Composite antigen polypeptide and echinococcosis vaccine

Abstract

The invention belongs to the technical field of immunobiology, and particularly relates to a composite antigen polypeptide and an echinococcosis vaccine, and the composite antigen polypeptide comprises an amino acid sequence having more than 80% of homology with the amino acid sequence of MHC-II P1, MHC-II P2 or MHC-I P3. According to the invention, a plurality of antigen epitopes are connected by adopting a linker, so that the composite polypeptide FL46 is successfully constructed, and the immunogenicity of the epitopes is enhanced; according to the invention, the Freund adjuvant and the polypeptide FL46 are jointly combined to form the FL46 polypeptide Freund adjuvant vaccine, the protection effect of the vaccine on mouse echinococcosis granulosa is verified, and a remarkable capsule reducing effect is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of immunobiology, and particularly relates to a complex antigen polypeptide and a hydatid disease vaccine. BACKGROUND

[0002] Hydatid disease, also known as echinococcosis, is a serious parasitic disease of human and animals caused by the larvae of echinococcus granulosus parasitizing in the liver, lung, spleen and other organs of human and animals, which seriously endangers human health and the development of animal husbandry. Echinococcosis is mainly divided into cystic echinococcosis (CE) and alveolar echinococcosis (AE), among which, cystic echinococcosis is the most common one with high morbidity and mortality.

[0003] Cystic echinococcosis mainly parasitizes in the liver, lung and spleen and other organs, causing harm to human or animals through mechanical compression, allergic reaction and toxin effect, among which, the liver is the most seriously affected organ. At present, in addition to the good effect of surgical removal of a single larger cyst, there is still a lack of effective treatment for scattered multiple and small cysts. Commonly used drugs such as albendazole and praziquantel have certain side effects, and preventive vaccination is an effective supplement to the comprehensive prevention and control of hydatid disease. The research of E. granulosus vaccine has experienced the development of crude antigen, cell vaccine, recombinant protein vaccine, DNA vaccine and polypeptide vaccine. The traditional vaccine prepared from E. granulosus secretions and metabolites has high production cost and infection risk; the recombinant protein vaccine such as commercial Eg95 is expensive and needs multiple immunization; the DNA vaccine may cause autoimmune diseases or insert mutations and other side effects. Epitope vaccine has the advantages of flexible design, strong specificity, no infection risk, easy production and storage, etc., and is a new direction of vaccine research. Multi-epitope vaccine can contain multiple dominant B or T cell epitopes of pathogens, and can simultaneously induce cellular immunity and humoral immunity of the body, overcoming the shortcomings of limited immune protection of single antigen. SUMMARY

[0004] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a complex antigen polypeptide for solving the problem of limited immune protection of single antigen in the prior art, and the present application also provides a hydatid disease vaccine. The present application connects multiple antigen epitopes by using a linker to successfully construct a complex polypeptide FL46, which enhances the immunogenicity of the epitope; the present application assembles a FL46 polypeptide Freund's adjuvant vaccine by using Freund's adjuvant and the polypeptide FL46, and verifies the protective effect of the vaccine on mice with cystic echinococcosis, which achieves significant cyst reduction.

[0005] To achieve the above-mentioned purposes and other related purposes,

[0006] In a first aspect of the present application, a complex antigen polypeptide is provided, which comprises an amino acid sequence having 80% or more homology with the amino acid sequence of MHC-IIP1, MHC-IIP2 or MHC-IP3.

[0007] The amino acid sequence of the MHC-IIP1 is shown in SEQ ID NO: 1.

[0008] The amino acid sequence of the MHC-IIP2 is shown in SEQ ID NO: 2.

[0009] The amino acid sequence of the MHC-IP3 is shown in SEQ ID NO: 3.

