CN110128544B - Hybrid peptide with immunoregulation and anti-inflammatory functions and preparation method and application thereof - Google Patents
Hybrid peptide with immunoregulation and anti-inflammatory functions and preparation method and application thereof Download PDFInfo
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- CN110128544B CN110128544B CN201910265006.4A CN201910265006A CN110128544B CN 110128544 B CN110128544 B CN 110128544B CN 201910265006 A CN201910265006 A CN 201910265006A CN 110128544 B CN110128544 B CN 110128544B
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Abstract
The invention relates to the technical field of genetic engineering and biological agents, in particular to a hybrid peptide with immunoregulation and anti-inflammatory functions, a preparation method and application thereof. The invention provides an immune anti-inflammatory hybrid peptide CTP which is obtained by hybridizing, optimizing and screening an antibacterial peptide CATH-2 and a thymosin TP5, wherein the amino acid sequence is shown as SEQ ID NO. 1. The hybrid peptide CTP has higher two-way 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, CTP can also inhibit the inflammatory reaction of the organism and relieve the injury 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 anti-inflammatory regulator, and has good application potential and value.
Description
Technical Field
The invention relates to the field of genetic engineering and biological agents, in particular to a hybrid peptide with immunoregulation and anti-inflammatory functions, 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. Thus, reduction or elimination of endotoxin, LPS, can reduce or eliminate the inflammatory response in a patient animal or human. 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 and environment-friendly active peptide with no side effect and simultaneously having the functions of immunoregulation, endotoxin digestion and anti-inflammation 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.
Cathelicidins are one of the largest families of antimicrobial peptides currently known and have been reported in vertebrates, including mammals, birds, reptiles, amphibians and fish. The Cathelicidin is composed of an N-terminal signal peptide region, a middle conserved cathelin structural domain and a C-terminal highly specific mature peptide region and plays an extremely important role in an animal innate immune system. In addition to broad-spectrum antibacterial activity, Cathelicidin also has excellent anti-inflammatory functions, such as chemotaxis of various immune cells, induction of mast cell degranulation and histamine release, regulation of macrophage transcription, promotion of wound healing, induction of apoptosis of variant cell lines, lymphocyte activation and the like. Poultry defensin 2(CATH-2) is an important representative polypeptide of the Cathelicidin family.
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 immunologically active 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 immunoregulation and anti-inflammatory functions, 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 functions of the polypeptide CATH-2 and the thymosin TP5, the invention utilizes a protein molecule design technology to carry out hybrid optimization of the polypeptide CATH-2 and the thymosin TP5, and finally obtains a novel immune anti-inflammatory hybrid peptide (IAIHP) named as CTP by screening and sequence optimization of the hybrid peptide, wherein the amino acid sequence of the hybrid peptide is shown as SEQ ID NO. 1. The hybrid peptide CTP has the functions of two maternal peptides, namely has bidirectional immunoregulation function: under normal or immune suppression state, the immune function of the organism can be improved; in the inflammatory state, CTP also inhibits the inflammatory response of the body and is more immunomodulatory and anti-inflammatory than the corresponding activities of the maternal peptides CATH-2 and TP 5.
Firstly, the invention provides a hybrid peptide with immunoregulation and anti-inflammatory functions, wherein the amino acid sequence of the hybrid peptide is shown as SEQ ID NO.1 or the amino acid sequence of the polypeptide with the same function obtained by replacing, deleting or inserting one or more amino acids in the amino acid sequence shown as SEQ ID NO. 1.
The derivative polypeptide of the hybrid peptide CTP with the same function obtained by modifying on the basis of the amino acid sequence shown as SEQ ID NO.1 comprises but is 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 technical means in the art and does not affect the inherent functions and activities of the polypeptide itself, and therefore, the above-mentioned CTP derivatives obtained by adding tag sequences at both ends of the hybrid peptide CTP 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: the substitution of Leu at position 6 to Ile does not have much effect on the function of the polypeptide.
