Use of sericin and sericin polypeptides

Abstract

The application relates to the field of immunology, and provides an application of silk sericin and silk polypeptide, researches on extraction and purification methods of silk sericin and polypeptide, finds that the protein and polypeptide can promote maturation of dendritic cells, enhance expression of MHCII and CD86 on the cell surface, enhance antigen presentation capacity, and stimulate lymphocyte T cell proliferation in cooperation with the dendritic cells; researches on a preparation method of nanoemulsion vaccine prepared by taking the silk sericin and polypeptide as an adjuvant and antigens, and detection finds that the silk sericin and polypeptide can improve the immune activation efficiency of the vaccine, obviously enhance the antigen-specific acquired immune response level in the body, and effectively inhibit the virus infection of the body and the replication level of the virus, so that the purpose of enhancing the vaccine in preventing or treating diseases is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of immunology, and particularly relates to application of silk fibroin and silk fibroin polypeptide. BACKGROUND

[0002] Vaccine generally refers to biological products for inducing safe immune response by using various antigens, so as to prevent various pathogenic microorganisms from invading the body. The vaccine is the most effective and economic intervention measure for viral infection of these public emergent infectious diseases.

[0003] At present, the mainstream vaccines are mainly attenuated vaccines, inactivated vaccines, recombinant protein vaccines and subunit vaccines. Such vaccines often have problems of poor immunogenicity, slow immune response and easy degradation. The vaccine adjuvant can reduce the use amount of the vaccine, non-specifically enhance or change the specific immune response of the body to the antigen, can induce the body to produce high-efficiency and long-term specific immune response, and is of great significance for the immunocompromised population such as the elderly and children.

[0004] Vaccination is the most effective specific preventive measure for viral infectious diseases, which can significantly enhance the body's resistance to specific pathogens, prevent the invasion and replication of viruses, or reduce the severity of infectious diseases. In recent years, in addition to traditional inactivated vaccines and attenuated vaccines, new vaccines such as recombinant subunit vaccines, synthetic peptide vaccines and nucleic acid vaccines have also rapidly increased. These vaccine antigens have smaller relative molecular mass, higher purity, and weaker immunogenicity, often requiring matching adjuvants to enhance their effects. Adjuvants can enhance the immunogenicity of antigens, enhance the stability of antigens in vaccines, change the intensity of immune responses, improve the level of innate and adaptive immune responses of the immunized organism to antigens, reduce the number of vaccinations and the amount of antigen, and reduce production costs. Therefore, the importance of adjuvants in the production of vaccines is self-evident. There are many types of adjuvants, which can be divided into three categories: immunomodulatory molecule adjuvants, antigen delivery adjuvants, and complex adjuvants, according to their functions and mechanisms. Currently, the most commonly used adjuvant is aluminum adjuvant, followed by emulsions, liposomes, saponins, mineral oils, proteins and polypeptides. Aluminum adjuvants are difficult to enter APC cells, difficult to affect the intracellular presentation of antigens and polypeptides, and cannot cross-present through major histocompatibility complex MHCII molecules to induce CD8+ T cell immune responses, which significantly limits the rapid immune response of therapeutic vaccines or vaccines. The unsaturated bonds in liposome adjuvants are usually easily oxidized, requiring higher preparation conditions, and are not easy to store during transportation, with a short shelf life and significant pain and other adverse reactions at the injection site. Many saponin vaccine adjuvants can cause serious local granulomas and hemolysis in the body during use, so they are mainly used for research on veterinary experimental vaccines. Cytokine adjuvants have poor stability during antigen preparation and are difficult to store, and most cytokines have a short half-life during vaccine action, making it difficult to maintain the ability to enhance immune responses. Freund's complete adjuvant and Freund's incomplete adjuvant are adjuvants prepared from inactivated tubercle bacilli, mineral oil and emulsifiers, which have very strong immune-enhancing effects and are currently unmatched by other adjuvants. However, the safety of Freund's complete adjuvant is very poor, and almost without exception, it causes tissue necrosis, connective tissue tumors, and allergic reactions at the injection site. Although incomplete Freund's adjuvant has strong ability to improve antigen-specific immune responses, the vaccine prepared by incomplete Freund's adjuvant has very high viscosity, which can cause significant pain and swelling at the injection site, and the swelling can last for a year. Studies have shown that incomplete Freund's adjuvant as a mineral oil substance can exist at the injection site for a long time, stimulating chronic inflammation and even connective tissue tumors.

[0005] Different vaccine adjuvants have different mechanisms to enhance immune responses, mainly stimulating and optimizing humoral and cellular immune responses, promoting the mutual transmission and contact between antigen and mucosal immune responses, reducing the amount of antigen required in vaccine formulation and the number of immunization in practical application, maintaining the basic molecular conformation of antigen, etc. The specific mechanisms of adjuvants to promote immune responses include: sustained release of antigen at injection site (antigen depot effect); up-regulation of various cytokines and chemokines; recruitment of immune cells to the injection site; enhanced antigen uptake and presentation; activation of antigen presenting cells (APCs), promoting their maturation and antigen transport to draining lymph nodes; activation of inflammasome, etc.

