Influenza b hla-b*1501 specific polypeptides and uses thereof

Abstract

The application discloses an influenza B HLA-B*1501 specific polypeptide and application thereof, and belongs to the field of immune detection. The application provides a specific polypeptide for influenza B, and the amino acid sequence of the specific polypeptide is ALIGASICF. A polypeptide-MHC tetramer is prepared by using the corresponding polypeptide. The polypeptide-MHC tetramer is used for detecting T cells of influenza B infection convalescents, and the positive rates are 9.94% and 8.03% respectively, which are obviously higher than 0.05% and 0.12% of the negative control group. The polypeptide-MHC tetramer effectively increases the affinity of the polypeptide-MHC and the specific T cell surface TCR, can be used as an effective tool for T cell evaluation, can be used for specific T cell separation and cloning, and can be combined with single cell sequencing technology to separate specific TCR and serve as a T cell activation reagent. The application has high application value in the T cell research of human groups after influenza B infection or vaccination.

Description

TECHNICAL FIELD

[0001] The present application relates to an influenza B specific polypeptide and its application, and belongs to the field of immune detection and vaccine. BACKGROUND

[0002] Influenza virus infects millions of people every year, causing recurrent respiratory illness in humans, resulting in about 250,000 to 500,000 severe infections and deaths, and having a major impact on human public health. Influenza virus belongs to the family of Orthomyxoviridae enveloped RNA viruses, including four genera A, B, C and D, among which influenza A (IAVs) and B (IBVs) can cause large-scale seasonal influenza epidemics. Influenza B virus presents a unique process in human evolution dynamics, since the 1970s, there are two parallel evolution pathways of influenza B, and in 1988, two antigenically distinct lineages, Yamagata and Victoria, were isolated from influenza B cases.

[0003] Vaccination is currently the most economical and effective method for preventing influenza virus infection. Currently marketed influenza vaccines include inactivated vaccines, attenuated live vaccines and protein vaccines, which have a certain protective effect against influenza virus epidemics. However, due to the frequent antigenic drift and / or antigenic shift of influenza virus haemagglutinin (HA), the antigenicity of vaccine strains does not match the epidemic strains, resulting in the current influenza vaccine being unable to effectively prevent seasonal and pandemic influenza. The development of a universal influenza vaccine that can induce broad and long-lasting immune responses has become an important trend in influenza vaccine development. T cells play a very important role in viral clearance, and the evaluation of T cell immune response is particularly important in the evaluation of vaccine immune effect.

[0004] MHC-tetramer technology is a technology that tetramerizes MHC monomer molecules, improves their affinity to TCR on T cells, and thus improves the sensitivity of detection. This technology can be applied to the detection of antigen-specific T lymphocytes, direct separation and cloning of T cells, isolation of specific TCR, in situ staining, etc., providing an efficient, rapid and sensitive detection means for a series of work related to cellular immune response. SUMMARY

[0005] The first object of the present application is to provide an influenza B HLA-B*1501 specific polypeptide, whose amino acid sequence is ALIGASICF.

[0006] The second object of the present application is to provide a polypeptide derivative which substitutes, or deletes, or adds one or several amino acids in the amino acid sequence of the influenza B specific polypeptide, and has the same antigenicity as the polypeptide.

[0007] A third object of the present application is to provide a polypeptide-MHC tetramer with high specificity and high sensitivity to T cells of a human population infected with or vaccinated against influenza B, said polypeptide-MHC tetramer being formed by biotinylated MHC-I and said influenza B specific polypeptide, or by biotinylated MHC-I and a derivative of said specific polypeptide.

[0008] A fourth object of the present application is to provide a method for preparing said polypeptide-MHC tetramer, comprising the following steps: (1) expressing MHC light chain and MHC heavy chain with E. coli; (2) diluting and recombining to prepare polypeptide / MHC complex; (3) preparing biotinylated polypeptide / MHC complex; (4) reacting biotinylated polypeptide / MHC complex with labeled streptavidin.

