CN107141355B

CN107141355B - HTNV (human immunodeficiency Virus) antigen epitope linear tandem polypeptide, epitope peptide-compound tetramer and application

Info

Publication number: CN107141355B
Application number: CN201710400546.XA
Authority: CN
Inventors: 马樱; 张赟; 袁斌; 唐康; 张宇丝; 张春梅; 方亮; 陈丽华; 杨琨; 金伯泉
Original assignee: Fourth Military Medical University FMMU
Current assignee: Fourth Military Medical University FMMU
Priority date: 2017-05-31
Filing date: 2017-05-31
Publication date: 2021-05-14
Anticipated expiration: 2037-05-31
Also published as: CN107141355A

Abstract

The invention provides an HTNV epitope linear tandem polypeptide. The linear tandem polypeptide can induce specific T cell response to play an immune protection role. The invention also discloses application of the epitope linear tandem polypeptide in preparation of a polypeptide vaccine. The invention also discloses an HLA-A02/epitope peptide-complex tetramer, wherein the tetramer has an epitope VV9 and fluorescein PE, and can be specifically combined with a corresponding TCR, so that the corresponding TCR can be detected by fluorescence. The invention also provides a preparation method of the tetramer and application of the tetramer in preparing a CTL detection reagent.

Description

HTNV (human immunodeficiency Virus) antigen epitope linear tandem polypeptide, epitope peptide-compound tetramer and application

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an HTNV (human immunodeficiency Virus) epitope linear tandem polypeptide, an epitope peptide-compound tetramer and application thereof.

Background

Nephrotic syndrome Hemorrhagic Fever (HFRS) is an acute infectious disease characterized by fever, hemorrhage, and severe impairment of renal function caused by infection with Hantavirus (HTV) of the bunyaviridae family. Among viral infectious hemorrhagic fever diseases, HFRS is a disease which is currently distributed most widely in the world, has the largest number of diseases and has great harm. By the end of 2010, 1893555 global cumulative report cases have the mortality rate of 15 percent, wherein 1585942 national report cases account for 83.75 percent of the global total cases. Hantaan virus (HTNV) is the prototype of Hantaan virus and one of the important pathogenic microorganisms in the protocol review range of banned biological weapon convention worldwide. In 1976, the first HTNV 76-118 strain isolated by Lijiawang et al was a representative strain of HTNV and one of the main pathogens causing HFRS in China. HFRS has wide distribution in China and high morbidity and mortality, is second to viral hepatitis and is a viral infectious disease with the greatest harm, and is specified as a B type infectious disease according to infectious disease prevention and treatment Law of the people's republic of China. The major clinical manifestations of HFRS include fever, systemic toxic symptoms, capillary damage and acute injury to renal function, with severe individuals often developing hypotensive shock and readily dying at this stage. Rodents are the natural hosts and major sources of infection for HTNV, and have a wide variety of modes of transmission, including respiratory, digestive, cutaneous, mucosal, vector, and vertical. The population has a relatively common susceptibility to HTNV, and is prone to outbreaks and epidemics under the conditions of dense population and large number of virus-carrying mice. At present, HFRS continuously appears in new epidemic areas in China, outbreaks occur at partial epidemic points of vast rural areas, cities and towns and forest areas, seriously harm the life and health of people in China, threaten and influence the development of industrial and agricultural production, economic development, foreign trade and tourism industry, and are one of the infectious diseases which are mainly prevented and treated by the nation.

HTNV is a single-strand negative-strand RNA virus, and the genome is located in the core part of the virus and comprises three gene segments, namely large (L), medium (M) and small (S), which respectively code RNA polymerase, envelope glycoproteins (Gn and Gc) and Nucleoprotein (NP). The total length of the S gene is 1.67-2.05 kb, and the coded HTNV-NP is a non-glycosylated protein, comprises 429 amino acid residues and has a molecular weight of about 50 kD. The primary structure of NP is highly conserved and has stronger immunogenicity, and a plurality of antigen epitopes exist, so that humoral immunity and cellular immune response of an organism can be induced. The M gene fragment has a total length of 3.6-3.7 kb, comprises 1135 amino acid residues, a co-translation cleavage site consisting of 5 amino acid residues (WAASA) is arranged in the middle part of the M gene fragment, two encoded precursor proteins Gn and Gc are cleaved in an endoplasmic reticulum and processed into two mature proteins Gn and Gc, and the molecular weights of the unglycosylated Gn and the Gc are respectively 64kD and 54 kD. The HTNV-Gn/Gc can stimulate the body to produce specific neutralizing antibodies, and T cell antigen sites exist to induce T cell immune response.

During viral infection, T cells exert pathogen-clearing effects by recognizing complexes formed by Major Histocompatibility Complex (MHC) -class i or ii molecules and antigenic peptide epitopes on the surface of Antigen Presenting Cells (APCs). CD8⁺T cells (cytotoxic T cells, CTL) are the main effector cells of antiviral immunity, can recognize viral antigens which have been treated and bound to MHC class I molecules and expressed on the surface of host cells by TCR, are 8-10 amino acids in length, kill target cells by secreting granzyme and perforin, or induce programmed death of target cells by receptor-ligand interaction (e.g., Fas-FasL), thereby eliminating cells which have been infected with viruses. And CD4⁺After T cells recognize antigen epitope combined with MHC-II molecule, the T cells are activated and differentiated into Th1 and Th2 helper T cell subsets which can not only be initiated by directly secreting cell factorsHas effects in resisting pathogen infection, and can be used for assisting CD8⁺T cell activation and helper B cell production of antibodies to limit viral replication and spread. In the study of specific T cell immune responses after HTNV infection, it was found that specific CD8 was present in the peripheral blood of HFRS patients caused by HTNV infection⁺T cell response and CD4⁺T cell response, infiltrating lymphocytes in the kidney of HFRS patients, especially CD8⁺T cells. The number of activated CTL in the acute-phase peripheral blood of the HFRS patient is obviously increased, the overall T cell frequency of IFN-gamma secretion in the acute phase and the epitope specific CTL response level are in negative correlation with the severity of the HFRS disease, and the fact that the HTNV specific T cell response plays an important role in the disease pathogenesis and prognosis of the HFRS disease is suggested. Therefore, there is a need to research and develop detection reagents capable of detecting HTNV-specific T cell responses.

In recent years, various preventive vaccines against hantavirus have been developed at home and abroad. HTNV vaccines produced in China are inactivated whole virus vaccines and are respectively derived from the culture of inner Mongolian rat kidney cells or golden hamster kidney cells, and the HTNV vaccines comprise white rat suckling rat brain purified vaccines, hamster kidney primary cell vaccines, Vero cell purified vaccines and the like. After being inoculated, the vaccines can stimulate the body to generate neutralizing antibodies, so that the vaccines play a positive role in preventing the generation and the epidemic of the HFRS, but the titer of the neutralizing antibodies generated by the virus inactivated vaccines by stimulating the body is usually not high, and the effect of inducing the generation of cellular immune response is relatively weak, so that the current situation seriously influences the effective prevention of people on the HFRS diseases, and further needs to explore more effective novel vaccines.

