Gene sequence constructs for gene therapy of AIDS virus infection.

Abstract

The present invention relates to a gene sequence construct for gene therapy of AIDS virus (HIV) infection. A gene sequence construct is constructed by arranging a gene coding sequence of each of single chain variable regions (scFv) of light and heavy chains of a monoclonal antibody against different binding site antigens involved in different steps of HIV infection to human CD4+ T cells, a gene coding sequence of each of single chain variable regions (scFv) of light and heavy chains of a monoclonal antibody binding to a CD4 receptor site, and a gene coding sequence of a polypeptide that inhibits the fusion of HIV with CD4+ T cell membrane downstream of a promoter and a secretory signal peptide coding sequence, thereby expressing a secretory antibody-like protein molecule encoded by a single gene. This recombinant single gene construct can be easily introduced into target tissue cells via a viral vector, and the expressed and secreted antibody or antibody-like protein molecule has polyantigen affinity and binds to multiple binding sites involved in different steps of HIV infection of human CD4+ T cells, thereby effectively and broadly blocking the progression of HIV infection in human CD4+ T cells and effectively avoiding the loss of inhibitory ability against HIV infection due to the occurrence of HIV escape mutations, thereby achieving long-term and even permanent therapeutic effects against HIV infection with a single injection.

Description

[Technical field]

[0001] This application claims priority to PCT Patent Application No. PCT / CN2021 / 115422, filed on August 30, 2021, the contents of which are incorporated in their entirety as part of this application.

[0002] The present invention belongs to the field of gene therapy / biomedicine technology, specifically relates to a gene sequence construct for gene therapy of AIDS virus (HIV) infection. The gene sequence construct can be used in gene therapy for HIV infection. The gene sequence construct can be used to express a polyphilic neutralizing antibody-like protein that has a broad spectrum and highly efficient neutralization of HIV virus activity both in vivo and in vitro, and can be used in clinical research of gene therapy drugs for HIV infection delivered from recombinant virus or non-virus vectors and in research and development of new drugs. [Background technology]

[0003] AIDS, caused by infection with the human immunodeficiency virus (HIV), is one of the world's most widespread infectious diseases. Of the 37.9 million people infected with HIV worldwide, approximately 770,000 die of AIDS each year. With the development of anti-AIDS drugs, the expected survival time of AIDS virus-infected people who are actively treated has been significantly extended, but the side effects and limitations of cocktail therapy, as well as the gradually emerging drug-resistant strains of HIV, still cause long-term economic and social burdens, significant decline in quality of life, and significant suffering for the vast majority of AIDS patients.

[0004] Therefore, despite the advances already made in the anti-AIDS field, there remains a need for the development of new drugs and methods for treating AIDS virus infection. Summary of the Invention

[0005] The present invention has created a series of recombinant viral vector-based gene therapy constructs for the treatment of AIDS virus infection, such as broadly neutralizing antibodies against multiple strains of HIV, and single-chain variable fragments (scFv) of neutralizing antibodies against the human CD4 receptor, combined with HIV membrane fusion inhibitor polypeptides, into a simple and efficient expression framework.

[0006] First, these gene sequence constructs for gene therapy of HIV infection contain gene coding sequences for one or more single-chain antibody molecules (including nanobodies) that have the ability to inhibit HIV infection and do not have a constant region, and gene coding sequences for one or more polypeptides (consisting of 2-50 amino acid residues) that have the ability to inhibit HIV infection, so as to express a fusion protein molecule containing an anti-HIV infection antibody molecule and a polypeptide, encoded by a single gene, and the fusion protein molecule has two or more types of action targets.

[0007] In particular, the single chain antibody molecule of said gene sequence construct comprises a heavy chain variable region and / or a light chain variable region.

[0008] wherein the light chain variable region comprises a light chain variable region of a κ or λ light chain.

[0009] The gene sequence construct comprises two or more gene coding sequences for a single-chain antibody molecule having the ability to inhibit HIV infection and having no constant region.

[0010] Alternatively, it comprises three or more gene coding sequences for a single-chain antibody molecule having the ability to inhibit HIV infection and no constant region.

[0011] Alternatively, it comprises four or more gene coding sequences for a single-chain antibody molecule having the ability to inhibit HIV infection and no constant region.

[0012] The gene sequence construct may contain two or more gene coding sequences for polypeptides capable of suppressing HIV infection, each of which consists of 2 to 50 amino acid residues.

[0013] Or, it contains three or more gene coding sequences for polypeptides having the ability to inhibit HIV infection.

[0014] Or, it contains four or more gene coding sequences for polypeptides having the ability to inhibit HIV infection.

[0015] In any one of the gene sequence constructs described above, the fusion protein molecule comprising an anti-HIV infection and single-chain antibody molecule having no constant region and a polypeptide, encoded by a single gene, has three or more action targets.

[0016] Alternatively, a fusion protein molecule comprising an anti-HIV infection, constant region-free single-chain antibody molecule and a polypeptide, encoded by a single gene, has four or more target molecules.

[0017] Alternatively, a fusion protein molecule comprising an anti-HIV infection, constant region-free single-chain antibody molecule and a polypeptide, encoded by a single gene, has five or more target molecules.

[0018] Alternatively, a fusion protein molecule comprising an anti-HIV infection, constant region-free single-chain antibody molecule and a polypeptide, encoded by a single gene, has six or more target molecules.

[0019] The gene sequence construct described in any one of the above comprises gene coding sequences for two or more single-chain antibody molecules having the ability to inhibit HIV infection and having no constant region, and gene coding sequences for one or more polypeptides having the ability to inhibit HIV infection.

[0020] In the gene sequence construct, the fusion protein molecule comprising an anti-HIV infection, constant region-free single-chain antibody molecule and a polypeptide, encoded by a single gene, has three or more action targets.

[0021] In the gene sequence construct described in any one of the above, the gene coding sequence for the single-chain antibody molecule having the ability to inhibit HIV infection and having no constant region is directly or indirectly connected in series with the gene coding sequence for the polypeptide having the ability to inhibit HIV infection via a coding sequence for a linker polypeptide.

[0022] The gene sequence construct described in any one of the above comprises two or more gene coding sequences for single-chain antibody molecules having the ability to inhibit HIV infection and having no constant region, and the gene coding sequences for the antibody molecules are directly or indirectly connected in series via a coding sequence for a linker polypeptide.

[0023] The gene sequence construct described in any one of the above contains two or more gene coding sequences for polypeptides capable of suppressing HIV infection, and the gene coding sequences for the polypeptides are directly or indirectly connected in series between each other via a coding sequence for a linker polypeptide.

[0024] In the gene sequence construct described in any one of the above, the gene coding sequence of the single-chain antibody molecule having the ability to inhibit HIV infection and having no constant region comprises a gene coding sequence of an anti-HIV-1-gp160 (or its cleavage products gp120 and gp41) antibody molecule.

[0025] In the gene sequence construct described in any one of the above, the gene coding sequence of one or more single-chain antibody molecules having the ability to suppress HIV infection and having no constant region comprises a gene coding sequence of an antibody molecule that binds to the human CD4 receptor site.

[0026] In the gene sequence construct described in any one of the above, the gene coding sequence for one or more polypeptides capable of suppressing HIV infection includes a gene coding sequence for a polypeptide that inhibits the fusion of HIV with a CD4+ T cell membrane.

[0027] The gene sequence construct described in any one of the above comprises gene coding sequences of two or more single-chain antibody molecules having the ability to inhibit HIV infection and having no constant region, and gene coding sequences of one or more polypeptides having the ability to inhibit HIV infection, wherein the gene coding sequences of the two or more single-chain antibody molecules having the ability to inhibit HIV infection and having no constant region comprise a gene coding sequence of an anti-HIV-1-gp160 antibody molecule (or its cleavage products gp120 and gp41) and a gene coding sequence of an antibody molecule that binds to the human CD4 receptor site, and the gene coding sequence of the one or more polypeptides having the ability to inhibit HIV infection comprises a gene coding sequence of a polypeptide that inhibits the fusion of HIV with the CD4+ T cell membrane.

[0028] The gene sequence construct according to any one of the above comprises (i) gene coding sequences of light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) and heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) of an anti-HIV-1-gp160 (or its cleavage products gp120 and gp41) monoclonal antibody, (ii) gene coding sequences of light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) and heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) of a monoclonal antibody that binds to the human CD4 receptor site, and (iii) a gene coding sequence of a polypeptide that inhibits the fusion of HIV with the membrane of a CD4+ T cell, wherein the coding sequences of the antibody light and heavy chains are connected in tandem directly or indirectly via the coding sequence of a linker polypeptide, regardless of the order.

[0029] The gene sequence construct according to any one of the above-mentioned aspects comprises (i) a gene coding sequence for the light chain variable region (VL) and heavy chain variable region (VH) of an anti-HIV-1-gp160 (or its cleavage products gp120 and gp41) monoclonal antibody, (ii) a gene coding sequence for the light chain variable region (VL) and heavy chain variable region (VH) of a monoclonal antibody that binds to the human CD4 receptor site, and (iii) a gene coding sequence for a polypeptide that inhibits the fusion of HIV with the CD4+ T cell membrane, wherein the coding sequences for the antibody light chain variable region (VL) and heavy chain variable region (VH) are linked in tandem, directly or indirectly, in any order, via a coding sequence for a linker polypeptide.

[0030] The gene sequence construct according to any one of the above aspects further comprises a promoter located upstream of the gene coding sequence for the single-chain antibody molecule having the ability to inhibit HIV infection and having no constant region, and the gene coding sequence for the polypeptide having the ability to inhibit HIV infection.

[0031] The gene sequence construct according to any one of the above aspects further comprises a gene coding sequence for a single-chain antibody molecule having the ability to inhibit HIV infection and having no constant region, and a secretory signal peptide coding sequence located upstream of the gene coding sequence for a polypeptide having the ability to inhibit HIV infection.

[0032] The gene sequence construct according to any one of the above, comprising: a gene coding sequence for a single-chain antibody molecule having a first ability to inhibit HIV infection and having no constant region; a gene coding sequence for a single-chain antibody molecule having a second ability to inhibit HIV infection and having no constant region; and a gene coding sequence for a polypeptide having an ability to inhibit HIV infection; The gene sequence construct comprises: VL2-linker-VH2-linker-VL1-linker-VH1-linker; or VL2-linker-VH2-linker-VL1-linker-VH1-linker-peptide inhibitor; or other combinations of VL2 and VH2 or VL1 and VH1 arranged in a different order in the construct.

[0033] Here, VL2 and VH2 are the variable region fragments of the light chain and heavy chain of a first antibody molecule, respectively, VL1 and VH1 are the variable region fragments of the light chain and heavy chain of a second antibody molecule, respectively, linker is a linker polypeptide, and peptide inhibitor is a polypeptide that suppresses HIV infection (e.g., a polypeptide that inhibits the fusion of HIV with the CD4+ T cell membrane).

[0034] The gene sequence construct may be VL2-linker-VH2-linker-VL1-linker-VH1, or a combination of VL2 and VH2 or VL1 and VH1 therein arranged in a different order.

[0035] The gene sequence construct is VL2-linker-VH2-linker-VL1-linker-VH1-linker-peptide inhibitor, or a construct in which the combination of VL2 and VH2 or VL1 and VH1 are arranged in a different order.

[0036] In said gene sequence construct, the protein sequences of VL2 and VH2 comprise SEQ ID NO:2, or a functional fragment thereof, or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0037] In said gene sequence construct, the protein sequence of VL1 and VH1 comprises SEQ ID NO:3, or a functional fragment thereof, or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0038] The gene sequence construct has a linker polypeptide sequence of GGGGS, (GGGGS) 2 , (GGGGS) 3 , (GGGGS)4 , (GGGGS) 5 , (GGGGS) 6 , Japanese (GGGGS) 7 , or other alternative linker polypeptide sequences.

[0039] In the above gene sequence construct, the polypeptide that inhibits the fusion of HIV with the CD4+ T cell membrane can be selected from membrane fusion-inhibiting polypeptides P52, C34, T20, and the like.

[0040] Here, the sequence of the membrane fusion-inhibiting polypeptide P52 comprises SEQ ID NO:5 or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto, the polypeptide sequence of C34 comprises SEQ ID NO:6 or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto, and the polypeptide sequence of T20 comprises SEQ ID NO:7 or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0041] Furthermore, the present invention also provides a viral vector genome comprising any of the above constructs, and a corresponding viral vector system comprising said genome.

[0042] The viral vector system may be a lentiviral vector system or an adeno-associated viral vector system.