[0009] In an embodiment of the present application, the C-terminal of the MHC heavy chain is linked to biotin.

[0010] In an embodiment of the present application, the C-terminal of the MHC is added with an amino acid sequence capable of linking biotin, which is GGGLNDIFEAQKIEWHE.

[0011] In an embodiment of the present application, in step (3), the polypeptide / MHC complex is combined with D-biotin under the catalysis of BirA enzyme.

[0012] In an embodiment of the present application, in step (3), the concentration of D-biotin is 400-600 μmol / L.

[0013] In an embodiment of the present application, in step (4), the labeled streptavidin is reacted according to a molar ratio of (4-6):(0.5-1.5).

[0014] In an embodiment of the present application, in step (4), the labeled streptavidin is reacted according to a molar ratio of 5:1.

[0015] In an embodiment of the present application, the method for preparing the polypeptide-MHC tetramer further comprises purification.

[0016] In an embodiment of the present application, the purification is purification of the prepared polypeptide / MHC complex by molecular sieve.

[0017] In one embodiment of the present application, the method is specifically: using E. coli to express MHC light chain and MHC heavy chain with C-terminal biotin, using dilution renaturation method to prepare polypeptide / MHC complex, purifying with superdex200, then binding with D-biotin under the catalysis of BirA enzyme to form biotinylated polypeptide / MHC complex, and then reacting with labeled streptavidin at a molar ratio of 5:1 to obtain polypeptide / MHC complex.

[0018] A fifth object of the present application is to provide a polypeptide vaccine, the active ingredient of which contains the influenza B HLA-B*1501 specific polypeptide and / or the influenza B HLA-B*1501 specific polypeptide derivative.

[0019] A sixth object of the present application is to provide an influenza B specific cellular immunity detection kit, the kit containing the influenza B HLA-B*1501 specific polypeptide and / or the influenza B HLA-B*1501 specific polypeptide derivative.

[0020] The present application also provides the use of the polypeptide-MHC tetramer.

[0021] In one embodiment of the present application, the use includes: preparing a vaccine; evaluating the T cell immune response of a human population infected or vaccinated with influenza B as an effective tool for T cell immunology evaluation; flow cytometry detection of corresponding immune cells, in situ staining of tissue sections, isolation and cloning of specific T cells, isolation of specific TCRs combined with single-cell sequencing technology, and use as a T cell activation reagent for a human population infected or vaccinated with influenza B.

[0022] Advantages:

[0023] The present application uses one polypeptide BM 58-66 The tetramer prepared by AF9 has high specificity and sensitivity, and can be used as an effective tool for T cell evaluation. Flow cytometry analysis shows that the positive rate of T cells of HLA-B*1501 volunteers detected by the polypeptide-MHC tetramer prepared by the method of the present application is 9.94% and 8.03%, respectively, which is significantly higher than that of the negative control group 0.05% and 0.12%. The technology can also be used for isolation and cloning of specific T cells of a human population infected or vaccinated with influenza B, isolation of specific TCRs combined with single-cell sequencing technology, and use as a T cell activation reagent. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The epitope polypeptide BM 58-66 Renaturation graph of AF9;

[0025] Figure 2HLA-B*1501 / BM 58-66 AF9-Tetramer staining of CD8 + T cell flow cytometry analysis results representative figure DETAILED DESCRIPTION

[0026] The present application will be further described by the following description of the drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0027] Unless otherwise specified, the reagents and materials used in the following examples are commercially available or can be prepared by known methods.

[0028] Example 1 Screening of influenza B specific polypeptides

[0029] A long peptide BM on the M protein of influenza B was predicted by polypeptide prediction software 55–70 IK16 sequence was predicted to obtain 14 short peptides of 8-11 amino acids, and the peptides of the M protein were mixed into a peptide library after the polypeptides were dissolved in DMSO. The PBMC of healthy people were cultured with the peptide library as a stimulant, and the cells were harvested after 9 days of culture.

