阅读说明：本技术 甲型流感病毒pb1蛋白t细胞表位多肽片段及其应用 (Influenza A virus PB1 protein T cell epitope polypeptide segment and application thereof ) 是由 孟颂东 王子豪 张含 郑华国 鞠莹 于 2020-04-16 设计创作，主要内容包括：本发明公开了甲型流感病毒PB1蛋白T细胞表位多肽片段及其应用。本发明首先提供了氨基酸序列为SEQ ID No.2或SEQ ID No.10或SEQ ID No.11的甲型流感病毒PB1蛋白T细胞表位多肽。本发明进一步提供了上述多肽在制备流感疫苗中的应用。本发明筛选并鉴定出甲型流感病毒PB1蛋白T细胞表位多肽片段,在接种该抗原表位时,以gp96蛋白作为免疫佐剂,极大激发病毒特异性T细胞免疫反应,能有效引起针对不同亚型的流感病毒株的交叉免疫保护,这为将来研发具有交叉保护功能的新型流感病毒疫苗奠定基础。(The invention discloses an influenza A virus PB1 protein T cell epitope polypeptide segment and application thereof. The invention firstly provides an influenza A virus PB1 protein T cell epitope polypeptide with an amino acid sequence of SEQ ID No.2 or SEQ ID No.10 or SEQ ID No. 11. The invention further provides application of the polypeptide in preparation of influenza vaccines. The invention screens and identifies the T cell epitope polypeptide segment of the influenza A virus PB1 protein, and when the epitope is inoculated, gp96 protein is used as an immunologic adjuvant, thereby greatly stimulating the virus specificity T cell immunologic reaction, effectively causing the cross immune protection aiming at influenza virus strains of different subtypes, and laying a foundation for developing novel influenza virus vaccines with the cross protection function in the future.)

1. A polypeptide or a pharmaceutically acceptable salt thereof, characterized by: the polypeptide is any one of the following:

A1) a polypeptide having an amino acid sequence of SEQ ID No. 2;

A2) a polypeptide having an amino acid sequence of SEQ ID No. 10;

A3) polypeptide with an amino acid sequence of SEQ ID No. 11.

2. A DNA molecule encoding the polypeptide of claim 1.

3. A recombinant vector, expression cassette or recombinant microorganism comprising the DNA molecule of claim 2.

4. Derivatives of the polypeptide of claim 1, characterized in that: the derivative is any one of the following compounds:

B1) a linker obtained by linking an amino-terminal protecting group to the amino-terminus of the polypeptide and/or a carboxyl-terminal protecting group to the carboxyl-terminus of the polypeptide;

B2) amino acid residues at the amino-terminal and/or carboxy-terminal of the polypeptide capable of reacting with H-2K^DA bound polypeptide;

B3) the oligopeptide is connected at the amino terminal and/or the carboxyl terminal of the polypeptide and can be connected with H-2K^DA conjugated polypeptide.

5, C1) or C2):

C1) a multimer formed from the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof;

C2) a multimer formed from the derivative of claim 4.

6. Use of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, or the DNA molecule of claim 2, or the recombinant vector, expression cassette or recombinant microorganism of claim 3, or the derivative of claim 4, or the multimer of claim 5, in the preparation of an influenza vaccine.

7. Use of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, or the DNA molecule of claim 2, or the recombinant vector, expression cassette or recombinant microorganism of claim 3, or the derivative of claim 4, or the multimer of claim 5, for the preparation of a medicament for the treatment and/or prevention of a disease caused by an influenza virus.

8. An influenza vaccine, characterized by: the active ingredient of the influenza vaccine is the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, or the derivative of claim 4, or the multimer of claim 5.

9. The influenza vaccine of claim 8, wherein: the influenza vaccine also comprises an immunologic adjuvant, and the immunologic adjuvant is gp96 protein.

10. The influenza vaccine of claim 9, wherein: the amino acid sequence of the gp96 protein is shown in SEQ ID NO. 9.

Technical Field

The invention belongs to the field of biological pharmacy, and particularly relates to an influenza A PB1 protein T cell epitope polypeptide fragment and application thereof.

Background

Influenza viruses spread rapidly, are widely prevalent and highly pathogenic, and are one of the most serious public health problems in the world today. In the 20 th century and 30 th century, human beings began to develop cognition and vaccines for influenza virus, however, the influenza virus still remains an important pathogen threatening the health of human beings and animals until now. Influenza viruses can be divided into several subtypes according to their surface antigens, Hemagglutinin (HA) and Neuraminidase (NA). To date, a total of 18 HA subtypes (H1 to H18) and 11 NA subtypes (N1 to N11) have been discovered. Influenza viruses vary in two major ways during infection of different hosts: rearrangements of genomic segments (fragments) and variations in the amino acids of the virus-encoded proteins. Due to the existence of the variation mechanisms, a large number of variant strains exist in the influenza virus, and great difficulty is caused in the prevention and control of the influenza virus. Vaccine immunization remains, without doubt, the primary method of preventing influenza. However, the vaccines currently used only have a protective effect against specific strains of virus that are very similar to their HA (hemagglutinin) and NA (neuraminidase) antigens, and are poorly protected against other strains that are not. Thus, existing vaccines lose their protective efficacy once a new subtype or strain of influenza virus has emerged. Obviously, due to the characteristics of "antigen conversion" and "antigen drift" of influenza viruses, the conventional inactivated and split influenza vaccines developed mainly for each subtype have been unable to effectively prevent and defend against new influenza virus infections. The emergence of new subtypes and the tendency to become prevalent, and in severe cases even influenza pandemics occur. Therefore, the development of influenza vaccines with cross-protection and long-term protection against multiple subtypes of influenza viruses has become a new focus of research.

The existing influenza vaccine immunization mainly generates antibodies aiming at virus HA, and HA is easy to be mutated, which is the root cause that the existing influenza vaccine can not effectively generate cross protection. Influenza virus HA antibody levels are considered a direct factor in the development of immune protection, however there is still controversy over their immune protective effect, with a 1:40 HAI antibody titer able to reduce 50% of infections in adults, but in children, a 1:110 HAI antibody titer able to reduce 50% of infections, whereas a 1:40 HAI antibody titer is only able to produce 20% protection. A large number of researches show that T cell immunity plays an important role in controlling influenza virus infection, and the immune protection effect is closely related to the T cell immunity level rather than the neutralizing antibody level. Influenza virus T cell immunity is mainly produced by internal proteins, such as M1, PB1, NP, etc., which contain T cell epitopes that are conserved in a variety of influenza a viruses relative to the surface proteins HA and NA. Research shows that a large number of influenza virus specific T cells exist in an organism due to periodic epidemics of influenza viruses A, H1N1, H3N2, B and the like, and the specific T cells can widely cross recognize T cell epitopes even derived from H5N1 viruses. For example, it has been demonstrated in a mouse infection model that T cell immune responses generated by infection with different subtypes of influenza viruses can cross-protect against other subtypes of influenza viruses. By infecting mice with the H1N1 virus and reinfection with the H3N2 virus, the T cell immune response in the respiratory tissues provides effective cross-protection against the invasion of the H7N7 virus, and the cross-response is initiated and effected within 3 days after the virus infection. In a ferret influenza virus infection model, H3N2(A/Brisbane/010/2007) virus infection can generate cross protection for H5N1(A/Indonesia/5/05) virus reinfection, and the cross protection effect has obvious correlation with influenza virus specific T cell frequency in PBMCs. Studies in avian infection models, re-infection with highly pathogenic H5N1 influenza virus 3-70 days after H9N2 influenza virus infection, allowed survival of chickens infected with H5N1 despite the absence of neutralizing antibodies that cross-react with H5N1 virus. Further studies have shown that CD8+ T cells produced in chickens infected with H9N2 virus when injected into non-immunized chickens effectively protect against infection with lethal doses of H5N1 virus, whereas injection of CD4+ T cells does not produce effective protection. In primate models, after seasonal H1N1(a/Kawasaki/173/2001) influenza virus infection, the specific T cells produced were able to exert an effect and clear the virus within 2 days of re-infection with 2009H1N1(a/California/04/2009) influenza virus, at which time the neutralizing antibodies had not been effectively activated. Studies in these animal infection models have shown well that T cell immune-mediated cross reactions can effectively protect animals from invasion by exposure to different influenza viruses again.