[0010] The major histocompatibility complex (MHC) is a group of highly polymorphic genes on the chromosome encoding proteins related to cell recognition and antigen presentation, and the MHC of human is called HLA complex. The complex epitope polypeptide FL46 prepared by this method can have high binding force and immunogenicity with HLA-I (MHC-I) and HLA-II (MHC-II) molecules at the same time, and can be used for the preparation of Echinococcus granulosus vaccine.

[0011] In an embodiment of the present application, the complex antigen polypeptide comprises an amino acid sequence having 80% or more homology with the amino acid sequence of MHC-IIP1, MHC-IIP2 and MHC-IP3.

[0012] In an embodiment of the present application, the complex antigen polypeptide comprises an amino acid sequence having 90% or more homology with the amino acid sequence of MHC-IIP1, MHC-IIP2 and MHC-IP3.

[0013] In an embodiment of the present application, the complex antigen polypeptide comprises MHC-IIP1, MHC-IIP2 and MHC-IP3.

[0014] In an embodiment of the present application, the complex antigen polypeptide comprises MHC-IIP1, MHC-IIP2 and MHC-IP3, and the number of MHC-IIP1 is at least 2.

[0015] In an embodiment of the present application, the first MHC-IIP1 and the second MHC-IIP1 in the complex antigen polypeptide are connected by a first KKK linker, the second MHC-IIP1 and MHC-II P2 are connected by a second KKK linker, and the MHC-IP3 and MHC-IIP2 are connected by a GPGPG linker.

[0016] In the embodiments of the present application, the amino acid sequence of the composite antigen polypeptide is shown as SEQ ID NO: 4.

[0017] In a second aspect of the present application, the composite antigen polypeptide is used for preparing a vaccine for Echinococcus granulosus.

[0018] In a third aspect of the present application, a vaccine for echinococcosis is provided, which comprises the composite antigen polypeptide.

[0019] In the embodiments of the present application, the vaccine for echinococcosis is a vaccine for Echinococcus granulosus.

[0020] In the embodiments of the present application, the vaccine for Echinococcus granulosus comprises:

[0021] the composite antigen polypeptide; and

[0022] Freund's complete adjuvant or Freund's incomplete adjuvant.

[0023] As described above, the composite antigen polypeptide and the vaccine for echinococcosis of the present application have the following beneficial effects:

[0024] (1) The composite antigen polypeptide of the present application uses a linker to connect three antigen polypeptides, which overcomes the limitation of limited immune protection of a single antigen and improves the immunogenicity of the vaccine;

[0025] (2) The composite antigen polypeptide of the present application can simultaneously induce cellular immunity and humoral immunity of the body, promote the body to produce a large amount of cytokines and antibodies, and improve the immune level of the body;

[0026] (3) The polypeptide Freund's adjuvant vaccine of the present application significantly reduces the size, number and weight of echinococcus after Echinococcus granulosus infection, and the reduction rate of the cyst weight is (59.16±1.83)%, which has a significant protective effect on Echinococcus granulosus infection. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the construction method of the composite epitope polypeptide FL46 of Echinococcus granulosus in Embodiment 1 of the present application.

[0028] Figure 2 is the HPLC analysis chart of the composite epitope FL46 of the present application.

[0029] Figure 3 is the MS analysis chart of the composite epitope FL46 of Embodiment 1.

[0030] Figure 4Figure 2 is a cell activity statistical graph of the CCK8 method for detecting the proliferation effect of FL46 on BMDCs in Example 2 of the present application, the FL46 protein stimulates BMDCs for 24 hours, the abscissa is the concentration of different concentrations of FL46 protein, and the ordinate is the cell activity. ns represents non-significant difference, * represents significant difference, and the number thereof represents the degree of significant difference.

[0031] Figure 5 Figure 3 is a cell activity statistical graph of the CCK8 method for detecting the proliferation effect of FL46 on U937 cells in Example 2 of the present application, the FL46 protein stimulates U937 cells for 24 hours, the abscissa and ordinate are the same as Figure 3 .