The derivative polypeptide of the hybrid peptide with the same function obtained by modifying on the basis of the sequence of the hybrid peptide shown in SEQ ID NO.1 still belongs to the protection scope of the patent.
The present invention also provides a gene encoding the hybrid peptide having immunoregulatory and anti-inflammatory functions.
Given the amino acid sequence of a hybrid peptide CTP, one skilled in the art can design a gene encoding a hybrid peptide CTP having a different nucleotide sequence according to the requirement for polypeptide expression based on the principle of codon degeneracy and codon usage bias of different species.
As an embodiment of the invention, the nucleotide sequence of the gene is shown as SEQ ID NO. 6. The gene shown as SEQ ID NO.6 is a heterozygous peptide CTP coding gene designed according to the codon preference of pichia pastoris.
The gene which is obtained by modifying the coding sequence shown as SEQ ID NO.6 and codes CTP or derivative peptide with the same function as CTP still belongs to the protection scope of the patent.
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 CTP 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 CTP 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.6 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 coding gene of the hybrid peptide CTP 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 heterozygous peptide CTP coding genes;
(3) culturing the recombinant engineering bacteria, and adding methanol to induce the expression of the heterozygosis peptide CTP;
(4) collecting culture liquid supernatant, and purifying to obtain the hybrid peptide CTP.
In-vivo and in-vitro tests prove that the hybrid peptide CTP can improve the immunocompetence of an organism in normal and immunodeficiency states, 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 inflammatory reaction induced by LPS can be inhibited, the expression level of cell factors is reduced, the damage of the inflammatory state to the weight and the intestinal tract of a mouse is relieved, and the immune anti-inflammatory two-way regulation effect is good.
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 the 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.
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 can be used for preventing and treating various inflammations including inflammatory reaction induced by LPS.
The invention also provides a product comprising said hybrid peptide or a hybrid peptide produced by said method.
The product is any one selected from medicines, health products and food or feed additives.
As an embodiment of the present invention, the present invention provides a pharmaceutical composition comprising the hybrid peptide or the hybrid peptide prepared by the method for preparing the hybrid peptide.
The medicine composition may have the hybrid peptide as the effective component or the hybrid peptide compounded with other active components to constitute the effective component of medicine composition.
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 CTP by hybridizing the antibacterial peptide CATH-2 and the thymosin TP5 for the first time and optimizing and screening, wherein the polypeptide CTP has the functions of two maternal peptides, namely has the function of bidirectional immune regulation, and has stronger immune regulation activity and anti-inflammatory activity compared with the corresponding activities of the maternal peptides CATH-2 and TP 5: 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; under the inflammatory state, CTP can also inhibit the inflammatory reaction of the organism and relieve the damage of the inflammatory reaction to tissues; meanwhile, CTP has the advantages of low cytotoxicity, high safety, simple preparation method and low cost, can be used as an ideal immunomodulator and an anti-inflammatory agent, 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 CTP, and the prepared hybrid peptide CTP has the bidirectional immunoregulation and anti-inflammatory activity and has no obvious toxic or side effect.
Drawings
FIG. 1 is a molecular docking diagram of a candidate hybrid peptide in example 1 of the present invention, wherein A is a molecular docking 3D simulation diagram of the hybrid peptide, and B is the energy absorbed or released by the hybrid peptide during the molecular docking process (positive values represent the absorbed energy, and negative values represent the released energy).
FIG. 2 is a flow chart showing the construction of recombinant expression vector pPICZ α A-CTP in example 1 of the present invention.
FIG. 3 shows the results of gel electrophoresis in PCR identification of the recombinant expression vector pPICZ α A-CTP in example 1 of the present invention, wherein M is a DNA molecular weight standard; lanes 1-5 are fragments of interest comprising the gene encoding the polypeptide CTP.
FIG. 4 shows the results of electrophoresis and mass spectrometry of polypeptide CTP expressed by Pichia pastoris engineering bacteria purified in example 1, wherein A is the result of SDS-PAGE electrophoresis, and Marker is the protein molecular weight standard; lane 1-2 shows the band of CTP, a target protein purified from the supernatant of 120h fermentation induced with methanol; b is a mass spectrum detection result diagram of the purified polypeptide CTP.