Compared with the traditional inactivated vaccine or attenuated live vaccine, the polypeptide vaccine has the advantages of high purity, stability among batches, low cost, high specificity and the like, and has good safety, strong protection capability, response type which can be effectively regulated and controlled, and can induce T cell immune response to play the role of resisting pathogenic microorganisms, thereby having higher research and application values. Polypeptide vaccines designed by applying the combined epitope at home and abroad have been reported in the aspects of infection resistance, tumor resistance and the like, and various synthetic peptide vaccines based on the polypeptide epitope enter the clinical research stage or are on the market. Synthetic epitope peptides are capable of binding directly to MHC molecules without the need for processing by APCs, and have the same effect as natural endogenous peptides in activating the immune system. However, because a single epitope peptide segment has short length, is easy to degrade in vivo and has weak immunogenicity, the epitope peptide segment can not be directly used as a vaccine to immunize organisms generally. In practical applications, in order to enhance the immunogenicity of epitope vaccines, various methods are usually required to be adopted to optimize the epitope vaccines or construct composite multi-epitope vaccines for use, and at the same time, safe and efficient immune adjuvants are used to improve the immune effect. The currently common polypeptide vaccine design methods comprise linear tandem of multiple antigen epitopes, multiple antigen polypeptide space patterns, lipopeptide patterns, epitope gene recombination expression patterns and the like.

The identification of the epitope of the HTNV structural protein is known to be an important basis for designing more efficient polypeptide vaccine molecules, and in the research of the epitope on the HTNV structural protein, a plurality of T cell epitopes on HTNV-NP and Gn/Gc primary structures and B cell epitopes with neutralizing activity, such as B cell epitope peptide recognized by HTNV specific neutralizing antibody 3D8, namely glycoprotein, have been reported⁸⁸²GFLCPEFPGSFRKKC⁸⁹⁶It has been identified that the HTNV-NP C-terminal polypeptides aa301-aa315, aa355-aa369 and aa415-aa429 can induce strong CD8⁺T cell responses, etc. We identified 5 novel HTNV-NP CD8 in previous work⁺T cell 9 peptide epitope and Human Leukocyte Antigen (HLA) -I molecule restriction thereof, HTNV-Gn/Gc 9 peptide CTL epitope with high affinity to HLA-A02 is predicted and screened by computer simulation software, and 7 HTNV-Gn/Gc 9 peptide CTL epitopes restricted by HLA-A02 are preliminarily identified and obtained in the peripheral blood of HFRS patients. The allele HLA-A02 in the HLA-I molecule is distributed most widely and has the highest frequency in Chinese population, so the HLA-A02 is the first choice of related molecule in the vaccine design research aiming at more specific and effective Chinese population, and is the most convenient model for carrying out related basic research.

T, B identification of cell epitopes provides the most important basis for design and construction of candidate molecules of polypeptide vaccines, but epitope peptides are often weak in immunogenicity alone, and epitope tandem polypeptides designed by combining multiple and multiple antigen epitopes show good immunogenicity and can induce strong T, B cell responses for a long time in polypeptide vaccine research of various infectious diseases. The linear concatenation of multiple epitopes is to directly construct a compound multivalent epitope vaccine with multiple epitope peptides by connecting a plurality of B cell or T cell epitopes obtained by prediction or identification end to end, the linear combination is generally artificially synthesized, wherein the general Th epitope has the capability of being not limited by MHC-II molecules, especially HLA-DR molecules, and the capability of inducing T cell response is more than one thousand times of that of the natural epitope. The method can effectively enhance the immunogenicity of the epitope polypeptide and can accurately quantify each epitope.

Because the incidence and mortality of HFRS caused by HTNV infection are high, but no specific prevention and treatment methods exist so far, the polypeptide vaccine becomes a new strategy for research on prevention and treatment of HTNV infection, and the key point of research is to design and synthesize an HTNV epitope tandem polypeptide macromolecule with stronger immunogenicity on the basis of identified HTNV specific epitope. The HTNV epitope tandem polypeptide vaccine molecule is established to induce specific T cell response to play an immune protection role, and can be used as an important means for effectively preventing HTNV infection. Therefore, the development of epitope peptide vaccines with high safety and high specificity for preventing and treating HTNV infection in chinese population is urgently needed.

Disclosure of Invention

The invention aims to provide an HTNV epitope linear tandem polypeptide and application thereof, wherein the linear tandem polypeptide can induce specific T cell response to play an immune protection role.

The present invention also provides an HLA-a 02/epitope peptide-complex tetramer capable of specifically binding to a corresponding TCR and having fluorescence, and uses thereof.

The invention is realized by the following technical scheme:

an HTNV epitope linear tandem polypeptide is a B + Th + CTL linear tandem polypeptide consisting of HTNV B cell epitope peptide GC15 with neutralization activity, universal Th epitope pan-DR epitope peptide and HLA-A x 02 limited HTNV-Gn/Gc CTL9 peptide epitope VV9, and the amino acid sequence of the polypeptide is shown as SEQ ID NO: 4, respectively.

The amino acid sequence of the HLA-A x 02 restricted HTNV-Gn/Gc CTL9 peptide epitope VV9 is shown in SEQ ID no: 1 is shown in the specification;

the amino acid sequence of the universal Th epitope pan DR epitope peptide is shown in SEQ ID No: 2 is shown in the specification;

the amino acid sequence of the HTNV B cell epitope peptide GC15 with the neutralizing activity is shown in SEQ ID no: 3 is shown in the specification;

wherein, in SEQ ID NO: 1 is connected with the amino acid at the N end of the epitope peptide shown in SEQ ID NO: 2 and the epitope peptide of SEQ ID NO: 3, epitope peptide represented by SEQ ID NO: 2 and the epitope peptide of SEQ ID NO: 3, three alanines are used as connecting peptides among the epitope peptides shown in SEQ ID NO: 3, the N end of the epitope peptide is connected with lysine-serine to form a leader sequence.

The invention also discloses application of the HTNV epitope linear tandem polypeptide in preparing a polypeptide vaccine for resisting Hantaan virus.

The polypeptide vaccine is induced CD8⁺Polypeptide vaccine for T cell proliferation.

The polypeptide vaccine is a polypeptide vaccine secreting IFN-gamma.

The polypeptide vaccine is induced CD8⁺T cell expresses polypeptide vaccine of CD107 a.

The polypeptide vaccine is induced CD8⁺T cell expresses the polypeptide vaccine of granzyme B.