[0006] An ideal vaccine adjuvant not only needs to efficiently activate immune responses, but also should have a clinically widely recognized safety. Currently, the mainstream of vaccine adjuvant is aluminum salt adjuvant, and more than 25 vaccines approved by FDA for human use contain aluminum salt adjuvant. The effect of aluminum adjuvant on cellular immune response, especially Th1 cells, is not obvious. When aluminum adjuvant interacts with APCs such as dendritic cells, it is difficult to promote antigen internalization, and aluminum adjuvant is difficult to enter APCs, affecting the intracellular presentation of antigen and polypeptide, and cannot cause CD8+ T cell immune response through MHCII molecule cross-presentation, which significantly limits the rapid immune response of therapeutic vaccines or vaccines. On the other hand, although aluminum adjuvant is a safe and effective vaccine adjuvant, its immune enhancement mechanism is still controversial. Currently, the main mechanisms of aluminum adjuvant are antigen depot effect and immune stimulation effect, and some scholars believe that aluminum adjuvant stimulates immune response by promoting partial apoptosis to release inflammatory factors. Freund's complete adjuvant and Freund's incomplete adjuvant belong to adjuvants prepared by inactivated Mycobacterium tuberculosis and mineral oil and emulsifying agent, which has very strong immune enhancement effect, and there is no other comparable adjuvant so far. However, the safety of Freund's complete adjuvant is very poor, and it almost causes tissue necrosis, connective tissue tumors, and side effects such as sensitization at the injection site. The toxicity of Freund's incomplete adjuvant is reduced after improvement, which can significantly enhance the stimulation of T cells to cause immune response. Although the incomplete Freund's adjuvant has strong ability to improve antigen-specific immune response, the vaccine prepared by incomplete Freund's adjuvant has very high viscosity, which can easily cause obvious pain and swelling at the injection site, and the swelling can last for one year. Studies have shown that incomplete Freund's adjuvant as a mineral oil substance can exist at the injection site for a long time, stimulate chronic inflammation, and even cause connective tissue tumors. Protein and polypeptide adjuvants are new types of vaccine adjuvants. It is found that a very small dose of vaccine can produce obvious immune response after two inoculations. HIV-coupled protein and polypeptide can significantly stimulate Th2 cytokine secretion and promote IgG1 and IgG2a production.

[0007] With the outbreak of the new crown vaccine, vaccine research continues to be in the hot, however, the clinical approved effective and safe vaccine adjuvant is few, so the research and development of adjuvant is more and more attention of researchers.

[0008] Silk glue is a spherical protein molecule secreted by silk gland cells, and silk glue protein is rich in serine amino acid sequences, accounting for more than 42%. Each serine residue in the polypeptide chain of silk glue protein contains a methanol group (-CH2OH) or an alcohol hydroxyl group (-OH), which determines that the silk glue protein has good water absorption; the methanol group is easily oxidized to aldehyde group and further oxidized to carboxyl group, so the product made of it has strong antioxidant activity. Silk glue protein has been confirmed to have low immunogenicity, promote cell proliferation, promote wound healing and repair of damaged skin, enhance cell antiviral ability, and its cross-linked silk glue hydrogel has good cell adhesion. The high-dose group of silk glue polypeptide silk protein peptide can significantly improve the phagocytosis of mononuclear phagocytes; effectively enhance and protect the function of the immune system damaged by immunity; within a certain concentration range, silk protein peptide can stimulate the proliferation ability of in vitro spleen lymphocytes. SUMMARY

[0009] In view of the fact that the research direction of silk glue protein and silk glue polypeptide as vaccine adjuvant has not been reported in the prior art, the present application provides a method for preparing and applying silk glue protein and silk glue polypeptide as vaccine adjuvant, studies the extraction and purification method of silk glue protein and polypeptide, and further explores the influence of the protein and polypeptide on the ability of dendritic cells to uptake antigens and present antigens; the preparation method of nanoemulsion vaccine prepared by silk glue protein and polypeptide as adjuvant and antigen, and further detects the effect of the vaccine on the immune response in the animal body.

[0010] The present application extracts silk fibroin with high natural activity and complete structure from silk, and separates, concentrates and freeze-dries the silk fibroin and polypeptide through a chromatographic column or dialysis, or directly synthesizes the polypeptide according to the amino acid sequence P1 (SEDSSEVDIDLGNLG), and incubates the stimulated dendritic cells in vitro, which has no toxic effect on the cells, promotes the maturation of the dendritic cells (DC), and enhances the expression level of MHCII and CD86. The silk fibroin / silk fibroin polypeptide and the antigen are prepared into an oil-in-water nanoemulsion vaccine, the antigen is uniformly wrapped and adhered, has good dispersibility and slow release, and can improve the ability of stimulating APC cells to phagocytize the antigen; the silk fibroin / silk fibroin polypeptide promotes the maturation of part of the DC and enhances the antigen presentation capacity, and the slow release of the antigen in the nanoemulsion vaccine can significantly improve the bioavailability of the antigen and continuously enhance the intensity of the immune effect. The DC stimulated by the silk fibroin / polypeptide proliferates and activates the lymph T cells, the DC secretes IL-12 to induce the differentiation of T0 cells into Th1 cells, the differentiated Th1 cells produce a large amount of interferon INF-γ, and the INF-γ promotes the maturation of the DC, and forms a positive feedback with the IL-12 secreted by the DC, thereby enhancing the immune effect of the lymphocytes.

[0011] Compared with the current vaccine adjuvant, the silk fibroin has high safety for cells or organisms, low immunogenicity, can promote the maturation of the dendritic cells and coordinate the proliferation of the lymph T cells, and significantly enhances the specific acquired immunity of the antigen in the body to achieve the purpose of resisting diseases such as viruses or tumors. Therefore, the silk fibroin and the polypeptide thereof can be applied to repeated vaccination of different vaccine antigens.

[0012] To solve the above technical problems, the present application adopts the following technical solutions:

[0013] The silk fibroin and the silk fibroin polypeptide are applied to improve the immune activation efficiency of the vaccine, and are used for preparing a preparation for reducing the use amount of the vaccine.

[0014] Preferably, the preparation is used for changing the specific immune response of the organism to the antigen.

[0015] Preferably, the preparation is used for enhancing the specific immune response of the organism to the antigen.

[0016] Preferably, the preparation is used for reducing the antigen demand amount in the vaccine formula or reducing the number of immunization times.

[0017] Preferably, the preparation is a vaccine adjuvant.

[0018] Preferably, the silk fibroin and the silk fibroin polypeptide are silk fibroin polypeptide P1, and the amino acid sequence is SEDSSEVDIDLGNLG.