FIG. 5 is a graph showing the effect of hybrid peptide CTP and its parent peptide on cell survival rate of mouse macrophage cells in example 2 of the present invention.
FIG. 6 shows the effect of the polypeptide of example 3 on the cytokine expression level of mouse macrophage RAW 264.7; wherein A is the expression level of TNF-alpha; b is the expression level of IFN-gamma; control represents the normal group, LPS represents the model group of LPS-induced inflammation, CATH2 represents test group 1 (normal condition with addition of CATH2 treatment), TP5 represents test group 2 (normal condition with addition of TP5 treatment), CTP represents test group 3 (normal condition with addition of CTP treatment), CATH2+ LPS represents test group 4 (addition of CATH2 treatment followed by induction of inflammation model with LPS), TP5+ LPS represents test group 5 (addition of TP5 treatment followed by induction of inflammation model with LPS), CTP + LPS represents test group 6 (addition of CTP treatment followed by induction of inflammation model with LPS); 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).
FIG. 7 is the immunomodulatory effect of the polypeptide CTP on cyclophosphamide induced immunosuppression in mice in example 4 of the present invention; wherein A is the influence of CTP on the body weight of an immunosuppressed mouse; b is the influence of CTP immunosuppression on mouse spleen; c is the influence of CTP on the thymus of an immunosuppressed mouse; d is the influence of CTP on the phagocytic activity of mouse macrophages; e is the influence of CTP on the release amount of mouse cytokine IFN-gamma; f is the influence of CTP on the release amount of mouse cytokine IL-6; control represents a blank group, CY represents a model group, and CTP + CY represents a test group; indicates significant difference from blank control group (p <0.5), and # indicates significant difference from model group (p < 0.5).
FIG. 8 is the inhibitory effect of polypeptide CTP on mouse inflammatory response in example 5 of the present invention; wherein A is the influence of CTP on the body weight of a mouse; b is the influence of the intestinal length of the CTP mouse; c is the influence of CTP on the integrity of jejunum tissues of the mice; d is the influence of CTP on the release amount of mouse cytokine IFN-gamma; e is the influence of CTP on the release amount of mouse cytokine IL-6; control represents blank group, LPS represents model group, CTP + LPS represents 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 the immune anti-inflammatory hybrid peptide CTP
1. Obtaining hybrid peptide sequences
Through researching the sequences, structures and the relation between the sequence structures and functions of the polypeptide CATH-2 (the sequence is shown as SEQ ID NO. 2) and the thymosin TP5 (the sequence is shown as SEQ ID NO. 3), the polypeptide CATH-2 and the thymosin TP5 are hybridized by utilizing a protein molecule design technology, and a plurality of candidate hybrid peptides are obtained. This example illustrates the screening process of the immune anti-inflammatory hybrid peptide CTP by taking candidate hybrid peptide CTP (shown in SEQ ID NO. 1), TP5-CATH2 (shown in SEQ ID NO. 4) and CATH2-TP 5' (shown in SEQ ID NO. 5) as examples.
The anti-inflammatory immune effect of each candidate hybrid peptide is preliminarily predicted by molecular docking of each candidate hybrid peptide with the MD-2 protein according to the grading condition of molecular docking by utilizing the characteristics of myeloid differentiation protein-2 (MD-2) (namely, the MD-2 is combined with TLR4 to endow TLR4 with the reactivity to various ligands including LPS, and the MD-2 can promote the expression of TLR4 and TLR2 and is closely related to the distribution of TLR4 in cells, so that the MD-2 is not only an auxiliary molecule of TLR4, but also a regulatory molecule in natural immunity and has wide biological functions in pathophysiological processes of infection, inflammation, immunity and the like).