Also, the generation of specific memory T cells plays a crucial role in long-lasting cross-protection against influenza viruses. For example, in 2009H1N1 influenza a pandemic, more than 300 volunteers were followed for health and their immune systems were heavily analyzed for responses to this novel influenza virus. It was found that specific CD8+ t (ctl) cells were generally present in higher blood levels in volunteers who did not present significant symptoms. Another recent study showed that among H7N9 critically infected patients, those who can be rapidly recovered and discharged within 2 to 3 weeks will develop high levels of H7N 9-specific CD8+ T cell responses early in the infection (about 1 to 2 weeks); patients who are discharged and have slower recovery more than 30 days need to have better specific T cell response at a later stage (about 4 weeks); while the patients who finally die due to infection only have low level of H7N9 specific immune response in the organism, and the response level of CD8+ T cells is lower, the research shows that in H7N9 severe infectors, memory CD8+ T cells are directly involved in the processes of patient recovery or death, and the response level is closely related to infection protection. The above shows that cellular immunity, especially T cell immunity, is an important defense line for the body against invading intracellular influenza viruses (including novel H1N1, H5N1, H7N9 viruses, etc.), virus-specific T cell-mediated immune response is crucial for controlling host susceptibility to influenza viruses and severity of infectious diseases, course outcome, mortality, especially when antibody reaction is unable to effectively control and neutralize viruses, T cell immunity plays an important role in immune protection, and T cell receptors expressed on cell surfaces recognize antigen polypeptides and major histocompatibility molecules complexes of infected cells, thereby recognizing and eliminating infected cells. In influenza pandemic outbreaks due to antigen drift or worldwide outbreaks due to the emergence of novel influenza viruses, antibody-mediated immune responses often do not effectively control the virus, and in these cases, T cells targeting relatively conserved proteins inside the virus can exert immune cross-protection, and in fact, most of currently known influenza virus CTL epitopes are conserved, which is crucial for immune cross-protection. Clinical researches show that cellular immunity is crucial to preventing and controlling influenza virus infection, improving the immunogenicity of cross protection of influenza vaccines, enhancing the cross protection capability of traditional vaccines on antigen-drifting influenza viruses, enhancing the immune protection of influenza vaccines on special crowds, reducing the dosage of the vaccines, shortening the immune response time after the immunization of the vaccines, enhancing the specificity and the memory of the immune response and the like.

The heat shock protein gp96 belongs to a member of the HSP90 family and is the only natural adjuvant molecule used for clinical self-treatment of tumors at present. Through analyzing clinical samples and immunological experiments, gp96 is proved to be directly combined with polypeptide epitope derived from virus, gp96 is found to be combined with MHC class I molecule restricted antigen peptide in hepatitis B virus infection, the combined molecule region is preliminarily clarified, the protein N end of gp96 is a part combined with polypeptide, and the combined antigen is presented to MHC class I molecule for CTL recognition through antigen presentation; simultaneously, gp96 interacts with TLR-2 (Toll-like receptor) and TLR-4 to mediate natural immunity, and further promotes the activation and proliferation of specific T cells. The researches lay a foundation for understanding an immune mechanism of gp96 activated T cells, reveal an immune amplification mechanism of gp96 through antigen presentation, and theoretically prove the feasibility of gp96 as a general T cell vaccine adjuvant. Researches find that the epitope in the current inactivated vaccine or the lytic vaccine of the influenza virus can only activate stronger antibody immunity and can not effectively activate T cell immunity, and the response level of vaccine specific CD8+ T cells (namely CTL and cytotoxic T cells) is increased by dozens of times after the heat shock protein gp96 is added as an adjuvant. The influenza virus epitope taking gp96 as adjuvant has cross immune protection capability, while a simple vaccine cannot effectively cause cross immune protection against different influenza virus strains, and the cross protection capability is closely related to the conserved antigen-specific CTL cell response in the influenza virus. The heat shock protein gp96, an important protein involved in immune regulation in the body, can help exogenous proteins or polypeptides to be presented by MHC class I molecules, and activate a strong CD8+ T cell immune response.

Epitopes (epitopes) are the special chemical groups in the antigenic molecule that determine the specificity of the antigen and are also the basic unit for the specific binding of TCR/BCR and antibodies, and play a critical role in the antigen. CTL cell epitopes can mediate the functions of cytotoxic lymphocytes and can start inflammatory factors, and play an important role in the virus elimination process. The vaccine is developed according to the influenza virus epitope, so that the protective antigen of the influenza virus can be effectively utilized, the specificity of immune response is enhanced, and the aim of preventing a plurality of strains can be achieved by selecting the antigen epitope with cross protection. The proteins reported to induce T cell immune response in vivo by influenza virus are mainly the internal proteins, M, NP, PB1, etc. At present, researchers test reported influenza virus T cell epitopes, and finally, a universal influenza vaccine aiming at the United states is designed by using 6 CD8+ T cell epitopes and 3 CD4+ T cell epitopes to cover 95% of influenza viruses in the United states. And 8 CD8+ T cell epitopes and 3 CD4+ T cell epitopes to design a universal influenza vaccine for the global scope, covering 88% of known influenza viruses globally, so as to prevent the possible outbreak of global large influenza in the future. Additional studies have shown that some epitopes induce strong activity but are not able to protect against viral challenge. Therefore, although epitope-based vaccines have many advantages, the mechanism and strength of the function of epitopes, the restriction of epitopes, the mechanism of coordination between epitopes and epitopes are still not completely understood, and even some identified epitopes are controversial. However, the epitope-based vaccine provides a brand new thought for the development of influenza vaccines, and is worthy of further research.

Therefore, screening and identifying the corresponding influenza functional epitope has important theoretical and practical values for deeply disclosing the infection and immune mechanism of the influenza virus, the cognition of the influenza functional epitope and the research of the epitope vaccine.

Disclosure of Invention

The technical problem to be solved by the invention is how to obtain the universal influenza vaccine with the cross protection function.

In order to solve the technical problems, the invention firstly provides an influenza A PB1 protein T cell epitope polypeptide or a medicinal salt thereof.

The T cell epitope polypeptide of the influenza A virus PB1 protein is shown in any one of the following:

A1) a polypeptide having an amino acid sequence of SEQ ID No. 2;

A2) a polypeptide having an amino acid sequence of SEQ ID No. 10;

A3) polypeptide with an amino acid sequence of SEQ ID No. 11.

Wherein the polypeptide is nonapeptide.

The polypeptide can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

The DNA molecule for encoding the T cell epitope polypeptide of the influenza A virus PB1 protein is also in the protection scope of the invention.

Recombinant vectors, expression cassettes or recombinant microorganisms comprising the above-described DNA molecules are within the scope of the present invention.

The invention further provides derivatives of the above polypeptides.

The derivative is shown as any one of the following:

B1) a linker obtained by linking an amino-terminal protecting group to the amino-terminus of the polypeptide and/or a carboxyl-terminal protecting group to the carboxyl-terminus of the polypeptide;

B2) amino acid residues at the amino-terminal and/or carboxy-terminal of the polypeptide capable of reacting with H-2K^DA molecularly bound polypeptide;

B3) the oligopeptide is connected at the amino terminal and/or the carboxyl terminal of the polypeptide and can be connected with H-2K^DA molecularly bound polypeptide.

In the above derivatives, the H-2K^DThe molecule consists of a heavy chain and a light chain, wherein the coding sequence of the heavy chain is shown as SEQ ID NO.4, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 5; the coding sequence of the light chain is shown as SEQ ID NO.6, and the amino acid sequence thereof is shown as SEQ ID NO. 7.

In the polypeptide or its pharmaceutically acceptable salt, or its derivative, the amino terminal of the polypeptide of the present invention may contain an amino terminal protecting group, wherein the amino terminal protecting group may be any one of acetyl, amino, maleoyl, succinyl, tert-butoxycarbonyl, or benzyloxy, or other hydrophobic group or macromolecular carrier group; the carboxyl end of the polypeptide of the invention may contain a carboxyl end protecting group, which may be any of amino, amido, carboxyl, or tert-butyloxycarbonyl or other hydrophobic group or macromolecular carrier group.

C1) Or C2) are also within the scope of the invention:

C1) a multimer formed from the above polypeptide or a pharmaceutically acceptable salt thereof;

C2) multimers formed from the above derivatives.

In order to solve the technical problems, the invention also provides application of the influenza A virus PB1 protein T cell epitope polypeptide or the medicinal salt thereof, the DNA molecule, the recombinant vector, the expression cassette or the recombinant microorganism, the derivative or the polymer in preparing an influenza vaccine.

The application of the influenza A virus PB1 protein T cell epitope polypeptide or the medicinal salt thereof, the DNA molecule, the recombinant vector, the expression cassette or the recombinant microorganism, the derivative or the polymer in preparing the medicament for treating or preventing the diseases caused by the influenza virus is also within the protection scope of the invention.

In the above application, the influenza virus is influenza a virus, specifically PR8 virus, WSN virus, H3N2 virus and/or H7N9 virus.

In order to solve the technical problems, the invention further provides an influenza vaccine.

The active component of the influenza vaccine provided by the invention is the influenza A virus PB1 protein T cell epitope polypeptide or the medicinal salt thereof, the derivative or the polymer.

The influenza vaccine further comprises an immunological adjuvant.

In the influenza vaccine, the immunologic adjuvant is gp96 protein, and the amino acid sequence of the gp96 protein is shown as SEQ ID No. 9.

