Tetrapeptide separated from bursa of Fabricius and application of tetrapeptide in immunoregulation

Abstract

The invention aims to provide a tetrapeptide separated from a bursa of Fabricius and an application of the tetrapeptide in immunoregulation, namely the tetrapeptide which has an immunoregulation effect and is separated from the bursa of Fabricius, and the amino acid sequence of the tetrapeptide is VVMK. The bursa of Fabricius tetrapeptide separated from the bursa of Fabricius extract is simple in structure, free of toxic and side effects and extremely weak in immunogenicity. The polypeptide disclosed by the invention has an induction effect on generation of an antibody, and can be used for regulating generation of cell factors and proliferation of lymphocytes and promoting cellular immune response. In addition, differentiation of the B cells is adjusted in a dose-dependent mode, and the method has wide application prospects such as application in the aspects of immunoregulation, treatment and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of functional polypeptides, and in particular relates to a tetrapeptide separated from a bursa of Fabricius and an application thereof in immune regulation. Background Art

[0002] The bursa of Fabricius (BF) is a less obvious sac-like structure attached to the dorsal anal region of the cloaca of birds. The bursa of Fabricus (BF) was first discovered by the Italian anatomist Hieronymus Fabricius in 1621, hence its name. Initially, Fabricius speculated that the hollow structure of the bursa was likely to be a container for storing semen. However, this is not the case. For hundreds of years, the function of the bursa has been puzzling many researchers. Since the bursa degenerates with sexual maturity, its size is negatively correlated with the testes and adrenal glands, so some researchers speculate that the BF must be some kind of endocrine gland or lymph gland related to growth and sexual development. As early as 1956, Glick et al. accidentally discovered that removing the bursa of Fabricius can reduce the production of antibodies to the Salmonella O antigen in young birds, but it did not attract much attention. Many years later, the importance of this work was properly treated, and mainstream immunologists also became very interested in the immune system of chickens. People finally reached a consensus that the bursa of Fabricius is an organ necessary for antibody (mediated) immunity, while the thymus, which also degenerates with sexual development, is an organ necessary for cell (mediated) immunity. Now, the bursa of Fabricius is confirmed to be a humoral central immune organ. It is connected and attached to the anal canal of birds by a duct. It is the place where B lymphocytes develop, differentiate and mature. The fully differentiated B lymphocytes are transferred from the BF to the peripheral lymphoid organs to live and reproduce, and dominate the immune function. Therefore, it is necessary for the occurrence of B lymphocytes (bursa-derived lymphocytes) and the antibody spectrum of birds.

[0003] BF is the place where B cells are induced to differentiate and mature. Lymphoid stem cells (B lymphocyte precursors) go from the bone marrow to BF, where mature B cells are induced to differentiate and participate in immune responses and are transferred to various peripheral lymphoid organs through the blood circulation and lymphatic circulation. Immunoglobulin (Ig) gene rearrangement is an early event in the development of the B cell line. The BF primordium begins to develop on the third day of the embryonic period. When the embryo develops to 8-14 days old, the BF primordium is formed by the anterior capsule stem cells. Before entering the BF follicle, the immunoglobulin L chain and H chain of the stem cell have been rearranged. At 12 days of age, IgM can be detected in BF. +Cells, after hatching, most (more than 90%) blastocyst cells are IgM+ cells. In the late embryonic period, B cells begin to enter peripheral immune tissues. BF degenerates at 6 months after hatching, and at this time, all B cells have entered peripheral immune tissues. Moriya et al. treated 10-day-old chicken embryos with testosterone, and detected the levels of J chain and μ chain in embryonic and chick serum, and found that the level of J chain decreased, while the level of μ chain did not change significantly during the embryonic period and after hatching. Mansikka et al. surgically removed BF at 60h of chicken embryo development, and still produced immunoglobulins to form B cells, but could not produce specific antibody responses. DNA sequence analysis showed that only a few B cell precursors differentiated into mature immunoglobulins to form B cells. Jankovic et al. could not eliminate humoral immunity or prevent the differentiation of Bu lymphocytes with specific origin from BF after removing BF primordium at 52-64h of embryonic development. Tong Liqi et al. reported that surgical removal of BF in Beijing duck embryos inhibited the development of the spleen, and the effects on peripheral lymphocytes and organogenesis were not significantly different in the middle and late embryos. Removal of BF after hatching cannot completely eliminate the production of antibodies, but can inhibit the production of specific antibodies accompanied by the secretion of IgM and IgG.