The molecular docking results of the candidate hybrid peptides CTP, TP5-CATH2 and CATH2-TP 5' are shown in A of figure 1, the energy change conditions of the three candidate hybrid peptides are shown in B of figure 1, and the three candidate hybrid peptides can release energy when being combined with MD-2, but the CTP releases the energy most, which indicates that the combination of the three candidate hybrid peptides with MD-2 is more stable and has better anti-inflammatory and immune activities. And (3) integrating other screening experiment results, and finally screening to obtain the hybrid peptide CTP, wherein the amino acid sequence of the hybrid peptide CTP is shown in SEQ ID No. 1.
2. Construction of recombinant expression vectors
Designing and synthesizing a coding gene (the sequence is shown as SEQ ID NO. 6) of the polypeptide CTP according to the amino acid sequence of the hybrid peptide CTP (polypeptide CTP for short) and the codon preference of pichia pastoris, adding a His label at the C end of the polypeptide CTP on the basis of the CTP coding gene sequence shown as SEQ ID NO.6, connecting the CTP coding gene sequence with the His label (shown as SEQ ID NO. 7) with an expression vector pPICZ alpha A, transforming the expression vector pPICZ alpha A-CTP to escherichia coli Top 10 competent cells, and constructing a recombinant expression vector pPICZ alpha A-CTP, wherein the construction process of the vector is shown as figure 2.
Extracting plasmids according to the operation method of a small plasmid extraction kit of Tiangen Biochemical technology (Beijing) Ltd, performing PCR amplification on a CTP gene by taking the extracted plasmids as a template, wherein an upstream primer is 5'-CCAGATGGGGTAGATTCTT-3'; a downstream primer: 5'-GTAACGTCCTTTCTCTTTG-3' are provided. The amplification product was subjected to agarose gel electrophoresis, observed under an ultraviolet lamp, and the conversion result was identified, as shown in fig. 3, showing that a band (96bp) of the CTP gene was present in the amplification 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 recombinant expression vector pPICZ alpha A-CTP is successfully constructed.
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 heterozygosis peptide CTP
Converting the recombinant expression vector pPICZ alpha A-CTP constructed in the step 3 into a Pichia pastoris GS115 strain, wherein the specific method is as follows:
mixing 5-10 mu g of linearized recombinant plasmid pPICZ alpha A-CTP with 200 mu l of competent cells of activated Pichia pastoris GS115, transferring the mixture into an electric rotating cup, carrying out ice bath for 5-30min, carrying out electric rotation for a plurality of seconds after ice bath (oscillation is avoided in the electric rotation process), then quickly placing the mixed solution on ice, quickly adding 1ml of ice-bath 1M sorbitol solution, suspending and uniformly mixing thalli, transferring the thalli into a 1.5ml EP tube, placing the thalli on ice, then adding 2ml of YPD liquid culture medium, and culturing for 3h at 30 ℃ to enable the strains to recover 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 in a YPD liquid culture medium containing 100 mu g/ml kanamycin for overnight culture, extracting plasmids by adopting a small-plasmid extraction medium-volume kit, and identifying a positive transformant through PCR verification to obtain the pichia pastoris engineering bacteria expressing the heterozygous peptide CTP.
5. Inducible expression of the hybrid peptide CTP
Selecting Pichia engineering bacteria expressing hybrid peptide CTP, inoculating into BMGY culture medium containing 100 ug/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 the induced expression of polypeptide CTP. 2ml of bacterial liquid is taken when methanol induction is carried out for 120h, centrifugation is carried out for 5min at 12000rpm, and supernatant is collected and stored at minus 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 CTP, the polypeptide CTP is successfully expressed, and the expression quantity of the polypeptide CTP can reach about 50mg/L through determination.