[0043] Here, the lentiviral vector system can contain the viral vector genome of any of the above constructs and other nucleotide sequences that encode and express a packaging kit necessary for lentivirus production, and is introduced into a production cell to produce lentiviral particles containing the above construct genome.

[0044] The adeno-associated virus vector system can contain the viral vector genome of any of the above constructs, as well as other nucleotide sequences that encode and express the packaging kit necessary for the production of adeno-associated virus, and is introduced into a producer cell to produce adeno-associated virus particles containing the genome of the above construct.

[0045] Any of the viral particles produced contain the genome of any of the constructs and are capable of expressing anti-HIV neutralizing antibody-like molecules after transduction of cells and can be administered to patients to suppress or prevent HIV infection.

[0046] The present invention also provides a pharmaceutical composition comprising said viral particles and a pharma- ceutically acceptable carrier or diluent, or cells transduced in vitro with said lentiviral particles, said transduced cells including, but not limited to, muscle cells, liver cells, or CD4+ T cells.

[0047] The virus particles or a pharmaceutical composition containing the virus particles are injected into the body to express an antibody molecule protein having two or more action targets, which is used in gene therapy for HIV infection, and the antibody molecule protein efficiently and broadly blocks the infection progression of HIV to human CD4+ T cells by binding to multiple binding sites related to different steps of HIV infection to human CD4+ T cells, so as to achieve long-term treatment for AIDS virus-infected individuals.

[0048] The present invention provides a method for inhibiting HIV infection, comprising administering the above-mentioned viral particle or pharmaceutical composition to a cell.

[0049] Here, the cells include muscle cells, liver cells, CD4+ T cells, and the like.

[0050] Said viral particle or pharmaceutical composition may transduce said cells in vitro or in vivo.

[0051] The present invention provides a method of treating HIV infection in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of a viral particle or pharmaceutical composition as described above.

[0052] Here, the subject includes an HIV-infected individual in an early stage, an HIV-infected individual undergoing cocktail drug therapy, or an HIV-infected individual with resistance to cocktail drug therapy.

[0053] The method of administering the drug to the subject is intramuscular injection of the viral particles or pharmaceutical composition.

[0054] Alternatively, the viral particles or pharmaceutical composition or CD4+ T cells transduced therewith are injected intravenously.

[0055] The virus particles and pharmaceutical composition injected into the body by the above-mentioned method can express anti-HIV protein molecules with multiple action targets and secrete them into the blood, acting on multiple HIV infection nodes, effectively blocking the HIV infection pathway and effectively avoiding the loss of inhibitory ability against HIV infection caused by the occurrence of HIV escape mutations, thereby achieving long-term (e.g., therapeutic efficacy lasts for one or several years) and even permanent therapeutic effects against HIV infection with a single injection.

[0056] The antibody-like molecule, consisting of multiple single-chain antibody variable region fragments (scFv) based on the above expression framework, shows effective blood drug concentrations after being delivered into mice via lentivirus and adeno-associated virus vectors, has in vitro cytological HIV-1 virus neutralizing activity, and simultaneously has broad-spectrum neutralizing ability against two types of CXCR4 and CCR5-tropic viruses, providing a highly potential technological route for the development of a broad-spectrum neutralizing antibody gene therapy drug for the anti-AIDS virus.

[0057] The scope of application of the present invention includes various forms of anti-AIDS virus gene therapy based on genetic expression of broadly neutralizing antibodies. [Brief description of the drawings]

[0058] The drawings of the present invention will now be described in detail. [Figure 1] FIG. 1 is a schematic diagram of the mature molecular structure of a predicted triphilic anti-HIV neutralizing antibody-like molecule existing alone. [Diagram 2] FIG. 1 is a schematic diagram showing the structure of the gene sequence of a Miphil anti-HIV neutralizing antibody-like molecule. [Diagram 3] FIG. 1 is a schematic diagram of the mature molecular structure of a predicted biphilic anti-HIV neutralizing antibody-like molecule existing alone. [Figure 4] FIG. 1 is a schematic diagram showing the structure of the gene sequence of a biphilic anti-HIV neutralizing antibody-like molecule. [Diagram 5] FIG. 1 is a schematic diagram of a gene construct in which the gene sequence of a biphilic (KL-BsHIV01-02) or triphilic (KL-BsHIV01-003-02) anti-HIV neutralizing antibody-like molecule was cloned into a lentivirus vector. [Figure 6] 1 is a graph of the latest generation lentiviral vector pKL-kan-lenti-CBH-WPRE applied to the present invention. [Figure 7] FIG. 1 is a schematic diagram of a gene construct in which the gene sequence of a biphilic (KL-BsHIV01-02) or triphilic (KL-BsHIV01-003-02) anti-HIV neutralizing antibody-like molecule was cloned into an adeno-associated virus vector. [Figure 8] 1 is a graph of the latest generation adeno-associated virus vector pAAV-MCS-CBH-WPRE applied to the present invention. [Figure 9]Detection of anti-HIV neutralizing antibody-like molecules by Western blot analysis. M, prestained protein size marker; 1 and 6, purified Flag-KL-BsHIV01-003-02; 2 and 7, purified KL-BsHIV01-003-02; 3-5 and 8-10, products after enzymatic cleavage of purified Flag-KL-BsHIV01-003-02 with enterokinase under different conditions. 1-5, primary antibody is rabbit Anti-KL-BsHIV01-003-02 multiplex antibody; 6-10, primary antibody is mouse Anti-DYKDDDDK (Flag-tag). [Figure 10] 1 shows the neutralizing activity of Miyoshi KL-BsHIV01-003 (having an Fc fragment) and KL-BsHIV01-003-02 (without an Fc fragment) anti-HIV neutralizing antibody-like molecules. [Figure 11] Expression of anti-HIV neutralizing antibodies in BALB / c mice after intramuscular injection of different doses of adeno-associated virus gene therapy vectors. The levels of anti-HIV neutralizing antibody-like molecules secreted into mouse serum were measured by ELISA. [Figure 12] Expression of anti-HIV neutralizing antibodies in BALB / c mice after intramuscular injection of different doses of adeno-associated virus gene therapy vectors. Neutralizing activity of anti-HIV neutralizing antibody-like molecules secreted into mouse serum. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] Broadly neutralizing antibodies (bNAbs) with HIV-1 neutralizing activity have been produced in a small proportion of HIV-1 infected individuals over the course of several years. They can efficiently bind to a broad range of HIV viral surface glycoproteins, neutralize the infectious activity of HIV to CD4+ T cells, and represent a promising method to prevent or combat HIV-1 infection. However, the specific mechanism of their production is unclear, and most HIV-1 infected individuals can only produce non-neutralizing antibodies. Currently, there have been no successful studies in inducing the production of broadly neutralizing antibodies in healthy subjects by standard immunization methods.

[0060] Without wishing to be bound by theory, in some embodiments, it is believed that recombinant-derived broadly neutralizing antibodies against HIV-1 virus can effectively reduce the viral load in the patient's body, and even if they cannot completely eliminate HIV in the body, they can contribute to controlling the progression of HIV infection to AIDS. Compared with small molecule anti-AIDS virus drugs, recombinant-derived neutralizing antibodies can also greatly reduce toxic side effects and increase patient compliance with administration. In clinical practice, recombinant broadly neutralizing antibodies can not only be used in vaccine replacement products in certain situations to prevent the occurrence of AIDS virus infection, but can also be used as antiviral drugs in different disease steps.

[0061] However, like highly efficient small molecule anti-AIDS virus drugs, specific broadly neutralizing antibodies are only directed against a single epitope on a single viral protein (HIV-gp160), and it is still difficult to avoid the escape phenomenon caused by viral mutation. In addition, the costs of developing, producing, and using broadly neutralizing antibodies are all much higher than those of small molecule anti-AIDS virus drugs, and there is no advantage to combination drugs. Therefore, the large number of broadly neutralizing antibody drugs for the anti-AIDS virus that are under research have been difficult to apply to clinical practice so far.

[0062] In order to address the above challenges, research and development of broadly neutralizing antibody drugs against the AIDS virus needs to make breakthroughs in the following two areas: 1. Research and develop biphilic or polyphilic neutralizing antibodies or antibody-like polymer drugs to cover multiple targets and avoid escape due to viral mutation. 2. Genetization of broadly neutralizing protein antibodies or antibody-like polymer drugs. Anti-AIDS virus infection gene therapy drugs delivered by recombinant viruses or non-viral vectors can stably express neutralizing antibodies or antibody-like polymers in vivo for a long period of time through genome integration or non-integration, so that a single treatment can be effective for a long period of time, even for life, and greatly reduce the production and use costs of polymer antibody drugs.

[0063] The present invention employs a polyphilic antibody molecular structure, i.e., two or more single-chain variable fragments (scFv) of monoclonal broadly neutralizing antibodies are connected in series by a linker polypeptide to form an antigen-binding region.

[0064] A specific embodiment includes constructing a series of antibody molecule gene constructs containing biphilic / triphilic antibody single chain variable fragments (scFv) and cloning them into recombinant adeno-associated virus and recombinant lentivirus vectors. Then, packaging the corresponding adeno-associated virus and lentivirus in 293T cells, and infecting 293T cells and differentiated and undifferentiated muscle cell lines with a certain biological titer of virus. The antibody molecules produced in the cell supernatant are quantitatively and qualitatively detected, and the neutralizing activity against HIV-1 wild type virus strains is detected in the infection activity assay of HIV-1 wild type virus strains and virus-sensitive reporter gene cell line TZM-bl.

[0065] The structure of the anti-HIV neutralizing antibody of the present invention, the composition of the gene sequence construct, and related terminology will be described in more detail below in combination with examples.

[0066] Antibody: may be an immunoglobulin, an antigen-binding fragment, or a protein molecule derived therefrom that can specifically recognize and bind to an antigen (e.g., HIV-1 gp41 antigen). In the present invention, "antibody" is broadly defined and includes various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, polyphilic antibodies (e.g., biphilic antibodies, triphilic antibodies), and antibody fragments, so long as they have specific antigen-binding activity. Specific examples of antibodies include complete immunoglobulins, as well as antibody variants and fragments that retain antigen-binding affinity. Examples of antibody fragments include, but are not limited to, variable region fragments (Fv), antigen-binding fragments (e.g., Fab, Fab', Fab'-SH, or F(ab')2 generated after hydrolysis by proteases), single-chain antibody molecules (e.g., scFv), diabodies, nanobodies, and polyphilic antibody fragments formed from a combination of antibody fragments. Antibody fragments include antigen-binding fragments generated by modification from complete antibodies, or antigen-binding fragments synthesized de novo using recombinant DNA technology.

[0067] A single-chain antibody (scFv) is a molecule obtained by genetic engineering, which contains one or more antibody light chain variable regions (VL) and heavy chain variable regions (VH), each fragment being connected in series with a suitable linker polypeptide to form a single fused single-chain molecule. The tandem order of VL and VH in a single-chain antibody molecule usually does not affect its antigen-binding function, so any single-chain antibody consisting of two tandem arrangements (VL-VH or VH-VL) can be used.

[0068] An antibody may have one or more antigen-binding sites. When an antibody has one or more antigen-binding sites, the binding sites may be the same or different. For example, a naturally occurring immunoglobulin has two identical antigen-binding sites, whereas a Fab fragment produced from an immunoglobulin by hydrolysis of papain has only one antigen-binding site, and a biphilic single-chain antibody (scFv) has two different antigen-binding sites.

[0069] Normally, a naturally produced immunoglobulin consists of a light chain and a heavy chain, which are connected by disulfide bonds. Immunoglobulin genes include gamma, alpha, delta, epsilon, mu, lambda, and kappa constant region genes, as well as numerous immunoglobulin variable region genes. There are two types of light chains, gamma and kappa. There are five main types of heavy chains (gamma, alpha, delta, epsilon, mu), which determine the functional typing of antibody molecules, and are IgG, IgA, IgD, IgE, and IgM, respectively.

[0070] Each heavy and light chain comprises one constant region and one variable region. VH represents the variable region of the heavy chain of an antibody, including the heavy chain variable region of an antigen-binding fragment Fv, scFv, or Fab. VL represents the variable region of the light chain of an antibody, including the light chain variable region of an antigen-binding fragment Fv, scFv, or Fab. In the following examples, VH and VL together play a role in specifically recognizing and binding to an antigen.

[0071] VH and VL are composed of three separate hypervariable regions (also called complementarity determining regions (CDRs)) and a frame region. The sequences of the frame regions of different light and heavy chains are relatively conservative within the same genus. The frame region of an antibody determines the position of the complementarity determining regions in the three-dimensional structure. The complementarity determining regions are mainly responsible for binding to the antigenic determinant cluster of an antigen. The three complementarity determining regions in the light chain are designated LCDR1, LCDR2, and LCDR3, respectively, from the N-terminus to the C-terminus. The three complementarity determining regions in the heavy chain are designated HCDR1, HCDR2, and HCDR3, respectively, from the N-terminus to the C-terminus.