[0019] A preparation method of sericin and silk polypeptide, comprising the following steps:

[0020] A, select cocoon to remove pupae and store at low temperature, cut cocoon in sterile environment, wash with water and dry at low temperature;

[0021] B, cocoon monolayer dispersion is sterilized by ultraviolet irradiation;

[0022] C, cut the cocoon into silk powder with a high-speed shearing machine;

[0023] D, dissolve sericin with urea method, filter and obtain supernatant;

[0024] E, add protein buffer solution to the supernatant and dialyze multiple times;

[0025] F, concentrate or freeze-dry after dialysis for standby, and obtain sericin;

[0026] G, take the sericin for chromatography column separation and purification, and collect eluate;

[0027] H, concentrate the eluate, freeze-dry for standby, and obtain the silk polypeptide.

[0028] The cocoon should be selected as raw material, and the damaged or seriously contaminated cocoon should be discarded.

[0029] In practical application, amino acid sequencing can also be performed on various polypeptides, and appropriate polypeptides can be directly synthesized artificially.

[0030] Preferably, in step A, the silkworm cocoons are stored at 4-8 DEG C, cut and washed, and then dried at 37-40 DEG C with air blowing; in step B, the intensity of the ultraviolet light is 1400 Lux, and the irradiation time is 30-60 minutes, after which the other side is turned over and the irradiation is continued for 30-60 minutes; in step C, the silkworm cocoons are weighed, and then rapidly sheared into silk powder using a high-speed shearing machine; in step D, the specific operation for dissolving the silk sericin protein by the urea method is as follows: in a sterile environment, the ratio of the silkworm cocoons to the sterile urea aqueous solution is 1:10-1:50, the concentration of the sterile urea aqueous solution is 5-10 mol / L, the temperature is kept at 37 DEG C, and oscillation is performed for 24-48 hours; the upper clear liquid is obtained by filtering through a 200-mesh screen, and then high-speed centrifugal filtration is performed at a speed of more than 5000 rpm at low temperature; the upper clear liquid is transferred to a new centrifugal tube, and repeated centrifugation is performed until the undissolved substances are completely removed, so as to obtain the supernatant; in step E, the specific operation for dialysis is as follows: according to the ratio of the silkworm cocoons to 1 mol / L Tris-HCl solution, 1 g:5-10 ml, the 1 mol / L Tris-HCl solution is added to the supernatant containing the silk sericin protein, and then taken into a dialysis bag, which is placed in sterile water for more than 10 times for dialysis, each time for 6 hours, and the dialysis is performed for more than 5 times; in step F, polyethylene glycol 6000 is used for concentration or freeze-drying; in step G, the specific operation for separation and purification by a chromatography column is as follows: the silk sericin protein is diluted to 5-10 mg / ml, and then separated by a Deae-anion exchange column or a non-polar macroporous adsorption resin; sterile water is used to elute the chromatography column at a speed of 1-3 BV / h, and the eluate is collected; in step H, the silk sericin polypeptide is obtained by performing vacuum concentration at 55 DEG C and freeze-drying.

[0031] A preparation method of a vaccine, comprising the following steps: mixing a silk sericin protein and / or silk sericin polypeptide solution, an antigen protein, a surfactant and water for injection to form an aqueous phase, mixing white oil for injection and egg yolk lecithin to form an oil phase, slowly adding the oil phase into the aqueous phase, performing high-pressure homogenization treatment after high-speed shearing, and performing sterilization filtration to obtain the vaccine.

[0032] Preferably, in the vaccine, the content of the silk sericin protein and / or silk sericin polypeptide is not less than 250 ug / ml; the vaccine comprises one or more of attenuated vaccines, inactivated vaccines, tumor vaccines and dendritic cell vaccines; the surfactant comprises one or more of butanediol and sodium oleate; and 0.22 um filter membranes are used for sterilization filtration.

[0033] The present application can be applied to therapeutic vaccines or prophylactic vaccines, the therapeutic vaccines usually refer to tumor vaccines, dendritic cell vaccines and other vaccines for treating diseases, the prophylactic vaccines usually refer to inactivated virus vaccines, attenuated virus vaccines, recombinant protein vaccines, subunit vaccines and other vaccines for preventing infections of pathogenic microorganisms such as viruses, compared with the prior art, the present application has the following beneficial effects:

[0034] The sericin and silk polypeptide described in the application have wide sources, and generally a large amount of protein liquid is left after cocoon spinning. The application can be combined with traditional silk production technology to recover high value-added product sericin solution and silk polypeptide solution, and develop by-products into non-toxic and harmless sericin solution, and further develop the vaccine adjuvant required by the application. According to the data published by China Silk Association, according to the national agricultural and rural work planning, during the "14th Five-Year" period, the standardization construction of modern agriculture is accelerated. In 2021, China's sericulture production increased steadily, with 1724 million pieces of cocoon, an increase of 2.74% over the previous year. In 2021, China's mulberry cocoon output was 71.72 million tons, an increase of 27,600 tons over the previous year, an increase of 4% over the same period. The study of the biological activity and potential biomedical application of silk protein will bring high economic and environmental benefits and promote the development of modern agriculture. Therefore, the source of sericin and silk polypeptide is stable and the production material cost is low.

[0035] Sericin has been proved to have low immunogenicity, no cell toxicity, can significantly promote cell proliferation, promote the healing and repair of damaged organs and skin, and appropriate amount of sericin or polypeptide can significantly improve the phagocytic function of macrophages, effectively enhance and protect the function of immune damaged immune system, and stimulate the proliferation ability of body lymphocytes. Therefore, the application aims at the existing problems of vaccine adjuvant, and combines sericin and silk polypeptide with different antigens, finds that sericin and polypeptide can promote the maturation of dendritic cells, prolong the survival period of dendritic cells, enhance the expression of MHCII and CD86 of dendritic cells, enhance the antigen presentation ability, and synergistically stimulate and enhance the proliferation ability of initial T lymphocytes. After the preparation of complex vaccine with antigen, the specific immune response ability of the body can be enhanced.