The concentration of the HTNV epitope linear tandem polypeptide in the polypeptide vaccine is 1 mu M.

The invention also discloses an HLA-A02/epitope peptide-compound tetramer, which comprises 4 monomers, wherein each monomer is formed by assembling an HLA-A02 molecular heavy chain and beta 2 microglobulin in vitro; the carbon end of the heavy chain of HLA-A02 molecule is connected with an activated biotin molecule; the HLA-A02 restricted HTNV-Gn/Gc CTL9 peptide epitope VV9 is bound to antigen peptide binding groove formed by heavy chain alpha 1 and alpha 2 structural domain of HLA-A02 molecule through anchoring amino acid; wherein the amino acid sequence of the HLA-A x 02 restricted HTNV-Gn/Gc CTL9 peptide epitope VV9 is as follows: VMASLVWPV (shown in SEQ. ID. NO: 1);

also comprises avidin marked with fluorescein PE, and biotin molecules of the four monomers are combined with the avidin.

The invention also discloses application of the HLA-A02/epitope peptide-compound tetramer in preparing a medicament reagent for detecting the frequency of the HTNV-Gn/Gc epitope peptide specific T cells;

binding the fluorescein-labeled HLA-A x 02/epitope peptide-complex tetramer with CTL cells, and detecting by a flow cytometer to distinguish the frequency of the HTNV-Gn/Gc epitope peptide specific T cells.

Compared with the prior art, the invention has the following beneficial technical effects:

the HTNV epitope linear tandem polypeptide provided by the invention is a 'B + Th + CTL' HTNV epitope linear tandem polypeptide consisting of an HTNV B cell epitope GC15 with neutralization activity, a general Th epitope PADRE and an HLA-A x 02 limited HTNV-Gn/Gc CTL9 peptide epitope VV9, wherein the N end of the linear tandem polypeptide is connected with lysine-serine (KSS) as a leader sequence, three alanines (AAA) are used as flexible linkers between the GC15 and the PADRE, and the VV9 is directly connected with the carbon end of the PADRE. Compared with a single HLA-A x 02 restricted HTNV-Gn/Gc CTL9 peptide epitope VV9, the epitope tandem polypeptide has the capacity of inducing epitope-specific CTL to generate higher level IFN-gamma, higher frequency killing medium and stronger proliferation of specific CTL, and induced T cell response can effectively reduce HLA-A2.1/K after HTNV infection^bHTNV antigen content in multiple organs of transgenic mice.

The HTNV epitope linear tandem polypeptide provided by the invention is applied to the preparation of polypeptide vaccine for resisting hantaan virus, and the prepared polypeptide vaccine can induce and generate stronger cytotoxic T cell response with CTL epitope peptide specificity.

The HLA-A02/epitope peptide-complex tetramer provided by the invention is marked with a fluorescein molecule and connected with VV9, VV9 can be specifically combined with a corresponding TCR, and then the corresponding TCR can be detected by fluorescence; the frequency of HTNV-Gn/Gc epitope peptide-specific T cells can be distinguished by flow cytometry detection.

Drawings

FIG. 1 is a graph of the results of flow cytometry experiments in which T2cell binding assays were performed to determine the epitope of HLA-A x 02 restricted HTNV-Gn/Gc CTL9 peptide VV 9.

Figure 2-1 is a scatter plot of HLA-a 02/epitope peptide-complex tetramer staining results.

FIG. 2-2 is a graph of VV 9-specific CTL frequency versus HFRS patient typing.

FIG. 3 is a diagram showing the design results of epitope linear tandem polypeptides.

FIG. 4 shows HLA-A2.1/K after immunization of ELISOPT detection of each polypeptide^bAnd (3) a frequency result chart of IFN-gamma secretion of epitope specific CTL in the spleen of the transgenic mouse.

FIG. 5-1 is a graph showing the results of the expression of CD107a by HTNV-Gn/Gc 9 peptide CTL epitope VV 9-specific CTL in spleen cells of mice with Tg after immunization with different polypeptides.

FIG. 5-2 shows CD8 in Tg mouse splenocytes after immunization with different polypeptides⁺CD107a⁺Statistical comparison of T cell frequency.

FIGS. 5-3 are graphs showing the results of CTL-specific CTL-expressed granzyme B of HTNV-Gn/Gc 9 peptide CTL epitope VV9 in splenocytes of mice with Tg after immunization with different polypeptides.

FIGS. 5-4 are graphs showing CD8 in splenocytes from different immunized Tg mice⁺Granular enzyme B⁺Statistical comparison of T cell frequency.

FIG. 6 shows HLA-A2.1/K detected by CFSE proliferation assay after immunization with different polypeptides^bCTL proliferation capacity result chart in spleen of transgenic mouse.

Detailed Description

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

The invention firstly utilizes a T2cell line binding test to obtain the peptide epitope and the amino acid sequence of the HTNV-Gn/Gc CTL9 with the highest HLA-A02 affinity in the sequence of the glycoprotein Gn/Gc of the HTNV 76-118 strain. Further selecting an HTNV-Gn/Gc CTL9 peptide epitope limited by HLA-A x 02, a B cell epitope with neutralizing activity and a general Th epitope, and designing a novel tandem polypeptide antigen based on the HTNV epitope by using a computer-assisted molecular design technology. The effectiveness and potential application value of the HTNV epitope tandem polypeptide vaccine are evaluated by designing the 'B + Th + CTL' epitope tandem polypeptide and evaluating the immunogenicity and the capability of inducing specific CTL response in a transgenic mouse.

1. Obtaining HLA-A02 highest affinity HTNV-Gn/Gc CTL9 peptide epitope

T2 cells are T-B hybridoma cell lines, and surface HLA-A02 molecules are positively expressed, but T2 cells are TAP molecule deficient cell lines, so endogenous antigens cannot be processed, and only exogenous antigen peptides can be presented. However, in the absence of exogenous antigen peptide, the expression of HLA-A02 molecules on the cell surface is very unstable and is easy to degrade quickly, when the antigen peptide is combined with the antigen peptide, the expression of HLA-A02 molecules on the cell surface is obviously enhanced, and the higher the affinity of the antigen peptide and the HLA-A02 molecules is, the higher the expression level of the HLA-A02 molecules on the cell surface of T2 is. Therefore, the increase of the expression level of HLA-A02 molecules on the surface of the T2cell line can visually reflect the affinity of the antigen peptide and the HLA-A02 molecules. The method comprises the following specific steps:

after the T2cell line is recovered, the cell line is cultured to logarithmic growth phase by 20% FCS RPMI 1640, and the cell density is adjusted to 2-4 multiplied by 10 by using serum-free medium RPMI 1640⁵Adding into sterile 24-well cell culture plate (0.5 mL/well, i.e. 1-2 × 10)⁵(ii) individual cells; respectively adding HTNV-Gn/Gc synthetic 9 peptides into experimental wells to enable the final concentration of the HTNV-Gn/Gc synthetic 9 peptides to reach 50 mu M, and simultaneously adding beta 2 microglobulin (beta 2-M) to achieve the final concentration of 2.5 mu g/mL; meanwhile, setting a blank control, namely setting a control hole which does not add HTNV-Gn/Gc synthesis 9 peptide and only adds T2cell line and beta 2-m; setting a positive control, namely adding the identified HLA-A x 02 restricted HTNV-NP 9 peptide CTL epitope peptide; 5% CO at 37 ℃₂Culturing for 18 h; RPMI 1640 washed the cells for 1 time, after centrifugation, the PE-labeled mouse anti-human HLA-A02 (HLA-A02-PE) was added thereto, 15. mu.L/cell, and incubated at 4 ℃ for 45 min; washing with FCM washing solution for 1 time, and detecting with flow cytometry; calculating the FI value as the FI value>1 is judged as a high affinity epitope, and the calculation formula is as follows:

FI ═ average fluorescence intensity of PE in experimental wells-average fluorescence intensity of PE in blank control wells/average fluorescence intensity of PE in blank control wells

The results of the experiments are shown in table 1 and in fig. 1.

TABLE 1T 2 screening of cell binding experiments to obtain the highest HLA-A02 affinity HTNV glycoprotein 9 peptides

FI: the fluorescence index.

^cFI ═ average PE fluorescence index loaded with peptide-average PE fluorescence index unloaded with peptide ═ based on

(average PE fluorescence index of unloaded peptide) FI ≥ 1 is high affinity epitope peptide

As shown in Table 1, the FI value of the positive control peptide, i.e. the HTNV-NP CTL epitope peptide aa129-aa137, after being combined with the T2cell line is more than 2, the FI value of the candidate HTNV-Gn/Gc CTL9 peptide epitope aa8-aa16(VMASLVWPV, VV9) is predicted to be 3.67, the FI value is the highest in the candidate peptides and is obviously higher than that of the positive control peptide, and the candidate peptide can be determined as the HLA-A2 highest affinity epitope.

Fig. 1 is a graph of the flow cytometry results of T2cell binding assay for determining HLA-a 02 highest affinity HTNV-Gn/Gc CTL9 peptide epitope VV9, including: isotype control (T2cell line with peptide) indicating the fluorescence intensity of HLA-a 02 molecules expressed on the surface of T2 cells not loaded with peptide; positive control (NP aa129-aa137), i.e. fluorescence intensity of HLA-A02 molecule expressed on the surface of T2cell after loading known HTNV-NP CTL epitope peptide aa129-aa 137; the experimental peptide (GP peptide indicated the fluorescence intensity of HLA-A02 molecule expressed on the surface of T2 cells after loading with the predicted HTNV-Gn/Gc CTL9 peptide epitope VV 9. Thus, the more the peak of the experimental peptide was shifted to the right compared to the peak of the isotype control, indicating that the peptide VV9 has a higher affinity for binding to HLA-a 02 molecules and is more likely to be an HLA-a 02 restricted HTNV Gn/Gc CTL9 peptide epitope.

2. HLA-A02/epitope peptide-complex tetramer staining identification of HLA-A02 restricted HTNV-Gn/Gc CTL9 peptide epitope

2.1 isolation of Peripheral Blood Mononuclear Cells (PBMC) from the peripheral blood of HFRS patients

According to the clinical diagnosis standard (serum anti-HTNV IgM antibody and clinical symptoms) of hemorrhagic fever with renal syndrome in China, peripheral blood of HFRS patients with different disease states (light, medium, heavy and dangerous) and different disease periods (fever period, hypotension shock period, oliguria period, diuresis period and recovery period) is aseptically collected, heparin sodium anticoagulation (50U/mL) is added, and different collection conditions are numbered. Diluting and uniformly mixing the collected peripheral blood with an equivalent serum-free RPMI 1640 culture solution, slowly superposing the diluted anticoagulated blood on a lymphocyte separation solution by using an elbow suction tube (10 mL of lymphocyte separation solution is added in a 50mL centrifuge tube in advance and purchased from Tianjin N-Letaiyang biological company), centrifuging at 2000rmp for 20min, carefully sucking a white cloud layer at the two layers of interfaces, washing with 5 times of volume of RPMI 1640, centrifuging at 1500rpm for 10min, and washing for 2 times to obtain PBMC in the peripheral blood of a HFRS patient; directly for subsequent use or resuspend cells in a frozen stock (90% FCS, 10% DMSO) and freeze in liquid nitrogen for use.

2.2 Flow Cytometry (FCM) screening of HLA-A02-positive HFRS patients

Resuscitating PBMC of HFRS patient, taking 5 × 10 cells from each experimental tube⁵After being washed by FCM washing liquor, 1 mu L of normal sheep serum is added and sealed for 10min at room temperature; respectively adding mouse anti-human HLA-A02-PE (10 muL/branch), and setting a control tube, namely adding mouse IgG1-PE (10 muL/branch) as isotype control for detecting HLA-A02; incubating at 4 ℃ for 30min, washing with FCM lotion for 2 times, centrifuging at 1000rpm for 5min, shaking and mixing HLA-A x 02 experiment tube, adding 300 μ L FCM stationary liquid, and performing on-machine detection.

2.3 HLA-A02/epitope peptide-complex tetramer staining to identify HTNV-Gn/Gc CTL9 peptide epitope VV9 and to detect its specific CTL frequency

The HLA-A02/epitope peptide-complex tetramer is a tetramer formed by binding four monomers of HLA-A02/epitope peptide-complex connected with biotin and an avidin molecule;

the HLA-A02/epitope peptide-complex monomer is formed by assembling a heavy chain of an HLA-A02 molecule with activated biotin molecules connected to the C end and beta 2 microglobulin (beta 2-m) in vitro, and then combining epitope peptide VV 9.

During preparation, the heavy chain of the HLA-A02 molecule and the beta 2-m are assembled in vitro (mainly realized by expression and purification of the heavy chain of the HLA-A02 molecule and the beta 2-m protein and renaturation and purification of the HLA-A02/peptide-monomer), and are combined with a specific HTNV-Gn/Gc CTL9 peptide epitope VV9 to form an HLA-A02/epitope peptide-complex monomer capable of being specifically combined with corresponding TCR, and then 1 activated biotin molecule is connected to the C end of the heavy chain. 4 monomers were bound to 1 avidin molecule, forming a tetramer. Because fluorescein PE is labeled on avidin, the fluorescein-labeled tetramer can be specifically combined with CTL specific to the HTNV-Gn/Gc CTL9 peptide epitope VV9, the frequency of T cells specific to the HTNV-Gn/Gc CTL9 peptide epitope VV9 can be detected by a flow cytometer, and the fluorescent tetramer is used for researching the frequency of the specific CTL.