[0032] Figure 6 Figure 5 is a mouse vaccine immunization scheme in Example 3, the mice are randomly divided into Freund's adjuvant group and polypeptide Freund's adjuvant group, and the mice in each group are injected subcutaneously on the back with Freund's adjuvant and FL46 polypeptide Freund's adjuvant complex, respectively.

[0033] Figure 7 Figure 6 is a MSD chip detection of the expression level of cytokines in the serum of mice at different time points after immunization in Example 4, including TNF-α, IL-2, IL-5, IL-12p70 and IL-10. The mouse orbital blood is taken, the serum is taken after centrifugation, and is sent to the company for detection, and the statistical graph ns represents non-significant difference, * represents significant difference, and the number thereof represents the degree of significant difference.

[0034] Figure 8 Figure 7 is an indirect ELISA method for detecting the IgG and IgG2a antibody titers in the serum of mice 2 weeks and 8 months after the last immunization in Example 5, the statistical graph ns represents non-significant difference, * represents significant difference, and the number thereof represents the degree of significant difference.

[0035] Figure 9 Figure 8 is a flow chart of the mouse vaccine immunization and echinococcus granulosus protoscoleces attack infection in Example 3, the protoscoleces are injected intraperitoneally after two weeks of immunization three times.

[0036] Figure 10 Figure 9 is a naked eye observation of the size and number of capsules in the mouse peritoneal cavity in Example 6.

[0037] Figure 11 Figure 10 is a statistical graph of the weight of the capsules in the mouse peritoneal cavity in Example 6. DETAILED DESCRIPTION

[0038] The following specific embodiments illustrate the embodiments of the present application, and those skilled in the art can easily understand other advantages and effects of the present application from the contents disclosed in the specification.

[0039] Example 1

[0040] Chemical synthesis and characterization of complex polypeptide FL46

[0041] Three T cell epitope antigen polypeptides P1, P2 and P3 are connected by appropriate linkers, a first KKK linker is used to connect a first HLA-II (MHC-II) molecule binding peptide P1 and a second HLA-II (MHC-II) molecule binding peptide P1, a second KKK linker is used to connect the second HLA-II (MHC-II) molecule binding peptide P1 and an HLA-II (MHC-II) molecule binding peptide P2, and a GPGPG linker is used to connect an HLA-I (MHC-I) molecule binding peptide P3 and the above-mentioned HLA-II (MHC-II) molecule binding peptide P2, to construct a complex epitope polypeptide FL46. Figure 1

[0042] The amino acid sequence of MHC-II P1 is shown as SEQ ID NO: 1, specifically FLQSEQLT.

[0043] The amino acid sequence of MHC-II P2 is shown as SEQ ID NO: 2, specifically AFIDTEVAAT.

[0044] The amino acid sequence of MHC-II P3 is shown as SEQ ID NO: 3, specifically FEQEMATAA.

[0045] The amino acid sequence of the complex epitope polypeptide FL46 is shown as SEQ ID NO: 4, specifically FLQSEQLTKKKFLQSEQLTKKKAFIDTEVAATGPGPGFEQEMATAA.

[0046] The complex epitope polypeptide FL46 is analyzed for purity and molecular weight by high performance liquid chromatography (HPLC) and mass spectrometry (MS). The high performance liquid chromatography (HPLC) detection result shows that the polypeptide peak value of FL46 complex polypeptide appears at 6.310 min, and the purity is 96.9% ( Figure 2 ); the molecular weight of the FL46 complex peptide analyzed by MS is 5044.8 g / mol ( Figure 3 ).