[0004] In 1976, Baba et al. found that crude BF extracts can restore BF in chicks that have had BF removed during the embryonic period, and can also restore the induction of immunoglobulin-specific antibodies. In 1977, Baba et al. reported that bursal extracts can restore the inhibition of bursal development by testosterone propionate (TP), increase the antibody level of chickens, and convert antibodies from IgM to IgG. In 1976, Brand et al. found that chick bone marrow cells were induced by BF extracts to express antigens, factors that promote early cell expression of antigens, and that the concentration of bursal extracts was negatively correlated with the ability to induce cell differentiation. In 1982, Eerol et al. reported that in vitro cultured BF epithelial cells and their serum-free culture supernatants have the ability to enhance lymphocyte differentiation. In addition, Miao De Nian et al. analyzed BF extracts using thin layer chromatography and found that BF extracts contained bursal proteins and no less than 8 different peptides or amino acid components. In recent years, with the continuous research on active peptides of bursal bursa, some active peptides with clear structures and functions have been continuously discovered.

[0005] Bursal anti-steroidogenic peptide (BASP) is another bursal active peptide that has been extensively studied. The humoral immune system may feedback regulate the thalamus-pituitary-gonadal axis of the newborn. Byrd et al. studied the effects of an acidic solution of bursal extract from prepubertal chickens on basal and LH-stimulated progesterone biosynthesis in granulosa cells isolated from the largest preovulatory follicles of the chicken ovary in vitro. The results showed that the 3-5 kDa crude extract significantly inhibited LH-stimulated progesterone secretion (P<0.05). Using reverse HPLC technology, a bursal active component was partially purified and had the ability to inhibit steroid peptides in vitro. This component inhibited the biosynthesis of LH-stimulated progesterone, with a maximum inhibition of about 79%. After removing the peptide, the granulosa cells had the ability to LH-stimulated progesterone biosynthesis similar to that of the control cells; and the bursal extract significantly inhibited the biosynthesis of progesterone stimulated by cAMP analogs. These data suggest that the antisteroidal peptide from the chicken bursa of Fabricius is a single heat-labile, N-terminally blocked peptide that has biological activity in granulosa cells (which are independent of the gonadotropin receptor).

[0006] Some progress has been made in the study of bursal active peptides, but at present, the understanding of bursal active peptides is still relatively superficial. There are still many aspects to be solved regarding their biological characteristics and functions, especially the application of known bursal active peptides is almost blank, and further research is needed. The bursa is currently the only recognized central humoral immune organ in the world. Due to the important immunomodulatory activity of the bursa and the extensive biological activity of bursal extracts. Therefore, the study of active substances in the bursa has a fairly broad field and a wide range of practical application significance. In the diagnosis and treatment of diseases, thymus active substances have played a pivotal role and are now widely used in the clinical treatment of hepatitis (hepatitis B, hepatitis C, etc.), tumors (cancer), and immunodeficiency research. However, with the in-depth study of the function and mechanism of action of bursal active peptides and continuous clinical and practical application experiments, in the near future, some bursal active peptides will be widely used in clinical medicine and production practice like thymosin. Therefore, bursal active peptides have broad application prospects, such as immunomodulation, immunotherapy and other fields. Summary of the invention

[0007] The purpose of the present invention is to provide a tetrapeptide separated from the bursa of Fabricius and its application in immunoregulation, that is, a tetrapeptide separated from the bursa of Fabricius with immunoregulatory effect, and its application in improving the immune ability of animals, improving the immune effect of vaccines and regulating B cell differentiation.