[0072] The VH and VL protein sequences of the present invention include, in addition to the sequences disclosed in the Examples below, any other sequence carrying a functional fragment thereof, or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identity thereto.

[0073] Antibody constant region fragment: A region in which the amino acid sequence of an immunoglobulin is relatively stable other than the variable region, which has a large variation in the amino acid sequence near the N-terminus, and includes the constant domain in the antigen-binding fragment (located at the N-terminus of the hinge region, including the light chain constant domain and the heavy chain constant domain) and the constant domain of the crystallizable fragment of the heavy chain (called the Fc fragment, located at the C-terminus of the hinge region). The Fc fragment usually refers to the last two constant region domains of immunoglobulins IgA, IgD, and IgG, or the last three constant region domains of IgE and IgM. The Fc fragment may include a part or all of the hinge region sequence located at its N-terminus.

[0074] Anti-HIV-1 neutralizing antibodies or antigen-binding fragments: The neutralizing antibodies or antigen-binding fragments specifically bind to HIV-1 envelope proteins (e.g., bind to gp41) and thereby inhibit biological functions associated with the HIV-1 envelope (e.g., the ability to bind to target receptors). In the following examples, the anti-HIV-1 neutralizing antibodies or antigen-binding fragments reduce the infectivity of HIV-1 virus strains of different tropisms to cells.

[0075] Biphilic or polyphilic antibodies: These antibodies are recombinant molecules consisting of two or more different antigen binding domains and therefore can bind to two or more different antigenic determinant clusters. Biphilic or polyphilic antibodies include molecules in which two or more different antigen binding domains are connected by chemical synthesis or genetic engineering methods. The antigen binding domains can be connected via a linker polypeptide. The antigen binding domains can be monoclonal antibodies, antigen binding fragments (e.g., scFv or Fab), or a combination of antigen binding domains of different origin.

[0076] Linker polypeptide: for connecting two protein molecules or fragments into one continuous single fusion molecule, for example, in the following examples, it connects two or more antibody molecules or antigen-binding fragments (e.g., scFv) to form a polyphilic antibody molecule having two or more antigen-binding sites, or connects the light chain variable region (VL) and heavy chain variable region (VH) of one antibody to form one single-chain antigen-binding sequence, or connects an antibody molecule or antigen-binding fragment to another effector molecule, for example, connects an antigen-binding fragment scFv to an HIV-1 membrane fusion inhibitor polypeptide to form one fusion protein. The linker polypeptide is usually rich in glycine (Gly or G) to improve the flexibility of the linker, and contains serine (Ser or S) or threonine (Thr or T) to improve solubility, for example, (GGGGS) of different lengths used in some of the following examples. n linker, where n may be 1 or more than 1. However, the linker polypeptide sequences used in the examples are not limited thereto and also include other alternative linker polypeptide sequences.

[0077] Antigenic determinant cluster: A specific chemical group or polypeptide sequence on a molecule that has antigenicity, i.e., is capable of eliciting a specific immune response in a host. An antibody specifically binds to a specific antigenic determinant cluster on a polypeptide, for example, in the following example, an antibody that can specifically bind to an antigenic determinant cluster on gp41.

[0078] HIV-1 envelope protein: HIV-1 envelope protein is first synthesized in the form of a precursor protein with a size of 845-870 amino acid residues, called HIV gp160. gp160 forms a homotrimer in the host cell, is glycosylated, sheared to remove the signal peptide, and is cleaved at 511 / 512 amino acid residues by a single intracellular protease to generate gp120 and gp41 polypeptide chains. gp120 and gp41 are associated in homotrimers as gp120 / gp41 protomers. Mature gp120 consists of 31-511 amino acid residues of HIV-1 envelope protein, is a highly N-glycosylated protein, and constitutes the majority of the domain exposed on the envelope surface of the HIV-1 envelope protein trimer. gp120 is responsible for binding to the human CD4 cell receptor and co-receptors (e.g., chemotactic factor receptors CCR5 or CXCR4). gp41 is composed of 512-860 amino acid residues of the HIV-1 envelope protein, and includes an intraenvelope domain, a transmembrane domain, and an extraenvelope domain. The extraenvelope domain of gp41 contains 512-644 amino acid residues and binds to gp120 to form a protomer, which together constitutes the HIV-1 envelope protein homotrimer. The domain protruding outside the envelope of the HIV-1 envelope protein homotrimer undergoes several structural rearrangements, from a blocking structure before fusion with the host cell membrane that can avoid antibody recognition, and an intermediate structure that binds to the human CD4 cell receptor and co-receptor, to a structure after membrane fusion.

[0079] HIV membrane fusion inhibitor polypeptide: Membrane fusion between the virus and the host cell is an important step in the infection of cells by HIV. After the glycoprotein gp120 / gp41 identical trimer on the HIV envelope binds to the human CD4 cell receptor and co-receptor, it undergoes several steps of conformational changes, and finally the gp41 trimer incorporated on the HIV envelope is inserted into the host cell membrane, completing the fusion of the virus and the host cell membrane, and the genetic material of HIV enters the cell. The HIV membrane fusion inhibitor polypeptide binds to the viral envelope protein to prevent the occurrence of the conformational changes required for the fusion of the virus with the host CD4 cell membrane, thereby inhibiting HIV infection of CD4 cells. The HIV membrane fusion inhibitor polypeptide used in the present invention includes the P52, C34, and T20 sequences disclosed in the following examples, or homologous sequences having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0080] Genetic sequence construct: A vector consisting of a recombinant polynucleotide sequence, in which an expression control sequence and a nucleic acid sequence to be expressed are connected. An expression vector contains sufficient cis-acting elements, and other expression elements are provided by the host cell. Expression vectors include all vectors in the present invention, including, for example, plasmids and viruses (e.g., recombinant lentiviruses and recombinant adeno-associated viruses) into which a recombinant polynucleotide sequence is incorporated. A vector contains a nucleic acid sequence (DNA or RNA) that allows it to replicate in a host cell, such as an origin of replication, one or more selectable marker genes, and other genetic structures disclosed in the present invention. A viral vector is a recombinant nucleic acid vector, and contains at least a portion of a nucleic acid sequence derived from one or more viruses. In some examples below, the viral vector contains one or more nucleic acid sequences encoding an antibody or antigen-binding fragment that can specifically bind to the disclosed HIV-1 gp160 and neutralize HIV-1. In some examples, the viral vector is a recombinant adeno-associated virus (AAV) vector or a recombinant lentivirus vector. These viral vectors are replication-defective vectors and require other helper plasmids or vectors carrying gene functions or components necessary for viral replication to amplify and package in cells to produce viral particles. The purified viral vectors or viral particles cannot be replicated in host cells during the course of treating patients.

[0081] Treatment of HIV infection: HIV is a virus that attacks the human immune system. It mainly attacks CD4 T lymphocytes, the most important cell in the human immune system, destroying them in large numbers, causing the body to lose immune function. Many patients in the acute HIV infection stage have flu-like symptoms 2-4 weeks after infection, which may last for several days to several weeks. There is a large amount of HIV in the blood of patients in the acute infection stage, and they are highly infectious. Then, they enter the asymptomatic chronic infection stage, also called the HIV latency stage. However, HIV in the patient's body is still active, continues to grow, and transmits HIV. The average latency period of HIV in the human body is 8-9 years, and during the HIV virus latency period, HIV-infected people can live and work for many years without any symptoms. However, if HIV-infected people do not undergo anti-HIV infection treatment, the HIV will progress through the latency stage to the most serious HIV infection stage, that is, the AIDS stage. AIDS patients have a high viral load, are easily able to transmit HIV to others, and have severely damaged immune systems, making the human body more susceptible to various diseases, leading to the development of malignant tumors and a high mortality rate.

[0082] At present, there is still no effective drug that can cure HIV infection in the whole world. The treatment goal at the current stage is to reduce the viral load in a sustained manner to the maximum extent, obtain the restructuring and maintenance of immune function, improve the quality of life, and reduce the HIV-related morbidity and mortality. The currently commonly used method of anti-HIV infection is antiretroviral combination therapy (i.e., cocktail therapy), which has significantly improved the therapeutic effect of anti-HIV and significantly improved the quality of life and prognosis of patients. For example, treatment is started after HIV infection is confirmed during the acute phase of HIV infection, i.e., the first few months after infection. However, the side effects and limitations of cocktail therapy, such as the need to maintain long-term and continuous compliance with medication, and the gradually emerging drug-resistant strains of HIV, still cause long-term economic and social burdens, significant decline in quality of life, and significant suffering to a vast number of AIDS patients.

[0083] Broadly neutralizing antibodies with HIV-1 neutralizing activity identified and isolated in a small proportion of HIV-infected individuals can efficiently bind to the glycoproteins on the surface of the HIV virus over a broad range, neutralizing the infectious activity of the HIV virus to human CD4+ T cells, and representing a promising method to prevent or fight HIV-1 infection. Compared with small molecule anti-HIV drugs, recombinantly derived neutralizing antibodies can significantly reduce toxic side effects and increase patient compliance. At present, the only anti-HIV neutralizing antibody in clinical use is Ibalizumab, which targets the CD4 receptor on the surface of human T cells and blocks the binding of the glycoproteins on the surface of the HIV virus to the CD4 receptor by binding to the CD4 receptor, thereby neutralizing the infectious activity of the virus to CD4+ T cells, and showing excellent therapeutic effects on multi-resistant patients infected with the AIDS virus.

[0084] However, a specific broadly neutralizing antibody is only directed against a single epitope of a single viral protein (HIV-gp160), so it is still difficult to avoid the escape phenomenon caused by viral mutation, and the development, production and use costs of broadly neutralizing antibodies are all much higher than those of small molecule anti-AIDS virus drugs, and there is no advantage in combination drugs. The present invention researches and develops biphilic and polyphilic neutralizing antibodies or antibody-like polymer drugs, and covers multiple targets to avoid escape caused by viral mutation, and gene-izes protein broadly neutralizing antibodies or antibody-like polymer drugs to make a breakthrough in the research and development of anti-HIV broadly neutralizing antibody drugs. Gene therapy drugs for anti-HIV infection delivered by recombinant viruses or non-viral vectors can stably express neutralizing antibodies or antibody-like polymers in vivo for a long period of time through genome integration or non-integration, and a single treatment can be effective for a long period of time (for example, the drug effect lasts for one or several years) and even for life, greatly reducing the production and use costs of polymeric antibody drugs.

[0085] The following examples are illustrative of the present invention, but the present invention is not limited to the following examples. EXAMPLES

[0086] 1. Design of a structure expressing HIV neutralizing antibodies The construction of anti-HIV neutralizing antibodies and antibody-like molecules applicable to the present invention includes the following steps: 1. Miphilic anti-HIV neutralizing antibody-like scFV molecule: The protein molecule consists of -signal peptide-anti-HIV-gp41 broadly neutralizing antibody single chain variable region fragment (VL-(ggggs) n linker-VH)-(ggggs) n Linker - Single chain variable region fragment of anti-human CD4 antibody (VL-(ggggs) n linker-VH)-(ggggs) n linker-HIV membrane fusion-inhibiting short peptide. The gene coding sequence shown is expressed in cells, translated into protein, and then secreted outside the cells. The mature molecular structure of the expected anti-HIV neutralizing antibody-like molecule is shown in Figure 1, and the gene construction is shown in Figure 2. 2. Biphilic anti-HIV neutralizing antibody-like scFv molecule: The protein molecule consists of a signal peptide and a single-chain variable region fragment of an anti-HIV-gp41 broadly neutralizing antibody (VL-(ggggs) n linker-VH)-(ggggs) n Linker - Single chain variable region fragment of anti-human CD4 antibody (VL-(ggggs) n The gene coding sequence shown is expressed in cells, translated into protein, and then secreted outside the cells. The mature molecular structure of the expected anti-HIV neutralizing antibody-like molecule is shown in Figure 3, and the gene structure is shown in Figure 4.