[0036] The present application is based on the optimization of traditional high-temperature water boiling method and solvent extraction method, and can obtain non-toxic, natural structure, high purity, easy absorption and metabolism of silk fibroin or polypeptide by simulating the solvent extraction method at human body temperature 37℃. The extract is directly applied to cell culture or injected into the body, so the biological safety of the protein or polypeptide is very high. The silk extraction pretreatment is particularly important. After removing the unqualified silkworm pupae, the particles attached to the surface are cleaned with sterile water, and after sufficient drying and water removal, the excessive dust and moisture in the silkworm cocoon are prevented from causing rapid reproduction of microorganisms. Then, the dried silkworm cocoon is sterilized by ultraviolet irradiation to kill a small amount of microorganisms attached to the silkworm cocoon, further ensuring the biological safety of the protein or polypeptide. The extraction process of the present application repeatedly verifies the extraction process of silk fibroin, and it is found that a small amount of lithium bromide residue still has a great toxic effect on immune cells. The protein or polypeptide obtained by high-temperature water boiling method or high-concentration alkali boiling method has serious hydrolysis and no obvious stimulating effect on in vitro cultured immune cells. Therefore, the present application adopts improved urea method for extraction, and the obtained protein or polypeptide has complete structure and strong activity, and has obvious stimulating effect on immune cells. Silk fibroin is easily dissolved in water, but the protein structure is easy to fold during dialysis and concentration, thereby producing irreversible denaturation and precipitation. Therefore, Tris-HCl protein buffer is added during dialysis to prevent the denaturation of silk fibroin during dialysis and concentration.

[0037] The protein / polypeptide extracted at human body temperature is more easily absorbed in the human body and plays a role on immune cells. The silk fibroin solution obtained by extraction is separated into different polarity protein fragments, and a plurality of silk fibroin polypeptides are found, wherein the silk fibroin polypeptide P1 (SEDSSEVDIDLGNLG), P2 (IILLLAAKFG) and P3 (KVIIKPLI) are polypeptides with known amino acid sequences, but the silk fibroin polypeptide P1 (SEDSSEVDIDLGNLG) has obvious stimulating proliferation effect on lymphocytes and obvious stimulating maturation effect on dendritic cells. Other silk fibroin polypeptide sequences silk fibroin polypeptide P2 (IILLLAAKFG) and silk fibroin polypeptide P3 (KVIIKPLI) have no obvious stimulating maturation effect on dendritic cells. Therefore, the present application directly entrusts a biological company to synthesize the polypeptide, and the synthesized polypeptide has the advantages of simple product obtainability, fast obtainment speed, simple process, less impurities, high purity and high safety.

[0038] This invention provides a method for preparing nanoemulsion vaccines using sericin / peptides, antigen proteins, and injectable oils. The vaccine is prepared as oil-in-water nanoparticles, allowing for better encapsulation and adhesion of the antigen proteins / peptides, resulting in better dispersibility and sustained release, and enhancing the ability of APC cells to phagocytose antigens. After the vaccine enters the body, the water-soluble sericin / peptides can rapidly promote the maturation of some dendritic cells (DCs) and enhance antigen presentation capabilities. Combined with the slow antigen release characteristic of nanoemulsion vaccines, this significantly improves antigen bioavailability and prolongs the intensity of the immune response. In vitro experimental data show that sericin / peptides can stimulate the proliferation and activation of T lymphocytes by DCs. DCs secrete IL-12 to induce T0 cells to differentiate into Th1 cells, playing a synergistic stimulatory role in the immunization process against specific antigens. The induced Th1 cells produce a large amount of interferon-γ, mediating the cytotoxic CD8+ T cell response. INF-γ can also form a positive feedback loop with interleukin IL-12 secreted by dendritic cells, thereby enhancing the immune response of lymphocytes. In addition, this method stabilizes the vaccine particle size at around 100nm and uses 0.22um micropore filtration to avoid high-temperature sterilization destroying the antigen, thus ensuring the safety and effectiveness of the vaccine. Attached Figure Description

[0039] Figure 1a SDS-PAGE image of sericin;

[0040] Figure 1b This is a schematic diagram illustrating the effect of sericin on the survival rate of BMDCs (mouse dendritic cells).

[0041] Figure 2a Schematic diagram of CD11c expression rate in cells detected by flow cytometry;

[0042] Figure 2b For the detection of CD11c by flow cytometry + Schematic diagram of CD86 and MHCII expression rates in cells;

[0043] Figure 2c This is a schematic diagram illustrating the effect of sericin / serice peptide on the expression of CD86 and MHCII in dendritic cells.

[0044] Figure 3a Schematic diagram of flow cytometry analysis for T lymphocyte isolation purity;

[0045] Figure 3b This is a schematic diagram illustrating the effect of sericin / serice peptide on dendritic cell-stimulated T cell proliferation.

[0046] Figure 3c This is a schematic diagram illustrating the effect of sericin / serice peptide on the stimulation of T cells by dendritic cells to secrete interferon-γ.

[0047] Figure 4a The time of immunization for mice is shown in the figure;

[0048] Figure 4b The IgG and IgGl antibody content of mice immunized with inactivated VSV antigen is shown in the figure;

[0049] Figure 4c The IgG and IgGl antibody content of mice immunized with OVA antigen is shown in the figure;

[0050] Figure 5 The blood VSV-eGfp expression of mice is shown in the figure. DETAILED DESCRIPTION

[0051] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings, so that those skilled in the art can better understand the present application and implement it. The embodiments are not intended to limit the present application. Any modification or replacement of the method, step or condition of the present application without departing from the spirit and essence of the present application shall fall within the scope of the present application. If not specifically indicated, the technical means used in the embodiments is the conventional means well known to those skilled in the art.