[0008] The present invention first provides a polypeptide, comprising:

[0009] 1) a polypeptide having an amino acid sequence of VVMK (SEQ ID NO: 1);

[0010] 2) A polypeptide derived from 1) by substituting, deleting or adding one or more amino acids in the amino acids in 1) and having the functions of the polypeptide in 1).

[0011] The above polypeptide is used to prepare vaccines or auxiliary preparations for improving animal immunity;

[0012] As a preferred embodiment, the above polypeptide is used to prepare antibodies;

[0013] As a preferred embodiment, the above polypeptide is also used to prepare a product for promoting B cell proliferation and / or differentiation.

[0014] Compared with the prior art, the present invention has the following advantages and significance:

[0015] 1. A new tetrapeptide with a molecular weight of 529.7 (m / z) and an amino acid sequence of VVMK was found in the bursa of Fabricius. This tetrapeptide was isolated from the bursa of Fabricius, the central humoral immune organ of poultry, for the first time, indicating that there are multiple active molecules in the bursa of Fabricius, which may play different immunomodulatory roles.

[0016] 2. It has obvious immunomodulatory effect in experimental animals. The above-mentioned bursal tetrapeptide can be used to verify its immunomodulatory effect on antibodies. According to the weight of the animal, 0.8-20 μg is used for each animal, mixed with the AIV vaccine, and injected into the immunized animal. It is characterized in that: when the active peptide is 20 μg / animal, it is used for animals according to the recommended dose of the vaccine, and the active peptide has a better effect. The bursal tetrapeptide not only has an inductive effect on humoral immunity (such as regulating the production of antibodies and lymphocyte proliferation), which is similar to the effect of bursal extracts reported in the past, but also has a strong regulatory effect on cell-mediated immune responses, which is rarely mentioned in previous reports, indicating that the bursa of Fabricius is an organic combined organ with a variety of biological regulatory molecules to coordinate and regulate immune responses. The present invention supplements the deficiencies in this regard and enriches the content in this regard.

[0017] 3. In vitro, bursal tetrapeptide can affect the growth and differentiation of B cells. Bursal tetrapeptide has the function of regulating B cell differentiation, thus providing new agents and methods for the treatment and prevention of immunodeficiency diseases, and is expected to play an important role in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Isolation and purification of bursal tetrapeptide, HPLC chromatogram, the arrow indicates the elution peak of BP-E4.

[0019] Figure 2:A graph of AIV antibody IgG determination, compared with the AIV immunization group, the difference is significant P < 0.05; the difference is extremely significant P < 0.01.

[0020] Figure 3 : The second immunization AIV antibody IgG determination chart, compared with the AIV immunization group, the difference was significant P < 0.05; the difference was extremely significant P < 0.01.

[0021] Figure 4 : Cytokine analysis chart, compared with AIV immunization group, the difference was significant P<0.05; the difference was extremely significant P<0.01.

[0022] Figure 5 : Cytokine analysis chart, compared with AIV immunization group, the difference was significant P<0.05; the difference was extremely significant P<0.01.

[0023] Figure 6 :Analysis of lymphocyte proliferation in mouse experiment. Compared with AIV immunization group, the difference was significant (P<0.05); the difference was extremely significant (P<0.01).

[0024] Figure 7 : DT-40 cell proliferation analysis chart. Compared with the AIV immunization group, the difference was significant (P<0.05); the difference was extremely significant (P<0.01). DETAILED DESCRIPTION

[0025] The present invention separates a new tetrapeptide from the bursal tissue fluid and identifies its amino acid structure composition through ultrafiltration molecular interception, molecular sieve, reverse phase high pressure liquid chromatography (HPLC) column chromatography, mass spectrometry and amino acid analysis. The immunoregulatory mechanism of the bursal tetrapeptide in animals is studied in depth and systematically, thereby providing a theoretical basis for the clinical application of the bursal active peptide and is expected to promote the development of avian immunology theory.