6. Purification of hybrid peptide CTP
When synthesizing CTP coding gene, histidine tag (6 × His) is added at C-terminal, so polypeptide CTP can be combined with Ni in Ni-NTA Sepharose chromatographic column2+And (3) combining, eluting the fermentation supernatant by using imidazole eluents with different concentrations, and collecting an elution peak sample, so that the separation and purification of the polypeptide CTP can be realized. Method for purifying Ni-NTA Sepharose chromatographic column by polypeptide CTP (computer to plate) by referring to using instruction of chromatographic column productCollecting samples of each elution peak, detecting the purification effect through Tricine-SDS-PAGE electrophoresis, wherein the molecular weight of CTP is 2.7KDa, and the result is shown in A of figure 4, which shows that the polypeptide CTP with higher purity is obtained through purification; the mass spectrometric detection result of the purified CTP is shown in fig. 4B, which indicates that the molecular weight of the purified polypeptide is consistent with the theoretical molecular weight of CTP.
Example 2 Effect of hybrid peptide CTP 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 condition of (1), adding a series of concentration gradients of CTP, CATH2 and TP5(0-100 mu g/mL), and after culturing for 24 hours, detecting the influence of the hybrid peptide CTP and the maternal peptides CATH2 and TP5 on the survival rate of mouse macrophages by using a CCK8 method. The result is shown in figure 5, the cytotoxicity of the polypeptide CTP is obviously reduced compared with that of the parent peptide CATH2, and the polypeptide CTP has no cytotoxicity at the concentration of 10 mug/mL, thereby being beneficial to subsequent research and application.
Example 3 immunomodulatory Activity of hybrid peptide CTP in mouse macrophages
Diluting the hybrid peptide CTP and the parent peptide CATH2 and TP5 with DMEM medium, preparing a polypeptide solution with the concentration of 10 mu g/mL, and detecting the influence of the CTP and the parent peptide CATH2 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 inflammation state. Respectively setting a normal group (Control), a model group (LPS), a test group 1(CATH2), a test group 2(TP5), a test group 3(CTP), a test group 4(CATH2+ LPS) and a test group 5(TP5+ LPS), wherein the normal group is not subjected to any treatment; test groups 1, 2 and 3 respectively add CATH2, TP5 or CTP solution with the final concentration of 10 mug/mL after overnight culture of cells; test groups 4, 5, and 6 were added with a final concentration of 10. mu.g/mL CATH2, TP5, or CTP solution after overnight cell culture, and one hour later, the model group and test groups 4, 5, and 6 were added with a final concentration of 100ng/mL LPS solution, respectively. The ELISA method is adopted to detect the cytokines TNF-alpha and IFN-gamma, the result is shown in figure 6, under the normal state, the hybrid peptide CTP can obviously improve the expression level of the mouse macrophage cytokines TNF-alpha (A in figure 6) and IFN-gamma (B in figure 6), and the expression level of the macrophage cytokines TNF-alpha and IFN-gamma in the CTP group is obviously higher than that in the CATH2 group and the TP5 group; but at the same time, when the cells are in an inflammatory state induced by LPS, CTP can obviously inhibit the expression of TNF-alpha (A of figure 6) and IFN-gamma (B of figure 6) cytokines, and the inhibition effect is better than that of the maternal peptides CATH2 and TP 5. Therefore, the polypeptide CTP has the two-way immunoregulation function, can enhance the immunocompetence of cells under the normal state and inhibit the inflammatory reaction of the cells under the inflammatory state, and has the immune anti-inflammatory effect superior to the maternal peptides CATH2 and TP 5.
EXAMPLE 4 immunomodulatory Effect of hybrid peptide CTP 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 polypeptide CTP on body weight and immune organ weight of 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 (CTP + CY): CTP (10mg/kg) and cyclophosphamide CY (100mg/kg) were injected.
Mice in the test group were administered polypeptide CTP (administered at a dose of 10mg/kg) by intraperitoneal injection for 14 days, once a day, and the blank group and the model group were administered with corresponding volumes of physiological saline. 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 A, B and C of fig. 7, and the results show that the body weight, spleen index and thymus index of the model group mice are all significantly reduced compared with those of 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 polypeptide CTP can promote the growth of the mice with low immune function and has protective effect on the immune organs of the mice with low immune function.