In the influenza vaccine, the mass ratio of the gp96 protein to the influenza a virus PB1 protein T cell epitope polypeptide can be 1: (0.5-10), specifically 3: 5.

The gp96 protein can be artificially synthesized, or can be obtained by synthesizing the coding gene (the sequence of the coding gene is shown in SEQ ID NO. 8) and then carrying out biological expression.

The gp96 protein is obtained by the secretory expression of insect cells infected by insect baculovirus, and specifically comprises the following steps:

1) construction of recombinant plasmid pFastBac containing gp96 Gene shown in SEQ ID NO.8^TM1-gp 96: the recombinant plasmid pFastBac^TM1-gp96 is pFastBac^TM1 between the EcoRI and XbaI cleavage sites and the gp96 gene fragment shown as SEQ ID NO.8, and the pFastBac^TM1 human gp96 gene expression vector obtained by keeping other sequences unchanged;

2) will pFastBac^TM1-gp96 is transfected into Sf9 insect cells, and Sf9 insect cells are induced to express to obtain gp96 protein.

The invention screens and identifies the T cell epitope polypeptide segment of the influenza A virus PB1 protein, and when the epitope is inoculated, gp96 protein is used as an immunologic adjuvant, thereby greatly stimulating the virus specificity T cell immunologic reaction, effectively causing the cross immune protection aiming at influenza virus strains of different subtypes, and laying a foundation for developing novel influenza virus vaccines with the cross protection function in the future.

Drawings

FIG. 1 shows PB181-89 polypeptide and H-2K^DResults of in vitro refolding (refolding) of complexes composed of heavy chain inclusion body protein and light chain β 2m inclusion body protein.

FIG. 2 shows the results of western blotting.

FIG. 3 shows the results of ELISPOT detection of polypeptide-specific CTL immune responses in splenocytes of polypeptide-immunized BALB/c mice.

FIG. 4 shows the body weight change of a polypeptide immunized BALB/c mouse subjected to a toxicity challenge experiment with PR8 virus, as compared with a negative control group.

FIG. 5 shows the survival rate of a polypeptide immunized BALB/c mouse subjected to a challenge test with PR8 virus compared with that of a negative control group.

FIG. 6 shows the body weight change of a polypeptide immunized BALB/c mouse subjected to a WSN virus challenge test as compared with a negative control group.

FIG. 7 shows the survival rate of the polypeptide immunized BALB/c mice after performing the virus challenge experiment of WSN virus compared with the negative control group.

FIG. 8 shows the weight change of a negative control group compared with a H3N2 virus challenge experiment performed after BALB/c mice are immunized with the polypeptide.

FIG. 9 shows the survival rate of the polypeptide immunized BALB/c mice after challenge experiment with H3N2 virus compared to the negative control group.

FIG. 10 shows the weight change of a negative control group compared with a H7N9 virus challenge experiment performed after BALB/c mice are immunized with the polypeptide.

FIG. 11 shows the survival rate of the polypeptide immunized BALB/c mice after challenge experiment with H7N9 virus compared to the negative control group.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

PBS buffer: 8g NaCl, 0.2g KCl, 3.625g Na₂HPO₄·12H₂O、0.24g KH₂PO₄Water was added to 1L to adjust pH 7.3.

Example 1 preparation of PB1 derived nonapeptide

The amino acid sequence of influenza A virus (PR8) PB1 is shown as SEQ ID NO.1, and the amino acid sequence is used as a sequence source of a synthetic polypeptide segment PB 181-89. Artificially synthesizing a polypeptide fragment PB181-89, randomly constructing point mutations (corresponding mutant polypeptide fragments a and b) except the PB181-89 anchored amino acids, and carrying out an experiment by using a negative control hepatitis B virus HBc82-90 peptide (polypeptide fragment HBc82-90 for short).

The amino acid sequences of these four polypeptides are shown below (from left to right in the nitrogen-to-carbon direction):

polypeptide fragment PB181-89 (SEQ ID NO. 2): GYAQTDCVL

Polypeptide fragment HBc82-90(SEQ ID NO. 3): RELVVSYVN

Mutant polypeptide fragment a (SEQ ID NO. 10): GYAQADCVL

Mutant polypeptide fragment b (SEQ ID NO. 11): GYAQTDAVL

The chemical synthesis of the polypeptide is realized by conventional amino acid condensation reaction, is synthesized by Gill Biochemical (Shanghai) Co., Ltd, and has purity of over 95% by HPLC detection.

Example 2 PB181-89 polypeptide fragment and H-2K^DBinding force of molecules

In-vitro refolding (refolding) experiment detection of polypeptide and H-2K^DBinding capacity of molecules

In the in vitro refolding experiments, if the polypeptide can react with H-2K^DThe molecules are combined, then the polypeptide, H-2K^DAnd beta 2 microglobulin (beta 2m) can form stable polypeptide-H-2K^D-beta 2m ternary complex, polypeptide-H-2K appearing in molecular sieve chromatogram^D- β 2m complex protein peak. H-2K^DThe molecule consists of a heavy chain and a light chain, wherein the coding sequence of the heavy chain is shown as SEQ ID NO.4, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 5; the coding sequence of the light chain is shown as SEQ ID NO.6, and the amino acid sequence thereof is shown as SEQ ID NO. 7.

(1) H-2K^DConstruction of heavy chain extracellular region expression plasmid

Will code for H-2K^DDNA of heavy chain extracellular region (the Genbank number is KF831066.1 from the 64 th-1080 th nucleotide at the 5' end) is inserted between the restriction enzyme HindIII restriction sites of pET-28 to obtain H-2K^DThe heavy chain extracellular region expression plasmid is named as H-2K^D-a HC plasmid.

Construction of mouse light chain beta 2m expression plasmid

DNA encoding murine β 2M (Genbank No. M84365.1 from nucleotide 61-357 from the 5' end) was inserted between the restriction sites of the restriction enzyme HindIII of pET-28 to obtain a light chain recombinant plasmid, which was designated as light chain β 2M expression plasmid.

(III) prokaryotic extraction of H-2K^DHeavy and light chain proteins

1. Reacting H-2K^Dthe-HC plasmid was transferred into BL21(DE3) competent cells (purchased from Tiangen Biochemical technology Co., Ltd., product No. CB105-02), and a single colony was picked from the plate and inoculated into 5ml of LB medium (tryptone 10g, yeast extract 5g, sodium chloride 10g, water to 1L) containing 100mg/ml ampicillin, activated in a small amount, and cultured for 10-12 hours. Then, the cells were inoculated into 2L of LB medium (37 ℃ C., 200 rpm) at an inoculum size of 1% and cultured in bulk (OD 0.4-0.6), IPTG (final concentration of 1mM) was added under aseptic conditions, and the cells were collected by centrifugation at 37 ℃ C. for 4-6 hours at 4 ℃ C., 7000rpm for 10 min.

2. The thallus obtained in the step 1 is resuspended by PBS, and ultrasonication is carried out under ice bath conditions (ultrasonication for 6s, interval of 12s, 99 times, 250W, multiple cycles) until the thallus suspension is transparent. Centrifuging at 12000rpm for 20min at 4 deg.C, collecting the bacteria liquid after ultrasonic treatment, and collecting white compact block in the precipitate as inclusion body.

3. Scraping thallus residue on the inclusion body obtained in the step 2 by using a glass rod, suspending the inclusion body by using an inclusion body cleaning solution (washing buffer), further purifying protein by using an ultrasonic cracking method (ultrasonic 4s, clearance 10s, 40 times and 250w), centrifuging at 4 ℃ and 12000rpm for 20min, and collecting precipitate. This step was repeated three times. The inclusion body pellet was then suspended in an inclusion body weight suspension (resuspension buffer) and sonicated once and centrifuged at 12000rpm for 20min at 4 ℃ to collect the inclusion bodies. Note: after each ultrasonic lysis and centrifugation, the bacterial residues on the surface need to be scraped off as much as possible by a glass rod.

4. Inverting the inclusion body obtained in the step 3, draining, weighing, dissolving with inclusion body dissolving solution (Dissolution Buffer) according to the proportion of 30mg/ml, and stirring and dissolving at 4 ℃ overnight. Centrifuging at 12000rpm at 4 deg.C for 20min to collect supernatant to obtain H-2K^DHeavy chain extracellular domain inclusion body protein solution (H-2K for short)^DHeavy chain inclusion body protein solution), subpackaging and freezing at-20 ℃.

Reacting H-2K^D-HC plasmidReplacing the inclusion body with a light chain beta 2m expression plasmid, and obtaining an inclusion body protein solution of the light chain beta 2m without changing other steps.

Wherein, the inclusion body cleaning solution (washing buffer): the solvent was water, the solute was 0.5% Triton-100, 50mM Tris pH8.0, 300mM NaCl, 10mM EDTA, 10mM DTT (now available);

inclusion body weight suspension (resuspension buffer): the solvent is water, the solute is 50mM Tris pH8.0, 100mM NaCl, 10mM EDTA, 10mM DTT (currently added);

inclusion body lysis Buffer: the solvent was water, the solute was 6M Gua-HCl (or 8M Urea), 10% glycerol, 50mM Tris pH8.0, 100mM NaCl, 10mM EDTA, 10mM DTT (now available).