[0047] Example 2

[0048] CCK8 method for in vitro detection of the proliferation effect of complex epitope polypeptide FL46 on BMDCs and U937 cell lines

[0049] (1) BMDCs proliferation

[0050] ① Primary bone myeloid dendritic cells (BMDCs) are obtained by dissecting and killing mice;

[0051] ​②Collect and separate BMDCs on the seventh day of culture, adjust the cell concentration to 1 x 10 6 6 / mL, and culture in a 96-well plate;

[0052] ③Add FL46 complex polypeptide (epitope polypeptide FL46) of different concentrations (20, 40, 80, 100, and 200 μg / mL) to the experimental group. Add an equal amount of PBS to the negative control group, and culture at 37°C in a 5% CO2 incubator for 24 h;

[0053] ④Add CCK8 reagent and culture in the incubator for 2-3 h

[0054] ⑤Detect the absorbance at 450 nm by an enzyme-labeled instrument, and calculate the cell viability.

[0055] (2) U937 proliferation

[0056] ①Adjust the density of U937 cells (monocyte cell line) to 1 x 10 6 6 / mL, and culture in a 24-well plate;

[0057] ②Add phorbolester (PMA) 100 ng / mL to each well, and place in a 37°C, 5% CO2 incubator for 48 h to induce cell transformation into macrophages, and discard the unattached cells;

[0058] ③Add FL46 complex polypeptide of different concentrations (200, 250, 300, 350, 400, 450, and 500 μg / mL);

[0059] ④The remaining operations are the same as those for BMDCs.

[0060] The results show that, compared with the PBS negative control group, the cell viability of primary BMDCs stimulated by FL46 complex polypeptide of different concentrations (20, 40, 80, 100, and 200 μg / mL) for 24 h has no statistical difference (P>0.05), indicating that FL46 complex polypeptide with a concentration lower than 200 μg / mL does not affect the viability of BMDCs and has no toxic effect on BMDCs Figure 4 ).

[0061] The U937 cell proliferation slightly increases (P<0.05) after being stimulated by FL46 complex polypeptide of different concentrations (200, 250 μg / mL) for 24 h, and the cell proliferation has no statistical difference (P>0.05) after being stimulated by FL46 complex polypeptide of concentrations (300, 350, 400, 450, and 500 μg / mL) for 24 h, indicating that FL46 complex polypeptide with a concentration lower than 500 μg / mL does not inhibit the proliferation of U937 cells and has no toxic effect on U937 cells Figure 5 ).

[0062] Example 3

[0063] Mouse immunization and challenge infection with granular protoscoleces

[0064] ① 6-8 weeks old C57BL / 6 female mice were randomly divided into polypeptide Freund's adjuvant group and Freund's adjuvant control group, 10 in each group;

[0065] ② 50 μg of polypeptide vaccine FL46 (complex epitope polypeptide FL46) was mixed with 50 μL of Freund's complete adjuvant (first immunization) or 50 μL of Freund's incomplete adjuvant (last two booster immunizations), respectively;

[0066] ③ C57BL / 6 mice were immunized by subcutaneous injection at multiple points on the back, with an interval of 2 weeks between each immunization.

[0067] ④ Two weeks after each immunization, the mice were taken blood from the orbit, and the serum was separated for detection of cytokine and antibody levels.

[0068] ⑤ Two weeks after the last immunization, the polypeptide vaccine group and the control group mice were challenged with 2000 granular protoscoleces by intraperitoneal injection, and were raised in SPF level animal room for 8 months.

[0069] Example 4

[0070] MSD chip detection of serum cytokine expression levels of polypeptide Freund's adjuvant immunized mice

[0071] ① The whole blood of mice 2 weeks after each immunization in Example 3 was taken by orbital blood collection method, centrifuged at 3000g for 20 min at 4°C, and the upper serum was collected.

[0072] ② 50 μL of 1x beads was added to each well of a 96-well plate, and then washed twice with washing buffer;

[0073] ③ 50 μL of standard, blank and serum samples were added to the 96-well plate, respectively, and incubated at room temperature for 1 h on a 850 rpm shaker, then washed 3 times with washing buffer;

[0074] ④ 25 μL of detection antibody (IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, KC / GRO, TNF-α) was added, and incubated at room temperature for 30 min on a 850 rpm shaker, then washed 3 times with washing buffer;

[0075] ⑤ 50 μL of 1x SA-PE was added, and incubated at room temperature for 10 min on a 850 rpm shaker, then washed 3 times;

[0076] ⑥ 125 μL of assay buffer was added, and incubated at room temperature for 30 s on a 850 rpm shaker;

[0077] V. The 96-well plate was placed in the Bio-Plex System for detection.