Specifically, HLA-a × 02/epitope peptide-complex tetramers were assigned to the company beijing spambo biotechnology.

The specific stimulation effect of the identified HLA-A x 02 restricted HTNV-Gn/Gc CTL9 peptide epitope VV9 on the T cells of the HFRS patient is detected by using IFN-gamma as a detection target, and the specific stimulation effect is as follows:

taking HLA-A02 positive HFRS patients or normal human PBMCs, each experimental tube 3-5 × 10⁶Washing with FCM washing solution for 1 time, centrifuging at 1000rpm for 5min, discarding supernatant, and mixing by vortex; taking out HLA-A02/epitope peptide-complex tetramer at 4 deg.C, centrifuging at 14000g for 5min, and collecting supernatant to avoid using precipitate to avoid non-specific staining. 10 μ L of HLA-A02/epitope peptide-complex tetramer was added to each tube and incubated for 10min at room temperature in the dark. Setting irrelevant HLA-A02/epitope peptide-complex tetramer control; setting homotype control tube, taking PBMC 5X 10⁵After washing with FCM washing solution, 5 mu L of each of mouse IgG1-PE, IgG1-PerCP-Cy5.5 and IgG1-APC is added; FCM washing solution is washed for 2 times, centrifuged for 5min at 1000rpm, the supernatant is discarded, vortexed and mixed, mouse anti-human CD8-PerCP-Cy5.5 and CD3-APC are added into the experimental tube, and vortexed and mixed. Incubating for 20min on ice in dark; FCM Wash 2 times, 1000rpm centrifugationAnd 5min, discarding the supernatant, and mixing by vortex. Adding 300 μ L FCM stationary liquid, mixing, detecting with flow cytometer, collecting at least 5 × 10⁵And (4) cells.

HLA-a × 02/epitope peptide-complex tetramer staining results showed that CTL specific to HTNV-Gn/Gc CTL9 peptide epitope VV9 could be detected in PBMCs of HFRS patients (as shown in fig. 2-1), and it was found that HTNV-Gn/Gc CTL9 peptide epitope VV9 specific CTL frequency was significantly higher in peripheral blood of HFRS mild/severe patients than in severe/critical patients (P ═ 0.028) (as shown in fig. 2-2).

FIGS. 2-1 and 2-2 are graphs showing the staining results of HLA-A02/epitope peptide-complex tetramer and a statistical comparison of CTL frequencies specific to the HTNV-Gn/Gc CTL9 peptide epitope VV9 in peripheral blood of different HFRS patients with different disease positive for HLA-A02. FIG. 2-1 shows the use of Flowjo software to gate out PBMC cell populations on SSC and FSC scattergrams, to gate out CD3 positive cell populations, and to gate out CD8⁺(abscissa) tetramer⁺(ordinate) the proportion of the double positive cell population, i.e. the specific CTL frequency of the HTNV-Gn/Gc CTL9 peptide epitope VV9 restricted by HLA-A x 02 in the peripheral blood of HFRS patients. The higher frequency indicates that the epitope induces a more potent CTL response in HFRS patients. FIG. 2-2 shows the comparison of CTL frequencies (ordinate) specific to the HTNV-Gn/Gc CTL9 peptide epitope VV9 in the peripheral blood of HFRS patients, which were divided into two groups (abscissa) of light/medium (Mild/model) and heavy/Critical (Severe/Critical) based on clinical data. The results show that the frequency of CTLs specific for the HTNV-Gn/Gc CTL9 peptide epitope VV9 is significantly higher in both mild/moderate patients than in heavy/critical patients (P ═ 0.028), suggesting that the identification of the resulting HLA-a × 02 restricted HTNV-Gn/Gc CTL9 peptide epitope VV 9-specific CTL response may play an immunoprotective role in the course of HFRS disease.

Design of 3 epitope tandem polypeptide and synthesis, purification and identification of polypeptide

3.1 design of epitope tandem Polypeptides

Design principle of epitope tandem polypeptide

When designing epitope tandem polypeptides, the selection and permutation combination of epitopes is the key for constructing epitope vaccines. Selecting T cell epitope with immunoprotection effect and B cell epitope with medium activityUniversal Th cell epitopes were introduced and designed with the addition of appropriate spacer sequences. T cell epitopes can enable the tandem polypeptides to effectively cause immune response, and meanwhile, the T cell epitopes are limited by MHC molecules and can cause immune response only after being recognized by the MHC molecules, so that the selection of the T cell epitopes is very important. The addition of spacer sequence, i.e. short and highly flexible hinge region, to design and effectively separate epitopes, avoid the generation of new epitopes and ensure the independent antigenicity of each epitope. Among the alleles of HLA, HLA-A02 is the most widely distributed and polymorphic class I molecule and gene family in Chinese population, and is also the absolute dominant gene in the HLA-A gene locus in Shanxi population, so that the identified HLA-A02 high affinity HTNV-Gn/Gc CTL9 peptide epitope VV9 capable of inducing immune protective response is selected as the preferred epitope peptide segment for designing HTNV novel epitope vaccine for Chinese population. Meanwhile, the HTNV specific neutralizing antibody 3D8 recognizes a B cell epitope, namely HTNV-Gn/Gc⁸⁸²GFLCPEFPGSFRKKC⁸⁹⁶(GC15) was identified earlier.

A universal Th epitope Pan DR epitope (Pan DR epitope, PADRE) was selected, the amino acid sequence of which is akxvaawttlkaaa (X ═ cyclohexylalanine, cyclohexylalanine). The tridecapeptide is not limited by MHC-II molecules, especially HLA-DR molecules, and has over one thousand times of capacity of inducing T cell response compared with natural epitope.

Design scheme of epitope tandem polypeptide

Constructing P1 peptide as HLA-A x 02 restriction HTNV-Gn/Gc CTL9 peptide epitope VV 9; the P2 peptide is an HTNV-Gn/Gc CTL9 peptide epitope VV9, and the N end of the peptide epitope is connected with a neutralizing active B cell epitope on Gn/Gc; the P3 peptide is a neutralizing active B cell epitope which is sequentially connected with a universal Th epitope PADRE and Gn/Gc on the N-terminal amino acid of an HTNV-Gn/Gc CTL9 peptide epitope VV 9. Three alanine AAA peptides were introduced as flexible linkers between two different epitopes, respectively, and the N-terminus of each peptide was linked to a leader sequence, lysine-serine (KSS) (as shown in fig. 3).