[0087] II. Gene sequence expressing HIV neutralizing antibodies Here, the single-chain variable region fragment (scFv) sequences of the anti-HIV-gp41 broadly neutralizing antibody are a signal peptide (see SEQ ID NO:1 for the protein sequence), an anti-HIV-1-gp41-MHER antibody (10E8v4-5R+100cF)-scFv (see SEQ ID NO:2 for the protein sequence), a single-chain variable region fragment scFv of an anti-human CD4 antibody (Ibalizumab) (see SEQ ID NO:3 for the protein sequence), an HIV membrane fusion inhibitor short peptide P52 (see SEQ ID NO:4 for the protein sequence), an HIV membrane fusion inhibitor short peptide C34 (see SEQ ID NO:5 for the protein sequence), and an HIV membrane fusion inhibitor short peptide T20 (see SEQ ID NO:6 for the protein sequence).

[0088] Third, two anti-HIV neutralizing antibody gene expression frameworks (shown in Figures 2 and 4) were constructed, as follows: 1. Anti-HIV biphilic neutralizing antibody-like molecule KL-BsHIV01-02 (protein sequence see SEQ ID NO:7) (DNA sequence see SEQ ID NO:8) comprising anti-HIV neutralizing antibody 10E8v4-5R+100cF-scFv (Figure 4); 2. Anti-HIV triple neutralizing antibody-like molecule KL-BsHIV01-003-02 (protein sequence see SEQ ID NO:9) comprising anti-HIV neutralizing antibody 10E8v4-5R+100cF-scFv (DNA sequence see SEQ ID NO:10) (Figure 2).

[0089] IV. Examples of HIV neutralizing antibody gene therapy vector constructs As shown in Figure 5, the monoclonal antibody molecule gene expression framework was cloned into the currently used latest generation lentiviral vector pKL-kan-lenti-CBH-WPRE (Figure 6) (DNA sequence see SEQ ID NO: 11) in the manner of multi-fragment recombination ligation. The lentiviral vector includes: 5'LTR, in which the promoter region of the LTR is replaced with the CMV promoter; ψ packaging signal; retroviral output element RRE; cPPT; promoter CBH; polynucleotide encoding the HIV neutralizing antibody fragment polypeptide; post-transcriptional regulatory element WPRE; PPT; ΔU3 3'LTR; and poly(A) signal. The neutralizing antibody gene expression framework designed in this embodiment (FIGS. 2 and 4) was synthesized by GenScript (Nanjing) Co. Ltd., and then cloned between the CBH promoter (sequence see SEQ ID NO: 22) and the multiple cloning site AgeI / EcoRV on the lentiviral vector frame pKL-kan-lenti-CBH-WPRE by a method of homologous recombination well known in the art (FIG. 6). After cloning was completed, the sequence information was confirmed by sequencing, and the plasmids were named pKL-Kan-lenti-CBH-KL-BsHIV01-02 (sequence see SEQ ID NO: 12) and pKL-Kan-lenti-CBH-KL-BsHIV01-003-02 (sequence see SEQ ID NO: 13).

[0090] As shown in Figure 7, the HIV neutralizing antibody gene expression frameworks CBH-KL-BsHIV01-02 and CBH-KL-BsHIV01-003-02 present in pKL-Kan-lenti-CBH-KL-BsHIV01 were respectively cloned between the multiple cloning sites AgeI / SalI of the latest generation adeno-associated virus vector pAAV-MCS-CBH-WPRE (sequence see SEQ ID NO: 14) currently applied in the manner of multi-fragment recombination junction (see Figure 8). The adeno-associated virus vector includes: AAV2 ITR; promoter CBH; polynucleotide encoding HIV neutralizing antibody fragment; WPRE and SV40 poly(A) signal; AAV2 ITR. The plasmids are designated pAAV-CBH-KL-BsHIV01-02-WPRE (sequence see SEQ ID NO:15), pAAV-CBH-KL-BsHIV01-003-02-WPRE (sequence see SEQ ID NO:16).

[0091] 5. Packaging and purification of viruses expressing HIV neutralizing antibodies Packaging of HIV neutralizing antibody gene therapy lentiviral vectors was carried out in 293T cell lines using lentiviral vectors (pKL-Kan-lenti-CBH-KL-BsHIV01-02, pKL-Kan-lenti-CBH-KL-BsHIV01-003-02). The HIV neutralizing antibody gene therapy lentiviral vectors constructed in the examples (pKL-Kan-lenti-CBH-KL-BsHIV01-02, pKL-Kan-lenti-CBH-KL-BsHIV01-003-02, envelope plasmid (pKL-Kan-Vsvg, the nucleotide sequence of which is shown in SEQ ID NO: 17) and packaging plasmids (pKL-Kan-Rev, the nucleotide sequence of which is shown in SEQ ID NO: 18; pKL-Kan-GagPol, the nucleotide sequence of which is shown in SEQ ID NO: 19) were used. After mixing the 293T cells with the HIV neutralizing antibody gene therapy lentivirus, the 293T cells were transfected with the HIV neutralizing antibody gene therapy lentivirus (purchased from American Type Culture Collection (ATCC), with the ATCC deposit number CRL-3216) at the same time. The transfection method was PEI cationic polymer-mediated eukaryotic cell transient transfection, the PEI cationic polymer was PEI-Max transfection reagent purchased from Polysciences (purchased from Polysciences, catalog number: 24765-1), the transfection procedure was performed with reference to the standardized procedure recommended by the manufacturer, and the transfection scale was a 15 cm cell culture dish. 48 hours after the transfection was completed, the lentivirus vector (transfected cell culture supernatant) was harvested and first centrifuged at 4000 rpm for 5 minutes at room temperature in a tabletop bucket centrifuge to remove cell fragments, and then further centrifuged at 4°C. The virus particles were precipitated by centrifugation at 10,000 g for 4 hours, and the centrifugation supernatant was removed. 1 mL of DMEM complete medium was then added to the virus particle precipitate, and the virus particles were resuspended using a microinjector. The prepared virus resuspension was aliquoted and frozen and stored at -80°C.

[0092] The HIV neutralizing antibody gene therapy AAV vector was packaged in 293T cell line using AAV expression vectors (pAAV-CBH-KL-BsHIV01-02-WPRE, pAAV-CBH-KL-BsHIV01-003-02-WPRE). The HIV neutralizing antibody gene therapy AAV vector constructed in the examples, capsid plasmid (AAV2 / 8, its nucleotide sequence shown in SEQ ID NO: 20) and packaging plasmid (pHelper, its nucleotide sequence shown in SEQ ID NO: 21) were mixed and transfected simultaneously into 293T cells, and the HIV neutralizing antibody gene therapy vector AAV was packaged in the 293T cell line. The transfection method was PEI cationic polymer-mediated transient transfection of eukaryotic cells, the PEI cationic polymer was PEI-Max transfection reagent purchased from Polysciences (purchased from Polysciences, catalog number: 24765-1), the transfection operation was performed with reference to the standardized operation recommended by the manufacturer, and the transfection scale was 15 cm cell culture dish. 7 h after the transfection was completed, the supernatant was aspirated and discarded, and replaced with 25 ml of toxic production medium. 120 hours after the transfection was completed, the supernatant and cells were collected and centrifuged at 4200 rpm for 10 min, and after the centrifugation was completed, the supernatant and cells were separated, and the lysate and ribozyme were added to the cells, lysed and digested for 1 h, and centrifuged at 10000 g for 10 min to obtain the lysate supernatant. The lysate supernatant and medium supernatant were purified by affinity chromatography and then frozen and stored at -80 ° C.

[0093] 6. Functional verification of HIV neutralizing antibodies expressed in the supernatant of cells transduced with lentiviral gene therapy vectors The packaged lentiviral vectors pKL-Kan-lenti-CBH-KL-BsHIV01-02 and pKL-Kan-lenti-CBH-BsHIV01-003-02 were infected into 293T cells at different MOIs. After 48 hours, the supernatants and aliquot cells were collected. The lentiviral vector infection copy number was detected by the probe method. 1. Collect 293T cells infected with lentiviral vectors, wash the cells with PBS, and then collect the cells by centrifugation at 4200 rpm for 5 minutes. Resuspend the cells in 20 μl of high-speed extraction solution (QE DNA Extraction Solution). Run the following program in a PCR machine to lyse the cells and extract total DNA.

[0094] [Table A]

[0095] The lentiviral copy number (VCN) of 293T cell infection is calculated by quantitative PCR using methods well known in the art.

[0096] 2. TZM-bl cells were plated at 2E4 cells / well. 50μL of different cell antibody expression supernatants with VCN of 1 were taken and diluted at different magnifications, then mixed with 50μL of HIV virus pAD-8 or pNL4-3, incubated at 37℃ for 30min, and added to TZM-bl cells. The wells with only pAD-8 or pNL4-3 were negative controls (NC), and the wells without HIV virus were blank. At 24 hours, the supernatant was discarded, 100μL of cell lysis solution was added, and the lysed cells were collected after 10min and centrifuged at 8000rpm for 5min. 100μL of firefly luciferase detection reagent was added to 50μL of lysed cells. RLU (relative light unit) was detected and measured with a multi-function microplate reader with chemiluminescence function. As shown in the results, in vitro cell tests showed that, when the infectious copy number was the same, antibodies capable of neutralizing the HIV virus were present in the culture supernatant of 293T cells transduced with the HIV-neutralizing antibody lentiviral gene therapy vector, and the neutralizing effect of the biphilic neutralizing antibody KL-BsHIV01-02 was significantly superior to that of KL-BsHIV01 (Table 1), and the neutralizing effect of the triphilic neutralizing antibody KL-BsHIV01-003-02 was significantly superior to that of KL-BsHIV01-003 (Table 2).

[0097] [Table 1]

[0098] [Table 2]

[0099] 7. Functional verification of HIV neutralizing antibodies expressed in the supernatant of cells transduced with AAV gene therapy vectors C2C12 myoblasts were cultured in 24-well cell culture plates with 1E5 cells per well. They were differentiated into myotubes in 2% horse serum medium. Packaged adeno-associated viruses pAAV-CBH-KL-BsHIV01, pAAV-CBH-KL-BsHIV01-02, pAAV-CBH-KL-BsHIV01-003, and pAAV-CBH-KL-BsHIV01-003-02 were infected into differentiated C2C12 cells according to different MOIs. Supernatants were collected after 96 hours.

[0100] TZM-bl cells were plated at 2E4 cells / well. The cell expression supernatants of KL-BsHIV01, KL-BsHIV01-02, KL-BsHIV01-003, and KL-BsHIV01-003-02, which also had an MOI of 8E4 vg, were taken, and the cell expression supernatants of the biphilic antibody KL-BsHIV01 and the triphilic antibody KL-BsHIV01-003, which did not contain an Fc segment, were taken in the same volume as the Fc segment-containing biphilic antibody KL-BsHIV01 and the triphilic antibody KL-BsHIV01-003, respectively, and mixed with 50 μL of HIV virus pAD-8 or pNL4-3, incubated at 37° C. for 30 min, and added to TZM-bl cells. The wells to which only pAD-8 or pNL4-3 was added were negative controls (NC), and the wells to which no HIV virus was added were Blank. At 24 hours, the supernatant was discarded, 100 μL of cell lysis solution was added, and after 10 minutes, the lysed cells were collected and centrifuged at 8000 rpm for 5 minutes. 100 μL of firefly luciferase detection reagent was added to 50 μL of lysed cells. RLU (relative light unit) was detected and measured using a multifunctional microplate reader with chemiluminescence function. As a result (Table 3), in the in vitro cell test, when the MOI is the same, the neutralizing effect of scFv KL-BsHIV01-02 and KL-BsHIV01-003-02 without Fc segment against HIV virus is significantly superior to KL-BsHIV01 and KL-BsHIV01-003 with Fc segment.

[0101] [Table 3]

[0102] 8. Expression of adeno-associated virus gene therapy vectors in BALB / c mice after intramuscular injection HIV neutralizing antibody AAV gene therapy vectors (pAAV-CBH-KL-BsHIV01-02-WPRE, pAAV-CBH-KL-BsHIV01-003-02-WPRE) were injected intramuscularly into the hind thigh muscle of mice at a dose of 2E11 vg / animal. Blood was collected every week and serum was separated.

[0103] TZM-bl cells were plated at 2E4 cells / well. The same volume of serum dilution was taken and mixed with 50μL of HIV virus pAD-8 or pNL4-3, then incubated at 37℃ for 30min, and added to TZM-bl cells. The wells with only pAD-8 or pNL4-3 were negative controls (NC), and the wells without HIV virus were blank. At 24 hours, the supernatant was discarded, 100μL of cell lysis solution was added, and after 10min, the lysed cells were collected and centrifuged at 8000rpm for 5min. 100μL of firefly luciferase detection reagent was added to 50μL of lysed cells. RLU (relative light unit) was detected and measured using a multi-function microplate reader with chemiluminescence function. As shown in the results (Table 4), in an in vitro cell test, after the addition of the same volume of HIV-neutralizing antibody, the neutralizing effect against HIV virus of the scFv alone KL-BsHIV01-02 and KL-BsHIV01-003-02, which do not contain the IgG1 constant region Fc segment, was significantly superior to that of KL-BsHIV01 and KL-BsHIV01-003, which have an Fc segment (this is the structure of an anti-HIV neutralizing antibody-like molecule in another patent application of the company).