(IV) in vitro renaturation of Inclusion bodies

1. The renaturation solution (refolding buffer) is filled into a beaker and placed on a magnetic stirrer, and the stirring speed of the rotor is preferably 1s per rotor revolution. Replacing the needle of a 5ml or 10ml syringe with the needle of a 1ml syringe, fixing the syringe after replacing the combination on a beaker, and sequentially adding a protein solution into the syringe to enable the protein solution to slowly drop by drop into a refolding buffer, wherein the specific sequence is as follows: first light chain beta 2m inclusion body protein solution, then PB181-89 polypeptide solution, again H-2K^DAdding heavy chain inclusion body protein solution with H-2K^DHeavy chain inclusion body protein: light chain β 2m inclusion body protein: PB181-89 polypeptide 1: 1: 3 in a molar ratio. H-2K^DThe heavy chain inclusion body protein is added for 3 times, the time interval of each addition is 8-10h, the PB181-89 polypeptide can be dissolved in 100-200 microliter dimethyl sulfoxide to obtain a PB181-89 polypeptide solution for one-time injection.

Wherein, refolding buffer: the solvent is water, the solute is 100mM Tris pH8.0, 400mM L-Arg HCl, 2mM EDTA, 5mM GSH (reducing glutathione, added after precooling solution), 0.5mM GSSG (oxidizing glutathione, added after precooling solution), 0.5mM PMSF (optional), and the reducing glutathione and the oxidizing glutathione are added after precooling solution.

2. Concentrating the renaturation solution after renaturation by using a concentration cup (optimally 12000rpm before adding the concentration cup, centrifuging for 20min at 4 ℃, removing precipitates as much as possible to reduce the possibility of blocking a concentration membrane by the precipitates), changing the solution to a molecular sieve buffer solution (20mM Tris, 50mM NaCl, pH8.0), changing the solution to a concentration tube after the volume of the solution is finally less than 50ml, continuously concentrating to be within 5ml, centrifuging for 10min at 12000rpm at 4 ℃, and taking a supernatant to prepare for loading. The prepared renaturation concentrate was injected into AKTAFPLC, and the renaturation effect was examined based on the results of the molecular sieve chromatography of superdex 20010/300 GL (GE Co., Ltd., product No. 17-5175-01). In the molecular sieve chromatography process, samples of each protein peak are collected and identified by denaturing polyacrylamide gel electrophoresis.

The renaturation results are shown in figure 1, and a clear complex protein peak appears at 85ml of a target position, which indicates that PB181-89 polypeptide and H-2K^DThe molecules have significant binding capacity.

Example 3 expression of Heat shock protein gp96 by an insect baculovirus expression System

1, pFastBac^TMConstruction of 1-gp96 plasmid

1. Design and synthesis of gp96 primer: according to the sequence (the sequence number is NM-003299, the gene sequence is shown as SEQ ID NO.8, and the coding amino acid sequence is shown as SEQ ID NO. 9) of human gp96 gene in GenBank as a template, EcoRI enzyme cutting sites are added at the 5 'end of gp96 gene, and XbaI enzyme cutting sites are added at the 3' end. The forward primer sequence is: 5' -GGAATTCATGGGCAGCAGCCATCAT-3' (EcoRI cleavage sites are underlined); the reverse primer sequence is as follows: 5' -GCTCTAGACTATTACAATTCATCTTTTTC-3' (the XbaI restriction site is underlined), Shanghai Bioengineering technology service company is entrusted to synthesize the primer, and the sequence of the primer is verified to be correct by sequencing.

2. Extracting mRNA of human liver cancer cell HepG2, and synthesizing cDNA through reverse transcription.

3. And (3) carrying out PCR amplification by using the cDNA in the step 2 as a template and using the forward primer and the reverse primer in the step 1 to obtain a PCR product of gp 96.

4. The PCR product of gp96 was digested with EcoRI and XbaI, and the digestion product was recovered.

5. Double digestion of pFastBac with EcoRI and XbaI^TM1 No-load plasmid (purchased from Invitrogen, cat #)10359-.

6. And (5) connecting the enzyme digestion product in the step (4) with the carrier skeleton in the step (5) to obtain a connection product.

7. The ligation product obtained in step 6 was transformed into E.coli DH10Bac competent cells (product number CL108-01, from Probiotics of Beijing Heizhao, Ltd.), and plasmids of recombinant E.coli were obtained by site-directed transfer. The forward primer sequence of the positive clone is verified by PCR amplification and is as follows: 5'-CCCAGTCACGACGTTGTAAAACG-3', the reverse primer sequence is: 5'-AGCGGATAACAATTTCACACAGG-3' are provided. If PCR produces a target band with a size of 4.7-5kb, it is a positive recombinant plasmid, which is then sequenced, and the correctly sequenced plasmid is named pFastBac^TM1-gp 96. The general formula of pFastBac^TM1 between the EcoRI and XbaI cleavage sites and the gp96 gene fragment shown as SEQ ID NO.8, and the pFastBac^TM1 human gp96 gene expression vector obtained by not changing other sequences.

Secondly, expressing gp96 protein by insect cells

Extraction of pFastBac^TM1-gp96, which was transfected into Sf9 insect cells using Cellfectin II reagent (available from Life technologies, cat # 10362-100). Sf9 insect cells transfected with plasmids were cultured for 72h, and the P1 virus was obtained by collecting cell supernatants, which showed that a generation of recombinant baculovirus (P1) had been released into the medium by cytopathic conditions. Adding appropriate amount of P1 poison into Sf9 monolayer (1 × 10)⁶cells/mL), culturing at 27 deg.C for 72h, centrifuging at 4000rpm for 5min, and collecting the supernatant to obtain the second generation virus (P2 virus). Adding appropriate amount of P2 toxin to 100ml of Sf9 (1.6X 10)⁶cells/mL) at 27 ℃ and 120rpm/min for 72h, and amplifying to obtain the third generation virus (P3 virus). The P3 toxin was subjected to a western blotting hybridization using (sc-393402) GRP 94Antibody (H-10) (available from Santa Cruz Biotechnology) and the results are shown in FIG. 2, indicating that the gp96 protein has been expressed in Sf9 cells.

In fresh Sf9 cells (1.5X 10)⁶cell/mL, 300mL) was added with an appropriate amount of P3 poison at 27 ℃,100 and 120rpm/min in the insert-XPRESS^TM Protein-free Insect Cells medium with L-Glutamine(CatalogNo. 12-730Q) medium, suspension culture at 27 ℃ and 100-. After 72 hours of infection, the suspension was centrifuged at 7000rpm for 20 minutes to give a clear supernatant which was filtered through a 0.22mm membrane filter and purified by HiTrap-Q Sepharose ion exchange chromatography and Superdex 20010/300 GL molecular sieves to give the relatively pure gp96 protein. The protein is identified by denaturing polyacrylamide gel electrophoresis. The gp96 protein collected above was exchanged with PBS buffer, concentrated, protein concentration was determined by BCA method, and finally the protein was dispensed and stored at-80 ℃.

Wherein the HiTrap-Q Sepharose ion exchange chromatography comprises the following steps: (a) loading the filtered supernatant into a chromatographic column at the flow rate of 1 ml/min; (b) 5ml of 200mM NaCl pH7.5 PBS (solvent is water, solute is 200mM NaCl, 0.2g/L KCl, 3.625g/L Na)₂HPO₄·12H₂O、0.24g/L KH₂PO₄) Washing the chromatographic column of step (a) at a flow rate of 1 ml/min; (c) 10ml of 300mM NaCl pH7.5 PBS (solvent is water, solute is 300mM NaCl, 0.2g/L KCl, 3.625g/L Na)₂HPO₄·12H₂O、0.24g/L KH₂PO₄) Washing the chromatographic column of step (b) at a flow rate of 1 ml/min; (d) 3ml of PBS (solvent is water, solute is 600mM NaCl, 0.2g/L KCl, 3.625g/L Na) with NaCl concentration of 600mM and pH7.5₂HPO₄·12H₂O、0.24g/L KH₂PO₄) Washing the chromatographic column in the step (c) at the flow rate of 1ml/min to obtain the eluent, namely the extract containing the gp96 protein.

The Superdex 20010/300 GL molecular sieve chromatography comprises the following steps: (a) concentrating the gp96 protein extract obtained by HiTrap-Q Sepharose ion exchange chromatography to 1ml of PBS solution by passing through 50Kd ultrafiltration tube (Merck Millipore, cat # UFC 905096); (b) loading (a) onto a chromatography column through a loading loop at a flow rate of 0.25 ml/min; (c) the column was washed with PBS buffer at a flow rate of 0.25ml/min to recover gp96 protein.