FIG. 3 is a diagram showing the design results of three groups of epitope tandem polypeptides. Wherein the P1 polypeptide is HLA-A x 02 restricted HTNV-Gn/Gc CTL9 peptide epitope VV9(VMASLVWPV), and is connected with a leader sequence KSS at the N end; the P2 peptide is HTNV-Gn/Gc CTL9 peptide epitope VV9, the N end of the peptide is connected with AAA through connection, and then is connected with a neutralizing active B cell epitope GC15(GFLCPEFPGSFRKKC) on Gn/Gc, and the N end of GC15 is connected with a leader sequence KSS; the P3 peptide is HTNV-Gn/Gc CTL9 peptide epitope VV9, N-terminal amino acid of which is sequentially connected with a general Th epitope PADRE (AKXVAAWTLKAAA), then is connected with a B cell epitope GC15 through a connecting AAA, and is connected with a leader sequence KSS at the N-terminal of GC 15.

3.2 Synthesis, purification and identification of epitope tandem Polypeptides

Shanghaijie peptide Biotechnology Co., Ltd was entrusted to synthesis and purification. Based on the amino acid sequences of the identified HLA-A x 02 restricted HTNV-Gn/Gc CTL9 peptide epitope VV9, the general Th epitope PADRE and the neutralizing activity B cell epitope on Gn/Gc, a Merrifield solid phase chemical synthesis scheme is adopted to fix the peptide chain extending from the carboxyl end to the amino end. Purifying the synthesized polypeptide by high performance liquid chromatography (WATERS 600), analyzing molecular mass and charge distribution by mass spectrometry (API2000), and performing low pressure rotary evaporation and ether precipitation on the purified product to obtain pure polypeptide product, and storing at-70 deg.C. Before use, 50. mu.L DMSO is used to dissolve the lyophilized powder, and PBS is added according to the molecular weight to make the final concentration (storage concentration) reach 1. mu.M.

4 polypeptide immune HLA-A2.1/K^bTransgenic mouse for detecting in vivo induced immune response capability

4.1 polypeptide immunization HLA-A2.1/K^bDetermination of transgenic mouse protocol

In order to further determine whether the epitope tandem polypeptide can induce more effective immune protection in vivo, different polypeptides are respectively applied to immunize HLA-A2.1/K^bTransgenic mice were studied in vivo. Meanwhile, the identified HLA-B x 35 restrictive HTNV-NP CTL epitope (aa131-aa139, VPILLKALY, VY9) is set as an irrelevant peptide control group, and a commercial bivalent inactivated vaccine for hemorrhagic fever with renal syndrome (Zhejiang Tianyuan biological medicine Co., Ltd.) is used for intramuscular injection as a positive control. 36 HLA-A2.1/K^bTransgenic mice (Tg) were purchased from third-military medical science and immunology laboratories and the grouping protocol was as follows:

①P1：VV9(VMASLVWPV)+gp96；

②P2：GC15(GFLCPEF PGSFR KKC)-VV9(VMASLVWPV)+gp96；

③P3：GC15(GFLCPEF PGSFR KKC)-PADRE(AKXVAAWTLKAAA)-VV9(VMASLVWPV)+gp96；

fourthly, irrelevant peptide VY9(VPILLKALY) + gp 96;

fifthly, inactivating the vaccine and attacking the virus (positive control);

sixthly, PBS + challenge (negative control).

The method is characterized in that an HTNV 9 peptide epitope/gp 96/Freund's adjuvant immunization scheme is adopted to immunize transgenic mice, the mice are divided into two groups according to a random principle, 6 mice are used in an experimental group, and the subcutaneous injection is carried out on 4-5 positions of the head and the back of each mouse at multiple points of 1: 1 Freund's complete adjuvant (sigma) and polypeptide (50 ug) + gp96(30 ug) (injection amount of about 40-50 ul per site) mixed together, and after primary immunization for 10 days and 20 days, incomplete Freund's adjuvant emulsifies the same amount of polypeptide and gp96, and mice are immunized in the same way. Control group an identical method was used to inject 1: 1 Freund's complete or incomplete adjuvant mixed with PBS buffer + gp96 (30. mu.g). The control group was injected with 1: 1 Freund's complete or incomplete adjuvant mixed with irrelevant 9 peptide (50. mu.g) + gp96 (30. mu.g). The method comprises three times of immunization, each time with an interval of 10 days, and 10 days after the last immunization, wherein each group takes 5 mice, takes the spleens of the mice to prepare single cell suspension, and lyses erythrocytes for further experimental detection.

4.2 ELISPOT test of the ability of epitope-specific effector CTL to secrete IFN-gamma in splenocytes of Tg mice after immunization

A commercial IFN-gamma ELISPOT kit (purchased from Mabtech) is adopted to identify whether HTNV-Gn/Gc CTL9 peptide epitope VV9 can stimulate PBMC to secrete IFN-gamma, and the following operations are carried out according to the instructions:

taking out the commercial pre-coated ELISPOT plate from 4 ℃, and washing for 3 times by using sterile deionized water; adding 10% FCS RPMI 1640 at 200. mu.L/well, and blocking at 37 ℃ for 2 h; removing the blocking solution, adding 1 × 10 solution per well⁶Adding HTNV-Gn/Gc CTL9 peptide epitope VV9 (the final concentration is 10 mu M) into spleen cells of each group of immunized transgenic mice, and incubating for 18-24 h at 37 ℃; setting positive and negative control, adding PHA (final concentration 10 mug/mL) or anti-CD 3mAb (final concentration 10 mug/mL) into the positive control hole, and only adding splenocytes into the negative control hole; washing with PBS for 6 times, adding 50 μ L of detection antibody diluted at a ratio of 1:200 into each well, and incubating at room temperature for 2 h; PBS cleaningAdding 100 mu L of BCIP/NBT color development liquid into each hole for 6 times, and developing for 1 hour at room temperature in a dark place; and washing the board with tap water, and naturally drying.

The number of IFN-. gamma.spot-forming cells was counted using a CTL ELISPOT reader. According to the report of the literature, the positive response definition standard is that the number of specks per hole is more than or equal to 50SFCs/10⁶Effector cells, and the number of spots is at least 3 times of the number of spots in the negative control wells. Normalized to SFCs/10, calculated according to the following formula⁶Cell: SFC/10⁶Cells ═ number of positive experimental wells-number of negative control wells/total number of effector cells added per well]×10⁶。

Compared with an HLA-A x 02 restricted HTNV-Gn/Gc CTL9 peptide epitope VV9(P1), the frequency of inducing the specific CTL of the VV9 epitope to secrete IFN-gamma is obviously increased (P <0.001) after the epitope tandem polypeptide P2 and the epitope tandem polypeptide P3 are immunized to Tg mice respectively, particularly the frequency of inducing the specific CTL to secrete the IFN-gamma is higher than that of inducing the specific CTL to secrete the IFN-gamma (P <0.001) by the epitope tandem polypeptide P2 (shown in figure 4), and the general Th epitope plays an important role in the process of assisting the CTL epitope to induce cellular immune response.