2. Effect of polypeptide CTP 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: CTP (10mg/kg) and cyclophosphamide CY (100mg/kg) were injected.
The administration modes of the blank group, the model group and the test group are the same as that in the 1, 24 hours after the last administration, the mice are killed after blood sampling, the mice are respectively soaked in 75% ethanol for 5-10s, 4mL of RPMI1640 added with heparin is injected into the abdominal cavity, the eluate is centrifuged, the supernatant is discarded, and the abdominal cavity macrophage is 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. 7D, and the results show that compared with the blank group, the phagocytic rate of the neutral erythrocytes of the model group is significantly reduced, cyclophosphamide can inhibit the phagocytic activity of macrophages of mice, and CTP can restore the phagocytic activity of macrophages of mice with low immune function, thereby enhancing the innate immunity of the mice with low immune function.
(3) Effect of polypeptide CTP on cytokine release amount of immunosuppressive 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: CTP (10mg/kg) and cyclophosphamide CY (100mg/kg) were injected.
The administration modes of the blank group, the model group and the test group are the same as those in the above 1, and 24 hours after the last administration, blood is taken from eyeballs, serum is separated, and the contents of cytokines (IFN-gamma, IL-6) in the mouse serum are detected by an ELISA method. The experimental results are shown in E, F of fig. 7, and the results show that the content of the cytokines (IFN-gamma and IL-6) in the mice of the model group is obviously reduced compared with that of the blank group; the administration of the polypeptide CTP 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 5 anti-inflammatory Effect of hybrid peptide CTP 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 polypeptide CTP 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 (CTP + LPS): CTP (10mg/kg), LPS (10 mg/kg).
Mice in the test group were administered polypeptide CTP (administration dose of 10mg/kg) by intraperitoneal injection for 7 days, and mice in the blank group and the model group were administered physiological saline in a volume corresponding to the administration dose 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 A, B and C of fig. 8, and the results show that the weight and the intestinal length of the model group mice are both significantly reduced compared with those of the blank group, and the intestinal villus morphology of the model group is found to be damaged through section observation, and meanwhile, the submucosa has obvious edema phenomenon, 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 polypeptide CTP can protect the damage of inflammatory reaction induced by LPS to the body weight and the intestinal tract of the mice.
2. Effect of polypeptide CTP on cytokine expression level of mice in 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: CTP (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 result is shown in D, E of fig. 8, and the result shows that the content of the cytokines (IFN-gamma and IL-6) of the model group mice is obviously increased compared with that of 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 polypeptide CTP, thereby inhibiting inflammatory reaction.
In conclusion, the hybrid peptide CTP has bidirectional immunoregulation effect. On one hand, CTP 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 CTP can be used for preventing and treating infection caused by low immune defense and immune monitoring functions. On the other hand, the polypeptide CTP can inhibit the inflammatory reaction of the organism, reduce the release amount of cytokines in the inflammatory state and relieve the damage of the inflammatory reaction to tissues such as intestinal tracts of animals and human beings. Therefore, the polypeptide CTP can be used for treating inflammatory diseases (such as enteritis and the like).
In addition, the invention also carries out functional experiments on derivatives of the polypeptide CTP, such as the CTP with amidated tail end and the CTP after corresponding substitution of the 6 th amino acid, and the result shows that the derivatives of the polypeptide CTP also have similar immune anti-inflammatory bidirectional regulation function with the polypeptide CTP.
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.
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Claims (11)
1. A hybrid peptide with immunoregulatory and anti-inflammatory functions 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. 6.
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 yeast is Pichia pastoris.
8. Use of a hybrid peptide according to claim 1 or a hybrid peptide produced by a method according to any one of claims 5 to 7 or a gene according to claim 2 or 3 or a biological material according to claim 4 for the preparation of an immunomodulating formulation.
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 agent.
10. Use according to claim 8 or 9, wherein the preparation is a medicament, nutraceutical, food or feed additive.
11. A product 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;
the product is any one selected from medicine, health product, food or feed additive.
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