[0052] Example 1

[0053] Urea extraction of sericin / sericin polypeptide

[0054] Select the appearance is complete and less pollution cocoon, select cocoon 24 hours and timely removal of pupae, and in sterile environment will cocoon cut, rinse twice and 40 ℃ air drying. The dried cocoon monolayer spread in 1400 Lux ultraviolet light irradiation half an hour, turn the broken cocoon to the other side and use ultraviolet light irradiation half an hour. 5 g of broken cocoon is put into the high speed shearing machine fast cutting into silk powder, take out and place in a glass cup. According to the cocoon and sterile urea solution ratio is 1:30, put into the concentration of 8 mol / L of sterile urea solution in each gram of cocoon fragments, 37 ℃ constant temperature oscillation 24 hours, 200 mesh filter take supernatant; Take supernatant low temperature high speed 5000 rpm above centrifugal filtration, take supernatant in a new centrifuge tube, repeat centrifugation until completely remove undissolved material. According to the cocoon quantity: 1 mol / L Tris-HCl solution = 1:5 ~ 1:10 (g:ml) ratio, then in the supernatant 1 mol / L Tris-HCl buffer (pH 9.0) and together into dialysis bag (MWCO 3500 Da) in room temperature with purified water dialysis, placed in 10 times volume of sterile water dialysis, each dialysis 6 hours, dialysis 6 times, and use magnetic stirrer, in order to accelerate the speed of dialysis, the solution pH value is neutral, confirm that urea solution has been completely removed. Dialysis is completed with PEG-6000 crystal solid outside the dialysis bag, the supernatant concentration concentration required protein concentration, obtain silk fibroin solution. Example every extraction cocoon dosage 1 g, can obtain 19 ml volume of 9.6 mg / ml silk fibroin solution, according to the China silk society reported: in raw silk, silk fibroin is about 70%, silk fibroin is about 22%. According to the cocoon contains silk fibroin 22% content calculation, the method of extracting silk glue yield is 82.9%. As shown in Figure 1a . The silk fibroin extracted by the present application shows two obvious bands after SDS electrophoresis, the molecular weight is mainly concentrated in 150 kDa-250 kDa, therefore, it is illustrated that the product extracted by the present application is relatively complete natural silk glue structure.

[0055] Example 2

[0056] Silk fibroin / silk fibroin polypeptide aqueous solution method extraction

[0057] The silk fibroin solution or lyophilized powder of Example 1 is diluted to 5 mg / ml, and chromatographically separated by a Deae-anion exchange column or a non-polar macroporous adsorption resin, with 0.05-0.4 mol / L pH 7.4 Tris-Hcl as the buffer solution, and the column is eluted with sterile water at a speed of 2 BV / h, and the eluate is collected, 100 ml each time as a component, and concentrated at 55°C under reduced pressure to obtain silk fibroin polypeptides of different polarity, and lyophilized to obtain silk fibroin polypeptide powder. The silk fibroin polypeptide powder is subjected to amino acid sequencing, and is ready for use. According to the obtained silk fibroin polypeptide amino acid sequence, the silk fibroin polypeptide P1 (SEDSSEVDIDLGNLG), the silk fibroin polypeptide P2 (IILLLAAKFG), and the silk fibroin polypeptide P3 (KVIIKPLI) with known amino acid sequences are directly synthesized by a biological company, and the experimental results are verified.

[0058] Example 3

[0059] Silk fibroin / silk fibroin polypeptide on immune cell toxicity

[0060] (1) Extraction and separation of BMDC cells

[0061] 1) C57BL / 6 mice (6-10 weeks old) are sacrificed by cervical dislocation, and all femurs and tibias are surgically removed, and the muscle tissue around the bones is removed as much as possible with scissors and forceps; note: do not damage the bones.

[0062] 2) The bones are moved to a clean bench, and immersed in a sterile petri dish containing 75% alcohol for 5 min for sterilization and disinfection, and then washed twice with sterile PBS.

[0063] 3) The bones are moved to another new petri dish containing serum-free RPMI-1640 medium, and the ends of the bones are cut off with scissors, and then PBS is drawn into a syringe, and the needle is inserted into the bone marrow cavity from both ends of the bone, and the bone marrow is repeatedly washed out into the culture dish until the bone is completely white.

[0064] 4) The bone marrow suspension is collected, and small fragments and muscle tissue are filtered out with a 200-mesh nylon net. The filtrate is centrifuged at 1200 rpm for 5 min, and the supernatant is discarded.

[0065] 5) After counting the mouse bone marrow cells obtained in step 4), the cells are resuspended in RPMI 1640 complete culture medium containing 10% FBS and adjusted to a cell concentration of 2x 10 5 / ml. Then, the cells are plated into 100 mm bacterial culture dishes (about 2 bacterial culture dishes per mouse), 10 ml of cells per dish, and recombinant mouse GM-CSF (20 ng / ml) is added, and cultured in a 37°C, 5% CO2 incubator; note: bacterial culture dishes are used here, not cell culture plates.

[0066] 6) On day 3, add another 10 ml of complete medium containing 20 ng / ml recombinant mouse GM-CSF to the culture dish.

[0067] 7) On day 6, replace 2 / 3 of the medium, i.e. collect the old medium, centrifuge and resuspend the cell pellet in complete medium containing 20 ng / ml recombinant mouse GM-CSF, then return the cell suspension to the original dish.

[0068] 8) Continue to culture to day 8, collect the suspended and semi-adherent cells by gentle pipetting, which are the in vitro induced dendritic cells for the next step experiment.

[0069] (2) CCK8 method for detecting the effect of sericin on the activity of dendritic cells.