[0078] The results show that the serum IL-2 level of the polypeptide Freund's adjuvant vaccine immunized mice is significantly higher than that of the Freund's adjuvant immunized mice after single immunization, and the expression amount is significantly increased with the increase of the number of immunization; the expression amounts of TNF-α, IL-5, IL-10 and IL-12p70 in the serum of the mice in the vaccine group are significantly increased two weeks after the last immunization of the polypeptide Freund's adjuvant vaccine. The cellular immunity level of the mice immunized by the FL46 vaccine is increased. Figure 7 )

[0079] Example 5

[0080] Detection of the titers of IgM, IgG and its subtypes in the serum of the mice immunized by the vaccine by indirect ELISA method

[0081] ①The whole blood of the mice immunized by the polypeptide Freund's adjuvant for 2 weeks and 8 months in the orbit of the eye in Example 3, and the whole blood of the mice immunized by the control Freund's adjuvant at the same time point were centrifuged at 3000 rpm for 15 min, and the upper serum was taken;

[0082] ②The antigen protein FL46 was diluted to 0.5 ug / ml with the antigen coating solution (absin) of PH 9.6, and 50 uL was added to each enzyme-labeled hole. It was placed in the wet box of the refrigerator at 4 degrees overnight; the coating solution was poured out, the remaining coating solution in the plate was wiped off with filter paper, 300 μL of PBST was added to each hole, and the PBST in the hole was discarded after washing for 3-5 min, and the residual PBST was wiped off with filter paper. Repeat the washing for three times;

[0083] ③250 μL of blocking solution (1% BSA) was added to each hole. 37°C blocking for 2 hours; pour out the blocking solution in the hole, wipe off the excess blocking solution in the hole with filter paper, and wash the plate with PBST for 3 times, the method is the same as above;

[0084] ④Primary antibody incubation: dilute the mouse serum with PBST at a ratio of 1:100, add the diluted mouse serum to the enzyme-labeled hole, 100 μL per hole. Place the ELISA plate in a 37°C incubator for 1.5 hours (note to set up negative control), pour out the primary antibody serum in the hole, wipe off the excess serum with filter paper, and wash the plate with PBST for 3 times, the method is the same as above;

[0085] ⑤Secondary antibody incubation: dilute the goat anti-mouse IgM, IgG, IgG2a and IgG1 labeled with horseradish peroxidase with PBST at a ratio of 1:10000, add to the enzyme-labeled hole, 50 μL per hole. Incubate at 37°C for 1 hour, wash the plate with PBST for 3 times, the method is the same as above.

[0086] ⑥Color development: add 50 μL of substrate solution (TMB) to each well, incubate at room temperature for 10 min in the dark; after color development, add 500 μL of stop solution to each well to stop the reaction;

[0087] ⑦Use the microplate reader to read the absorbance at 450 nm.

[0088] The results show that 32 weeks after the last immunization, the absorbance of total IgG and IgG2a in the serum of the polypeptide Freund's adjuvant group of mice is (0.42±0.16) and (0.19±0.15) respectively, which is significantly higher than that of the Freund's adjuvant group (0.09±0.09), (0.02±0.02). It is shown that after the polypeptide Freund's adjuvant immunization of mice, the mice produce humoral immunity, the immune level of the mice is increased, and the antibody expression can last for 8 months.

