Bursal heptapeptide and preparation method and application thereof
阅读说明：本技术 一种法氏囊七肽及其制备方法和应用 (Bursal heptapeptide and preparation method and application thereof ) 是由 刘晓东 董旭峰 王述柏 刘旭 秦志华 于 2020-11-04 设计创作，主要内容包括：本发明公开了一种从法氏囊中发现一种新的七肽,氨基酸序列为SEQ ID NO:1 APKPRKK,该七肽是第一次从禽类的中枢体液免疫器官中分离出来。本发明还提供了上述法氏囊七肽的分离方法。本发明提供的法氏囊七肽在实验动物体内具有明显的免疫调节作用,法氏囊七肽对体液免疫具有诱导效应(如调节抗体的产生和分型。(The invention discloses a novel heptapeptide found from bursa of Fabricius, the amino acid sequence of the heptapeptide is SEQ ID NO. 1APKPRKK, and the heptapeptide is separated from central humoral immune organs of poultry for the first time. The invention also provides a separation method of the bursa of Fabricius heptapeptide. The bursa of Fabricius heptapeptide provided by the invention has an obvious immunoregulation effect in an experimental animal body, and has an induction effect (such as generation and typing of a regulatory antibody) on humoral immunity.)
1. The bursa of Fabricius heptapeptide is characterized in that the amino acid sequence is SEQ ID NO 1 APKPRKK.
2. The method of preparing the bursa of Fabricius heptapeptide of claim 1, comprising:
1) taking the bursa of Fabricius tissues of healthy young chickens, grinding the bursa of Fabricius tissues in normal saline, wherein the proportion of bursa of Fabricius to normal saline is 2: 3; repeatedly freezing and thawing for three times, centrifuging for 60 minutes at the temperature of 0-10 ℃ of 10000-16000g, filtering by adopting an ultrafiltration membrane with the cutoff molecular weight of 1000Da, and carrying out vacuum freeze drying to obtain a bursa of fabricius heptapeptide freeze-dried sample;
2) diluting the freeze-dried sample with PBS, centrifuging at 12000g for 10 min, filtering with a 0.22 μm filter membrane, and freeze-drying to obtain a secondary freeze-dried sample;
3) diluting the secondary freeze-dried sample with the solution B, centrifuging at 12000g for 10 minutes, and then filtering with a 0.22um filter membrane; purifying and analyzing the filtrate by reverse high performance liquid chromatography, and harvesting an active peak with the retention time of 10 minutes to obtain the active peptide; the solution B is an aqueous solution containing 2% of acetonitrile and 0.065% of trifluoroacetic acid.
3. Use of the bursa of Fabricius heptapeptide of claim 1 for the preparation of an immunopotentiator.
The technical field is as follows:
the invention relates to the technical field of biological products, in particular to bursa of Fabricius heptapeptide with an immunoregulation effect and application thereof in immunity.
Background art:
the Bursa of Fabricus (BF) was first discovered by Hieromonas Fabricus in 1961, and the shape and location of the bursa of Fabricus was also well-defined, hence the name of Bursa of Fabricus. The bursa of Fabricius is a central lymph organ of the specific humoral immunity of the poultry, is positioned at the back side of a cloaca, has a sac-shaped cecum end and is opened in the cloaca, and a neck part of the bursa of Fabricius faces the front end of a body and a short handle is positioned towards the back lower part of the body. Early researchers observed that bursa of Fabricius gradually degenerated with sexual maturation, so it has been considered to be an organ involved in reproductive physiology. Since Glick et al discovered that removal of the bursa of Fabricius reduced antibody production by chicks to Salmonella "O" antigen in 1956, bursa of Fabricius was identified as the central lymphoid organ specific to birds as the earliest site for B cell colonization and proliferation by which B cells migrate to peripheral lymphoid organs and perform immune functions.
In 1960, Glick et al reported that injection of a physiological saline suspension of a 9 hour treatment with acetone for 6 times of the bursa of Fabricius crude extract increased the level of antibodies to Sheep Red Blood Cells (SRBC) in chicks from which bursa of Fabricius was removed. Baba et al 1976 reported that injection of chicken bursa of Fabricius extract into bursa of Fabricius excised chicks resulted in IgG production and increased antibody levels in these chickens. In 1976, Brand found that bursa of Fabricius extracts induced the expression of Th-1 antigen by chicken bone marrow cells, and that factors promoting the expression of Bu-1 antigen by early B cells were present, and that the lower the concentration of bursa extract, the stronger the ability to induce B cell differentiation. In 1981, Shunhua et al studied the regeneration of bursa of Fabricius with chicken embryo bursa extract. In 1982, Eerola et al reported that cultured bursal epithelial cells in vitro and serum-free culture supernatants exhibited biological properties that promote differentiation of B lymphocytes. Audhya et al first reported in 1986 that 1.3mg of Bursin (BS) was isolated from 160g of chicken bursa of Fabricius. The mass spectrometric identification analysis revealed that BS was a substance having a tripeptide arrangement structure with the amino acid sequence Lys-His-Gly-NH2, wherein Lys: His: Gly: NH2 was 1: 0.9: 1: 0.8, and was shown to induce differentiation of B cell precursors and specifically increase cAMP and cGMP levels in human Daudi B cells, while being weak in T cells. When stimulated with BS at a concentration of 100ug/ml, the levels of cAMP and cGMP in B lymphocytes reached a maximum of 8-15pmol/ml, whereas in T lymphocytes only the cGMP level was elevated, not the cAMP level. This is the first structurally defined active factor obtained from bursa of Fabricius and involved in B-cell development.
Two bursal heptapeptide BP7 with immune enhancement function are respectively found in Chinese invention patent applications with publication numbers CN109134613A and CN 101830968A.
The invention content is as follows:
the invention discovers a new bursal heptapeptide for the first time, and compared with the two bursal heptapeptides discovered in the prior art, the discovered BP7 can obviously improve the immune efficacy of the vaccine and can also obviously promote the differentiation of B cells.
The invention provides the following technical scheme:
the invention provides a novel bursal heptapeptide, the amino acid sequence of which is SEQ ID NO 1 APKPRKK.
The invention also provides a preparation method of the bursa of Fabricius heptapeptide, which comprises the following steps:
1) taking the bursa of Fabricius tissues of healthy young chickens, grinding the bursa of Fabricius tissues in normal saline, wherein the proportion of bursa of Fabricius to normal saline is 2: 3; repeatedly freezing and thawing for three times, centrifuging for 60 minutes at the temperature of 0-10 ℃ of 10000-16000g, filtering by adopting an ultrafiltration membrane with the cutoff molecular weight of 1000Da, and carrying out vacuum freeze drying to obtain a bursa of fabricius heptapeptide freeze-dried sample;
2) diluting the freeze-dried sample with PBS, centrifuging at 12000g for 10 min, filtering with a 0.22 μm filter membrane, and freeze-drying to obtain a secondary freeze-dried sample;
3) diluting the secondary freeze-dried sample with the solution B, centrifuging at 12000g for 10 minutes, and then filtering with a 0.22um filter membrane; purifying and analyzing the filtrate by reverse high performance liquid chromatography, and harvesting an active peak with the retention time of 10 minutes to obtain the active peptide; the solution B is an aqueous solution containing 2% of acetonitrile and 0.065% of trifluoroacetic acid.
The invention further provides application of the bursa of Fabricius heptapeptide in preparation of an immunopotentiator.
The preparation method of the bursa of Fabricius heptapeptide provided by the invention has the following beneficial effects:
1. a novel heptapeptide is found in bursa of Fabricius, the amino acid sequence of which is SEQ ID NO:1APKPRKK, and the heptapeptide is separated from the central humoral immune organs of the poultry for the first time.
2. Has obvious immunoregulation effect in experimental animals, and the bursal heptapeptide has induction effect (such as regulating antibody generation and typing) on humoral immunity and can also obviously promote B cell differentiation.
Description of the drawings:
FIG. 1: high performance liquid chromatogram for the isolation and purification of bursal heptapeptide, indicated by the arrow, BP7
The elution peak of (4);
FIG. 2 is a mouse lymphocyte proliferation analysis chart, compared with a blank control group, the difference is significant (P is less than 0.01);
FIG. 3 is an AIV antibody IgG assay with significant differences (P < 0.01) compared to the AIV immune group;
FIG. 4 is a cytokine profile showing significant differences (P < 0.01) compared to the blank control;
FIG. 5 is a schematic diagram showing the results of B cell differentiation experiments.
The specific implementation mode is as follows:
the following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention more readily understood by those skilled in the art, and thus will more clearly and distinctly define the scope of the invention.
Example 1, isolation and characterization of bursal heptapeptide:
4000 g of bursa of Fabricius (AA broiler chicken, affiliated farm of Shanghai agricultural academy of sciences) of healthy 4-6 weeks old chickens are taken, crushed by 6000ml of normal saline, mixed evenly and repeatedly frozen and thawed for three times. After centrifugation at 12000g for 60 minutes at 4 ℃ the supernatant was transferred to another tube and centrifuged once again. Then ultrafiltered through 1000Da ultrafiltration membrane, and the filtrate is lyophilized. The lyophilized samples were diluted with PBS, centrifuged at 12000g for 10 min, then filtered through a 0.22um filter and lyophilized. The lyophilized sample was diluted with solution B (2% acetonitrile, 0.065% trifluoroacetic acid), centrifuged at 12000g for 10 minutes, and then filtered through a 0.22um filter. The filtrate was analyzed by reversed phase high performance liquid purification, and the activity peak with a retention time of 10 minutes was harvested (see FIG. 1). The harvested eluate was analyzed by MALDI-TOF-MS for the amino acid sequence APKPRKK (SEQ ID NO:1), which was first isolated from bursa of Fabricius.