[0026] The present invention is described in detail below with reference to the embodiments.

[0027] Example 1, separation and identification of bursal protein tetrapeptide:

[0028] Take 4000 grams of the bursa of Fabricius from a healthy 4-6 week old chicken (AA broiler, farm affiliated to Shanghai Academy of Agricultural Sciences), crush and mix with 6000 ml of physiological saline, and freeze-thaw repeatedly three times. Centrifuge at 12000g for 60 minutes at 4°C, transfer the supernatant to another tube, and centrifuge again. Then ultrafilter through a 1000Da ultrafiltration membrane, and freeze-dry the filtrate. The freeze-dried sample was diluted with PBS, centrifuged at 12000g for 10 minutes, then filtered with a 0.22μm filter membrane and freeze-dried. The freeze-dried sample was diluted with solution B (2% acetonitrile, 0.065% trifluoroacetic acid), centrifuged at 12000g for 10 minutes, and then filtered with a 0.22μm filter membrane. The filtrate was purified and analyzed by reverse HPLC, and the active peak with a retention time of 21.25 minutes was harvested (see Figure 1 The eluate was analyzed by MALDI-TOF-MS, and the molecular weight was 529.7, and the amino acid sequence was VVMK (SEQ ID NO: 1), which was the first time that the gene was isolated from the bursa of Fabricius.

[0029] The solid phase chemical synthesis method is used to synthesize tetrapeptides with the same amino acid structure, with a purity of more than 97%.

[0030] Example 2: Application of BP-E4 as an immunopotentiator

[0031] 1. BP-E4 synthesis

[0032] The bursal peptide VVMK was synthesized by solid phase chemical synthesis method with a purity of more than 98%.

[0033] 2. Product Usage

[0034] 4-6 week old BALB / c mice (purchased from Yangzhou University Experimental Animal Center) were immunized twice with an inactivated avian influenza vaccine (H9 subtype NJ01 strain, purchased from Shandong Huahong Bioengineering Co., Ltd.) and 1, 0.2, 0.04 mg / Kg of bursal tetrapeptide. Blood was collected 14 days after the two immunizations, serum was separated, and the changes in antibody IgG were detected to analyze the regulatory function of bursal tetrapeptide on humoral immunity (see Table 1). Through this experiment, it can be found that bursal tetrapeptide can increase antibody levels in mice and has the function of regulating cellular immune response.

[0035] Table 1: Mouse immunization schedule

[0036]

[0037] 2) Antibody IgG detection, using the indirect ELISA method, the specific operation method is: 14 days and 28 days after immunization, blood was collected and serum was separated to detect specific subtype antibodies. The AIV antigen was coated on the strips, 100μl / well, incubated at 4℃ overnight, or at 37℃ for 2 hours, washed three times, and then blocked with 1% BSA at 37℃ for 2 hours, or at 4℃ overnight. Then serum was added, diluted 1:100, incubated at 37℃ for 2 hours, and then HRP-labeled goat anti-mouse IgG was added at 1:1000, incubated at 37℃ for 2 hours, after sufficient washing, TMB substrate colorimetric solution was added, incubated at 37℃ for 10-15 minutes, and then 2MH2SO4 was added to terminate the reaction, and the reading was at 450nm. The analysis results showed that 1mg / Kg of Fabricius tetrapeptide (BP-E4) could significantly regulate the level of antibody IgG in the first immunization, and 1mg / Kg and 0.2mg / Kg could significantly regulate the level of antibody IgG in the second immunization ( Figure 2 , Figure 3 ).