[0104] [Table 4]

[0105] 9. Identification of cells expressing HIV-neutralizing antibodies Lentiviral plasmid carrying Flag-KL-BsHIV01-003-02 was transiently transfected into 293T cells, and after 48 h, the supernatant was collected, and Flag-KL-BsHIV01-003-02 was purified using anti-DYKDDDDK (i.e., Flag-tag) G1 Affinity Resin (GenScript, L00432-10). The resulting Flag-KL-BsHIV01-003-02 was enzymatically cleaved with enterokinase (novoprotein, PE001-01A). Flag-KL-BsHIV01-003-02 before and after enzymatic cleavage was subjected to Western blotting assay using rabbit Anti-KL-BsHIV01-003-02 or mouse Anti-DYKDDDDK (GenScript, A00187-100) (Figure 9). Western blotting assay confirmed that the HIV neutralizing antibody KL-BsHIV01-003-02 bearing either a Flag tag or no Flag tag was expressed in cells and secreted into the supernatant, and had a molecular weight consistent with prediction.

[0106] 10. Identification of the activity of HIV neutralizing antibodies expressed in cells and purified TZM-bl cells were plated at 2E4 cells / well. Dilutions of purified antibodies were mixed with 50μL of HIV virus pAD-8 or pNL4-3, incubated at 37℃ for 30min, and added to TZM-bl cells. Wells with only pAD-8 or pNL4-3 were negative controls, and wells without HIV virus were blank. After 24h of incubation, the supernatant was discarded, 100μL of cell lysis solution was added, and lysed cells were collected after 10min and centrifuged at 8000rpm for 5min. 100μL of firefly luciferase detection reagent was added to 50μL of lysed cells. RLU (relative light unit) was detected and measured using a multi-function microplate reader with chemiluminescence function. As a result, in the cell test, the anti-HIV triphilic neutralizing antibody without Fc fragment (KL-BsHIV01-003-02) was similar to the triphilic neutralizing antibody with Fc fragment (KL-BsHIV01-003), and both had strong HIV neutralizing activity (Figure 10), and the IC50 of both neutralizing HIV-1 virus strain pAD-8 (CCR5-specific) was 3.1 pM and 1.2 pM, respectively, and the IC50 of both neutralizing HIV-1 virus strain pNL4-3 (CXCR4-specific) was 0.2 pM and 0.6 pM, respectively. These two HIV neutralizing antibodies of the present invention have stronger neutralizing activity against HIV than the previously reported broadly neutralizing antibodies against HIV.

[0107] 11. Expression of adeno-associated virus gene therapy vectors in BALB / c mice after intramuscular injection Different doses of AAV gene therapy vector (pAAV-CBH-KL-BsHIV01-003--02WPRE) were injected intramuscularly into the hind thigh muscle of mice. Blood was collected at regular intervals and serum was separated.

[0108] 1. ELISA: The antibody concentration expressed in the serum was detected. The synthesized HIV MHER polypeptide was coated on an ELISA PLATE (Corning, Catalog No.: 42592), and the supernatant-expressed and purified quantified KL-BsHIV01-003-02 standard was used as the primary antibody, rabbit anti-HIV membrane fusion inhibitor short peptide multi-antibody was used as the secondary antibody, and HRP-labeled goat anti-rabbit IgG (KPL, 5220-0283) was used as the tertiary antibody. TMB was used for color development, and the OD value at 450 nm was detected using a microplate reader. The ELISA results (Figure 11) show that the anti-HIV neutralizing antibody adeno-associated virus vector can persistently and effectively express HIV neutralizing antibodies in mice.

[0109] 2. TZM-bl cells were plated at 2E4 cells / well. Each serum dilution diluted 200 times was mixed with 50μL of HIV virus strain pNL4-3, then incubated at 37℃ for 30min, and added to TZM-bl cells. The wells with only pNL4-3 added were negative controls, and the wells without HIV virus were blanks. After overnight incubation, the supernatant was discarded, 100μL of cell lysis solution was added, and the lysed cells were collected after 10min and centrifuged at 8000rpm for 5min. 100μL of firefly luciferase detection reagent was added to 50μL of lysed cells. RLU (relative light unit) was detected and measured using a multi-function microplate reader with chemiluminescence function. As a result, in the cell test, it was shown that the HIV neutralizing antibodies expressed in mouse serum had excellent neutralizing activity against the HIV virus strain pNL4-3 after 200-fold dilution, and the activity increased with increasing dose (Figure 12).

[0110] SEQ ID NO: 1 MARPLCTLLLLMATLAGALA SEQ ID NO: 2 SELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVLGGGGSGGGGSGGGGSEVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWGQGTLVIVSS SEQ ID NO: 3 DIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKRTVAGGGGSGGGGSGGGGSQVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSS SEQ ID NO: 4 WEQKIEELLKKAEEQQKKNEEELKKLEK SEQ ID NO: 5 YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF SEQ ID NO: 6 LLEQENKEQQNQSEEILSHILSTYNNIERDWEMW

[0111] SEQ ID NO: 7 MARPLCTLLLLMATLAGALASELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVLGGGGSGGGGSGGGGSEVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWGQGTLVIVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKRTVAGGGGSGGGGSGGGGSQVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSS

[0112] SEQ ID NO: 8

[0113] SEQ ID NO: 9 MARPLCTLLLLMATLAGALASELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVLGGGGSGGGGSGGGGSEVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWGQGTLVIVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKRTVAGGGGSGGGGSGGGGSQVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSWEQKIEELLKKAEEQQKKNEEELKKLEK

[0114] SEQ ID NO: 10

[0115] SEQ ID NO: 11 TGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGACCGGTACGCGTGCCTCGGATCCTCCAGTGTGGTGTGCAGATATCCAGCACAGTCCCGGGCCGAGTCTAGACGTTTAAACCCGCTGATCAGGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCA CCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCG GCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCGCTAGCGTCGACCATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGGCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCCCTCGACATGGCAGTCTAGCACTAGTGCGGCCGCAGATCTGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAA

[0116] SEQ ID NO: 12 GGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGAGCTTGATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCCAGTTTACTTTGCAGGGCTTCCCAACCTTACCAGAGGCCTGCGCCGCGGCCAGCTGGCTAGCAATTCCCGGGTTAACTCTAGAGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACAT CTCCCCCACTCACAGTGACCCGGAATCCCTCGACATGGCAGTCTAGCACTAGTGCGGCCGCAGATCTGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAA

[0117] SEQ ID NO: 13 GTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTACCGGTCGCCACCATGGCGAGACCCCTGTGCACATTACTTCTGTTGATGGCTACCCTGGCAGGCGCCCTCGCCAGCGAGCTGACACAGGACCCTGCCGTGTCCGTGGCCCTGAAGCAGACCGTGACAATCACCTGCAGAGGCGATTCCCTGAGATCCCACTACGCCTCCTGGTACCAGAAGAAGCCTGGCCAGGCCCCCGTGCTGCTGTTTTACGGCAAGAATAACCGCCCCAGCGGCATCCCCGATAGATTTTCCGGCAGCGCCTCCGGCAACAGAGCCAGCCTGACAATCACCGGCGCCCAGGCCGAGGACGAGGCTGATTACTACTGCAGCTCCAGAGATAAGAGCGGCAGCAGACTGTCCGTGTTTGGCGGCGGCACCAAGCTGACCGTGCTCGGAGGAGGAGGAAGCGGAGGAGGAGGCTCAGGCGGCGGCGGCTCTGAGGTGAGGCTGAGAGAGTCCGGCGGCGGCCTGGTGAAGCCCGGAGGATCTCTGAGGCTGTCCTGCTCCGCCTCCGGCTTCGATTTTGACAATGCCTGGATGACCTGGGTGAGACAGCCCCCTGGCAAGGGCCTGGAGTGGGTGGGAAGGATCACAGGCCCCGGCGAGGGCTGGTCCGTGGATTACGCCGAGTCCGTGAAGGGCAGGTTCACAAT GCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCGCTAGCGTCGACCATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGGCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCCCTCGACATGGCAGTCTAGCACTAGTGCGGCCGCAGATCTGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGG TTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAA

[0118] SEQ ID NO: 14 AAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAG TTTGTAACCATCTAGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGTTTAAACAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

[0119] SEQ ID NO: 15 ACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATAT GAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAG TCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTACGCGTCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTACCGGTCGCCACCATGGCGAGACCCCTGTGCACATTACTTCTGTTGATGGCTACCCTGGCAGGCGCCCTCGCCAGCGAGCTGACACAGGACCCTGCCGTGTCCGTGGCCCTGAAGCAGACCG。 TGACAATCACCTGCAGAGGCGATTCCCTGAGATCCCACTACGCCTCCTGGTACCAGAAGAAGCCTGGCCAGGCCCCCGTGCTGCTGTTTTACGGCAAGAATAACCGCCCCAGCGGCATCCCCGATAGATTTTCCGGCAGCGCCTCCGGCAACAGAGCCAGCCTGACAATCACCGGCGCCCAGGCCGAGGACGAGGCTGATTACTACTGCAGCTCCAGAGATAAGAGCGGCAGCAGACTGTCCGTGTTTGGCGGCGGCACCAAGCTGACCGTGCTCGGAGGAGGAGGAAGCGGAGGAGGAGGCTCAGGCGGCGGCGGCTCTGAGGTGAGGCTGAGAGAGTCCGGCGGCGGCCTGGTGAAGCCCGGAGGATCTCTGAGGCTGTCCTGCTCCGCCTCCGGCTTCGATTTTGACAATGCCTGGATGACCTGGGTGAGACAGCCCCCTGGCAAGGGCCTGGAGTGGGTGGGAAGGATCACAGGCCCCGGCGAGGGCTGGTCCGTGGATTACGCCGAGTCCGTGAAGGGCAGGTTCACAATCTCCAGGGATAACACCAAGAACACCCTGTACCTGGAGATGAACAACGTGAGGACAGAGGATACCGGCTACTACTTTTGCGCCAGAACAGGCAAGTACTACGACTTTTGGTTCGGCTACCCCCCTGGCGAGGAGTACTTCCAGGATTGGGGCCAGGGCACCCTGGTCATTGTGTCCAGCGGCGGCGGCGGCAGTGGCGGCGGCGGAAGCGGCGGCGGCGGCTCTGGCGGCGGCGGCAGCGGCGGCGGCGGCTCCGACATCGTGATGACCCAGTCTCCTGATAGCCTGGCCGTGAGCCTGGGCGAGAGAGTGACAATGAACTGTAAGTCTAGCCAGAGCCTGCTGTACTCCACCAACCAGAAGAATTACCTGGCCTGGTATCAGCAG AAGCCTGGCCAGTCCCCAAAGCTGCTGATCTATTGGGCATCTACAAGGGAGAGCGGAGTGCCAGACAGATTCAGCGGATCCGGATCTGGAACCGACTTCACCCTGACAATCTCCTCTGTGCAGGCCGAGGACGTGGCCGTGTACTATTGCCAGCAGTACTATAGCTACAGGACATTCGGCGGCGGCACCAAGCTGGAGATCAAGCGCACCGTGGCCGGAGGAGGAGGATCTGGCGGAGGAGGGTCCGGCGGCGGCGGCTCCCAGGTGCAGCTGCAGCAGAGCGGACCAGAGGTGGTGAAGCCTGGAGCCTCCGTGAAGATGTCTTGTAAGGCCAGCGGCTACACCTTCACATCCTATGTGATCCACTGGGTGAGGCAGAAGCCAGGACAGGGACTGGACTGGATCGGCTACATCAACCCTTATAATGATGGCACCGACTACGATGAGAAGTTTAAGGGCAAGGCCACCCTGACATCCGATACCAGCACATCCACCGCCTATATGGAGCTGAGCTCCCTGCGGTCTGAGGACACAGCCGTGTACTATTGCGCCAGAGAGAAGGATAACTACGCAACCGGAGCATGGTTCGCATATTGGGGACAGGGTACCCTGGTCACCGTGTCTAGCTAGGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGG CTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCGAATTCGTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATCTAGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGTTTAAACAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