Example 4 stimulation of BALB/c transgenic mouse specific CTL (cytotoxic T cell) production by Polypeptides

Gp96 used for animal immunization in this example was gp96 protein expressed by the insect cells prepared in example 3. The polypeptide used for immunization of animals is the polypeptide of example 1: the animal immunity polypeptide of the experimental group is a polypeptide fragment PB181-89, and the amino acid sequence of the polypeptide fragment is shown as SEQ ID NO. 2; the animal immunity polypeptide of the negative control group is hepatitis B virus HBc82-90 peptide (namely polypeptide fragment HBc82-90), and the amino acid sequence is shown as SEQ ID NO. 3.

One, group immunization

BALB/c mice (purchased from Jackson laboratory) at 4-6 weeks of age were divided into groups of 10 mice each weighing 18-20g and immunized separately. Before immunization, gp96 and a polypeptide (PB181-89 or HBc82-90) for immunization are assembled in an assembly solution to obtain an immunogen solution. The solvent of the assembly solution is water, and the solutes and their concentrations are as follows: NaCl 8g/L, KCl 0.2g/L, KH₂PO₄ 0.24g/L,Na₂HPO₄·12H₂O3.63 g/L; assembling conditions are as follows: the mixture was allowed to stand at 55 ℃ for 10min and at room temperature for 30 min. Obtaining 4 immunogen solutions according to the method, wherein solutes in the first immunogen solution are gp96 and HBc82-90, and the mass ratio of HBc82-90 to gp96 is 5: 3; in the second immunogen solution, solutes are gp96 and PB181-89, and the mass ratio of PB181-89 to gp96 is 5: 3; in the third immunogen solution, solutes are gp96 and PB181-89, and the mass ratio of PB181-89 to gp96 is 1: 2; in the fourth immunogen solution, solutes are gp96 and PB181-89, and the mass ratio of PB181-89 to gp96 is 10: 1.

The immunization mode is subcutaneous injection, the first immunization time is the first week of the experiment, the second immunization time is the second week of the experiment, and the third immunization time is the fourth week of the experiment. Each mouse had the same volume of immunogen solution per injection, 0.2 mL.

First group (negative control group): the first immunity polypeptide fragment HBc82-90(50 microgram/piece), gp96(30 microgram/piece); the second immunization polypeptide fragment HBc82-90(50 microgram/piece), gp96(30 microgram/piece); third immunity polypeptide fragment HBc82-90(50 microgram/piece), gp96(30 microgram/piece);

second group (experimental group): the first immunity polypeptide PB181-89 (50 microgram/one), gp96(30 microgram/one); the second immunity polypeptide PB181-89 (50 microgram/piece), gp96(30 microgram/piece); third immunity polypeptide PB181-89 (50 microgram/one), gp96(30 microgram/one);

third group (experimental group): the first immunity polypeptide PB181-89 (15 microgram/one), gp96(30 microgram/one); the second immunity polypeptide PB181-89 (15 microgram/one), gp96(30 microgram/one); third immunity polypeptide PB181-89 (15 microgram/one), gp96(30 microgram/one);

fourth group (experimental group): the first immunity polypeptide PB181-89(300 microgram/piece), gp96(30 microgram/piece); the second immunity polypeptide PB181-89(300 microgram/piece), gp96(30 microgram/piece); the third immunity polypeptide PB181-89(300 microgram/piece), gp96(30 microgram/piece);

fifth group (epitope-only immunization group): first immunity polypeptide PB181-89 (50 microgram/mouse); second immunity polypeptide PB181-89 (50 microgram/mouse); third immunopolypeptide PB181-89 (50. mu.g/mouse);

sixth panel (gp 96 immunised alone): first gp96(30 microgram/stick); second immunization gp96(30 microgram/mouse); gp96 was immunized a third time (30. mu.g/mouse).

Analysis of immune-related factors

After the immunization, splenocytes from mice were taken for enzyme-linked immunospot assay (ELISPOT assay kit from BD, product No. 551083, protocol see kit manufacturer's instructions), and the results are shown in fig. 3: mouse splenocyte ELISPOT results were compared between treatment groups as follows, second group: PB181-89 VS HBc82-90 is 373.10 + -40.66 VS 8.47 + -12.01, and P is less than 0.001; third group: PB181-89 VS HBc82-90 is 257.67 + -74.22 VS 8.47 + -12.01, and P is less than 0.001; and a fourth group: PB181-89 VS HBc82-90 is 359.57 + -42.08 VS 8.47 + -12.01, and P is less than 0.001; (vii) a fifth group of PB181-89 VS HBc 82-90: 46.90 ± 44.07VS 8.47 ± 12.01, P < 0.05: a sixth group: gp96 VS HBc82-90 equals 27.7 + -38.32 VS 8.47 + -12.01, P > 0.05. The negative control group (the first group) and the gp96 immune group alone (the sixth group) have few T cells with specific IFN-gamma secretion, and the experimental group (the first group, the second group and the third group) and the epitope immune group alone (the fifth group) can cause strong specific IFN-gamma secretion, which indicates that the polypeptide fragment PB181-89 can stimulate strong cytotoxic T cell immune response in mice, and the gp96 and the negative control polypeptide fragment HBc82-90 can not activate the cytotoxic T cell immune response.

Example 5 challenge following polypeptide immunization of BALB/c mice

Gp96 used for animal immunization in this example was gp96 protein expressed by the insect cells prepared in example 3, and the polypeptide used for animal immunization was the polypeptide of example 1: the animal immunity polypeptide of the PB1 polypeptide group is a polypeptide fragment PB181-89, and the amino acid sequence of the polypeptide fragment is shown as SEQ ID NO. 2; the animal immunity polypeptide of the mutant polypeptide a group is a mutant polypeptide fragment a, and the amino acid sequence of the fragment is shown in SEQ ID NO. 10; the animal immunity polypeptide of the mutant polypeptide group b is a mutant polypeptide fragment b, and the amino acid sequence of the mutant polypeptide fragment b is shown in SEQ ID NO. 11; the animal immunity polypeptide of the negative control group is hepatitis B virus HBc82-90 peptide (namely polypeptide fragment HBc82-90), and the amino acid sequence is shown as SEQ ID NO. 3.

One, group immunization

BALB/c mice (purchased from Jackson laboratory) at 4-6 weeks of age were divided into groups of 40 mice each weighing 18-20g and immunized separately. Before immunization, gp96 used for immunization and a polypeptide (PB181-89 or HBc82-90 or mutant polypeptide fragment a or mutant polypeptide fragment b) were assembled in an assembly solution to obtain an immunogen solution. The solvent of the assembly solution is water, and the solutes and their concentrations are as follows: NaCl 8g/L, KCl 0.2g/L, KH₂PO₄ 0.24g/L,Na₂HPO₄·12H₂O3.63 g/L; assembling conditions are as follows: the mixture was allowed to stand at 55 ℃ for 10min and at room temperature for 30 min. Obtaining 4 immunogen solutions according to the method, wherein solutes in the first immunogen solution are gp96 and HBc82-90, and the mass ratio of HBc82-90 to gp96 is 5: 3; in the second immunogen solution, solutes are gp96 and PB181-89, and the mass ratio of PB181-89 to gp96 is 5: 3; in the third immunogen solution, the solute is gp96 and a mutant polypeptide fragment a, and the mass ratio of the mutant polypeptide fragment a to gp96 is 5: 3; in the fourth immunogen solution, the solute is gp96 and the mutant polypeptide fragment b, and the mass ratio of the mutant polypeptide fragment b to gp96 is 5: 3.

The immunization mode is subcutaneous injection, the first immunization time is the first week of the experiment, the second immunization time is the second week of the experiment, and the third immunization time is the fourth week of the experiment. Each mouse had the same volume of immunogen solution per injection, 0.2 mL.

First group (negative control group): the first immunity polypeptide HBc82-90(50 microgram/one), gp96(30 microgram/one); the second immunity polypeptide HBc82-90(50 microgram/one), gp96(30 microgram/one); third immunity polypeptide HBc82-90(50 microgram/one), gp96(30 microgram/one);

second group (PB181-89 Polypeptides group): the first immunity polypeptide PB181-89 (50 microgram/one), gp96(30 microgram/one); the second immunity polypeptide PB181-89 (50 microgram/piece), gp96(30 microgram/piece); third immunity polypeptide PB181-89 (50 microgram/one), gp96(30 microgram/one);

third group (mutant polypeptide group a): the first immunization polypeptide mutant polypeptide fragment a (50 microgram/piece), gp96(30 microgram/piece); the second immunization polypeptide mutant polypeptide fragment a (50 microgram/piece), gp96(30 microgram/piece); third immunization polypeptide mutant polypeptide fragment a (50 microgram/piece), gp96(30 microgram/piece);

fourth group (mutant polypeptide group b): the first immunization polypeptide mutant polypeptide fragment b (50 microgram/piece), gp96(30 microgram/piece); the second immunization polypeptide mutant polypeptide fragment b (50 microgram/piece), gp96(30 microgram/piece); the third immunization polypeptide mutant polypeptide fragment b (50 microgram/piece), gp96(30 microgram/piece).

Second, PR8 toxicity challenge experiment

And (3) taking 10 mice in each group after immunization in the step one, and carrying out anesthesia by using chloral hydrate, namely, intravenously injecting the chloral hydrate according to the weight of the mice, wherein the injection dose is 375mg/kg of the weight. Under the condition of anesthesia, the mice are subjected to nose dropping of PR8 virus liquid (namely influenza A virus A/Puerto Rico/8/1934(H1N1) which is described in non-patent document "Penngxinxin. mechanism research of host cell microRNA library for regulating and controlling the replication of the influenza A virus. in Microbiol research institute of Chinese academy of sciences. Ph of sciences. 2018, 6 months.) to ensure that the system is at 50 mu l and the dosage of PR8 is 10⁴PFUs/mouse. The status of the mice was observed every day and the body weight of the mice was recorded for 14 days, and the results are shown in fig. 4, in which the body weight of the mice in the first group (negative control group) rapidly and continuously decreased and died between day 6 and day 12; in contrast, the second group (PB181-89 polypeptides)Group), third group (mutant polypeptide a group) and fourth group (immune mutant polypeptide b group) although their body weights also experienced an initial drop, their body weights reached a minimum on day 9 or day 10 and subsequently recovered. Survival of the mice was calculated and the results are shown in fig. 5, with the first group (negative control group) of mice dying 1 at day 6, 3 at day 10, 3 at day 11, 3 at day 12, and the group having all died by day 12 post infection; in contrast, the mice of the second group (PB181-89 polypeptide group), the third group (mutant polypeptide a group) and the fourth group (immune mutant polypeptide b group) survived all the day 14 after virus infection, and all the mice were significantly different (p < 0.0001) compared with the mice of the first group (negative control group).

Third, WSN challenge experiment

And (3) taking 10 mice in each group after immunization in the step one, and carrying out anesthesia by using chloral hydrate, namely, intravenously injecting the chloral hydrate according to the weight of the mice, wherein the injection dose is 375mg/kg of the weight. Under anesthesia, mice were subjected to nasal drops of WSN Virus fluid (i.e., Influenza A Virus A/WSN/1933(H1N1), described in non-patent documents "Tian X, Zhang K, Min J, Chen C, Cao Y, Ding C, Liu W, Li J. Metabological Analysis of Influenaza A viruses A/WSN/1933(H1N1) infested A549 Cells reduced First Cycle of viral infection. viruses 2019 Oct. (11)") at a WSN dose of 10 μ l⁴PFUs/mouse. The status of the mice was observed every day and the body weight of the mice was recorded for 14 days, and the results are shown in fig. 6, in which the body weight of the mice in the first group (negative control group) rapidly and continuously decreased and died between the 10 th and 12 th days; in contrast, the second (PB181-89 polypeptide group), third (mutant polypeptide a group) and fourth (immuno-mutant polypeptide b group) mice, although also undergoing an initial drop in body weight, reached a minimum on day 10 and then recovered. Survival of the mice was calculated and the results are shown in fig. 7, with 4 mice from the first group (negative control group) dying on day 10, 3 dying on day 11, 3 dying on day 12, and all dying by day 12 after infection; while the second group of immune PB181-89 polypeptides and the third group of immune mutant polypeptides a both died 1 on the 10 th day after the infection of the virus,fourth group of immune mutant polypeptide b mice in the observation of the virus infection after 14 days, all mice survived, compared with the negative control group, the difference was significant (p < 0.0001).

H3N2 challenge experiment

And (3) taking 10 mice in each group after immunization in the step one, and carrying out anesthesia by using chloral hydrate, namely, intravenously injecting the chloral hydrate according to the weight of the mice, wherein the injection dose is 375mg/kg of the weight. Under anesthesia, mice were subjected to nasal drip of H3N2 virus fluid (i.e., influenza A virus A/JIANGXI/206/2005(H3N2) described in non-patent document "Penngxinxin. mechanism study of host cell microRNA library for regulating replication of influenza A virus. Microbiol. institute of Chinese academy of sciences. Ph. 2018, 6.Y.) at a dose of H3N2 of 10 at 50. mu.l⁴PFUs/mouse. The status of the mice was observed every day and the body weight of the mice was recorded for 14 days, and the results are shown in fig. 8, in which the body weight of the mice in the first group (negative control group) rapidly and continuously decreased and died between day 10 and day 13; in contrast, the second (PB181-89 polypeptide group), third (mutant polypeptide a group) and fourth (immuno-mutant polypeptide b group) mice, although also undergoing an initial drop in body weight, reached a minimum on day 9 or 10 and then recovered. Survival of the mice was calculated and the results are shown in fig. 9, with the first group (negative control group) of mice dying 2 on day 10, 2 on day 11, 8 on day 12 after infection, and the group of mice having all died by day 12 after infection; in contrast, 2 mice in the second group (PB181-89 polypeptide group) and the fourth group (immune mutant polypeptide b group) died at 11 days and 13 days after virus infection respectively, and were significantly different (p < 0.001) compared with the control group, and the mice in the third group (mutant polypeptide a group) were all alive at 14 days after virus infection and were significantly different (p < 0.0001) compared with the negative control group.

Fifth, H7N9 toxicity challenge experiment

And (3) taking 10 mice in each group after immunization in the step one, and carrying out anesthesia by using chloral hydrate, namely, intravenously injecting the chloral hydrate according to the weight of the mice, wherein the injection dose is 375mg/kg of the weight. Under anesthesia, H7N9 virus fluid (i.e., influenza A virus) was administered to mice(A/Shanghai/02/2013(H7N9)) described in non-patent documents "Xu J, Li S, Wang X, Liu J, Shan P, Zhou Y, ZHao J, Wang Z, Xu C, Chen M, Chen Z, ZHao K, Qu D.systematic and mucosal immune response induced by the JY-adjuvanted nasal spray H7N9 vaccine in micro. 7(1): 140.') of the nasal drip, the dosage of the H7N9 is 10 under the condition of ensuring that the system is 50 mu l⁴PFUs/mouse. The status of the mice was observed every day and the body weight of the mice was recorded for 14 days, and the results are shown in fig. 10, in which the body weight of the first group (negative control group) rapidly and continuously decreased and died between day 9 and day 11; in contrast, the second (PB181-89 polypeptide group), third (mutant polypeptide a group) and fourth (immuno-mutant polypeptide b group) mice, although also undergoing an initial drop in body weight, reached a minimum on day 9 or 10 and then recovered. Survival of the mice was calculated and the results are shown in fig. 11, with 3 mice from the first group (negative control group) dying on day 9, 3 mice dying on day 10, 4 mice dying on day 11, and the group having all died by day 11 post infection; and 3 mice in the third group of immune mutant polypeptide a and the fourth group of immune mutant polypeptide b die at 12 days and 11 days after virus infection respectively, and have significant difference (p is less than 0.001) compared with the control group, and the mice in the second group of immune PB181-89 polypeptide group survive completely at 14 days after virus infection and have significant difference (p is less than 0.0001) compared with the negative control group.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

SEQUENCE LISTING

<110> institute of microbiology of Chinese academy of sciences

<120> influenza A virus PB1 protein T cell epitope polypeptide segment and application thereof

<130> GNCFY200610

<160> 11

<170> PatentIn version 3.5

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<213> Artificial Sequence (Artificial Sequence)

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Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn

1 5 10 15

Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His

20 25 30

Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln

35 40 45

Tyr Ser Glu Lys Ala Arg Trp Thr Thr Asn Thr Glu Thr Gly Ala Pro

50 55 60

Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser

65 70 75 80

Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu

85 90 95

Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Ile Glu Thr Met Glu

100 105 110

Val Val Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr

115 120 125

Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala

130 135 140

Asn Thr Ile Glu Val Phe Arg Ser Asn Gly Leu Thr Ala Asn Glu Ser

145 150 155 160

Gly Arg Leu Ile Asp Phe Leu Lys Asp Val Met Glu Ser Met Lys Lys

165 170 175

Glu Glu Met Gly Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg

180 185 190

Asp Asn Met Thr Lys Lys Met Ile Thr Gln Arg Thr Ile Gly Lys Arg

195 200 205

Lys Gln Arg Leu Asn Lys Arg Ser Tyr Leu Ile Arg Ala Leu Thr Leu

210 215 220

Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala

225 230 235 240

Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu

245 250 255

Thr Leu Ala Arg Ser Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro

260 265 270

Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys

275 280 285

Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Leu Thr Ile Thr Gly

290 295 300

Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala

305 310 315 320

Met Ile Thr Tyr Met Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val

325 330 335

Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly

340 345 350

Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile

355 360 365

Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser

370 375 380

Thr Arg Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Ile Glu Gly Thr

385 390 395 400

Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser

405 410 415

Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Lys Arg Tyr Thr

420 425 430

Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala

435 440 445

Leu Ile Val Asn Ala Pro Asn His Glu Gly Ile Gln Ala Gly Val Asp

450 455 460

Arg Phe Tyr Arg Thr Cys Lys Leu His Gly Ile Asn Met Ser Lys Lys

465 470 475 480

Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe

485 490 495

Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe

500 505 510

Gly Val Ser Gly Ser Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr

515 520 525

Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala

530 535 540

Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg

545 550 555 560

Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Ile

565 570 575

Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Ala Gly Leu Leu Val Ser

580 585 590

Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu

595 600 605

Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Gln Gly Arg Leu

610 615 620

Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Met

625 630 635 640

Asn Asn Ala Val Met Met Pro Ala His Gly Pro Ala Lys Asn Met Glu

645 650 655

Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg

660 665 670

Ser Ile Leu Asn Thr Ser Gln Arg Gly Val Leu Glu Asp Glu Gln Met

675 680 685

Tyr Gln Arg Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser

690 695 700

Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser

705 710 715 720

Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys

725 730 735

Lys Glu Glu Phe Thr Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu

740 745 750

Leu Arg Arg Gln Lys

755

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<213> Artificial Sequence (Artificial Sequence)