FIG. 4 shows HLA-A2.1/K after ELISOPT detection of each epitope polypeptide immunization^bAnd (3) a frequency result chart of IFN-gamma secretion of epitope specific CTL in the spleen of the transgenic mouse. Wherein the abscissa is each immunization group, and the P2 polypeptide (KSS-GFLCPEFPGSFRKKC-AAA-VMASLVWPV), the P3 polypeptide (KSS-GFLCPEFPGSFRKKC-AAA-AKXVAAWTLKAAA-VMASLVWPV), the P1 polypeptide (KSS-VMASLVWPV), the unrelated peptide (VPILLKALY), the HTNV inactivated vaccine and the PBS immunization group are arranged in sequence. The ordinate represents the frequency of IFN- γ secretion by splenocytes from mice of each immunization group under the stimulation of the polypeptide. SFC (spot forming cell) is the number of spot forming cells.

4.3 frequency detection of polypeptide specific effect CTL secretion killing medium induced by different design group epitope tandem polypeptides in vitro

The method is characterized in that the intracellular cytokine staining technology is applied to detect the frequency of polypeptide specific effect CTL secretion killing medium induced by each group of epitope tandem polypeptides in vitro, and the specific operation is as follows:

after the immunized Tg mouse splenocytes are incubated with corresponding tandem polypeptide or single peptide and CD28/CD49d monoclonal antibody (BD company, used for the production of T cell cytokines with the specificity of antigen peptide) for 1 hour, then an intracellular protein transport inhibitor Brefeldin A (BFA) is added for incubation for 5 hours, after membrane rupture, killing medium mAbs such as granzyme B (granzyme B) and CD107a and the like, CD3mAb and CD8mAb (BD company) are used for staining, and after fixation, the frequency of the generation of HTNV-Gn/Gc CTL9 peptide epitope VV9 specific intracellular killing medium in the CTLs is analyzed by a flow cytometer.

The CTLs specific to HTNV-Gn/Gc CTL9 peptide epitope VV9 in spleen cells of mice with Tg respectively immunized by the three polypeptides (P1, P2 and P3) can express CD107a (shown in figure 5-1) and granzyme B (shown in figure 5-3) to different degrees. Statistical comparison analysis shows that compared with Tg mice immunized by HTNV-Gn/Gc CTL9 peptide epitope VV9(P1), the Tg mice immunized by the tandem polypeptide P3 have CD8 in splenocytes⁺CD107a⁺The frequency of T cells was elevated, but the statistical difference was not significant; CD8 in Tg mouse splenocytes after immunization with tandem polypeptide P3⁺CD107a⁺T cell frequency was significantly higher than that of the irrelevant peptide control group and PBS group (P)<0.05) (as shown in fig. 5-2). Compared with Tg mice immunized by HTNV-Gn/Gc CTL9 peptide epitope VV9(P1), the tandem polypeptides P2 and P3 are respectively immunized to obtain the Tg mice with CD8 in splenocytes⁺Granular enzyme B⁺The frequency of T cells is increased to different degrees, in particular to CD8 in splenocytes of mice immunized by the tandem polypeptide P3⁺Granular enzyme B⁺The frequency of T cells is obviously higher than that of HTNV-Gn/Gc CTL9 peptide epitope VV9(P1) immunized mice (P1)<0.01), also higher than the control group of unrelated peptide (VY9) and the PBS group immunized mice (P)<0.05) (as shown in fig. 5-4). The results indicate that the tandem polypeptide P3 can induce HTNV-Gn/Gc CTL9 peptide epitope VV9 specific CTL to generate a higher frequency of killing medium and a stronger killing effect in Tg mice than P1.

FIG. 5-1, FIG. 5-2, FIG. 5-3 and FIG. 5-4 show HLA-A2.1/K after intracellular cytokine staining for detecting the immunity of each epitope polypeptide^bThe result of frequency of epitope specific CTL secretion killing medium in spleen of transgenic mouse is shown in FIG. 5-1 and FIG. 5-3, wherein the result is shown by FCM dotted plot, and the first row is sequentially from left to right via P1 polypeptide (KSS-VMASLVWPV) and P2 polypeptideThe level of a killing medium secreted or expressed by the spleen cells of the mice after the immunization of the peptide (KSS-GFLCPEFPGSFRKKC-AAA-VMASLVWPV) and the P3 polypeptide (KSS-GFLCPEFPGSFRKKC-AAA-AKXVAAWTLKAAA-VMASLVWPV) is arranged in the second row from left to right, the level of the killing medium secreted or expressed by the spleen cells of the mice after the immunization of the irrelevant peptide (VPILLKALY), the HTNV inactivated vaccine and the PBS is arranged in sequence, the abscissa is the fluorescence intensity of CD8 expression, the ordinate of the graph in figure 5-1 is the fluorescence intensity of CD107a expression, and the ordinate of the graph in figure 5-3 is the fluorescence intensity of granzyme B expression. The upper right quadrant, CD8, in each scatter plot set⁺CD107a⁺Or CD8⁺Granular enzyme B⁺Cell frequency is a detection and statistical indicator. FIG. 5-2 and FIG. 5-4 are each mice of each immunization group on the abscissa, and FIG. 5-2 is CD8 expressing CD107a on the ordinate⁺T cell frequency, FIG. 5-4 ordinate CD8 secreting granzyme B⁺T cell frequency. Compared with the P1 polypeptide, the P3 polypeptide immune group can induce CD8⁺T cells secrete and express higher levels of granzyme B and CD107 a.

4.4 detection of in vitro induced polypeptide epitope specific effect CTL proliferation ability of different design group epitope tandem polypeptides

CFSE staining is used for detecting the in vitro induced polypeptide epitope specific effect CTL proliferation capacity of each group of epitope tandem polypeptides, and the specific operation is as follows:

tg mouse spleen cells (1X 10) after immunization of each set of polypeptide epitopes with 5. mu.M CSFE (Eugene Co.) labeling⁶) After washing, HTNV-Gn/Gc CTL9 peptide epitope VV9 is added for co-incubation, peptide stimulation is not added to serve as a negative control group, and PHA stimulation is used as a positive control group. After 5 days of culture, the cells were stained with CD3 and CD8 fluorescein-labeled mAb, fixed, and analyzed by flow cytometry for the position and peak pattern of CSFE positive peak, and the proliferation potency of HTNV-Gn/Gc CTL9 peptide epitope VV9 specific CTL on specific antigen peptide stimulation in splenocytes was examined.