[0089] Example 6

[0090] The number, size and weight of echinococcus of mice immunized with the vaccine after echinococcus challenge infection

[0091] ①Take the polypeptide Freund's adjuvant and the mice immunized with the Freund's adjuvant 8 months after the last immunization in Example 3, and anesthetize the mice by intraperitoneal injection of 0.1% sodium pentobarbital, and execute them by cervical dislocation;

[0092] ②Open the abdominal cavity, and observe the size and number of the cysts in the abdominal cavity of the mice in the two groups by naked eyes;

[0093] ③Gently take out all the cysts, weigh the cysts of each mouse, and count the weight.

[0094] The results show that compared with the mice immunized with the Freund's adjuvant, the size and number of the cysts of the mice in the polypeptide Freund's adjuvant group are significantly reduced, and the weight reduction rate of the cysts is (59.16±1.83)% Figures 10-11 ). It is shown that the FL46 polypeptide vaccine has a protective effect on echinococcus infection, and can reduce the echinococcus infection level.

[0095] In summary, the linker is used to connect multiple antigen epitopes in the application, and a composite polypeptide FL46 is successfully constructed, and the immunogenicity of the epitopes is enhanced; the Freund's adjuvant and the polypeptide FL46 are jointly constructed into the FL46 polypeptide Freund's adjuvant vaccine in the application, and the protective effect of the vaccine on the mice echinococcosis is verified, and a significant cyst reduction effect is achieved. Therefore, the application effectively overcomes various shortcomings in the prior art and has a high industrial utilization value.

[0096] The above embodiments are only illustrative of the principles of the present application and its efficacy, and are not intended to limit the present application. Any modification or change made by any person skilled in the art without departing from the spirit and scope of the present application shall be covered by the claims of the present application.

Claims

1. A composite antigen polypeptide, characterized in that, The complex antigen polypeptide comprises an amino acid sequence homologous to the amino acid sequence of MHC-IIP1, MHC-IIP2 or MHC-IP3 by 80% or more; The amino acid sequence of the MHC-IIP1 is shown as SEQ ID NO:

1. The amino acid sequence of the MHC-IIP2 is shown as SEQ ID NO:

2. The amino acid sequence of the MHC-IP3 is shown as SEQ ID NO:

3.

2. The composite antigen polypeptide of claim 1, wherein, The complex antigen polypeptide comprises an amino acid sequence homologous to the amino acid sequence of MHC-IIP1, MHC-IIP2 and MHC-IP3 by 80% or more.

3. The composite antigen polypeptide of claim 1, wherein: The complex antigen polypeptide comprises an amino acid sequence homologous to the amino acid sequence of MHC-IIP1, MHC-IIP2 and MHC-IP3 by 90% or more.

4. The composite antigen polypeptide of claim 1, wherein: The complex antigen polypeptide comprises MHC-IIP1, MHC-IIP2 and MHC-IP3.

5. The composite antigen polypeptide of claim 1, wherein: The complex antigen polypeptide comprises MHC-IIP1, MHC-IIP2 and MHC-IP3, and the number of MHC-IIP1 is at least 2.

6. The composite antigen polypeptide of claim 5, wherein: The first MHC-IIP1 and the second MHC-IIP1 in the complex antigen polypeptide are connected by a first KKK linker, the second MHC-IIP1 and the MHC-ⅡP2 are connected by a second KKK linker, and the MHC-IP3 and the MHC-IIP2 are connected by a GPGPG linker.

7. The composite antigen polypeptide of claim 6, wherein: The amino acid sequence of the complex antigen polypeptide is shown as SEQ ID NO:

4.

8. A vaccine for echinococcosis, characterized by, The hydatid disease vaccine comprises the complex antigen polypeptide according to any one of claims 1-7.

9. A vaccine for hydatid disease according to claim 8, characterised in that, The hydatid disease vaccine is a Echinococcus granulosus vaccine.

10. The vaccine for hydatidosis according to claim 9, characterized in that, The Echinococcus granulosus vaccine comprises: The complex antigen polypeptide according to any one of claims 1-7; and Freund's complete adjuvant or Freund's incomplete adjuvant.

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