[0038] Table 2: Data table of immune serum antibodies

[0039]

[0040] Table 3: Secondary immune serum antibody data table

[0041]

[0042]

[0043] 3) Cytokine test, using ELISA method, using kits (Shanghai, Langton) to detect the content of IL-4 and IFN-γ in serum. One week after the second immunization, blood was collected to separate serum. Operation was performed according to the instructions. In the mouse experiment, IL-4 and IFN-γ in the 1mg / Kg polypeptide experimental group were significantly higher than those in the vaccine control group ( Figure 4 , Figure 5 ). This indicates that 1 mg / kg of bursal tetrapeptide can not only increase the level of IFN-γ, a cytokine representing the Th1 type of immune response, but also significantly increase the level of IL-4, a cytokine representing the Th2 type.

[0044] Table 4: Secondary immune serum IL-4 data table

[0045]

[0046] Table 5: Secondary immune serum IFN-γ data table

[0047]

[0048]

[0049] 3. Splenocyte proliferation test

[0050] In 2 nd One week after boost immunization, chicken blood lymphocytes were aseptically separated and dispensed into 96-well cell plates (10 7 After stimulating with AIV antigen for 48 hours, 10 μg / well CCK-8 (Sigma, USA) was added and allowed to act for 4 hours. The OD450nm reading was used to analyze the results. As a result, 1 mg / Kg and 0.2 mg / Kg of BP-E4 significantly increased the activity of spleen lymphocytes ( Figure 6 ).

[0051] Table 6: Splenocyte proliferation data

[0052]

[0053] Example 3, DT-40 cell proliferation test

[0054] DT40 cells have the same biological characteristics as chicken bursal pre-B lymphocytes, and can provide an ideal cell model for studying the mechanism of action of bursal active peptides on B cells. In this study, DT40 cells were used as an avian pre-B cell model, and bursal active peptide VVMK (SEQ ID NO: 1) was used to stimulate DT40 cells at concentrations of 100ug / ml, 20ug / ml, and 4ug / mg, respectively. After 24 hours of stimulation, 10μg / well CCK-8 (Sigma, USA) was added and acted for 4 hours, and the OD450nm reading was analyzed. The results showed that compared with the PBS control group, 20ug / ml BP-E4 could significantly increase the activity of DT-40 cells, 100ug / ml BP-E4 increased the activity of DT-40 cells extremely significantly, and 4ug / mL BP-E4 could enhance the activity of DT-40 cells but not significantly ( Figure 7 ).

[0055] Table 7: DT-40 cell proliferation test table

[0056]

[0057] In summary, the present invention separates bursal tetrapeptide from bursal extract, and the polypeptide has an inducing effect on antibody production, and can also regulate the production of cytokines, the proliferation of lymphocytes, and promote cellular immune response. In addition, the polypeptide also regulates the differentiation of B cells in a dose-dependent manner, in terms of immune regulation and treatment.

Claims

1. A polypeptide, characterized in that The polypeptide comprises: 1) a polypeptide having an amino acid sequence of SEQ ID NO: 1; 2) A polypeptide derived from 1) by replacing, deleting or adding one or more amino acids in the amino acids in 1) and having the functions of the polypeptide in 1).

2. The polypeptide according to claim 1, characterized in that The polypeptide is isolated from the bursa of Fabricius.

3. Use of the polypeptide according to claim 1 in preparing a vaccine or an immunopotentiator for improving animal immunity.

4. A vaccine, characterized in that The vaccine is prepared using the polypeptide according to claim 1 as an antigen.

5. Use of the polypeptide according to claim 1 in preparing antibodies.

6. Use of the polypeptide according to claim 1 in the preparation of a product for promoting B cell proliferation and / or differentiation.

7. A product for promoting B cell proliferation and / or differentiation, characterized in that: The preparation contains the polypeptide according to claim 1 at a pharmacologically effective concentration.

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