Compared with an unstimulated group, the three peptides (P1, P2 and P3) are respectively immunized to Tg mouse spleen cell HTNV-Gn/Gc CTL9 peptide epitope VV9 specific to CD8⁺T cells all showed different degrees of proliferative capacity under polypeptide stimulation, wherein 2.2% of epitope-specific CD8 in splenocytes was induced by mice immunized with HLA-A x 02 restricted HTNV-Gn/Gc CTL9 peptide epitope VV9(P1)⁺T cell productionProliferation effect, 11.0 percent and 27.0 percent of epitope specificity CD8 are respectively found in spleen cells of Tg mice immunized by epitope tandem polypeptides P2 and P3⁺Significant proliferation of T cells occurred, and the proliferation frequency was also much higher than that of the irrelevant peptide (VY9) control group (3.9%), vaccine immunized group (2.7%) and PBS group (1.7%) immunized mice (fig. 6). This result suggests that the tandem polypeptides P2 and P3, particularly P3, induced higher frequency of proliferation of VV9 epitope-specific CTLs than VV9 epitope (P1) in Tg mice.

FIG. 6 shows HLA-A2.1/K after detection of each epitope tandem polypeptide immunization by CFSE proliferation experiment^bCTL proliferation capacity result chart in spleen of transgenic mouse. FCM histograms are used for CFSE fluorescence intensity on the abscissa and cell number on the ordinate. The first row is the mouse spleen cell proliferation capacity after being immunized by P2 polypeptide (KSS-GFLCPEFPGSFRKKC-AAA-VMASLVWPV), P3 polypeptide (KSS-GFLCPEFPGSFRKKC-AAA-AKXVAAWTLKAAA-VMASLVWPV) and P1 polypeptide (KSS-VMASLVWPV) from left to right, and the second row is the mouse spleen cell proliferation capacity after being immunized by irrelevant peptide (VPILLKALY), HTNV inactivated vaccine and PBS from left to right. No stimuli line in each group indicates the proliferation level of mouse splenocytes without any condition stimulation, Peptide or vaccine stimuli line indicates the proliferation level of mouse splenocytes under the condition of each group of corresponding polypeptide or inactivated vaccine stimulation, and PHA line indicates the proliferation level of mouse splenocytes under the condition of PHA stimulation. Wherein under the condition of no stimulation, the average fluorescence intensity of the CFSE labeled mouse splenocytes is strongest, and the proliferation and division of splenocytes are strongest under the condition of PHA non-specific stimulation, so the average fluorescence intensity of CFSE is obviously reduced. The spleen cells of mice in each group stimulated by the polypeptide are proliferated to different degrees, so that the average fluorescence intensity of CFSE is reduced to different degrees, particularly the P3 polypeptide immune group (27%) is most obvious and is higher than those of P1 polypeptide and P2 polypeptide immune groups.

By selecting HTNV B cell epitope GC15, general Th epitope PADRE and HTNV-Gn/Gc CTL9 peptide epitope VV9 limited by HLA-A x 02, epitope tandem polypeptide "B + CTL" (KSS-GFLCPEFPGSFRKKC-AAA-VMASLVWP) or "B + Th + CTL" (KSS-GFLCPEFPGSFRKKC-AAA-AKXVAAWTLKAAA-VMASLVWPV) is designed, compared with single CTL epitope of VV9, the epitope tandem polypeptide has the capacity of inducing epitope specific CTL to generate higher level IFN-gamma, killing medium with higher frequency and proliferation of specific CTL. The sequence and the construction scheme of the epitope tandem peptide can be used as a candidate vaccine molecule for designing and researching an HTNV polypeptide vaccine, can provide important theoretical and experimental basis for designing a novel HTNV epitope peptide vaccine, and has good development and application prospects in the field of HFRS specific immunotherapy.

Sequence listing

<110> the fourth military medical university of the Chinese people liberation army

<120> HTNV (human immunodeficiency Virus) antigen epitope linear tandem polypeptide, epitope peptide-compound tetramer and application

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Val Met Ala Ser Leu Val Trp Pro Val

1 5

<210> 2

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Ala Lys Cha Val Ala Ala Trp Thr Leu Lys Ala Ala Ala

1 5 10

<210> 3

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Gly Phe Leu Cys Pro Glu Phe Pro Gly Ser Phe Arg Lys Lys Cys

1 5 10 15

<210> 4

<211> 43

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Lys Ser Ser Gly Phe Leu Cys Pro Glu Phe Pro Gly Ser Phe Arg Lys

1 5 10 15

Lys Cys Ala Ala Ala Ala Lys Cha Val Ala Ala Trp Thr Leu Lys Ala

20 25 30

Ala Ala Val Met Ala Ser Leu Val Trp Pro Val

35 40

Claims

1. An HTNV epitope linear tandem polypeptide, comprising: the tandem polypeptide is a B + Th + CTL linear tandem polypeptide consisting of HTNV B cell epitope peptide GC15 with neutralization activity, universal Th epitope pan DR epitope peptide and HTNV-Gn/Gc CTL9 peptide epitope VV9 limited by HLA-A x 02, and the amino acid sequence of the tandem polypeptide is shown as SEQ ID NO: 4 is shown in the specification;

2. The use of the HTNV epitope linear tandem polypeptide of claim 1 in the preparation of a polypeptide vaccine against Hantaan virus.

3. Use according to claim 2, characterized in that: the polypeptide vaccine is induced CD8⁺Polypeptide vaccine for T cell proliferation.

4. Use according to claim 2, characterized in that: the polypeptide vaccine is induced CD8⁺T cells secrete IFN-gamma polypeptide vaccine.

5. Use according to claim 2, characterized in that: the polypeptide vaccine is induced CD8⁺T cell expresses polypeptide vaccine of CD107 a.

6. Use according to claim 2, characterized in that: the polypeptide vaccine is induced CD8⁺T cell expresses the polypeptide vaccine of granzyme B.

7. Use according to claim 2, characterized in that: the concentration of the HTNV epitope linear tandem polypeptide in the polypeptide vaccine is 1 mu M.

8. An HLA-A02/epitope peptide-complex tetramer, characterized by: comprises 4 monomers, each monomer is formed by assembling an HLA-A02 molecular heavy chain and beta 2 microglobulin in vitro; the carbon end of the heavy chain of the HLA-A02 molecule is connected with an activated biotin molecule; the HLA-A02 restricted HTNV-Gn/Gc CTL9 peptide epitope VV9 is bound to antigen peptide binding groove formed by heavy chain alpha 1 and alpha 2 structural domain of HLA-A02 molecule through anchoring amino acid; wherein, the amino acid sequence of the HLA-A x 02 restricted HTNV-Gn/Gc CTL9 peptide epitope VV9 is shown in SEQ ID no: 1 is shown in the specification;

9. Use of the HLA-a x 02/epitope peptide-complex tetramer of claim 8 for the preparation of a pharmaceutical agent for detecting T cell frequency specific to HTNV-Gn/Gc epitope peptide; the method is characterized in that: