CN106478802A - TNF α protein B cell epitope, the multiple antigenic peptide containing this epi-position and application - Google Patents

Abstract

The present invention provides a B cell epitope of TNF-α protein, a multi-antigen peptide based on the epitope, combined with an 8-branched peptide conformation synthesized by the MAP scheme, and an application in preparing a vaccine for ulcerative colitis. Creatively applied the MAP design scheme to the immunology research field of small molecule self-antigens, and screened out the B cell dominant epitope of human TNF-α protein through a large number of experiments. On this basis, the MAP design scheme was used to obtain the 8-branched peptide conformation with lysine as the core matrix. The 8-branched peptide conformation polypeptide belongs to the autologous epitope vaccine, so it has the advantages of convenient administration and less frequent injections. It has the advantage of being able to induce the production of antibodies; and has the advantages of safety, good tolerance, and both prevention and treatment. It is expected to provide an important theoretical basis for the research and development of autoantigen MAP vaccines, and also provide a new clinical treatment for ulcerative colitis. train of thought.

Description

TNF-α protein B cell epitope, multi-antigen peptide containing this epitope and application

technical field

The invention belongs to the field of immunology and gastroenterology, and specifically relates to a B cell epitope of TNF-α protein, and a multi-antigen peptide in an 8-branched peptide conformation synthesized based on the epitope in combination with the MAP scheme, and in the preparation of ulcerative Use in colitis vaccines.

Background technique

TNF-α is a cytokine with complex biological activities. In addition to killing tumor cells in vivo and in vitro, it also has multiple functions such as inducing inflammatory responses, antiviral infection, regulating body immunity, and promoting cell proliferation and activation. However, studies have shown that when TNF-α is overexpressed, it will synergistically produce a variety of pathological injuries, such as inflammatory bowel disease, infectious disease, dyscrasia, etc.; at the same time, TNF-α is also a therapeutic target The human body's own antigens can neutralize the excessive TNF-α in the body, which can alleviate the above-mentioned pathological damage. At present, there are two main immunotherapy methods for such human autoantigens: the first method is to inject patients with monoclonal antibodies to directly neutralize the passive immunization of autoantigens in the body: such as anti-TNF-α monoclonal antibody (infliximab) Anti-infliximab) is currently the most commonly used biological agent for passive immunotherapy. Infliximab is a synthetic chimeric monoclonal anti-TNF-α antibody composed of 75% human and 25% murine that binds to membrane-bound TNF-α in monocyte-macrophages and activated T cells or with The free TNF-α in the plasma binds and neutralizes it, thereby achieving the effect of reducing TNF-α. It is now believed that infliximab can be used for the treatment of patients with severe active Crohn's disease (Crohn disease, CD) who do not respond to or cannot tolerate hormones and immunosuppressants, and who are not suitable for surgery; The treatment of ulcerative colitis (Ulcerative colitis, UC) also has a relatively positive effect. However, Infliximab has defects such as large dosage, high production cost, long-term repeated use, and easy hypersensitivity reactions; in addition, in response to the defects of this type of monoclonal antibody drugs, Nature magazine also published an article titled "Are Super Antibody Drugs Can it be tamed?" The article pointed out that the potential harm of the immune response induced by this type of drug has become a key factor limiting its use. The second method of immunotherapy is to immunize patients with autologous vaccines with autoantigen epitopes to generate polyclonal antibodies against autologous molecules in the body: in view of the low production cost of the vaccine, easy to use, and only a small amount of injection can induce Antibodies, and can overcome the defects of antibodies and soluble receptor drugs. Therefore, from passively accepting monoclonal antibody drugs to seeking active immune vaccines (i.e. autologous vaccines) against human self-proteins, has become the mainstay of anti-self molecular immunotherapy. new direction of development.

The hottest field of autovaccine research is epitope vaccine research. Epitope vaccines include T cell epitope vaccines and B cell epitope vaccines. The research and development of epitope vaccines can provide a better treatment for various diseases. However, since autologous molecules belong to self-antigens with small molecular weight and single structure, they often result in reduced immunogenicity and cannot induce ideal immune responses in vivo, especially It is the humoral immune response, which is the main reason why there is relatively little research on B cell epitope vaccines. In order to solve this problem, short-peptide vaccines often use the method of epitope polypeptide cross-linking carrier protein to improve their immunogenicity, but because the carrier protein itself is also a macromolecular foreign antigen, most of the antibodies induced are often directed against the carrier protein rather than the carrier protein. target epitope.

In recent years, in the field of tumor-associated antigen (TAA) research, some scholars have proposed a multi-antigenic peptide (muptile antigenic peptides, MAP) design scheme, using lysine (K) with a small molecular weight and weak immunogenicity As the core matrix, several (generally 4 or 8) monomeric peptides with the same or different epitopes are coupled together to form a dendritic structure. This design mode can not only simulate the conformation of the natural epitope well, but also activate humoral immunity and induce high-titer, high-affinity antibodies without coupling carrier proteins. Therefore, the branched multi-epitope complex peptide designed by the MAP scheme is expected to improve the quality of peptide vaccines and remove the defects of carrier proteins.

The MAP design scheme is mainly used in the research of constructing T cell and B cell epitope multi-antigen peptides of macromolecular antigens such as tumor-associated antigens, and has many applications in the research of heparanase. Compared with macromolecular antigens, self-antigens have defects such as natural immune tolerance, smaller molecular weight, shorter epitope peptides, more difficult to screen, and less likely to cause humoral immune responses. Therefore, researchers in this field have never applied the MAP design scheme. on self-small molecule antigens. However, based on a large amount of previous research on B cell epitope MAP vaccines, we successfully constructed a TNF-α B cell epitope vaccine with strong immunogenicity with the support of the National Natural Science Foundation of China in the early stage.

Contents of the invention

The purpose of the present invention is to address the deficiencies in the prior art, to provide a B cell epitope of TNF-α protein, and based on the epitope, combined with the multi-antigen peptide of the 8-branched peptide conformation synthesized by the MAP scheme, and to prepare ulcerative Use in colitis vaccines.

The purpose of the present invention is achieved through the following technical solutions: a B cell epitope of TNF-α protein, the amino acid sequence of the epitope is VMVPRRKAGTSKS or VCFTLNKSQSNQE. The dominant epitope is VCFTLNKSQSNQE.

A multi-antigen peptide for ulcerative colitis, the multi-antigen peptide is designed by MAP, uses lysine (K) with small molecular weight and weak immunogenicity as the core matrix, and is coupled with an epitope polypeptide whose sequence is VCFTLNKSQSNQE Together, a multi-antigenic peptide in an 8-branched peptide conformation is formed.

The application of a B cell epitope of human TNF-alpha protein whose amino acid sequence is VMVPRRKAGTSKS or VCFTLNKSQSNQE in preparing ulcerative colitis vaccine.

Application of an ulcerative colitis multi-antigen peptide in preparation of ulcerative colitis vaccine.

The beneficial effect of the present invention is that: the present invention creatively applies the MAP design scheme to the immunological research field of small molecule self-antigens, and through a large number of tests, predicts, screens, and identifies those with good hydrophilicity, accessibility, and plasticity, And the B cell dominant epitope of human TNF-α protein is located in the protein extended structure or random coiled structure on the secondary structure. On this basis, the MAP design scheme was applied to obtain the 8-branched peptide conformation with lysine as the core matrix. Compared with the existing technology, the peptide vaccine with the 8-branched peptide conformation has the following advantages:

TNF-α B cell epitope MAP vaccine belongs to autologous antigen epitope vaccine, so it has the advantages of convenient administration and less frequency, and only a small amount of injection can induce the production of antibodies; and it has relatively low price, good safety and tolerance, Advantages of both prevention and control. Therefore, if TNF-αMAP vaccine can be used to prevent the aggravation and recurrence of ulcerative colitis, it will certainly improve the compliance of patients with ulcerative colitis, improve the quality of life and save medical expenses. " and General Secretary Xi Jinping's concept of "Healthy China" are expected to provide an important theoretical basis for the research and development of autoantigen MAP vaccines, and also provide new ideas for the clinical treatment of ulcerative colitis.

Description of drawings

Figure 1 is a graph of DNAstar prediction results of human TNF-α B cell epitope;

Figure 2 is a graph of the prediction results of human TNF-α B cell epitope Bcepred;

Figure 3 is a schematic diagram of the structure of a candidate 8-branched peptide MAP;

Figure 4 is a schematic diagram of the binding forces of MAP1 and MAP2 to commercial TNF-α antibodies;

Figure 5 is a graph showing the specificity results of each MAP antibody detected by Western blotting;

Figure 6 is a dynamic change diagram of mouse serum specific antibody titer after MAP immunization;

Figure 7 is a schematic diagram of the MAP antibody weakening the killing effect of TNF-α on L929 cells;

Figure 8 is a schematic diagram of MPO activity in colon tissue of mice inoculated with MAP vaccine, showing that MPO activity is significantly reduced;

Figure 9 is a schematic diagram of the pathological damage of the colon tissue of mice inoculated with the MAP vaccine, showing that the pathological damage was significantly reduced after the inoculation of the MAP vaccine;

Figure 10 is a schematic diagram of the expression of E-cadherin in the colon tissue of mice inoculated with MAP vaccine, indicating that the expression of E-cadherin was significantly reduced after inoculation with MAP vaccine.

detailed description

Example 1: Screening of dominant epitopes of human TNF-α B cell epitopes and construction of multiple antigenic peptides.

①Search the NCBI GenBank database to obtain the amino acid sequence of human TNF-α protein; use DNAStar, GOLDENKEY (Academy of Military Medical Sciences) software and the computer vaccine design tool Bcepred on the Internet to analyze and obtain the most antigenic amino acid sequence information in the hydrophilic region and B cell epitope; according to the prediction of antigenic epitope, select a peptide rich in hydrophilic amino acids with a length of about 6-25 amino acids, while avoiding more than 4 consecutive adjacent hydrophobic amino acid residues and containing more than 2 and a half Peptides of cystine, so as not to form complex multimers through disulfide bonds.

② Analysis software (Figure 1) and Internet design tool (Figure 2) prediction results. Taking 13 amino acid residues as a group, analyze its hydrophilicity, accessibility, plasticity and antigenicity index to evaluate the antigenicity of the highly hydrophilic region of human TNF-α molecule. Finally, several analysis methods were combined, and the overlapping areas of the results were taken to determine that the peptides composed of amino acids 22-34 (VMVPRRKAGTSKS) and amino acids 54-66 (VCFTLNKSQSNQE) had good hydrophilicity, accessibility and plasticity. The secondary structure is located in the protein stretch structure or random coil structure, and it is a candidate epitope for B cells of TNF-α protein.

③Using lysine (K) as the core matrix, using the MAP design scheme, Hangzhou Zhongpei Biochemical Co., Ltd. was entrusted to couple the C-terminals of the 22-34 (VMVPRRKAGTSKS) and 54-66 (VCFTLNKSQSNQE) amino acids in an 8-branched peptide conformation Linked to the amino group of the core matrix lysine, the TNF-αB cell candidate epitope MAP polypeptides were prepared by HPLC separation and purification, mass spectrometry analysis and peptide amino acid sequencing, and were named MAP1 and MAP2 respectively. Schematic diagram of the structure of MAP1 and MAP2 See Figure 3.

④ Screening of candidate MAPs is as follows:

(1) The binding ability of MAP antibody and MAP in vitro: 1) Use different MAPs with a concentration of 10 μg/mL respectively, and the diluent is carbonate buffer solution with pH 9.6; 2) Coat the microwell reaction plate, each well 100 μL, overnight at 4°C; 3) Wash once with PBS buffer, add 1% BSA to each well, 1-2 hours at 37°C; 4) Wash once with PBS, pat dry; 5) Add primary antibody (PBS 1: 4000 diluted MAP antibody), added to the coated reaction plate, 100 μL per well, and PBS was used as a control. Incubate at 37°C for 60 minutes; 6) Add goat anti-rabbit IgG-HRP diluted with 1% BSA, 100 μL per well, and incubate at 37°C for 60 minutes; 7) Add chromogenic agent OPD, incubate at 37°C for 10-20 minutes; 8) Stop adding solution, 50 μl per well; 8) read at a wavelength of 492nm, and the results are expressed as OD mean values. As shown in Figure 4, the binding force of MAP2 polypeptide to TNF-α whole protein antibody is significantly stronger than that of MAP1.