The heptapeptide with the same amino acid structure is synthesized by a solid phase chemical synthesis method, and the purity is over 97 percent.
Example 2 use of BP7 as an immunopotentiator
1. BP7 Synthesis
Adopts a solid phase chemical synthesis method to synthesize bursa of Fabricius heptapeptide (APKPRKK), and the purity is more than 98 percent.
2. Product use
BALB/c mice (purchased from the center of laboratory animals at Yangzhou university) 4-6 weeks old were immunized twice in combination with 40. mu.g/ml bursal heptapeptide, and 14 days after the immunization, blood was collected, serum was separated, changes in antibody IgG were detected, and the humoral immunity-modulating function of bursal heptapeptide was analyzed (see Table 1). Through the experiment, the bursa of Fabricius heptapeptide can improve the antibody level of the mouse and has the function of regulating the cellular immune response.
Table 1: mouse immunization procedure
2) The antibody IgG detection adopts an indirect ELISA method, and the specific operation method comprises the following steps: blood was collected and serum was isolated for detection of specific subtype antibodies at 14 days, 28 days and 42 days after immunization, respectively. Will 102.5EID50The strips were coated with 100. mu.l/well of inactivated AIV antigen, incubated overnight at 4 ℃ or for 2 hours at 37 ℃ and washed three times, then blocked with 1% BSA at 37 ℃ for 2 hours or overnight at 4 ℃. Then adding serum according to the ratio of 1: 10-1: 105Four gradients, acting at 37 ℃ for 2 hours, then adding HRP-labeled goat anti-mouse IgG diluted 1:1000 respectively, acting at 37 ℃ for 2H, fully washing, adding a TMB substrate developing solution, acting at 37 ℃ for 10-15 min, and then adding 2M H2SO4End, 450nm reading, analysis results.
The results showed that bursal heptapeptide (BP7) significantly modulated the level of antibody IgG (see FIG. 2).
3) Splenocyte proliferation assay at 2nd1 week after boost immunization, mouse blood lymphocytes were aseptically isolated and plated onto 96-well cell plates (10)5Individual cells/well). After 48 hours of stimulation with AIV antigen, 100. mu.g/well MTT (Sigma, USA) was added for 4 hours, the supernatant was discarded, 100. mu.l/well DMSO (Sigma, USA) was added, OD570 nm was read, and the results were analyzed. As a result, BP7 increased the activity of spleen lymphocytes, as shown in FIG. 3.
4) Cytokine assay, ELISA method, kit (RD, USA) is used to detect IL-4 and IFN-gamma content in serum. Serum was isolated 1 week after the second immunization. Operating according to the instruction. The result shows that BP7 can promote the secretion of cell factor, and the IL-4 and IFN-gamma amount in chicken experiment is obviously higher than that in vaccine control group. As shown in FIG. 4, bursal heptapeptide not only increased the level of the cytokine IFN-. gamma.representing the Th1 type immune response, but also significantly increased the level of the cytokine IL-4 representing the Th2 type. In mouse experiments, IL-4 was significantly higher than that of vaccine control group (see FIG. 4)
Example 3B cell differentiation assay
1. BP7 Synthesis
Adopts a solid phase chemical synthesis method to synthesize bursa of Fabricius heptapeptide (APKPRKK), and the purity is more than 98 percent.
2. Product use
Obtaining 1 × 106 cells/ml bone marrow mononuclear cells (C57/BL 6 mice are selected), adding IL-7(10ng/ml), and subpackaging on 6-hole cell plates;
② test group BP7(1g/ml-25g/ml) is stimulated for 7 days, and control group BSA (1g/ml) is stimulated;
thirdly, judging the basis of the result: counting colonies of greater than 40 cells;
the results are shown in FIG. 5, where BP7 significantly improved colony formation of B cells and was dose dependent.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Sequence listing
<110> Qingdao agricultural university
<120> bursa of Fabricius heptapeptide, and preparation method and application thereof
<130> 11531
<141> 2020-11-04
<160> 1
<170> SIPOSequenceListing 1.0
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<211> 7
<212> PRT
<213> bursa
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Ala Pro Lys Pro Arg Lys Lys
1 5