[0070] Collect the dendritic cells cultured to day 6, carefully pipette into a single cell suspension, adjust the cell density to 5 x 10 4 cells / mL, inoculate in a 96-well plate, 100 uL of cell suspension per well, dilute different concentrations of sericin and polypeptide solution to 100 uL (final concentrations are 100, 500, 1000, 1500 and 2000 ug / mL) with RPMI-1640 medium respectively. After 24 h culture in a 37℃ 5% CO2, saturated humidity incubator, directly add 10% cck8, shake well, 37℃ 5% CO2, saturated humidity incubator for 4 h, detect the absorbance A value of each well at 450 nm wavelength. Results Figure 1b As shown, sericin has no obvious growth inhibition or promotion on dendritic cells, and the higher the final concentration of sericin, the results show a weak increase in survival rate. In theory, dendritic cells are highly differentiated cells and no longer replicate and subculture, and it is possible that sericin stimulates some stem cells and other cells to produce weak proliferation. Therefore, sericin / polypeptide has no toxicity to BMDC (mouse dendritic cells) and has high biological safety.

[0071] Example 4

[0072] Sericin / sericin polypeptide promotes the expression of CD86 and MHC II of BMDC (mouse dendritic cells)

[0073] Collect the BMDC (mouse dendritic cells) in Example 3, carefully pipette into a single cell suspension, adjust the cell density to 1 x 10 6cells / mL were inoculated in 6-well plates, 2 mL of cell suspension per well, and 2 mL of RPMI 1640 complete culture medium or a series of concentrations of silk fibroin and polypeptide solution (final concentration 1000 μg / mL), LPS (1 μg / ml) were added respectively. After 24 h of continuous culture at 37°C in a 5% CO2 incubator, the cells were collected, centrifuged at 1500 rpm for 5 min, and washed once with PBS. Then, fluorescently labeled monoclonal antibodies FITC-CD11c and APC-CD86 or FITC-CD11c and PE-MHC II were added for double staining, and the mixture was incubated at 4°C in the dark for 30 min. After washing with PBS for 3 times, 400 μL of PBS was added to resuspend the cells, and the expression of CD86 and MHC II was determined by flow cytometry. The results are shown in Table 1. Figure 2a It is shown that the positive expression rate of dendritic cells FITC-CD11c is as high as 85.1%, indicating that the purity of BMDC cells extracted by the method of the present application is relatively high. The expression of costimulatory molecules such as CD86 and CD80 and antigen-presenting molecules such as MHC I and MHC II will be up-regulated to a certain extent during the maturation of BMDC. For example, Figure 2b and 2c It is shown that the expression of CD11c + The expression of dendritic cell CD86 and MHC II is significantly up-regulated, and there is a significant difference compared with the blank group without administration. The expression of the silk fibroin experimental group is stronger than that of the silk polypeptide experimental group. Therefore, silk fibroin and silk polypeptide P1 can promote the maturation of BMDC and the expression of antigen-presenting molecules to a certain extent, and the high-temperature boiling method of silk fibroin and silk polypeptides P2 and P3 have no obvious effect.

[0074] Example 5

[0075] Effect of silk fibroin / silk polypeptide on activation of T lymphocytes by dendritic cells

[0076] (1) Extraction and separation of mouse spleen T lymphocytes

[0077] 1) Nylon wool pre-treatment: Take 2 grams of nylon wool out of the package and break it up as much as possible. Then boil the nylon wool in deionized water for 20 minutes, and let it cool for 20 minutes. Repeat this process 6 times. After the nylon wool is completely dry, tear it into single fibers again. The goal is to make the nylon wool as fluffy as possible by boiling it repeatedly so that it can better absorb B cells. Weigh 0.5 grams of the treated nylon wool and carefully put it into a 10 mL glass syringe that has been sterilized. The volume of the filling should be about 6-7 mL, and the filling should be loose, because the tightness of the nylon wool affects the recovery rate of T cells. After adding a large amount of PBS to balance the column, wrap it with aluminum foil, and place it in a suitable container. Sterilize it in a pressure cooker for 15 minutes. Fix the sterilized nylon wool column vertically on a support in a clean bench, cover the top with aluminum foil to prevent contamination. Cut a 4 cm long disposable blood transfusion tube with a speed control chute. Sterilize it with anhydrous alcohol and connect it to the lower end of the syringe. Adjust the flow rate to about 3-4 mL / min. First, pour in the non-protein RPMI1640 culture solution to balance it. Close the valve for a certain period of time, then open the valve and discard the cell culture solution. Repeat this process several times to clean the nylon wool. Close the valve. Try to balance the nylon wool with the culture solution as much as possible. Then, add the same volume of complete RPMI1640 culture solution (all the above culture media should be preheated). Finally, close the blood transfusion tube when the culture solution level is close to the column filling page.

[0078] 2) Take 4 C57 mice, kill them by breaking their necks, and soak them in 75% ethanol for about 5 minutes.

[0079] 3) In a clean bench, cut open the left abdominal skin of the mouse to expose the abdominal cavity, and find and remove the spleen. Put the spleen in a PBS solution and use ophthalmic scissors to remove the surrounding connective tissue. Place the spleen on a 200-mesh cell sieve and grind it with a glass syringe plunger.

[0080] 4) Rinse the cell sieve into a petri dish with PBS, and collect the cell suspension in a 10 mL centrifuge tube. Wash the cells at 1500 rpm for 5 minutes, discard the supernatant, and add 4 mL of RPMI1640 to resuspend the cells.

[0081] 5) Carefully and slowly add the cell suspension to the 4 mL Ficoll solution along the wall of the tube at a ratio of 1:1, making sure the boundary is clear. Centrifuge at 2000 rpm for 20 minutes, with slow acceleration and deceleration. Take out the centrifuge tube, and the liquid is divided into 4 layers from top to bottom. The first layer is the RPMI1640 culture solution, the second layer is the cloudy lymphocyte layer, the third layer is the colorless Ficoll solution, and the fourth layer is the red blood cell layer.