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Gly Tyr Ala Gln Thr Asp Cys Val Leu

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<213> Artificial Sequence (Artificial Sequence)

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Arg Glu Leu Val Val Ser Tyr Val Asn

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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aagtcgctaa tcgccgacca gtgcgatggc accctgcacg ctgctcctgc tgttggcggc 60

cgccctggcc cccactcaga cccgcgcggg cccacattcg ctgaggtatt tcgtcaccgc 120

cgtgtcccgg cccggcctcg gggagccccg gttcatcgct gtcggctacg tggacgacac 180

gcagttcgtg cgcttcgaca gcgacgcgga taatccgaga tttgagccgc gggcgccgtg 240

gatggagcag gaggggccgg agtattggga ggagcagaca cagagagcca agagcgatga 300

gcagtggttc cgagtgagcc tgaggaccgc acagagatac tacaaccaga gcaagggcgg 360

ctctcacacg ttccagcgga tgttcggctg tgacgtgggg tcggactggc gcctcctccg 420

cgggtaccat cagttcgcct acgacggccg cgattacatc gccctgaacg aagacctgaa 480

aacgtggacg gcggcggaca cggcggcgct gatcaccaga cgcaagtggg agcaggctgg 540

tgatgcagag tattacaggg cctacctaga gggcgagtgc gtggagtggc tccgcagata 600

cctggagctc gggaatgaga cgctgctgcg cacagattcc ccaaaggccc atgtgaccta 660

tcaccccaga tctcaagttg atgtcaccct gaggtgctgg gccctgggct tctaccctgc 720

tgatatcacc ctgacctggc agttgaatgg ggaggacctg acccaggaca tggagcttgt 780

agagaccagg cctgcagggg atggaacctt ccagaagtgg gcagctgtgg tggtgcctct 840

tgggaaggag cagaattaca catgccatgt gcaccataag gggctgcctg agcctctcac 900

cctgagatgg aagcttcctc catccactgt ctccaacacg gtaatcattg ctgttctggt 960

tgtccttgga gctgcaatag tcactggagc tgtggtggct tttgtgatga agatgagaag 1020

gaacacaggt ggaaaaggag tgaactatgc tctggctcca ggctcccaga cctctgatct 1080

gtctctccca gatggtaaag tgatggttca tgaccctcat tctctagcgt gaagacagct 1140

gcctggagtg gacttggtga cagacaatgt cttcacacat ctcctatgac atccagagcc 1200

ctcagttctc tttagtcaag tgtctgatgt tccctgtgag cctatggact caaagtgaag 1260

aactgtggag cccagtccac ccctccacac cagcaccctg tccctgcact gctctgtctt 1320

cccttccaca gccaaccttg ctggttcagc caaacactgg gggacatctg cagcctgtca 1380

gctccatgct accctgacct gcagctcctc acttccacac tgagaatagt aatttgaatg 1440

taaccttgat tgttatcatc ttgacctagg gctgatttct tgttaatttc atgcttagag 1500

gttttgtttg tttgtttgat ttgttttttt tttttgaaga aataaatgat agatgaataa 1560

<210> 5

<211> 275

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Pro His Ser Leu Arg Tyr Phe Val Thr Ala Val Ser Arg Pro Gly Leu

1 5 10 15

Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln Phe

20 25 30

Val Arg Phe Asp Ser Asp Ala Asp Asn Pro Arg Phe Glu Pro Arg Ala

35 40 45

Pro Trp Met Glu Gln Glu Gly Pro Glu Tyr Trp Glu Glu Gln Thr Gln

50 55 60

Arg Ala Lys Ser Asp Glu Gln Trp Phe Arg Val Ser Leu Arg Thr Ala

65 70 75 80

Gln Arg Tyr Tyr Asn Gln Ser Lys Gly Gly Ser His Thr Phe Gln Arg

85 90 95

Met Phe Gly Cys Asp Val Gly Ser Asp Trp Arg Leu Leu Arg Gly Tyr

100 105 110

His Gln Phe Ala Tyr Asp Gly Arg Asp Tyr Ile Ala Leu Asn Glu Asp

115 120 125

Leu Lys Thr Trp Thr Ala Ala Asp Thr Ala Ala Leu Ile Thr Arg Arg

130 135 140

Lys Trp Glu Gln Ala Gly Asp Ala Glu Tyr Tyr Arg Ala Tyr Leu Glu

145 150 155 160

Gly Glu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Leu Gly Asn Glu

165 170 175

Thr Leu Leu Arg Thr Asp Ser Pro Lys Ala His Val Thr Tyr His Pro

180 185 190

Arg Ser Gln Val Asp Val Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr

195 200 205

Pro Ala Asp Ile Thr Leu Thr Trp Gln Leu Asn Gly Glu Asp Leu Thr

210 215 220

Gln Asp Met Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe

225 230 235 240

Gln Lys Trp Ala Ala Val Val Val Pro Leu Gly Lys Glu Gln Asn Tyr

245 250 255

Thr Cys His Val His His Lys Gly Leu Pro Glu Pro Leu Thr Leu Arg

260 265 270

Trp Lys Pro

275

<210> 6

<211> 357

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atggctcgct cggtgaccct ggtctttctg gtgcttgtct cactgaccgg cctgtatgct 60

atccagaaaa cccctcaaat tcaagtatac tcacgccacc caccggagaa tgggaagccg 120

aacatactga actgctacgt aacacagttc cacccgcctc acattgaaat ccaaatgctg 180

aagaacggga aaaaaattcc taaagtagag atgtcagata tgtccttcag caaggactgg 240

tctttctata tcctggctca cactgaattc acccccactg agactgatac atacgcctgc 300

agagttaagc atgtcagtat ggccgagccc aagaccgtct actgggatcg agacatg 357

<210> 7

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Met Ala Arg Ser Val Thr Leu Val Phe Leu Val Leu Val Ser Leu Thr

1 5 10 15

Gly Leu Tyr Ala Ile Gln Lys Thr Pro Gln Ile Gln Val Tyr Ser Arg

20 25 30

His Pro Pro Glu Asn Gly Lys Pro Asn Ile Leu Asn Cys Tyr Val Thr

35 40 45

Gln Phe His Pro Pro His Ile Glu Ile Gln Met Leu Lys Asn Gly Lys

50 55 60

Lys Ile Pro Lys Val Glu Met Ser Asp Met Ser Phe Ser Lys Asp Trp

65 70 75 80

Ser Phe Tyr Ile Leu Ala His Thr Glu Phe Thr Pro Thr Glu Thr Asp

85 90 95

Thr Tyr Ala Cys Arg Val Lys His Ala Ser Met Ala Glu Pro Lys Thr

100 105 110

Val Tyr Trp Asp Arg Asp Met

115

<210> 8

<211> 2349

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gacgatgaag ttgatgtgga tggtacagta gaagaggatc tgggtaaaag tagagaagga 60