[0030] According to the intensity of IFN-γ production, CD8 + T epitopes were predicted by Elispot technology. Elispot technology has strong specificity and can be used for the preparation of polypeptide vaccines, the research and development of diagnostic reagents, and can be used as an effective tool for T cell immunology evaluation, and can be used for the evaluation of T cell immune response of human population after influenza B infection or vaccination.

[0031] Example 2 Preparation of polypeptide-MHC tetramer

[0032] (1) Preparation of polypeptide / MHC complex:

[0033] 1) Add amino acid sequence GGGLNDIFEAQKIEWHE capable of connecting biotin to the C-terminal of HLA-B*1501 protein to construct recombinant plasmid pET28a-HLA-B*1501-Bio, link B2m gene (Genbank accession number AAA39668.1) with the vector to construct recombinant vector pET28a-B2m, and transform the plasmid into E. coli BL21 to obtain recombinant bacteria E. coli / pET28a-B2m and E. coli / pET28a-HLA-B*1501-Bio;

[0034] 2) The E. coli BL21 carrying plasmid in step 1) was cultured at 37°C and induced protein expression by adding 1 mmol / L IPTG, and the bacterial cells were collected and ultrasonically broken. After high-speed centrifugation (12000 rpm, 10 min), the precipitate was dissolved in a lysis buffer (6 mol / L guanidine hydrochloride, 10% glycerol, 50 mmol / L Tris pH 8.0, 100 mmol / L NaCl, 10 mmol / L EDTA) to obtain the heavy chain HLA-B*1501-Bio and the light chain β2m;

[0035] 3) The polypeptide BM with the sequence ALIGASICF was refolded in a refolding buffer (100 mmol / L Tris pH 8.0, 400 mmol / L arginine, 2 mmol / L EDTA) by using the dilution refolding method, so as to form an MHC complex. 58-66 AF9 and the heavy chain and the light chain were simultaneously refolded to form an MHC complex;

[0036] 4) The refolded sample obtained in step 3) was concentrated by using an ultrafiltration cup and a 10 kDa filter membrane, and the concentrated replacement solution was exchanged into an exchange buffer (20 mmol / L Tris-HCl, 50 mmol / L NaCl, pH 8.0); after the sample was taken out, centrifugation was performed at 12000 rpm for 10 min at 4°C, and the supernatant was transferred into an ultrafiltration tube and concentrated to about 0.5-1 ml. The sample was subjected to polypeptide / MHC complex purification by passing through a superdex200 molecular sieve, and the results are shown in Figure 1 .

[0037] (2) Biotinylation of the polypeptide / MHC molecule

[0038] 1) The polypeptide / MHC complex protein sample purified by the molecular sieve in step (1) was collected in an ultrafiltration concentration tube, concentrated to about 300 μL, and reacted with D-biotin under the catalysis of BirA enzyme, and incubated overnight at 4°C to obtain a biotinylated protein sample.

[0039] 2) The biotinylated protein sample was centrifuged and subjected to biotinylated complex purification by passing through a superdex200 molecular sieve to remove excess biotin.

[0040] 3) The purified polypeptide / MHC complex was concentrated to about 500 μL by using an ultrafiltration concentration tube, and the sample was taken for a gel shift test to verify the biotinylation effect.

[0041] Sample preparation:

[0042] A. 2 μL streptavidin + 8 μL molecular sieve buffer.

[0043] B. 8 μL biotinylated polypeptide / MHC sample + 2 μL 20 mg / mL streptavidin;

[0044] C. 8 μL biotinylated polypeptide / MHC sample + 2 μL molecular sieve buffer;

[0045] The three samples are incubated on ice for 30 min to 2 h and then subjected to SDS-PAGE identification.

[0046] The biotinylated MHC can bind with streptavidin to form a macromolecule, so that its band lags in SDS-PAGE. By comparing the ratio of MHC content of (C-B) / C, the effect of biotinylation can be determined, that is, how much proportion of MHC is well biotinylated. The biotinylation effect of the technical scheme of the present application is about 70%.