[0082] 6) Carefully aspirate the lymphocyte layer, add RPMI1640, and wash twice at 1000 rpm for 5 minutes. Resuspend with complete RPMI1640 culture solution, and adjust the cell density to 5x106 cells / ml, and the cell viability was 99% by trypan blue staining. The sample cell suspension was prepared for nylon wool column.

[0083] 7) The cell suspension was poured into the syringe, and the nylon wool column was covered. The sample cell suspension was controlled at 5 x 10 6 cells / ml, and the sample volume was generally 1 / 3-1 / 5 of the column volume. After the sample was added, a small amount of culture solution was added, and the slide was closed.

[0084] 8) The glass syringe was covered with aluminum foil, and was placed in a 37°C, 5% CO2 incubator for 1 hour. During this process, the column must be kept vertical.

[0085] 9) The column was taken out of the incubator and placed on the clean bench, and the aluminum foil was removed. The slide and the transfusion tube were sterilized with a 75% alcohol cotton ball, and an appropriate volume of preheated culture medium at 37°C was added. The slide was opened to control the flow rate at 1-2 drops / min, and about 5 ml of the culture medium was discharged. At this time, the culture medium contained T cells, and the culture medium was collected.

[0086] 10) The collected cell suspension was washed with complete RPMI1640 at 1000 rpm for 5 min, and was prepared into a 1 x 10 6 cells / ml cell suspension.

[0087] 11) The purity and survival rate of the isolated T cells were determined. Flow cytometry was used to detect the expression rate of CD3 on the surface of the isolated cells.

[0088] ① The cell suspension prepared in the previous step was adjusted to a cell concentration of 1 x 10 5 cells / ml, and was divided into two EP tubes. The tubes were washed twice with PBS, and a 1 x 10 5 cells / ml cell suspension was prepared with PBS.

[0089] ② One tube was not added with fluorescent-labeled antibody, and the other tube was added with 0.1 uL of anti-CD3-FITC;

[0090] ③ Shake, incubate at 4°C in the dark for 30 min, centrifuge for 5 min, discard the supernatant, and wash twice with cold PBS (PBS solution 1 mL, gently blow, mix well, centrifuge, discard the supernatant);

[0091] ⑤ The expression level was detected by flow cytometry.

[0092] After the above experimental steps, the number of viable cells detected by trypan blue after the T lymphocytes were separated by the nylon wool column was more than 95%. The results are shown in Figure 3a, and the purity of the T lymphocytes was 46.4%.

[0093] (2) C57BL / 6 mouse spleen T lymphocytes as the reaction cells, T cell suspension was added to 96 cell plate. After LPS or different concentrations of silk fibroin / silk fibroin polypeptide were used to stimulate BMDC, OVA was added to the final concentration of 1 ug / ml as an antigen, and the stimulated BMDC and T cells were mixed at a ratio of 1:10 and co-cultured in a 37°C, 5% CO2 incubator for 72h. Each sample was repeated 3 wells. Then 10uL of CCK8 reagent was added to each well, and after 4h of continuous culture, the OD value was measured on the microplate reader at 450nm and 650nm wavelength (dual wavelength detection).

[0094] Stimulation index (SI) = (stimulated well OD450-stimulated well OD650) / (no antigen stimulation well OD450-no antigen stimulation well OD650).

[0095] The results are shown in Figure 3b After mixing and co-culturing the OVA-silk fibroin or silk fibroin polypeptide P1-stimulated dendritic cells with T cells, the stimulation index of the silk fibroin group and the silk fibroin polypeptide P1 group increased significantly, and there was a significant difference; the stimulation index had a certain concentration-dependent relationship with silk fibroin / silk fibroin polypeptide, and the stimulation index was the highest in the silk fibroin experimental group with a final concentration of 500ug / ml. Overall, there was no significant difference between the silk fibroin and silk fibroin polypeptide experimental groups, and both had a significant effect on stimulating and activating T cell proliferation. In the cell stimulation experiment, dendritic cells secrete IL-12 to induce Th1 cell differentiation, and play a synergistic stimulation role in the immune process to specific antigens, and induce a large number of interferon INF-γ produced by differentiated Th1 cells, and INF-γ can also secrete interleukin IL-12 from primary dendritic cells to form a positive feedback between each other, thereby enhancing the immune effect of T cells. The results are shown in Figure 3c . Silk fibroin / silk fibroin polypeptide induces dendritic cell maturation, which in turn stimulates and activates T cells to produce a certain amount of INF-γ, thereby enhancing the immune effect.

[0096] Example 6

[0097] Vaccine preparation and animal immunization

[0098] (1) Vaccine preparation

[0099] According to the prescription of vaccine preparation, the oil and water phases are weighed separately. Oil phase: white oil for injection, 45%~55%. Emulsifier: lecithin 2.9967g, 1.2%~3%. Water phase: water for injection, 45%~55%. Water-soluble surfactant: sodium oleate, 0.03%. Lecithin is added to the oil phase and slowly stirred to dissolve. Silk fibroin, antigen protein and sodium oleate are added to the water phase and slowly stirred to dissolve at 37°C. The water phase and oil phase are heated to the same temperature, 37°C. The water phase is placed under a high-speed shear machine, and the shear machine head position is adjusted so that the first hole below the head is above the liquid level. The shear machine speed is adjusted to 5~7krpm. After adjusting the speed, the oil phase is slowly added to the oil phase, and shearing is performed for 10~15min. After shearing, the shear machine is turned off, and the initial emulsion is taken out and diluted to obtain an initial emulsion with an average particle size of about 1um~10um. High-pressure homogenization: the initial emulsion is subjected to high-pressure homogenization, and the initial emulsion is subjected to the effects of cavitation, laminar flow and turbulent flow of the homogenizer to produce strong shearing and homogenization to achieve further particle fragmentation and emulsification. The homogenization pressure is 300~400bar, the cycle number is 2~4 times, the homogenization pressure is 600~700bar, and the cycle number is 6 times. The oil-in-water nanoemulsion obtained by homogenization has a stable emulsion particle size of about 100nm, a PI value of 0.05~0.1, and a translucent state. The emulsion is judged to be a qualified process if it does not separate or emulsify after long storage, and finally filtered through a 0.22um filter and stored at low temperature of -20~4°C for standby.

[0100] (2) Animal immunization

[0101] Forty-eight adult mice weighing 18~22g were evenly divided into 12 groups, 4 mice in each group, half male and half female. Each group prepared a vaccine preparation according to the ratio of antigen to silk fibroin / silk fibroin polypeptide in Table 1. Immunization injection was performed according to the order of Table 1, 0.1ml each time. The immunization route was muscle injection, and the second immunization was performed with the same dose two weeks later. The growth of mice was observed.

[0102] Table 1 Test mouse grouping and immunization injection

[0103]

[0104] The results are as follows Figure 4b 、 4cAs shown, the content of IgG and IgG1 in the experimental group mice was significantly increased compared with the PBS group and the negative control group, and the absorbance value was significantly different, P < 0.05 (wherein the height of the columnar chart represents the average value, and each point represents the antibody content value of each mouse). The silk fibroin / silk fibroin polypeptide has little difference in the ability to improve antigen-specific immune response with the mature Freund's incomplete adjuvant on the market; reducing the amount of Freund's incomplete adjuvant, silk fibroin / silk fibroin polypeptide mixed with Freund's incomplete adjuvant can completely and significantly increase antigen-specific immune response. The addition of silk fibroin / silk fibroin polypeptide group, OVA and inactivated VSV immunization IgG and IgG1 increased by about 30-50%. At present, the immunopotentiating effect of Freund's incomplete adjuvant is very strong, but it is easy to cause injection tissue pain and swelling, and even cause side effects such as hoof tissue tumor. Therefore, silk fibroin / silk fibroin polypeptide has good effect on enhancing antigen-specific immune response, and can significantly improve the activation efficiency of vaccine immunization, and is a good immune adjuvant.

[0105] Example 7

[0106] VSV challenge experiment

[0107] After the mice in Example 6 were immunized twice with VSV inactivated virus vaccine, 10 9 PFU / ml, 0.2ml virus solution was injected. The virus challenge method was nasal infection. After 7 days, blood samples were collected for detection of VSV-eGFP relative expression, with Gapdh as the internal reference. The higher the relative expression, the more VSV replication. As shown in Figure 5 , the VSV inactivated vaccine containing silk fibroin or Freund's incomplete adjuvant can promote the production of strong antigen-specific immune response in mice. When infected with VSV again, a large number of antibodies are produced to neutralize the virus, and the body can quickly produce an immune response to inhibit the invasion of the virus. Therefore, the challenge experiment shows that silk fibroin can significantly enhance the antigen-specific immune response in the body, effectively protecting the body from virus invasion and infection.

[0108] In summary, the present application provides a use of sericin and sericin polypeptide, a preparation method and application method of vaccine adjuvant, sericin is obtained by urea solvent low-temperature extraction separation method, sericin polypeptide P1 is obtained by chromatography column adsorption, elution separation method or amino acid sequence synthesis method. The sericin / sericin polypeptide has no toxicity to immune cells such as dendritic cells, can promote the maturation of dendritic cells, promote the expression of dendritic cell B7 molecules CD86 and antigen presenting molecules MHCII, and thus enhance the effect of antigen presentation to T lymphocytes. The T lymphocyte activation experiment proves that the sericin / sericin polypeptide can obviously activate T lymphocytes and enhance INF-γ secretion, thereby forming a positive feedback with IL-12 secreted by dendritic cells to enhance immune response. The in vivo experiment of mice further proves that the sericin / sericin polypeptide can obviously enhance the specific immune response of antigen whether used alone or combined with other adjuvants. In the experiment, the sericin and sericin polypeptide are prepared into vaccine emulsion with OVA egg white protein model antigen and VSV inactivated virus antigen, the sericin / sericin polypeptide promotes the maturation of DC to enhance the antigen presentation effect, stimulates lymph T cells to proliferate in cooperation with dendritic cells, and combines the adhesion and slow release of nano-emulsion antigen to enhance the phagocytic ability of APC, so that the sericin / sericin polypeptide has good effect of enhancing antigen specific immunity as a vaccine adjuvant. The protein / polypeptide adjuvant is suitable for being applied to vaccines prepared with different antigens, and is suitable for 2-3 times of repeated vaccination.

[0109] The above only shows the preferred embodiments of the present application, and of course cannot limit the scope of the present application, so the equivalent changes made according to the claims of the present application still fall within the scope of the present application.

Claims

1. The application of a sericin or sericin polypeptide in the preparation of a formulation that enhances the body's specific immune response to an antigen, characterized in that, The formulation is a vaccine adjuvant; the antigen is an inactivated OVA or VSV virus; the amino acid sequence of the sericin polypeptide is SEDSSEVDIDLGNLG.

2. A method for preparing a vaccine containing sericin or sericin polypeptide as described in claim 1, characterized in that, The procedure includes the following steps: mixing a sericin or sericin polypeptide solution, an antigen protein OVA, a surfactant, and water for injection to form an aqueous phase; mixing injectable white oil and egg yolk lecithin to form an oil phase; slowly adding the oil phase to the aqueous phase; performing high-speed shearing followed by high-pressure homogenization; and filtering for sterilization to obtain the vaccine. The vaccine contains sericin or sericin polypeptide at a content of not less than 250 ug / ml. The surfactant includes one or more of butylene glycol and sodium oleate.

3. The method for preparing the vaccine as described in claim 2, characterized in that, The filtration and sterilization process uses a 0.22µm filter membrane.

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