tcaaggacgg atgatgaagt agtacagaga gaggaagaag ctattcagtt ggatggatta 120

aatgcatcac aaataagaga acttagagag aagtcggaaa agtttgcctt ccaagccgaa 180

gttaacagaa tgatgaaact tatcatcaat tcattgtata aaaataaaga gattttcctg 240

agagaactga tttcaaatgc ttctgatgct ttagataaga taaggctaat atcactgact 300

gatgaaaatg ctctttctgg aaatgaggaa ctaacagtca aaattaagtg tgataaggag 360

aagaacctgc tgcatgtcac agacaccggt gtaggaatga ccagagaaga gttggttaaa 420

aaccttggta ccatagccaa atctgggaca agcgagtttt taaacaaaat gactgaagca 480

caggaagatg gccagtcaac ttctgaattg attggccagt ttggtgtcgg tttctattcc 540

gccttccttg tagcagataa ggttattgtc acttcaaaac acaacaacga tacccagcac 600

atctgggagt ctgactccaa tgaattttct gtaattgctg acccaagagg aaacactcta 660

ggacggggaa cgacaattac ccttgtctta aaagaagaag catctgatta ccttgaattg 720

gatacaatta aaaatctcgt caaaaaatat tcacagttca taaactttcc tatttatgta 780

tggagcagca agactgaaac tgttgaggag cccatggagg aagaagaagc agccaaagaa 840

gagaaagaag aatctgatga tgaagctgca gtagaggaag aagaagaaga aaagaaacca 900

aagactaaaa aagttgaaaa aactgtctgg gactgggaac ttatgaatga tatcaaacca 960

atatggcaga gaccatcaaa agaagtagaa gaagatgaat acaaagcttt ctacaaatca 1020

ttttcaaagg aaagtgatga ccccatggct tatattcact ttactgctga aggggaagtt 1080

accttcaaat caattttatt tgtacccaca tctgctccac gtggtctgtt tgacgaatat 1140

ggatctaaaa agagcgatta cattaagctc tatgtgcgcc gtgtattcat cacagacgac 1200

ttccatgata tgatgcctaa atacctcaat tttgtcaagg gtgtggtgga ctcagatgat 1260

ctccccttga atgtttcccg cgagactctt cagcaacata aactgcttaa ggtgattagg 1320

aagaagcttg ttcgtaaaac gctggacatg atcaagaaga ttgctgatga taaatacaat 1380

gatacttttt ggaaagaatt tggtaccaac atcaagcttg gtgtgattga agaccactcg 1440

aatcgaacac gtcttgctaa acttcttagg ttccagtctt ctcatcatcc aactgacatt 1500

actagcctag accagtatgt ggaaagaatg aaggaaaaac aagacaaaat ctacttcatg 1560

gctgggtcca gcagaaaaga ggctgaatct tctccatttg ttgagcgact tctgaaaaag 1620

ggctatgaag ttatttacct cacagaacct gtggatgaat actgtattca ggcccttccc 1680

gaatttgatg ggaagaggtt ccagaatgtt gccaaggaag gagtgaagtt cgatgaaagt 1740

gagaaaacta aggagagtcg tgaagcagtt gagaaagaat ttgagcctct gctgaattgg 1800

atgaaagata aagcccttaa ggacaagatt gaaaaggctg tggtgtctca gcgcctgaca 1860

gaatctccgt gtgctttggt ggccagccag tacggatggt ctggcaacat ggagagaatc 1920

atgaaagcac aagcgtacca aacgggcaag gacatctcta caaattacta tgcgagtcag 1980

aagaaaacat ttgaaattaa tcccagacac ccgctgatca gagacatgct tcgacgaatt 2040

aaggaagatg aagatgataa aacagttttg gatcttgctg tggttttgtt tgaaacagca 2100

acgcttcggt cagggtatct tttaccagac actaaagcat atggagatag aatagaaaga 2160

atgcttcgcc tcagtttgaa cattgaccct gatgcaaagg tggaagaaga gcccgaagaa 2220

gaacctgaag agacagcaga agacacaaca gaagacacag agcaagacga agatgaagaa 2280

atggatgtgg gaacagatga agaagaagaa acagcaaagg aatctacagc tgaaaaagat 2340

gaattgtaa 2349

<210> 9

<211> 803

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Arg Ala Leu Trp Val Leu Gly Leu Cys Cys Val Leu Leu Thr Phe

1 5 10 15

Gly Ser Val Arg Ala Asp Asp Glu Val Asp Val Asp Gly Thr Val Glu

20 25 30

Glu Asp Leu Gly Lys Ser Arg Glu Gly Ser Arg Thr Asp Asp Glu Val

35 40 45

Val Gln Arg Glu Glu Glu Ala Ile Gln Leu Asp Gly Leu Asn Ala Ser

50 55 60

Gln Ile Arg Glu Leu Arg Glu Lys Ser Glu Lys Phe Ala Phe Gln Ala

65 70 75 80

Glu Val Asn Arg Met Met Lys Leu Ile Ile Asn Ser Leu Tyr Lys Asn

85 90 95

Lys Glu Ile Phe Leu Arg Glu Leu Ile Ser Asn Ala Ser Asp Ala Leu

100 105 110

Asp Lys Ile Arg Leu Ile Ser Leu Thr Asp Glu Asn Ala Leu Ser Gly

115 120 125

Asn Glu Glu Leu Thr Val Lys Ile Lys Cys Asp Lys Glu Lys Asn Leu

130 135 140

Leu His Val Thr Asp Thr Gly Val Gly Met Thr Arg Glu Glu Leu Val

145 150 155 160

Lys Asn Leu Gly Thr Ile Ala Lys Ser Gly Thr Ser Glu Phe Leu Asn

165 170 175

Lys Met Thr Glu Ala Gln Glu Asp Gly Gln Ser Thr Ser Glu Leu Ile

180 185 190

Gly Gln Phe Gly Val Gly Phe Tyr Ser Ala Phe Leu Val Ala Asp Lys

195 200 205

Val Ile Val Thr Ser Lys His Asn Asn Asp Thr Gln His Ile Trp Glu

210 215 220

Ser Asp Ser Asn Glu Phe Ser Val Ile Ala Asp Pro Arg Gly Asn Thr

225 230 235 240

Leu Gly Arg Gly Thr Thr Ile Thr Leu Val Leu Lys Glu Glu Ala Ser

245 250 255

Asp Tyr Leu Glu Leu Asp Thr Ile Lys Asn Leu Val Lys Lys Tyr Ser

260 265 270

Gln Phe Ile Asn Phe Pro Ile Tyr Val Trp Ser Ser Lys Thr Glu Thr

275 280 285

Val Glu Glu Pro Met Glu Glu Glu Glu Ala Ala Lys Glu Glu Lys Glu

290 295 300

Glu Ser Asp Asp Glu Ala Ala Val Glu Glu Glu Glu Glu Glu Lys Lys

305 310 315 320

Pro Lys Thr Lys Lys Val Glu Lys Thr Val Trp Asp Trp Glu Leu Met

325 330 335

Asn Asp Ile Lys Pro Ile Trp Gln Arg Pro Ser Lys Glu Val Glu Glu

340 345 350

Asp Glu Tyr Lys Ala Phe Tyr Lys Ser Phe Ser Lys Glu Ser Asp Asp

355 360 365

Pro Met Ala Tyr Ile His Phe Thr Ala Glu Gly Glu Val Thr Phe Lys

370 375 380

Ser Ile Leu Phe Val Pro Thr Ser Ala Pro Arg Gly Leu Phe Asp Glu

385 390 395 400

Tyr Gly Ser Lys Lys Ser Asp Tyr Ile Lys Leu Tyr Val Arg Arg Val

405 410 415

Phe Ile Thr Asp Asp Phe His Asp Met Met Pro Lys Tyr Leu Asn Phe

420 425 430

Val Lys Gly Val Val Asp Ser Asp Asp Leu Pro Leu Asn Val Ser Arg

435 440 445

Glu Thr Leu Gln Gln His Lys Leu Leu Lys Val Ile Arg Lys Lys Leu

450 455 460

Val Arg Lys Thr Leu Asp Met Ile Lys Lys Ile Ala Asp Asp Lys Tyr

465 470 475 480

Asn Asp Thr Phe Trp Lys Glu Phe Gly Thr Asn Ile Lys Leu Gly Val

485 490 495

Ile Glu Asp His Ser Asn Arg Thr Arg Leu Ala Lys Leu Leu Arg Phe

500 505 510

Gln Ser Ser His His Pro Thr Asp Ile Thr Ser Leu Asp Gln Tyr Val

515 520 525

Glu Arg Met Lys Glu Lys Gln Asp Lys Ile Tyr Phe Met Ala Gly Ser

530 535 540

Ser Arg Lys Glu Ala Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys

545 550 555 560

Lys Gly Tyr Glu Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys

565 570 575

Ile Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala

580 585 590

Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg

595 600 605

Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp

610 615 620

Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu

625 630 635 640

Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly

645 650 655

Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp

660 665 670

Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn

675 680 685

Pro Arg His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp

690 695 700

Glu Asp Asp Lys Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr

705 710 715 720

Ala Thr Leu Arg Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly

725 730 735

Asp Arg Ile Glu Arg Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp

740 745 750

Ala Lys Val Glu Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu

755 760 765

Asp Thr Thr Glu Asp Thr Glu Gln Asp Glu Asp Glu Glu Met Asp Val

770 775 780

Gly Thr Asp Glu Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu Lys

785 790 795 800

Asp Glu Leu

<210> 10

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gly Tyr Ala Gln Ala Asp Cys Val Leu

1 5

<210> 11

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Gly Tyr Ala Gln Thr Asp Ala Val Leu

1 5