte subpopulation development and activation, and is an important immunomodulator.

With the continuous and deep research on the structure, function and action mechanism of anti-inflammatory peptides and immune enhancing peptides, researchers have tried to design bidirectional immune modulating peptides with higher safety and stronger regulatory activity by using protein engineering methods. Research has reported that by hybridizing different types of polypeptides, a novel multifunctional polypeptide is obtained or different immunologically active peptides and anti-inflammatory peptides are hybridized to obtain a novel anti-inflammatory and modulatory agent.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a hybrid peptide with the functions of regulating immunity, resisting oxidation, resisting inflammation and detoxifying, and a preparation method and application thereof

In order to achieve the purpose, the technical scheme of the invention is as follows: on the basis of carrying out a great deal of research on the sequences, structures and the relationship between the sequence structures and the functions of the polypeptide LL-37 and the thymosin TP5, the invention applies a protein molecule design technology to carry out hybrid optimization of the polypeptide LL-37 (the amino acid sequence is shown as SEQ ID NO. 3) and the thymopentin TP5 (the amino acid sequence is shown as SEQ ID NO. 4), and finally obtains a novel immune detoxification hybrid peptide, namely LTP, the amino acid sequence of which is shown as SEQ ID NO.1 by screening and sequence optimization of the hybrid peptide. Hybrid peptide LTP has the functions of two maternal peptides, namely the bidirectional immunoregulation function: under normal or immune suppression state, the immune function of the organism can be improved; in the presence of inflammation or endotoxin, LTP also has antioxidant, endotoxin eliminating, and inflammatory reaction inhibiting effects.

Firstly, the amino acid sequence of the detoxication hybrid peptide LTP provided by the invention is shown as SEQ ID NO.1 or the amino acid sequence of the detoxication hybrid peptide LTP is obtained by replacing, deleting or inserting one or more amino acids in the amino acid sequence shown as SEQ ID NO.1 and has the same functional polypeptide.

The derivative polypeptides of the hybrid peptide LTP with the same function obtained by modification on the basis of the amino acid sequence shown in SEQ ID NO.1 include, but are not limited to, the following polypeptides:

(1) a polypeptide obtained by adding a protein tag sequence to the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID No.1, for example: polypeptide obtained by adding His label containing 6 His residues at C end or N end of amino acid sequence shown as SEQ ID NO. 1; or polypeptide obtained by adding GST or C-Myc label to C terminal or N terminal of amino acid sequence shown in SEQ ID NO. 1;

it will be understood by those skilled in the art that the addition of tag sequences at both ends of the polypeptide for the purpose of easy purification, polypeptide labeling, etc. is a routine technique in the art and does not affect the inherent functions and activities of the polypeptide itself, and therefore, the LTP derivatives obtained by adding tag sequences at both ends of the hybrid peptide LTP as shown in SEQ ID NO.1 are also within the scope of the present invention.

(2) Polypeptides obtained by conservative amino acid substitution of one or more amino acid sequences in the amino acid sequence shown in SEQ ID No.1, such as: replacement of Leu at position 15 with Ile does not result in a substantial change in protein function.

The invention also provides a gene for coding the hybrid peptide with the functions of immunoregulation, oxidation resistance, endotoxin digestion and anti-inflammation.

Given the amino acid sequence of hybrid peptide LTP, one skilled in the art can design genes encoding hybrid peptide LTP with different nucleotide sequences based on the principle of codon degeneracy and codon usage bias of different species according to the need for polypeptide expression.

As an embodiment of the invention, the nucleotide sequence of the gene is shown as SEQ ID NO. 2. The gene shown as SEQ ID NO.2 is a hybrid peptide LTP coding gene designed according to the codon preference of pichia pastoris. Gene sequences encoding proteins of the same function having at least 80%, 85%, 90%, 95%, 98% or 99% homology to SEQ ID No.2 are also within the scope of the present invention.

Further, the present invention also provides a biomaterial containing the gene encoding the hybrid peptide, which comprises a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a phage vector, a viral vector or a host cell.

The host cell comprises an animal cell, a plant cell or a cell line and a microbial cell.

Further, the present invention provides a method for preparing the hybrid peptide, comprising: introducing a gene encoding the hybrid peptide into a host cell and expressing the hybrid peptide.

Preferably, the preparation method comprises the following steps: connecting the encoding gene of the hybrid peptide LTP with an expression vector to construct a recombinant expression vector, and introducing the recombinant expression vector into a host cell by a transgenic method to obtain the host cell introduced with the LTP encoding gene.

The transgenic method includes heat stress transformation, electric transformation, transfection and the like.

The host cell includes but is not limited to animal and plant cells and microbial cells.

Preferably, the host cell is yeast, more preferably pichia pastoris.

When pichia is adopted as a host cell, the encoding gene with a sequence optimized by pichia codon preference as shown in SEQ ID NO.2 is adopted to express the hybrid peptide, so that the expression quantity is better.

As an embodiment of the invention, the preparation of the hybrid peptide takes pichia pastoris as a host, takes an expression vector pPICZ alpha A as a vector, and expresses the hybrid peptide through methanol induction, and the preparation method specifically comprises the following steps:

(1) connecting the encoding gene of the hybrid peptide LTP to an expression vector pPICZ alpha A to construct a recombinant expression vector;

(2) transforming the recombinant expression vector into pichia pastoris GS115 to construct recombinant engineering bacteria introduced with hybrid peptide LTP coding genes;

(3) culturing the recombinant engineering bacteria, and adding methanol to induce the expression of the hybrid peptide LTP;

(4) collecting culture supernatant, and purifying to obtain hybrid peptide LTP.

In-vivo and in-vitro experiments prove that the hybrid peptide LTP can improve the immunocompetence of a normal organism and an immunodeficiency state, improve the expression quantity of cell factors, promote the growth of a mouse and relieve the damage of immunodeficiency to the spleen and the thymus of the mouse; in addition, in the inflammatory reaction process, the anti-inflammatory and anti-inflammatory immune peptide can inhibit inflammatory reaction induced by LPS, enhance the oxidation resistance of an organism, reduce the expression level of cell factors, relieve the damage of inflammatory states to the weight and intestinal tracts of mice, and has good immune anti-inflammatory two-way regulation effect.

Based on the above functions, the present invention provides the use of the hybrid peptide or the hybrid peptide prepared by the preparation method, or the gene encoding the hybrid peptide or the biomaterial containing the gene encoding the hybrid peptide in the preparation of an immunomodulatory agent.

Preferably, the immunomodulatory agent is an immunopotentiator.

The invention also provides application of the hybrid peptide or the hybrid peptide prepared by the preparation method or the coding gene of the hybrid peptide or the biological material containing the coding gene of the hybrid peptide in preparing an anti-inflammatory preparation or a digestion/anti-endotoxin preparation.

The preparation provided by the invention comprises medicines, health products and food or feed additives.

The immunopotentiator can be used for preventing and treating various immunosuppression including body immunosuppression caused by Cyclophosphamide (CY).

The anti-inflammatory agent or endotoxin-digesting agent can be used for the prevention and treatment of various inflammations including inflammatory reaction induced by LPS or endotoxemia.

The invention also provides a product which is a medicament, a health product and a food or feed additive, and the product comprises the hybrid peptide TP5 or the hybrid peptide prepared by the preparation method of the hybrid peptide.

In the product, the hybrid peptide can be used as an active ingredient or compounded with other active ingredients to form the active ingredients of medicaments, health-care products and food or feed additives.

Preferably, the pharmaceutical composition further comprises a carrier or an auxiliary material acceptable in the pharmaceutical field.

The invention has the beneficial effects that: the invention obtains the immune anti-inflammatory hybrid peptide LTP by hybridizing LL-37 and TP5 for the first time, optimizing and screening, the polypeptide LTP has the functions of two parent peptides, namely has the bidirectional immune regulation function, and compared with the corresponding activities of the parent peptides LL-37 and TP5, the immune regulation activity, the antioxidant activity, the anti-inflammatory activity and the endotoxin digestion activity are stronger: under the normal or immune suppression state, the immune function of the organism can be obviously improved, and the damage of the immune suppression to the organism can be protected; in an inflammatory state, LTP can also inhibit the inflammatory reaction of the organism and relieve the damage of the inflammatory reaction to tissues; meanwhile, LTP has the advantages of low cytotoxicity, high safety, convenient preparation and low cost, can be used as an ideal immunomodulator, antioxidant, anti-inflammatory agent and endotoxin antidote, is widely applied to the fields of medicine, food, health care, feed, nutrition and the like of human and animals, and has great application value.

The preparation method of the hybrid peptide provided by the invention can realize the large-scale and high-efficiency preparation of the hybrid peptide LTP, and the prepared hybrid peptide LTP has the activities of bidirectional immunoregulation, endotoxin digestion and antioxidation, and has no obvious toxic or side effect.

Drawings

FIG. 1 is a diagram of molecular docking of candidate hybrid peptides in example 1 of the present invention, wherein A is a diagram of a 3D simulation of molecular docking of hybrid peptides, and B is a diagram of the energy absorbed or released during molecular docking of hybrid peptides (positive values represent absorbed energy, negative values represent released energy).

FIG. 2 is a flow chart of the construction of the recombinant expression vector pPICZ α A-LTP in example 1 of the present invention.

FIG. 3 shows the result of gel electrophoresis in PCR identification of the recombinant expression vector pPICZ α A-LTP of example 1 of the present invention, wherein M is the DNA molecular weight standard; lanes 1-3 are fragments of interest comprising the gene encoding the polypeptide LTP.

FIG. 4 shows the results of electrophoresis and mass spectrometry of the purified Pichia pastoris engineered bacteria expressed hybrid peptide LTP in example 1 of the present invention, wherein A is the result of SDS-PAGE electrophoresis, and M is the protein molecular weight standard; lanes 1-2 are bands of purified LTP after 144h fermentation supernatant induced with methanol; b is a mass spectrum detection result graph of the purified hybrid peptide LTP.

FIG. 5 shows the neutralizing activity of hybrid peptide LTP and its parent peptides LL-37 and TP5 on LPS in example 2 of the present invention, wherein LTP, LL-37 and TP5 represent polypeptides LTP, LL-37 and TP5, respectively, and PMB represents polymyxin B.

FIG. 6 shows the effect of hybrid peptide LTP and its parent peptides LL-37 and TP5 on cell survival of mouse macrophages in example 3 of the present invention.

FIG. 7 is a graph showing the effect of hybrid peptide LTP on the cytokine expression level of mouse macrophage RAW264.7 in example 4 of the present invention; wherein, A is the expression level of TNF-alpha; the B picture shows the expression level of IFN-gamma; control represents normal group, LPS represents model group of LPS-induced inflammation, LL-37 represents test group 1 (under normal conditions with addition of LL-37 treatment), TP5 represents test group 2 (under normal conditions with addition of TP5 treatment), LTP represents test group 3 (under normal conditions with addition of LTP treatment), LL-37+ LPS represents test group 4 (after addition of LL-37 treatment, model of inflammation induced by LPS), TP5+ LPS represents test group 5 (after addition of TP5 treatment, model of inflammation induced by LPS), LTP + LPS represents test group 2 (under LPS-induced inflammation with addition of LTP treatment); represents that the two are compared with a significant difference (p <0.5), and represents that the two are compared with a very significant difference (p < 0.01).

FIGS. 8A to 8F are graphs showing the immunomodulatory effects of hybrid peptide LTP on cyclophosphamide-induced immunosuppressed mice in example 5 of the present invention, respectively; wherein, FIG. 8A is the effect of LTP on the body weight of immunosuppressed mice; FIG. 8B is a graph of the effect of spleen in LTP immunosuppressive mice; FIG. 8C is the effect of LTP on the thymus of immunosuppressed mice; FIG. 8D is the effect of LTP on phagocytic activity of mouse macrophages; FIG. 8E is a graph showing the effect of LTP on the amount of IFN-. gamma.released from mouse cytokines; FIG. 8F is a graph showing the effect of LTP on the release of mouse cytokine IL-6; control represents blank group, CY represents model group, and LTP + CY represents test group; indicates significant difference from blank control group (p <0.5), and # indicates significant difference from model group (p < 0.5).

FIGS. 9A-9E are graphs showing the inhibition of inflammatory responses in mice by hybrid peptide LTP of example 6 of the present invention; wherein, fig. 9A is the effect of LTP on mouse body weight; FIG. 9B is the effect of intestinal length in LTP mice; FIG. 9C is a graph of the effect of LTP on the integrity of intestinal tissue in mice; FIG. 9D is a graph showing the effect of LTP on the amount of IFN-. gamma.released from mouse cytokines; FIG. 9E is a graph showing the effect of LTP on the release of mouse cytokine IL-6; control represents blank group, LPS represents model group, LTP + LPS represents test group; indicates significant difference from blank control group (p <0.5), and # indicates significant difference from model group (p < 0.5).

FIGS. 10A-10C are graphs showing the antioxidant effect of hybrid peptide LTP in example 7 of the present invention on mice; wherein, FIG. 10A is the effect of LTP on mouse serum MDA levels; FIG. 10B is a graph showing the effect of LTP on mouse serum SOD activity; FIG. 10B is a graph showing the effect of LTP on the CAT activity in mouse serum, wherein Control represents a blank group, LPS represents a model group, and LTP + LPS represents a test group; indicates significant difference from blank control group (p <0.5), and # indicates significant difference from model group (p < 0.5).

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available, unless otherwise specified, wherein E.coli competent cell Top 10, Pichia pastoris GS115 and expression vector pPICZ. alpha.A were purchased from Invitrogen corporation.

EXAMPLE 1 preparation of an immune anti-inflammatory hybrid peptide LTP

1. Obtaining hybrid peptide sequences

Through researching the sequences, structures and the relation between the sequence structures and functions of the polypeptide LL-37 and the thymopentin TP5, the protein molecule design technology is utilized to carry out the hybridization of the polypeptide LL-37 and the thymopentin TP5, and candidate hybrid peptides LTP, TP5-LL-37 and LL-37-TP5 'are obtained, and the amino acid sequences of the candidate hybrid peptides LTP, TP5-LL-37 and LL-37-TP 5' are respectively shown as SEQ ID NO.1, SEQ ID NO.5 and SEQ ID NO. 6. Myeloid differentiation protein-2 (MD-2) binds to TLR4, conferring TLR4 reactivity to various ligands (including LPS); second, MD-2 promotes the expression of TLR4 and TLR2 and is closely associated with the intracellular distribution of TLR 4. Thus, MD-2 is not only an accessory molecule for TLR4, but also a regulatory molecule in innate immunity, and may have a broader range of biological functions in pathophysiological processes such as infection, inflammation, immunity, and the like. The research predicts the anti-inflammatory immune effect by using the scoring condition of the molecular docking of the hybrid peptide and MD-2, and the result is shown in figure 1, and the three candidate hybrid peptides can release energy when combined with MD-2, but the LTA releases more energy, which shows that the combination is more stable and has better anti-inflammatory immune activity, so that the hybrid peptide LTP is finally obtained, and the amino acid sequence of the hybrid peptide LTP is shown in SEQ ID No. 1.

2. Construction of recombinant expression vectors

According to the amino acid sequence of hybrid peptide LTP (polypeptide LTP for short) and pichia pastoris codon preference, a coding gene (the sequence is shown as SEQ ID NO. 2) of the polypeptide LTP is designed and synthesized, the coding gene is connected with an expression vector pPICZ alpha A and is transformed into escherichia coli Top 10 competent cells to construct a recombinant expression vector pPICZ alpha A-LTP, and the construction process of the vector is shown as figure 2.

Extracting plasmid according to the operation method of small plasmid extraction kit of Tiangen Biotechnology (Immunity) limited company, performing PCR amplification on LTP gene by using the extracted plasmid as a template,

5'-GGTACCATTGGTAAGGAATT-3' as upstream primer; a downstream primer: 5'-GATGATGATGAGTAACATCC-3' are provided. The amplified product was subjected to agarose gel electrophoresis, observed under an ultraviolet lamp, and the transformation result was identified, as shown in FIG. 3, showing that a band (120bp) of LTP gene was present in the amplified product. Sequencing the recombinant plasmid further identified whether the insertion of the desired fragment was correct and the fidelity of the inserted fragment was checked. Sequencing proves that the construction of the recombinant expression vector pPICZ alpha A-LTP is successful.

3. Preparation of Pichia competent cells

Inoculating single colony of Pichia pastoris GS115 into 3-5ml YPD (2% tryptone, 2% glucose, 1% yeast powder, pH 7.0) liquid culture medium, performing shake culture at 30 deg.C for about 12 hr, inoculating into 100ml YPD liquid culture medium at volume ratio of 1:100-1:50, performing shake culture at 30 deg.C to OD₆₀₀The culture was stopped at about 1.3-1.5. The culture was transferred to a centrifuge tube, left on ice for 10 minutes, and then centrifuged at 1500 Xg for 5 minutes at 4 ℃ to collect the cells. The cells were gently suspended in precooled sterile water, left on ice for 15-30 minutes, and then centrifuged at 1500 Xg for 5 minutes at 4 ℃. The supernatant was discarded, 10ml of a pre-cooled sterile sorbitol solution (1M) was added, and the cells were resuspended to obtain a competent cell suspension. 200. mu.l of each tube was dispensed into Pichia pastoris GS115 competent cells for use immediately or stored at-70 ℃.

4. Construction of Pichia pastoris engineering bacteria for expressing hybrid peptide LTP

The recombinant expression vector pPICZ alpha A-LTP constructed in the step 3 is converted into a Pichia pastoris GS115 strain, and the specific method is as follows:

5-10 mu g of linearized recombinant plasmid pPICZ alpha A-LTP is mixed with 200 mu l of competent cells of activated Pichia pastoris GS115, the mixture is transferred into an electric rotating cup and is subjected to ice bath for 5-30min, the mixture is subjected to electric rotation for a plurality of seconds after ice bath (oscillation is avoided during the electric rotation), then the mixed solution is quickly placed on ice, 1ml of 1M sorbitol solution subjected to ice bath is quickly added, the thalli are suspended and uniformly mixed, transferred into a 1.5ml EP tube and placed on the ice, then 2ml of YPD liquid culture medium is added, and the mixture is cultured for 3h at 30 ℃ so that the strains are recovered and form resistance. After the recombinant bacteria became resistant, 200. mu.l of the culture broth was spread on YPDS plates containing 100. mu.g/ml kanamycin, and the plates were inverted and cultured overnight at 30 ℃. And (3) selecting a single colony growing on the YPDS plate, inoculating the single colony into a YPD liquid culture medium containing 100 mu g/ml kanamycin for overnight culture, extracting plasmids by adopting a small-plasmid extraction medium-amount kit, and identifying a positive transformant through PCR verification to obtain the pichia pastoris engineering bacteria expressing the hybrid peptide LTP.

5. Inducible expression of hybrid peptide LTP

Selecting Pichia engineering bacteria expressing hybrid peptide LTP, inoculating into BMGY medium containing 100 μ g/ml kanamycin, shake culturing at 30 deg.C and 180-₆₀₀When the number is 2-6, the thalli are collected by low-speed centrifugation; resuspending the cells to OD in BMMY Medium₆₀₀The culture was carried out at 30 ℃ and 200rpm with shaking at 1. During the shaking culture process, methanol is added every 24h to a final concentration of 5% for induced expression of polypeptide LTP. 2ml of the bacterial liquid is taken when the methanol is induced for 144 hours, centrifuged for 5min at 12000rpm, and the supernatant is collected and stored at-20 ℃. And (3) carrying out Tricine-SDS-PAGE electrophoresis detection on the collected culture supernatant, wherein an electrophoresis result shows that the culture supernatant contains polypeptide LTP, the polypeptide LTP is successfully expressed, and the expression quantity of the polypeptide LTP can reach about 46mg/L through determination.

6. Purification of hybrid peptide LTP

In the synthesis of LTP coding gene, histidine tag (6 × His) is added at C-terminal, so that polypeptide LTP can be combined with Ni-NTA Sepharose columnNi of (2)²⁺And (3) combining, eluting the fermentation supernatant by using imidazole eluents with different concentrations, and collecting elution peak samples to realize separation and purification of the polypeptide LTP. According to the Ni-NTA Sepharose chromatographic column purification method of the polypeptide LTP, referring to the use instruction of a chromatographic column product, collecting each elution peak sample, detecting the purification effect through Tricine-SDS-PAGE electrophoresis, wherein the molecular weight of the LTP is 3.6kDa, and the result is shown in A of figure 4, and the result shows that the polypeptide LTP with higher purity is obtained through purification; the mass spectrometric detection of purified LTP is shown in FIG. 4B, which indicates that the molecular weight of the purified polypeptide is consistent with the theoretical molecular weight of LTP. The nucleotide sequence of LTP containing a restriction enzyme site terminator and an N-terminal His tag is shown in SEQ ID NO. 7.

EXAMPLE 2 neutralization of LPS by hybrid peptide LTP

The hybrid peptide LTP and its parent peptides LL-37 and TP5 were dissolved and diluted in pyrogen-free endotoxin test water to different concentrations of solutions (0-64. mu.g/mL), and 100. mu.L of each of the above concentrations of LTP polypeptide solutions was mixed with LPS (1 EU/mL). After incubation at 37 ℃ for 30min, neutralization of LPS by polypeptides LTP, LL-37 and TP5 was detected by a chromogenic limulus kit, and polymyxin B (PMB) was used as a control. As shown in FIG. 5, the polypeptide LTP has high LPS neutralizing activity, which is equivalent to polymyxin B, and at a concentration of 8. mu.g/mL, the neutralizing rate of the hybrid peptide LTP on LPS is close to 100%, and the neutralizing activity is significantly higher than that of the parent peptide LL-37 and TP 5.

Example 3 Effect of hybrid peptide LTP on mouse macrophage cell survival

Taking macrophage RAW264.7 in logarithmic growth phase to inoculate in a 96-well plate, wherein the initial cell culture density is 1 multiplied by 10⁴one/mL, 100. mu.L per well, 5% CO at 37 ℃₂After culturing overnight under the conditions of (1), adding a series of concentration gradient LTP, LL-37 and TP5(0-100 mu g/mL) solutions respectively, and after culturing for 24h, detecting the influence of polypeptide LTP on the survival rate of mouse macrophages by using a CCK8 method. The results are shown in FIG. 6, where the cytotoxicity of the hybrid peptide LTP is significantly reduced compared to the parent peptide LL-37, and the survival rate of macrophages is greater than 80% in the concentration range of 0-100. mu.g/mL, indicating that the cytotoxicity of LTP is lower and higherThe safety of (2).

Example 4 immunomodulatory Activity of hybrid peptide LTP in mouse macrophages

Diluting the hybrid peptide LTP and the parent peptides LL-37 and TP5 thereof by a DMEM medium to prepare a polypeptide solution with the concentration of 10 mu g/mL, and detecting the influence of the hybrid peptide LTP and the parent peptides LL-37 and TP5 on the secretion of cytokines such as TNF-alpha, IFN-gamma and the like of mouse macrophage RAW264.7 under normal state and LPS-induced inflammatory state. Respectively setting a normal group (Control), a model group (LPS), a test group 1(LL-37), a test group 2(TP5), a test group 3(LTP), a test group 4(LL-37+ LPS), a test group 5(TP5+ LPS) and a test group 6(LTP + LPS), wherein the normal group is not subjected to any treatment;

test groups

1, 2, and 3 were each added to a final concentration of 10. mu.g/mL LL-37, TP5, or LTP solution after overnight cell culture;

test groups

4, 5 and 6 were each added to an LL-37, TP5 or LTP solution at a final concentration of 10. mu.g/mL after overnight cell culture, and one hour later, to the model and

test groups

4, 5 and 6 was added an LPS solution at a final concentration of 100 ng/mL. The results of using Elisa method to detect the cytokines TNF-alpha and IFN-gamma are shown in figure 7, the hybrid peptide LTP can significantly improve the expression level of the cytokines TNF-alpha (A in figure 7) and IFN-gamma (B in figure 7) of mouse macrophages in a normal state, and the expression level of the cytokines TNF-alpha and IFN-gamma of macrophages in the LTP group is significantly higher than that of LL-37 group and TP5 group. At the same time, however, when the cells are in an LPS-induced inflammatory state, LTP can also remarkably inhibit the expression of TNF-alpha (A in figure 7) and IFN-gamma (B in figure 7) cytokines, and the inhibition effect is better than that of the parent peptides LL-37 and TP 5. Therefore, the hybrid peptide LTP has a bidirectional immunoregulation effect, can enhance the immunocompetence of cells under a normal state and inhibit the inflammatory reaction of the cells under an inflammatory state, and has an immune anti-inflammatory effect superior to that of the parent peptides LL-37 and TP 5.

Example 5 immunomodulatory effects of hybrid peptide LTP on immunosuppressive mice

In the embodiment, the animal experiment is carried out by adopting C57BL/6 male mice (with the weight of 20-22 g, purchased from Beijing Wintoli laboratory animal technology Co., Ltd.), the whole experiment process refers to the guiding principle of European laboratory animal ethics committee (86/609/EEC), and the approval of the Chinese agriculture university laboratory animal ethics committee is obtained. The animal feeding environment is clean, the environment temperature is 22 +/-2 ℃, the humidity is 50% -55%, and the illumination is 8: 00-20: 00. The mice are raised in 6-8 cages and can freely eat and drink water.

1. Effect of hybrid peptide LTP on body weight and immune organ weight in immunosuppressed mice

36 healthy male mice were randomly divided into 3 groups of 12 mice each. Divide into blank group (Control): physiological saline; model group (CY): cyclophosphamide CY (100mg/kg) was injected; test group (LTP + CY): LTP (10mg/kg) and cyclophosphamide CY (100mg/kg) were injected.

The mice in the test group were administered with the hybrid peptide LTP (administered at a dose of 10mg/kg) by intraperitoneal injection for 14 consecutive days, and the blank group and the model group were administered with the corresponding volumes of physiological saline once a day. From the 8 th day of administration, mice in the model group and the test group were subjected to intraperitoneal injection of cyclophosphamide at 100mg/kg, and mice in the blank group were administered with the same amount of physiological saline, and were injected every other day for 4 times to prepare animal models with low immune function. After the last administration, the mice were sacrificed by dislocation of cervical vertebrae, the body weights of the mice were recorded, the spleen and the thymus were taken and weighed as wet weights, respectively, and the spleen index and the thymus index of the mice were calculated (spleen index ═ mouse spleen weight/body weight, thymus index ═ mouse thymus weight/body weight). The results are shown in fig. 8A, fig. 8B and fig. 8C, and the results indicate that the body weight, spleen index and thymus index of the mice in the model group are all significantly reduced compared with those in the blank group, which indicates that cyclophosphamide can inhibit the growth of the mice, reduce the immune organ index and inhibit the immunity; the body weight, spleen index and thymus index of the mice in the test group are all restored to the normal level of the blank group, which shows that the hybrid peptide LTP can promote the growth of the mice with low immune function and has protective effect on immune organs of the mice with low immune function.

2. Effect of hybrid peptide LTP on macrophage phagocytic Activity in immunosuppressed mice

36 healthy male mice were randomly divided into 3 groups of 12 mice each. The method is divided into a blank group: physiological saline; model group: cyclophosphamide CY (100mg/kg) was injected; test groups: LTP (10mg/kg) and cyclophosphamide CY (100mg/kg) were injected.

Blank set, model set and trialThe administration method of the test group was the same as that in the step 1, and 24 hours after the last administration, the mice were blood-collected and sacrificed, and then soaked in 75% ethanol for 5-10s, 4mL of RPMI1640 supplemented with heparin was injected into the abdominal cavity, the eluate was centrifuged, the supernatant was discarded, and the abdominal macrophages were obtained by separation. 0.5mL of 10% fetal bovine serum-containing RPMI1640 was added to the pellet and resuspended, and the cell concentration was adjusted to 5X 10⁶one/mL, seeded in 96-well plates at 37 ℃ with 5% CO₂Culturing for 3h in the environment, discarding the supernatant, adding neutral red physiological saline solution, cracking the cells after 10min, and detecting the absorbance at the wavelength of 540 nm. The experimental results are shown in fig. 8D, and the results show that compared with the blank group, the phagocytic rate of the neutrophils in the model group is significantly reduced, cyclophosphamide can inhibit the phagocytic activity of macrophages of mice, and LTP can be added to restore the phagocytic activity of macrophages of mice with low immune function, so as to enhance the innate immunity of the mice with low immune function.

(3) Effect of hybrid peptide LTP on cytokine Release amounts in immunosuppressed mice

The administration method of the blank group, the model group and the test group was the same as that described in step 1, and 24 hours after the last administration, blood was collected from the eyeball, and serum was separated, and the contents of cytokines (IFN-. gamma., IL-6) in the mouse serum were measured by ELISA. The experimental results are shown in fig. 8E and fig. 8F, and the results show that the contents of the cytokines (IFN-gamma, IL-6) in the model group mice are significantly reduced compared with the blank group; the administration of the hybrid peptide LTP can obviously improve the content of cell factors (IFN-gamma and IL-6) in the serum of an immunosuppressed mouse, thereby improving the immunocompetence of the mouse with low immune function.

Example 6 anti-inflammatory Effect of hybrid peptide LTP on mice with inflammatory states

In the embodiment, the animal experiment is carried out by adopting C57BL/6 male mice (with the weight of 20-22 g, purchased from Beijing Wintoli laboratory animal technology Co., Ltd.), the whole experiment process refers to the guiding principle of European laboratory animal ethics committee (86/609/EEC), and the approval of the Chinese agriculture university laboratory animal ethics committee is obtained. The animal feeding environment is clean, the environment temperature is 22 +/-2 ℃, the humidity is 50% -55%, and the illumination is 8: 00-20: 00. The mice are raised in 6-8 cages and can be fed with food and water.

1. Effect of hybrid peptide LTP on body weight and intestinal tract of mice in inflammatory State

36 healthy male mice were randomly divided into 3 groups of 12 mice each. Divide into blank group (Control): physiological saline; model group (LPS): LPS (10 mg/kg); test group (LTP + LPS): LTP (10mg/kg), LPS (10 mg/kg).

The mice in the experimental group were administered with the hybrid peptide LTP (administered at a dose of 10mg/kg) by intraperitoneal injection for 7 days, and the mice in the blank group and the model group were administered with a corresponding volume of physiological saline once a day. After the last administration for 1H, mice in the model group and the test group were subjected to intraperitoneal injection of LPS (10mg/kg), the blank group was administered with an equal amount of physiological saline, the mice were sacrificed by cervical dislocation after 6H, the weight and intestinal length of the mice were recorded, and the jejunum of the mice was taken for H & E staining and section observation. The experimental results are shown in fig. 9A, fig. 9B and fig. 9C, and the results show that the body weight and the intestinal length of the mice in the model group are both significantly reduced compared with those in the blank group, and the intestinal villus morphology of the mice in the model group is found to be damaged through section observation, and meanwhile, a remarkable edema phenomenon appears in the submucosa, which indicates that the inflammatory reaction induced by LPS can cause the weight reduction, the intestinal atrophy and the injury of the mice; the body weight and intestinal length of the mice in the test group are restored to the normal level of the blank group, the intestinal villus form and the edema phenomenon are obviously improved, and the hybrid peptide LTP can protect the damage of the inflammatory reaction induced by LPS to the body weight and the intestinal tract of the mice.

2. Effect of hybrid peptide LTP on cytokine expression levels in mice with inflammatory states

36 healthy male mice were randomly divided into 3 groups of 12 mice each. The method is divided into a blank group: physiological saline; model group: LPS (10 mg/kg); test groups: LTP (10mg/kg), LPS (10 mg/kg).

The administration modes of the blank group, the model group and the test group are the same as that in the 1, LPS (10mg/kg) is injected into the abdominal cavity of the mice of the model group and the test group after 1 hour of the last administration, the blank group is given with the same amount of physiological saline, blood is taken from eyeballs after 6 hours, serum is separated, and the contents of the cytokines (IFN-gamma and IL-6) in the serum of the mice are detected by adopting an ELISA method. The experimental results are shown in fig. 9D and fig. 9E, and the results show that the contents of the cytokines (IFN- γ and IL-6) in the model group mice are significantly increased compared with the blank group; and the test group can obviously inhibit the expression quantity of cytokines (IFN-gamma and IL-6) induced by LPS by administering the hybrid peptide LTP, thereby inhibiting inflammatory reaction.

Example 6 antioxidant Effect of hybrid peptide LTP on mice with inflammatory states

1. Scavenging ability of hybrid peptide LTP for DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) free radical

Mixing 1.5mL of sample solution with an equivalent amount of 1mmol/L of DPPH ethanol solution, carrying out a dark reaction at 25 ℃ for 30min, and measuring the light absorption value Asample at 517 nm; replacing a DPPH ethanol solution with equal amount of absolute ethanol, mixing the solution with 1.5mL of a sample for reaction, and measuring the light absorption value Asample blank at the wavelength of 517 nm; the absorbance Acontrol of a mixture of 1.5mL of DPPH ethanol and 1.5mL of water was measured at 517nm without adding a sample solution to be measured. The test result shows that the hybrid peptide LTP has stronger DPPH clearance, and the clearance of the hybrid peptide LTP is 63.26 +/-5.69% at 10 mg/mL.

2. Scavenging ability of hybrid peptide LTP for superoxide anion

Adding 0.1ml sample to be tested into 2.8ml Tris-HCl-EDTA (pH 8.2) buffer solution, keeping temperature in 25 deg.C water bath for 10min, adding 0.1ml 3.0mM pyrogallol solution, rapidly mixing, measuring light absorption value at 325nm every 30s, and ending 5 min. And (4) making a regression equation of the change of the light absorption value along with time, and solving the slope of the regression equation. The superoxide anion scavenging capacity is calculated by the formula:

capacity to clear (%) - (V)_Control-V_{Sample (I)})/V_Control×100％

In the formula, V_ControlIs the control pyrogallol autoxidation rate (delta A/min); v_{Sample (I)}Is a sample group oThe rate of triphenol oxidation (. DELTA.A/min).

The test result shows that the hybrid peptide LTP has stronger superoxide anion scavenging capacity, and the clearance of the hybrid peptide LTP is 44.57 +/-3.14% at 10 mg/mL.

3. Reducing power of hybrid peptide LTP

1.0ml of the sample was mixed with 1.0ml of a sodium phosphate solution (pH6.6) and 1.0ml of 1% potassium ferricyanide, and incubated at 50 ℃ for 20 min. 1.0ml of 10% TCA solution was added and centrifuged at 5000 Xg for 10 min. Mixing 2.0ml of supernatant with 2.0ml of deionized water and 0.4ml of 0.1% ferric chloride, standing at room temperature for 10min, and measuring light absorption value at 700 nm. The test result shows that the hybrid peptide LTP has better reducing power, and the reducing power of the hybrid peptide LTP is 0.54 +/-0.07 at 10 mg/mL.

4. Effect of hybrid peptide LTP on serum MDA level and SOD and CAT activities in mice with inflammatory State

The administration method of the blank group, the model group and the test group is the same as that in the above 1, after 1 hour of the last administration, LPS (10mg/kg) is injected into the abdominal cavity of the mice in the model group and the test group, the blank group is given with the same amount of physiological saline, blood is taken from the eyeball after 6 hours, serum is separated, the concentration of Malondialdehyde (MDA) in the serum of the mice and the activities of superoxide dismutase (SOD) and Catalase (CAT) are measured, and the measurement method is operated according to the instruction of a kit (provided by Nanjing Biotechnology Ltd.). The experimental results are shown in fig. 10A, fig. 10B and fig. 10C, and the results show that the serum MDA level of the model mouse is significantly increased, and the activities of SOD and CAT are significantly reduced compared with the blank group; and the MDA level in the serum of the mice in the test group is obviously reduced, and the SOD and CAT activities are obviously improved.

In conclusion, the hybrid peptide LTP has the functions of bidirectional immunoregulation, oxidation resistance and endotoxin digestion. On one hand, LTP can enhance the immunocompetence of the organism in a normal state or an immune function inhibition state, promote the phagocytosis capability of macrophages to foreign matters in the aspect of the innate immunity of the organism, improve the expression level of cytokines and protect the damage of immunosuppression to the organism. Therefore, the polypeptide LTP can be used for preventing and treating infection caused by low immune defense and immune monitoring functions.

On the other hand, the hybrid peptide LTP can digest endotoxin, inhibit inflammatory reaction of organisms, reduce the release amount of cytokines in an inflammatory state, and relieve the damage of the inflammatory reaction to tissues such as intestinal tracts of animals and humans. Therefore, the hybrid peptide LTP can be used for treating inflammatory diseases (such as enteritis and the like).

And the oxidative stress is relieved by regulating and controlling the activity of the antioxidant enzyme in the host cell, so that the activity of the antioxidant enzyme in the host cell tends to a normal level.

In addition, the hybrid peptide LTP has good DPPH clearance rate, superoxide anion scavenging capacity and reducing power, and shows that the hybrid peptide can scavenge free radicals in a body so that the body is not further damaged by the free radicals. Meanwhile, the hybrid peptide LTP can also enhance the activities of antioxidant enzymes SOD and CAT in mice, enhance the capability of organisms in clearing active oxygen and free radicals, has certain tolerance to active oxygen in the environment and further plays a role in antioxidation. The hybrid peptide LTP can reduce the content of MDA in mice and reduce membrane damage caused by oxidative stress. Therefore, the hybrid peptide LTP can also be used for preparing antioxidant drugs.

The invention also carries out functional experiments on derivatives of hybrid peptide LTP, such as LTP with amidated terminal and LTP after corresponding substitution of 16 th amino acid, and the results show that the derivatives of hybrid peptide LTP also have similar immune anti-inflammatory bidirectional regulation function with the hybrid peptide LTP.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of agriculture in China

<120> a hybrid peptide with the functions of regulating immunity, resisting oxidation, resisting inflammation and detoxifying, and a preparation method and application thereof

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Claims

1. An immune oxidation-resistant detoxification hybrid peptide, which is characterized in that the amino acid sequence of the hybrid peptide is shown as SEQ ID NO. 1.

2. A gene encoding the hybrid peptide of claim 1.

3. The gene of claim 2, wherein the nucleotide sequence of the gene is shown as SEQ ID No. 2.

4. Biomaterial containing the genes of claim 2 or 3, characterized in that it is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a phage vector, a viral vector or a host cell.

5. A method of preparing the hybrid peptide of claim 1, comprising: introducing the gene of claim 2 or 3 into a host cell and expressing the hybrid peptide.

6. The method of claim 5, wherein the host cell is a yeast.

7. The method of claim 6, wherein the host cell is Pichia pastoris.

8. Use of the hybrid peptide of claim 1 or the gene of claim 2 or 3 or the biomaterial of claim 4 in the preparation of an immunomodulatory preparation using the method of any one of claims 5 to 7.

9. Use of the hybrid peptide of claim 1 or the hybrid peptide produced by the method of any one of claims 5 to 7 or the gene of claim 2 or 3 or the biomaterial of claim 4 for the preparation of an anti-inflammatory or anti-endotoxin digestion/release formulation.

10. Use according to claim 8 or 9, wherein the preparation is a medicament, nutraceutical, food or feed additive.

11. A product which is a pharmaceutical, nutraceutical, food or feed additive comprising a hybrid peptide according to claim 1 or a hybrid peptide produced by a method according to any one of claims 5 to 7.