[0047] (3) Tetramerization of biotinylated MHC molecules:

[0048] The biotinylated MHC molecules are concentrated, and the biotinylated MHC molecules are tetramerized according to the molar ratio of streptavidin to polypeptide / MHC complex of 1:5. The streptavidin is a streptavidin with a fluorescent label, and is incubated at 4°C overnight to prepare BM 58-66 AF9 tetramer.

[0049] Example 3 Application of polypeptide / MHC tetramer in T cell analysis

[0050] The high affinity and high specificity of the B-type influenza specific polypeptide / MHC tetramer are used to detect T cells of people infected with B-type influenza and people immunized with a vaccine, evaluate the cellular immune effect of people infected with B-type influenza, convalescents and people immunized with a vaccine, and separate and clone T cells. The T cell immune level of convalescents infected with B-type influenza is evaluated by the following steps:

[0051] 1) Select PBMCs of convalescents infected with B-type influenza with HLA-B*1501 typing, stimulate the cultured PBMCs with a synthetic peptide library as a stimulant, and culture for 9 days;

[0052] 2) Harvest the cultured cells, and wash twice with FACS buffer / staining buffer (PBS+0.5% BSA);

[0053] 3) Cell surface molecule staining. Add antibodies (such as FITC-CD8, APC-CD4, PerCp-CD3, PE-Tetramer). Incubate at 4°C for 30 min;

[0054] 4) Wash. Centrifuge and wash twice with 200 μL FACS buffer;

[0055] Flow analysis was performed by using a cell flow meter. The results are shown in Figure 2 Figure 2. Polypeptide-MHC tetramer BM 58-66 The positive rates of AF9 in the influenza B virus infected convalescents were 9.94% and 8.03%, respectively, which were significantly higher than the negative control groups of 0.05% and 0.12%.

[0056] The polypeptide or polypeptide derivative prepared above is dissolved in water phase or oil phase adjuvant, diluted to a suitable concentration, and filtered to remove bacteria. Alternatively, emulsification is performed to prepare a vaccine.

[0057] Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the application. Therefore, the scope of the present application should be defined by the claims.

Claims

1. An influenza B HLA-B*1501 specific polypeptide characterised in that, The amino acid sequence of which is ALIGASICF.

2. A polypeptide-MHC tetramer, characterized in that, The biotinylated MHC-I binds to the influenza B HLA-B*1501 specific polypeptide of claim 1.

3. A method for the production of a polypeptide-MHC tetramer according to claim 2, characterized in that, The method comprises the following steps: (1) expressing MHC light chain and MHC heavy chain by using E. coli; (2) diluting and recombining to prepare polypeptide / MHC complex; (3) preparing biotinylated polypeptide / MHC complex; and (4) reacting the biotinylated polypeptide / MHC complex with labeled streptavidin.

4. The method of claim 3, wherein, The C terminal of the MHC heavy chain is connected with biotin.

5. The method of claim 3, wherein, In step (3), the polypeptide / MHC complex is combined with D-biotin under the catalysis of BirA enzyme.

6. The method of claim 5, wherein, In step (4), the biotinylated polypeptide / MHC complex is reacted with labeled streptavidin according to a molar ratio of (4-6):(0.5-1.5).

7. A vaccine comprising a polynucleotide of claim 1. The active ingredient contains the influenza B HLA-B*1501 specific polypeptide of claim 1.

8. An influenza B virus-specific cellular immunity test kit, characterized by comprising: The active ingredient contains the influenza B HLA-B*1501 specific polypeptide of claim 1.

9. Use of the polypeptide-MHC tetramer of claim 2 in the preparation of a reagent for evaluating T cell immunity after influenza B infection or vaccination, flow cytometry detection of immune cells in vivo, in situ staining of organism tissue sections, isolation and cloning of specific T cells, or isolation of specific TCR.

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