(2) MAP antibody specificity test: 1×10 ⁹ H22 cells (this cell line stably and highly expresses TNF-α, purchased from Huazhong University of Science and Technology) after centrifugation and washing were added to 1 mL of ice-cold suspension buffer and vortexed Centrifuge at 3000r/min at 4°C for 5 minutes; discard the supernatant, add an equal volume of 2×SDS as soon as possible to extract the total protein; use the total protein to make SDS-PAGE, and use commercial TNF-α protein as a control; Western blot adopts chemiluminescence method, and the specific steps are carried out according to the kit instructions. The antibody used is a commercial anti-TNF-α whole protein antibody (purchased from Cilag AG Ltd, Switzerland) or a purified anti-TNF-α B cell epitope MAP antibody The bands obtained by immunofluorescence observation were compared with the kDa value of the bands obtained by the commercialized whole protein antibody to identify the specificity of the purified antibody obtained, and GAPDH was selected as the internal reference. Commercial antibodies have a very clear band at a molecular weight of about 17kD; both MAP1 and MAP2 have clear bands at a molecular weight of about 17kD. After semi-quantitative analysis, the gray value of the MAP2 band is significantly higher than that of MAP1. According to the instructions of the commercial antibody, the 17kD is TNF-α protein. However, the TNF-α recombinant protein antiserum and the negative control had no obvious bands at the corresponding positions, as shown in FIG. 5 .

(3) To test the ability of MAP to stimulate the proliferation of human mononuclear cells (PBMC) and release IFN-β in vitro: the classic standard 3H-TdR incorporation test was adopted. During the transformation of lymphocytes into lymphoblasts stimulated by specific antigens, the synthesis of DNA increases significantly, and the degree of transformation is positively correlated with the synthesis of DNA. 3H-TdR) marker, added to the culture system, that is, taken up by transformed lymphocytes and incorporated into DNA molecules. After the culture was terminated, the radiation amount of 3H-TdR incorporated in the lymphocytes was measured to judge the transformation degree of the lymphocytes. The results showed that MAP multi-antigen peptides could significantly stimulate PBMC proliferation and release IFN-β in vitro, and the ability of MAP2 to stimulate PBMC to release IFN-β was significantly stronger than that of MAP1.

Comprehensively analyze the results of MAP antibody and MAP in vitro affinity test, MAP antibody specificity test, MAP vaccine in vitro stimulation of human mononuclear cell (PBMC) proliferation and IFN-β release, and screen and identify amino acids 54-66 (VCFTLNKSQSNQE) is the dominant epitope of TNF-αB cells. Therefore, the multi-antigen peptide MAP2, which is designed by MAP, uses lysine with small molecular weight and weak immunogenicity as the core matrix, and is coupled with the epitope polypeptide with the sequence VCFTLNKSQSNQE to form MAP with an 8-branched peptide conformation. Multiple antigenic peptide vaccines of B cell epitopes.

Example 2: Verification of the in vivo and in vitro immunological effects of the multi-antigen peptide constructed by the method described in Example 1.

Due to the high homology between rats and human TNF-α, this embodiment takes rats as the research object, screens and identifies the dominant epitope according to the method described in Example 1, and then uses its dominant epitope to synthesize The TNF-α B cell epitope MAP multi-antigen peptide vaccine was used to verify the immunological effect of rats in vivo and in vitro, so as to prove the positive effect of the multi-antigen peptide with 8-branched peptide conformation constructed by the present invention in human and mouse ulcerative colitis.

Among them, the in vitro effect research includes: the effect of MAP antibody on the killing of mouse fibroblast L929 by TNF-α in vitro; the effect of MAP antibody on the dissolution of 3H-TdR-labeled liver cancer cell HCC97H by TNF-α in vitro. The in vivo effect research includes: the effect of TNF-α B cell epitope MAP vaccine on the disease activity index of model mice; the effect of MAP vaccine on serum CRP, ESR, DAO and other levels of model mice; The effect of MAP vaccine on the pathological changes of colonic mucosa, the expression of E-cadherin in colonic tissue and the activity of MPO in colonic tissue in model mice. details as follows:

① MAP2 subcutaneous multi-point immunization of BALB/c mice, once every 2 weeks, a total of 4 times, Freund's incomplete adjuvant and Th universal auxiliary short peptide were used to boost the immunization, and the orbital blood method was used in batches 10 days after each immunization Animal serum was collected, and the MAP antibody titer in the serum of each group (MAP2, TNF-α whole protein and PBS control group) was measured and compared by standard indirect ELISA method. Specific antibodies can be detected 2 weeks after the first immunization, and the antibody titer of MAP2 immunization is 1:130,000 after 6 weeks, and reaches the highest peak after 8 weeks, and the average antibody titer is 1:140,000 , TNF-α control group and PBS control group had no specific antibody production, see Figure 6. The specific antibody induced by MAP2 was separated and purified by Sephadase column chromatography to collect IgG, and Hangzhou Zhongpei Biochemical Co., Ltd. was entrusted to use antibody affinity chromatography to separate and purify the specific polyclonal antibody contained in the antiserum; after purification The average antibody concentration obtained by Coomassie Brilliant Blue was 28.6 mg/dL. .

② After the intervention of MAP antibody, observe the killing effect of TNF-α on mouse fibroblast L929 cells in vitro: adjust the mouse L929 cells in good growth state to a cell suspension of 10 ⁶ /mL, add to each well of a 96-well culture plate 100 μL of cell suspension, 5% CO2, culture overnight at 37°C; discard the supernatant, add TNF-α to each well, and MAP antibody 5% CO2, culture at 37°C for 16 hours, discard the supernatant, add 30 μL to each well at a concentration of 500 μg/ Stain with mL crystal violet for 3-5 minutes, carefully wash away the crystal violet with running water, shake off the residual water, add 100 μL of decolorization solution to each well, and measure the absorbance at 570 nm with a microplate reader. The results are shown in Figure 7, with the increase of the concentration of MAP antibody, the killing effect of TNF-α on mouse fibroblast L929 cells was gradually weakened (P<0.05).

③After the intervention of MAP antibody, observe the dissolution rate of TNF-α on 3H-TdR-labeled human primary liver cancer cells HCC97H in vitro: TNF-α receptors are widely distributed in various tumor cells, and TNF-α on the surface of some tumor cell membranes The combination of α receptor and TNF-α can lead to the death of these tumor cells, and the biological activity of TNF-α can be reflected by detecting the killing ability of tumor cells. HCC97H liver cancer cells were first labeled with 3H-TdR, and 3H-TdR was released outside the cells after being killed. The 3H-TdR released by tumor cells showed that after the intervention of MAP antibody, the lysis rate of TNF-α on HCC97H cells was significantly reduced.

③Verification of immunological effects in vivo:

A. Establishment of mouse UC model: refer to the method of Kihara N et al. after repeated exploration and improvement, a DSS BALB/c mouse UC model with a relatively uniform disease burden was established. The mice in the blank control group were intragastrically administered with the same amount of distilled water every day as comparison.

B. Select 30 male BALB/c UC model mice with a body weight of 18-20 grams, 5-6 weeks old, and divide them into 3 groups. There is no statistical difference in body weight between groups. The first group of 10 rats, the immunization method is subcutaneous multi-point immunization with MAP vaccine, the immunization dose is 50 μg/rat, Freund’s complete adjuvant is used for the first immunization, and Freund’s incomplete adjuvant is used for the last three booster immunizations, adjuvant and antigen Mix well at 1:1, immunize once every other week, and immunize 4 times in total; 10 rats in the second group are subcutaneously immunized with 50 μg of lysine matrix per rat, and the immunization frequency is the same as that of the first group. The third group of 10 rats was immunized by subcutaneous multi-point immunization, and the immunization dose was 100 μL of normal saline per rat, and the immunization frequency was the same as that of the first group. The average disease activity index of the mice in each group was recorded weekly from 4 weeks after immunization, and the results showed that the disease activity index of the mice in the experimental group was significantly lower than that in the matrix group or the normal saline control group. Five experimental animals in each group were sacrificed at 4 weeks and 8 weeks after immunization. Mice were fasted for 12 h before specimen collection and had free access to water. Ketamine hydrochloride and Sumianxin were formulated as an anesthetic at a ratio of 1:1, and the mice were anesthetized by intraperitoneal injection at a dose of 1 mg/kg, and the mice were to be killed. After anesthesia, the supine position was fixed:

i. Serum: Use sterile forceps to remove the eyeball of the mouse, and repeatedly collect blood from the orbit with a siphon tube, take about 1ml of blood, put it into a centrifuge tube moistened with 1% heparin, let it stand at room temperature for 30min, and centrifuge at 3000r/min at 4°C for 10min , collect the supernatant, divide into 2 sterile Eppendorf tubes, store at -80°C, and use to detect the contents of serum D-LAC, ESR, CRP and DAO. The results suggested that after the mice with ulcerative colitis were inoculated with MAP vaccine, the contents of D-LAC, ESR, CRP and DAO in the serum were significantly reduced.

ⅱ. Bacterial culture of mesenteric lymph nodes and observation of bacterial translocation: open the abdomen quickly after blood collection, take about 200 mg of mesenteric lymph nodes under sterile conditions, put them into a sterile grinder, add 2 mL of sterile saline, grind by hand for 2 minutes, and take 100 ul The grind was inoculated onto a blood agar petri dish. The results showed that the positive rate of bacterial culture in the mesenteric lymph nodes of ulcerative mice vaccinated with MAP vaccine was significantly lower than that of the control group, suggesting that the translocation of intestinal bacteria was alleviated after vaccination.

Ⅲ. Colon tissue: expose the abdominal cavity tissue, carefully separate the colon (from the ileocecal to the anus), cut the intestinal segment longitudinally along the mesentery, rinse with ice saline, wash away the contents of the intestinal cavity, and take a colon tissue 2 cm away from the anus 1 cm , fixed in 10% formalin, routinely embedded in paraffin, sliced at 4 μm in length, 3 slices in total, used for H&E staining and immunohistochemical staining of tissue sections. Immediately after that, 10 mg of colonic tissue was taken, quickly put into an Eppendorf tube, placed in an insulated tank filled with liquid nitrogen, and then stored in a -80°C refrigerator for the determination of MPO activity in the colonic tissue. The results are shown in Figure 8. MPO activity in the colon tissue of mice inoculated with MAP vaccine (MP group) was significantly lower than that of matrix group (M group) and TH control group.

iv. Observation of colon tissue sections: After H&E staining, observe and compare the pathological changes of mouse colon tissue under a light microscope (×100, ×400). The results are shown in Figure 9. Nuclear cell infiltration and gland destruction were significantly reduced.

v. Immunohistochemical detection of E-cadherin in colon tissue: using the SP method, the antibodies used are rabbit anti-E-cadherin monoclonal antibody and biotin-labeled goat anti-rabbit IgG working solution, observe under a microscope and take pictures. Cell membranes stained in brownish yellow were positive. As shown in Figure 10, the expression of E-cadherin in the colon tissue of mice with ulcerative colitis inoculated with MAP vaccine was significantly reduced.

From the above research data, it can be seen that the multi-antigen peptide vaccine with 8-branched peptide conformation constructed by the present invention has a positive effect in preventing and treating the exacerbation and recurrence of ulcerative colitis.

The present invention illustrates the detailed features and methods of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed features and methods, that is, it does not mean that the present invention can only be implemented depending on the above-mentioned detailed features and methods. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of selected materials and steps in the present invention, addition of auxiliary materials and steps, selection of specific methods, etc., all fall within the protection scope and disclosure of the present invention. within range.

Claims (5)

1. A B cell epitope of TNF-α protein, characterized in that the amino acid sequence of the epitope can be VMVPRRKAGTSKS or VCFTLNKSQSNQE. 2. The B cell dominant epitope of TNF-alpha protein according to claim 1, characterized in that the amino acid sequence of the epitope is preferably VCFTLNKSQSNQE. 3. A multi-antigen peptide for ulcerative colitis, characterized in that the multi-antigen peptide is designed through MAP, uses lysine as the core matrix, and is coupled with an epitope polypeptide whose sequence is VCFTLNKSQSNQE to form 8 branches Multiple antigenic peptides in peptide conformation. 4. The use of the epitope according to claim 1 in the preparation of ulcerative colitis vaccine. 5. The application of a multi-antigenic peptide according to claim 3 in the preparation of ulcerative colitis vaccine.