[0120] SEQ ID NO: 16 CTCCACCAACCAGAAGAATTACCTGGCCTGGTATCAGCAGAAGCCTGGCCAGTCCCCAAAGCTGCTGATCTATTGGGCATCTACAAGGGAGAGCGGAGTGCCAGACAGATTCAGCGGATCCGGATCTGGAACCGACTTCACCCTGACAATCTCCTCTGTGCAGGCCGAGGACGTGGCCGTGTACTATTGCCAGCAGTACTATAGCTACAGGACATTCGGCGGCGGCACCAAGCTGGAGATCAAGCGCACCGTGGCCGGAGGAGGAGGATCTGGCGGAGGAGGGTCCGGCGGCGGCGGCTCCCAGGTGCAGCTGCAGCAGAGCGGACCAGAGGTGGTGAAGCCTGGAGCCTCCGTGAAGATGTCTTGTAAGGCCAGCGGCTACACCTTCACATCCTATGTGATCCACTGGGTGAGGCAGAAGCCAGGACAGGGACTGGACTGGATCGGCTACATCAACCCTTATAATGATGGCACCGACTACGATGAGAAGTTTAAGGGCAAGGCCACCCTGACATCCGATACCAGCACATCCACCGCCTATATGGAGCTGAGCTCCCTGCGGTCTGAGGACACAGCCGTGTACTATTGCGCCAGAGAGAAGGATAACTACGCAACCGGAGCATGGTTCGCATATTGGGGACAGGGTACCCTGGTCACCGTGTCTAGCGGCGGAGGAGGAAGCGGAGGAGGCGGCTCTGGCGGAGGAGGTTCCGGAGGAGGCGGAAGCGGCGGAGGAGGCTCTTGGGAGCAGAAGATCGAGGAGCTGCTGAAGAAGGCCGAGGAGCAGCAGAAGAAGAATGAGGAGGAGCTGAAGAAGCTGGAGAAGTAGGTCGACGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTG。 TCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCGAATTCGTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATCTAGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGTTTAAACAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

[0121] SEQ ID NO: 17 GCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGAGCTTGATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCCAGTTTACTTTGCAGGGCTTCCCAACCTTACCAGAGGCCTGCGCCGCGGCCAGCTGGCTAGCAATTCCCGGGTTAACTCTAGAGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAG TGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACC CCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCA GCCTCCCCTCGAAGCTTACATGTGGTACCGAGCTCGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACCCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGAGAAGTAACAGGGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAAT CCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGCACGTGAGATCTGAATTCTGACACTATGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGGGGTGAATTGCAAGTTCACCATAGTTTTTCCACACAACCAAAAAGGAAACTGGAAAAATGTTCCTTCTAATTACCATTATTGCCCGTCAAGCTCAGATTTAAATTGGCATAATGACTTAATAGGCACAGCCTTACAAGTCAAAATGCCCAAGAGTCACAAGGCTATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTCACTACTTGTGATTTCCGCTGGTATGGACCGAAGTATATAACACATTCCATCCGATCCTTCACTCCATCTGTAGAACAATGCAAGGAAAGCATTGAACAAACGAAACAAGGAACTTGGCTGAATCCAGGCTTCCCTCCTCAAAGTTGTGGATATGCAACTGTGACGGATGCCGAAGCAGTGATTGTCCAGGTGACTCCTCACCATGTGCTGGTTGATGAATACACAGGAGAATGGGTTGATTCACAGTTCATCAACGGAAAATGCAGCAATTACATATGCCCCACTGTCCATAACTCTACAACCTGGCATTCTGACTATAAGGTCAAAGGGCTATGTGATTCTAACCTCATTTCCATGGACATCACCTTCTTCTCAGAGGACGGAGAGCTATCATCCCTGGGAAAGGAGGGCACAGGGTTCAGAAGTAACTACTTTGCTTATGAAACTGGAGGCAAGGCCTGCAAAATGCAATACTGCAAGCATTGGGGAGTCAGACTCCCATCAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAAGGG。 ACATAAAGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGCAAACAGCTAATGCACATTGGCAACAGCCCCTGATGCCTATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAGAGGCTTGATTTGGAGGTTAAAGTTTTGCTATGCTGTATTTTAGTCGACCATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGGCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCCCTCGACATGGCAGTCTAGCACTAGTGCGGCCGCAGATCTGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAA

[0122] SEQ ID NO: 18 GGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAAGCGCTTTTGAAGCTCGGATCCGAACAAACGACCCAACACCCGTGCGTTTTATTCTGTCTTTTTATTGCCGATCCCCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGC GGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGAGCTTGATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCCAGTTTACTTTGCAGGGCTTCCCAACCTTACCAGAGGCCTGCGCCGCGGCCAGCTGGCTAGCAATTCCCGGGTTAACTCTAGAGAA TGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACAATAAACGCCATTTGACCATTCACCACATTGGTGTGCACCTCCAAGCTCGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCCCTCGAAGCTAGTCGATTAGGCATCTCCTATGGCAGGAAGAAGCGGAGACAGCGACGAAGACCTCCTCAAGGCAGTCAGACTCATCAAGTTTCTCTATCAAAGCAACCCACCTCCCAATCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCCTTAGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTACAATATTGGAGTCAGGAGCTAAAGAATAGTGCTGTTAGCTTGCTCAATGCCACAGCTATAGCAGTAGCTGAGGGGACAGATAGGGTTATAGAAGTAGTACAAGAAGCTTGGCACTGGCCGTCGTTTTACA ACGTCGTGATCTGAGCCTGGGAGATCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCAGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTGTCGACCATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGGCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCCCTCGACATGGCAGTCTAGCACTAGTGCGGCCGCAGATCTGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCG GTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAA

[0123] SEQ ID NO: 19 GGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAAGCGCTTTTGAAGCTCGGATCCGAACAAACGACCCAACACCCGTGCGTTTTATTCTGTCTTTTTATTGCCGATCCCCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCC TGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCCCTCGAAGCTTACATGTGGTACCGAGCTCGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACCCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGAGAAGTAACAGGGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGC。 ACGTGAGATCTGAATTCGAGATCTGCCGCCGCCATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGACACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCAAATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGTAGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATCAAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGAATGGGATAGAGTGCATCCAGTGCATGCAGGGCCTATTGCACCAGGCCAGATGAGAGAACCAAGGGGATCAGACATCGCTGGAACTACTAGTACCCTTCAGGAACAAATAGGATGGATGACACATAATCCACCTATCCCAGTAGGAGAAATCTATAAAAGATGGATAATCCTGGGA TTAAATAAAATAAGAATGTATAGCCCTACCAGCATTCTGGACATAAGACAAGGACCAAAGGAACCCTTTAGAGACTATGTAGACCGATTCTATAAAACTCTAAGAGCCGAGCAAGCTTCACAAGAGGTAAAAAATTGGATGACAGAAAACCTTGTTGGTCCAAAATGCGAACCCAGATTGTAAGACTA TTTTAAAAGCATGGACCAGGAGCGACACTAGAAGAAAATGATGACAGCATGTCAGGGAGTGGGGGGACCGGCCATAAAGCAAGAGTTTTGGCTGAAGCAATGAGCCAAGTAACAAATCCAGCTACCATAATGATACAGAAAGGCAATTTTAGGAACCAAAGAAAGACTGTTAAGTGTTTCCAATTGTGG CAAAGAAGGCACATAGCCAAAAATTGCAGGGCCCCTAGGAAAAAGGCTGTTGGAAATGTGGAAAAGGAAGGACACCCAAATGAAAGATTGTACTGAGAGAGACAGGCTAATTTTTTAGGGAAGATCTGGCCTTCCCACAAGGGAAGGCCAGGGAATTTTCTTCAGAGCACACAAGCCCAGCCAAGCCCCACAGAAGAGCTTCAGGTTTGGGAAGAGACAACAACTCCCTCTCAGAAGCAGGAGCCGATAGACAAGGAACTGTATCCTTTAGCTCCCTCAGATCACTCTTTGGCAGCGACCCCCTCGTCTACAATAAAGATAGGGGGGCAATTAAAGGAAGCTCTATTAGATACTGGTGCTGACGACACAGTATTTAGAGA AATGAATTTGCCAGGAAGATGGAAACCAAAAATGATAGGGGGAATTGGAGGTTTTATCAAAGTAAGACAGTATGATCAGATACTCATAGAAATCTGCGGACATAAAGCTATAGGTACAGTATTAGTAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAGATTGGCTGCACTTTAAATTTTCCCATTAGTCCTATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGATGGCCCAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAGCATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGAAAAATTTCAAAAATTGGGCCTGAAAATCCATACAATACTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAAATGGAGAAAATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGATTTCTGGGAAGTTCAATTAGGAATACCACATCCTGCAGGGTTAAAACAGAAAAAATCAGTAACAGTACTGGATGTGGGCGATGCATATTTTTCAGTTCCCTTAGATAAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTATAAACAATGAGACACCAGGGATTAGATATCAGTACAATGTGCTTCCACAGGGATGGAAAGGATCACCAGCAATATTCCAGTGTAGCATGACAAAAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATAGTCATCTATCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAACTGAGACAACATCTGTTGAGGTGGGGATTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGCCTATAGTGCTGCCAGAAAAGGACAGCTGGACTGTCAATGACATACAGAAATTAGTGGGAAAATTGAATTGGGCAAGTCAG。 ATTTATGCAGGGATTAAGTAAGGCAATTATGTAAACTTCTTAGGGGAACCAAAGCACTAACAGAAGTAGTACCACTAACAGAAGAAAGCAGAGCTAGAACTGGCAGAAAACAGGGAGATTCTAAAGAACCGGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAATACAGAAGGAGGCAAGGCCAATGGACATATCAAATTTCAAGAGCCATTTAAAAAATCTGAAAACAGGAAGTATGCAAGAAGGGTGCCCACACTAATGATGTGAAACAATTAACAGAGGCAGTACAAAAAATAGCCACAGAAGCATAGTAATATGGGGAAAGACTCCTAAATTTAAATTACCCATACAAAAGGAAACATGGGAAGCATGGTGGACAGAGTATTGGCAAGCCACCAC TGGATTCCTGAGTGGGAGTTTGTCAATACCCCTCCCCTTAGTGAAGTTATGGTACCAGTTAGAGAAGAACCCATAATATAGGAGCAGAAACTTTCTATGTAGATGGGGCAGCCAATAGGGAAACTAAATTAGGAAAAGCAGGATATGTAACTGACAGAGGAAGACAAAAAGTTGTCCCCTAACGGACAACAAATCAGAAGACTGAGTTACAAGCAATTCATCTAGCTTTGCAGGATTCGGGATTAGAAGTAAACATAGTGACAGACTCACAATATGCATTGGGAATCATTCAAGCACAACCAGATAAGGTGAATCAGAGTTAGTCAGTCAAATAATAGAGACAGTTAATAAAAAAGGAAAAGTAGCTACCTGCATGGTACCAGCACACAAAGGAATTGGAGAAATGAACAAGTAGATGAATGAACAAGTAGAT AAATTGGTCAGTGCTGGAATCAGGAAAGTACTATTTTTAGATGGAATAGATAAGGCCCAAGAAGAACATGAGAAATATCACAGTAATTGGAGAGCAATGGCTAGTGATTTTAACCTACCCTGTAGTAGCAAAAAATAGTAGCCAGCTGTAGATAAATGTCAGCTAAAAGGGGAAGCCATGCATGGACAAGTAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAAGGAAAAGTTATCTTGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGAGACAGGGCAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCCAGTAAAAACAGTACATACAGACAAGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGCGGGGATCAA GCAGGAATTTGGCATTCCCTCAATCCGCAGTCACAAGGAGTAATAGAATCTATGAATAAAGAATTAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAAACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAAGAAAGGGGGGGATTGGGGGACAGTGCAGGGAAAGATATAGATAGCAACACACATACAAACTAAGAATTACAAAAATTACAAAAATTCAAATTTTCCGGTTTTACAGGGACAGCAGAGATCCAGTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAGGGCAGTAGTAATACAAGATAATAGTGACATAAAGTAGTGGCCAAGAAAAAGCAAAGATCATCAGGGATTATGGGAAAACAGATGGCAGGTGATGATTGTGTGGCAA GTAGACAGGATGAGGATTAACACATGGAATTCCGGAGCGGCCGCAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTCCGCGGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC AATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAAGGGAATGTGGGAGGTCAGTGCATTTAAAACATAAAGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGCAAACAGCTAATGCACATTGGCAACAGCCCCTGATGCCTATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAGAGGCTTGATTTGGAGGTTAAAGTTTTGCTATGCTGTATTTTACATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGGCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCCCTCGACATGGCAGTCTAGCACTAGTGCGGCCGCAGATCTGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGTCGACCATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTC ACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGGCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCCCTCGACATGGCAGTCTAGCACTAGTGCGGCCGCAGATCTGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAA

[0124] SEQ ID NO: 20 ATGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCGGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGGAGGCTCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGTTTTGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGAGCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAGGTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGTGGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCC ACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGGTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTG CAGCAAGCGCTGGAACCCCGAGATCCAGTACACCTCCAACTACTACAAATCTACAAGTGTGGACTTTGCTGTTAATACAGAAGGCGTGTACTCTGAACCCCGCCCCATTGGCACCCGTTACCTCACCCGTAATCTGTAATTGCCTGTTAATCAATAAACCGGTTGATTCGTTTCAGTTGAACTTTGGTCTCTGCGAAGGGCGAATTCGTTTAAACCTGCAGGACTAGAGGTCCTGTATTAGAGGTCACGTGAGTGTTTTGCGACATTTTGCGACACCATGTGGTCACGCTGGGTATTTAAGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGCCGCCAAGCCGAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGACTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGG TATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGA CAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAA AAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTT GGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGATCGAGGTCGACGGTATCGGGGGAGCTCGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGCCGCC

[0125] SEQ ID NO: 21GGTACCCAACTCCATGCTTAACAGTCCCCAGGTACAGCCCACCCTGCGTCGCAACCAGGAACAGCTCTACAGCTTCCTGGAGCGCCACTCGCCCTACTTCCGCAGCCACAGTGCGCAGATTAGGAGCGCCACTTCTTTTTGTCACTTGAAAAACATGTAAAAATAATGTACTAGGAGACACTTTCAATAAAGGCAAATGTTTTTATTTGTACACTCTCGGGTGATTATTTACCCCCCACCCTTGCCGTCTGCGCCGTTTAAAAATCAAAGGGGTTCTGCCGCGCATCGCTATGCGCCACTGGCAGGGACACGTTGCGATACTGGTGTTTAGTGCTCCACTTAAACTCAGGCACAACCATCCGCGGCAGCTCGGTGAAGTTTTCACTCCACAGGCTGCGCACCATCACCAACGCGTTTAGCAGGTCGGGCGCCGATATCTTGAAGTCGCAGTTGGGGCCTCCGCCCTGCGCGCGCGAGTTGCGATACACAGGGTTGCAGCACTGGAACACTATCAGCGCCGGGTGGTGCACGCTGGCCAGCACGCTCTTGTCGGAGATCAGATCCGCGTCCAGGTCCTCCGCGTTGCTCAGGGCGAACGGAGTCAACTTTGGTAGCTGCCTTCCCAAAAAGGGTGCATGCCCAGGCTTTGAGTTGCACTCGCACCGTAGTGGCATCAGAAGGTGACCGTGCCCGGTCTGGGCGTTAGGATACAGCGCCTGCATGAAAGCCTTGATCTGCTTAAAAGCCACCTGAGCCTTTGCGCCTTCAGAGAAGAACATGCCGCAAGACTTGCCGGAAAACTGATTGGCCGGACAGGCCGCGTCATGCACGCAGCACCTTGCGTCGGTGTTGGAGATCTGCACCACATTTCGGCCCCACCGGTTCTTCACGATCTTGGCCTTGCTAGACTGCTCCTTCAGCGCGCGCTGCCCGTTTTCGCTCGTCACATCCATTTCAATCACGTGCTCCTTATTTATCATAATGCTCCCGTGTAGACACT。TAAGCTCGCCTTCGATCTCAGCGCAGCGGTGCAGCCACAACGCGCAGCCCGTGGGCTCGTGGTGCTTGTAGGTTACCTCTGCAAACGACTGCAGGTACGCCTGCAGGAATCGCCCCATCATCGTCACAAAGGTCTTGTTGCTGGTGAAGGTCAGCTGCAACCCGCGGTGCTCCTCGTTTAGCCAGGTCTTGCATACGGCCGCCAGAGCTTCCACTTGGTCAGGCAGTAGCTTGAAGTTTGCCTTTAGATCGTTATCCACGTGGTACTTGTCCATCAACGCGCGCGCAGCCTCCATGCCCTTCTCCCACGCAGACACGATCGGCAGGCTCAGCGGGTTTATCACCGTGCTTTCACTTTCCGCTTCACTGGACTCTTCCTTTTCCTCTTGCGTCCGCATACCCCGCGCCACTGGGTCGTCTTCATTCAGCCGCCGCACCGTGCGCTTACCTCCCTTGCCGTGCTTGATTAGCACCGGTGGGTTGCTGAAACCCACCATTTGTAGCGCCACATCTTCTCTTTCTTCCTCGCTGTCCACGATCACCTCTGGGGATGGCGGGCGCTCGGGCTTGGGAGAGGGGCGCTTCTTTTTCTTTTTGGACGCAATGGCCAAATCCGCCGTCGAGGTCGATGGCCGCGGGCTGGGTGTGCGCGGCACCAGCGCATCTTGTGACGAGTCTTCTTCGTCCTCGGACTCGAGACGCCGCCTCAGCCGCTTTTTTGGGGGCGCGCGGGGAGGCGGCGGCGACGGCGACGGGGACGACACGTCCTCCATGGTTGGTGGACGTCGCGCCGCACCGCGTCCGCGCTCGGGGGTGGTTTCGCGCTGCTCCTCTTCCCGACTGGCCATTTCCTTCTCCTATAGGCAGAAAAAGATCATGGAGTCAGTCGAGAAGGAGGACAGCCTAACCGCCCCCTTTGAGTTCGCCACCACCGCCTCCACCGATGCCGCCAACGCGCCTACCACCTTCCCCGTCGAGGCACCCCCGCTTGAGGAGGAGGA。 AGTGATTATCGAGCAGGACCCAGGTTTTGTAAGCGAAGACGACGAGGATCGCTCAGTACCAACAGAGGATAAAAAGCAAGACCAGGACGACGCAGAGGCAAACGAGGAACAAGTCGGGCGGGGGGACCAAAGGCATGGCGACTACCTAGATGTGGGAGACGACGTGCTGTTGAAGCATCTGCAGCGCCAGTGCGCCATTATCTGCGACGCGTTGCAAGAGCGCAGCGATGTGCCCCTCGCCATAGCGGATGTCAGCCTTGCCTACGAACGCCACCTGTTCTCACCGCGCGTACCCCCCAAACGCCAAGAAAACGGCACATGCGAGCCCAACCCGCGCCTCAACTTCTACCCCGTATTTGCCGTGCCAGAGGTGCTTGCCACCTATCACATCTTTTTCCAAAACTGCAAGATACCCCTATCCTGCCGTGCCAACCGCAGCCGAGCGGACAAGCAGCTGGCCTTGCGGCAGGGCGCTGTCATACCTGATATCGCCTCGCTCGACGAAGTGCCAAAAATCTTTGAGGGTCTTGGACGCGACGAGAAACGCGCGGCAAACGCTCTGCAACAAGAAAACAGCGAAAATGAAAGTCACTGTGGAGTGCTGGTGGAACTTGAGGGTGACAACGCGCGCCTAGCCGTGCTGAAACGCAGCATCGAGGTCACCCACTTTGCCTACCCGGCACTTAACCTACCCCCCAAGGTTATGAGCACAGTCATGAGCGAGCTGATCGTGCGCCGTGCACGACCCCTGGAGAGGGATGCAAACTTGCAAGAACAAACCGAGGAGGGCCTACCCGCAGTTGGCGATGAGCAGCTGGCGCGCTGGCTTGAGACGCGCGAGCCTGCCGACTTGGAGGAGCGACGCAAGCTAATGATGGCCGCAGTGCTTGTTACCGTGGAGCTTGAGTGCATGCAGCGGTTCTTTGCTGACCCGGAGATGCAGCGCAAGCTAGAGGAAAC GTTGCACTACACCTTTCGCCAGGGCTACGTGCGCCAGGCCTGCAAAATTTCCAACGTGGAGCTCTGCAACCTGGTCTCCTACCTTGGAATTTTGCACGAAAACCGCCTCGGGCAAAACGTGCTTCATTCCACGCTCAAGGGCGAGGCGCGCCGCGACTACGTCCGCGACTGCGTTTACTTATTTCTGTGCTACACCTGGCAAACGGCCATGGGCGTGTGGCAGCAATGCCTGGAGGAGCGCAACCTAAAGGAGCTGCAGAAGCTGCTAAAGCAAAACTTGAAGGACCTATGGACGGCCTTCAACGAGCGCTCCGTGGCCGCGCACCTGGCGGACATTATCTTCCCCGAACGCCTGCTTAAAACCCTGCAACAGGGTCTGCCAGACTTCACCAGTCAAAGCATGTTGCAAAACTTTAGGAACTTTATCCTAGAGCGTTCAGGAATTCTGCCCGCCACCTGCTGTGCGCTTCCTAGCGACTTTGTGCCCATTAAGTACCGTGAATGCCCTCCGCCGCTTTGGGGTCACTGCTACCTTCTGCAGCTAGCCAACTACCTTGCCTACCACTCCGACATCATGGAAGACGTGAGCGGTGACGGCCTACTGGAGTGTCACTGTCGCTGCAACCTATGCACCCCGCACCGCTCCCTGGTCTGCAATTCGCAACTGCTTAGCGAAAGTCAAATTATCGGTACCTTTGAGCTGCAGGGTCCCTCGCCTGACGAAAAGTCCGCGGCTCCGGGGTTGAAACTCACTCCGGGGCTGTGGACGTCGGCTTACCTTCGCAAATTTGTACCTGAGGACTACCACGCCCACGAGATTAGGTTCTACGAAGACCAATCCCGCCCGCCAAATGCGGAGCTTACCGCCTGCGTCATTACCCAGGGCCACATCCTTGGCCAATTGCAAGCCATCAACAAAG CCCGCCAAGAGTTTCTGCTACGAAAGGGACGGGGGGTTTACCTGGACCCCCAGTCCGGCGAGGAGCTCAACCCAATCCCCCCGCCGCCGCAGCCCTATCAGCAGCCGCGGGCCCTTGCTTCCCAGGATGGCACCCAAAAAGAAGCTGCAGCTGCCGCCGCCGCCACCCACGGACGAGGAGGAATACTGGGACAGTCAGGCAGAGGAGGTTTTGGACGAGGAGGAGGAGATGATGGAAGACTGGGACAGCCTAGACGAAGCTTCCGAGGCCGAAGAGGTGTCAGACGAAACACCGTCACCCTCGGTCGCATTCCCCTCGCCGGCGCCCCAGAAATTGGCAACCGTTCCCAGCATCGCTACAACCTCCGCTCCTCAGGCGCCGCCGGCACTGCCTGTTCGCCGACCCAACCGTAGATGGGACACCACTGGAACCAGGGCCGGTAAGTCTAAGCAGCCGCCGCCGTTAGCCCAAGAGCAACAACAGCGCCAAGGCTACCGCTCGTGGCGCGGGCACAAGAACGCCATAGTTGCTTGCTTGCAAGACTGTGGGGGCAACATCTCCTTCGCCCGCCGCTTTCTTCTCTACCATCACGGCGTGGCCTTCCCCCGTAACATCCTGCATTACTACCGTCATCTCTACAGCCCCTACTGCACCGGCGGCAGCGGCAGCGGCAGCAACAGCAGCGGTCACACAGAAGCAAAGGCGACCGGATAGCAAGACTCTGACAAAGCCCAAGAAATCCACAGCGGCGGCAGCAGCA GGAGGAGGAGCGCTGCGTCTGGCGCCCAACGAACCCGTATCGACCCGCGAGCTTAGAAATAGGATTTTTCCCACTCTGTATGCTATATTTCAACAAAGCAGGGGCCAAGAACAAGAGCTGAAAATAAAAAACAGGTCTCTGCGCTCCCTCACCCGCAGCTGCCTGTATCACAAAAGCGAAGATCAGCTTCGGCGCACGCTGGAAGACGCGGAGGCTCTCTTCAGCAAATACTGCGCGCTGACTCTTAAGGACTAGTTTCGCGCCCTTTCTCAAATTTAAGCGCGAAAACTACGTCATCTCCAGCGGCCACACCCGGCGCCAGCACCTGTCGTCAGCGCCATTATGAGCAAGGAAATTCCCACGCCCTACATGTGGAGTTACCAGCCACAAATGGGACTTGCGGCTGGAGCTGCCCAAGACTACTCAACCCGAATAAACTACATGAGCGCGGGACCCCACATGATATCCCGGGTCAACGGAATCCGCGCCCACCGAAACCGAATTCTCCTCGAACAGGCGGCTATTACCACCACACCTCGTAATAACCTTAATCCCCGTAGTTGGCCCGCTGCCCTGGTGTACCAGGAAAGTCCCGCTCCCACCACTGTGGTACTTCCCAGAGACGCCCAGGCCGAAGTTCAGATGACTAACTCAGGGGCGCAGCTTGCGGGCGGCTTTCGTCACAGGGTGCGGTCGCCCGGGCGTTTTAGGGCGGAGTAACTTGCATGTATTGGGAATTGTAGTTTTTTTAAAATGGGAAGTGACGTATCGTGGGAAAACGGAAGTGAAGATTTGAGGAA GTTGTGGGTTTTTTGGCTTTCGTTTCTGGGCGTAGGTTCGCGTGCGGTTTTCTGGGTGTTTTTTGTGGACTTTAACCGTTACGTCATTTTTTAGTCCTATATATACTCGCTCTGTACTTGGCCCTTTTTACACTGTGACTGATTGAGCTGGTGCCGTGTCGAGTGGTGTTTTTTAATAGGTTTTTTTACTGGTAAGGCTGACTGTTATGGCTGCCGCTGTGGAAGCGCTGTATGTTGTTCTGGAGCGGGAGGGTGCTATTTTGCCTAGGCAGGAGGGTTTTTCAGGTGTTTATGTGTTTTTCTCTCCTATTAATTTTGTTATACCTCCTATGGGGGCTGTAATGTTGTCTCTACGCCTGCGGGTATGTATTCCCCCGGGCTATTTCGGTCGCTTTTTAGCACTGACCGATGTTAACCAACCTGATGTGTTTACCGAGTCTTACATTATGACTCCGGACATGACCGAGGAACTGTCGGTGGTGCTTTTTAATCACGGTGACCAGTTTTTTTACGGTCACGCCGGCATGGCCGTAGTCCGTCTTATGCTTATAAGGGTTGTTTTTCCTGTTGTAAGACAGGCTTCTAATGTTTAAATGTTTTTTTTTTTGTTATTTTATTTTGTGTTTAATGCAGGAACCCGCAGACATGTTTGAGAGAAAAATGGTGTCTTTTTCTGTGGTGGTTCCGGAACTTACCTGCCTTTATCTGCATGAGCATGACTACGATGTGCTTGCTTTTTTGCGCGAGGCTTTGCCTGATTTTTTGAGCAGCACCTTGCATTTTATATCGCCGCCCATGCAACAAGCTTACATAGGGGCTACGCTGGTTAGCATAGCTCCGAGTATGCGTGTCATAATCAGTGTGGGTTCTTTTGTCATGGTTCCTGGCGGGGAAGTGGCCGCGCTGGTCCGTGCAGACCT GCACGATTATGTTCAGCTGGCCCTGCGAAGGGACCTACGGGATCGCGGTATTTTTGTTAATGTTCCGCTTTTGAATCTTATACAGGTCTGTGAGGAACCTGAATTTTTGCAATCATGATTCGCTGCTTGAGGCTGAAGGTGGAGGGCGCTCTGGAGCAGATTTTTACAATGGCCGGACTTAATATTCGGGATTTGCTTAGAGACATATTGATAAGGTGGCGAGATGAAAATTATTTGGGCATGGTTGAAGGTGCTGGAATGTTTATAGAGGAGATTCACCCTGAAGGGTTTAGCCTTTACGTCCACTTGGACGTGAGGGCAGTTTGCCTTTTGGAAGCCATTGTGCAACATCTTACAAATGCCATTATCTGTTCTTTGGCTGTAGAGTTTGACCACGCCACCGGAGGGGAGCGCGTTCACTTAATAGATCTTCATTTTGAGGTTTTGGATAATCTTTTGGAATAAAAAAAAAAAAACATGGTTCTTCCAGCTCTTCCCGCTCCTCCCGTGTGTGACTCGCAGAACGAATGTGTAGGTTGGCTGGGTGTGGCTTATTCTGCGGTGGTGGATGTTATCAGGGCAGCGGCGCATGAAGGAGTTTACATAGAACCCGAAGCCAGGGGGCGCCTGGATGCTTTGAGAGAGTGGATATACTACAACTACTACACAGAGCGAGCTAAGCGACGAGACCGGAGACGCAGATCTGTTTGTCACGCCCGCACCTGGTTTTGCTTCAGGAAATATGACTACGTCCGGCGTTCCATTTGGCATGACACTACGACCAACACGATCTCGGTTGTCTCGGCGCACTCCGTACAGTAGGGATCGCCTACCTCCTTTTGAGACAGAGACCCGCGCTACCATACTGGAGGATCATCCGCTGCTGCCCGAATGTAACACTTTGACAATGCACAACGTGAGTTACGTGCGAGGTCTTCCCTGCAGTGTGGGATTTACGCT GATTCAGGAATGGGTTGTTCCCTGGGATATGGTTCTGACGCGGGAGGAGCTTGTAATCCTGAGGAAGTGTATGCACGTGTGCCTGTGTTGTGCCAACATTGATATCATGACGAGCATGATGATCCATGGTTACGAGTCCTGGGCTCTCCACTGTCATTGTTCCAGTCCCGGTTCCCTGCAGTGCATAGCCGGCGGGCAGGTTTTGGCCAGCTGGTTTAGGATGGTGGTGGATGGCGCCATGTTTAATCAGAGGTTTATATGGTACCGGGAGGTGGTGAATTACAACATGCCAAAAGAGGTAATGTTTATGTCCAGCGTGTTTATGAGGGGTCGCCACTTAATCTACCTGCGCTTGTGGTATGATGGCCACGTGGGTTCTGTGGTCCCCGCCATGAGCTTTGGATACAGCGCCTTGCACTGTGGGATTTTGAACAATATTGTGGTGCTGTGCTGCAGTTACTGTGCTGATTTAAGTGAGATCAGGGTGCGCTGCTGTGCCCGGAGGACAAGGCGTCTCATGCTGCGGGCGGTGCGAATCATCGCTGAGGAGACCACTGCCATGTTGTATTCCTGCAGGACGGAGCGGCGGCGGCAGCAGTTTATTCGCGCGCTGCTGCAGCACCACCGCCCTATCCTGATGCACGATTATGACTCTACCCCCATGTAGGCGTGGACTTCCCCTTCGCCGCCCGTTGAGCAACCGCAAGTTGGACAGCAGCCTGTGGCTCAGCAGCTGGACAGCGACATGAACTTAAGCGAGCTGCCCGGGGAGTTTATTAATATCACTGATGAGCGTTTGGCTCGACAGGAAACCGTGTGGAATATAACACCTAAGAATATGTCTGTTACCCATGATATGATGCTTTTTAAGGCCAGCCGG GGAGAAAGGACTGTGTACTCTGTGTGTTGGGAGGGAGGTGGCAGGTTGAATACTAGGGTTCTGTGAGTTTGATTAAGGTACGGTGATCAATATAAGCTATGTGGTGGTGGGGCTATACTACTGAATGAAAAATGACTTGAAATTTTCTGCAATTGAAAAATAAACACGTTGAAACATAACATGCAACAGGTTCACGATTCTTTATTCCTGGGCAATGTAGGAGAAGGTGTAAGAGTTGGTAGCAAAAGTTTCAGTGGTGTATTTTCCACTTTCCCAGGACCATGTAAAAGACATAGAGTAAGTGCTTACCTCGCTAGTTTCTGTGGATTCACTAGAATCGATGTAGGATGTTGCCCCTCCTGACGCGGTAGGAGAAGGGGAGGGTGCCCTGCATGTCTGCCGCTGCTCTTGCTCTTGCCGCTGCTGAGGAGGGGGGCGCATCTGCCGCAGCACCGGATGCATCTGGGAAAAGCAAAAAAGGGGCTCGTCCCTGTTTCCGGAGGAATTTGCAAGCGGGGTCTTGCATGACGGGGAGGCAAACCCCCGTTCGCCGCAGTCCGGCCGGCCCGAGACTCGAACCGGGGGTCCTGCGACTCAACCCTTGGAAAATAACCCTCCGGCTACAGGGAGCGAGCCACTTAATGCTTTCGCTTTCCAGCCTAACCGCTTACGCCGCGCGCGGCCAGTGGCCAAAAAAGCTAGCGCAGCAGCCGCCGCGCCTGGAAGGAAGCCAAAAGGAGCGCTCCCCCGTTGTCTGACGTCGCACACCTGGGTTCGACACGCGGGCGGTAACCGCATGGATCACGGCGGACGGCCGGATCCGGGGTTCGAACCCCGGTC GTCCGCCATGATACCCTTGCGAATTTATCCACCAGACCACGGAAGAGTGCCCGCTTACAGGCTCTCCTTTTGCACGGTCTAGAGCGTCAACGACTGCGCACGCCTCACCGGCCAGAGCGTCCCGACCATGGAGCACTTTTTGCCGCTGCGCAACATCTGGAACCGCGTCCGCGACTTTCCGCGCGCCTCCACCACCGCCGCCGGCATCACCTGGATGTCCAGGTACATCTACGGATTACGTCGACGTTTAAACCATATGATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGT GGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAA GTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAAC CAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGATGGATCC

[0126] SEQ ID NO:22 CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGT

Claims

1. A gene sequence construct for gene therapy of AIDS virus (HIV) infection, comprising: the gene coding sequence for one or more single-chain antibody molecules having the ability to inhibit HIV infection and having no constant region, and the gene coding sequence for one or more polypeptides (consisting of 2 to 50 amino acid residues) having the ability to inhibit HIV infection, so as to express a fusion protein molecule containing an anti-HIV antibody molecule and a polypeptide, the fusion protein being encoded by a single gene; The fusion protein molecule has two or more targets of action. The gene sequence construct.

2. The single-chain antibody molecule is a nanobody, or the single-chain antibody molecule comprises a heavy chain variable region and / or a light chain variable region; Optionally, the light chain variable region comprises a light chain variable region of a kappa or lambda light chain. The gene sequence construct of claim 1.

3. (a) The gene coding sequence of the single-chain antibody molecule having the ability to inhibit HIV infection and having no constant region and the gene coding sequence of the polypeptide having the ability to inhibit HIV infection are directly or indirectly linked in series via the coding sequence of a linker polypeptide. (b) comprising two or more gene coding sequences for single-chain antibody molecules having the ability to inhibit HIV infection and having no constant region, the gene coding sequences for the antibody molecules being directly or indirectly connected in series via a coding sequence for a linker polypeptide; and / or (c) comprising two or more gene coding sequences for polypeptides having the ability to suppress HIV infection, wherein the gene coding sequences for the polypeptides are directly or indirectly connected in series via a coding sequence for a linker polypeptide; A gene sequence construct according to claim 1 or 2.

4. (i) The gene coding sequence of one or more single-chain antibody molecules having the ability to suppress HIV infection and having no constant region comprises a gene coding sequence of an antibody molecule of anti-HIV-1-gp160 (or its cleavage products gp120 and gp41), (ii) the gene coding sequence of one or more single-chain antibody molecules capable of inhibiting HIV infection and lacking a constant region comprises a gene coding sequence of an antibody molecule that binds to the human CD4 receptor site; and / or (iii) the gene coding sequence for one or more polypeptides capable of suppressing HIV infection includes a gene coding sequence for a polypeptide that inhibits the fusion of HIV with the CD4+ T cell membrane; The gene sequence construct of claim 1.

5. The antibody comprises two or more gene coding sequences for single-chain antibody molecules having the ability to inhibit HIV infection and having no constant region, and one or more gene coding sequences for polypeptides having the ability to inhibit HIV infection, The gene coding sequences for the two or more single-chain antibody molecules having the ability to inhibit HIV infection and having no constant region comprise a gene coding sequence for an antibody molecule of anti-HIV-1-gp160 (or its cleavage products gp120 and gp41) and a gene coding sequence for an antibody molecule that binds to the human CD4 receptor site; The gene sequence construct according to claim 1, wherein the gene coding sequence for one or more polypeptides capable of inhibiting HIV infection includes a gene coding sequence for a polypeptide that inhibits the fusion of HIV with the CD4+ T cell membrane.

6. (A) (i) Gene coding sequences for the light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) and heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) of an anti-HIV-1-gp160 (or its cleavage products gp120 and gp41) monoclonal antibody; (ii) gene coding sequences for the light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) and heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) of a monoclonal antibody that binds to the human CD4 receptor site; (iii) a gene coding sequence for a polypeptide that inhibits fusion of HIV with the CD4+ T cell membrane; The coding sequences for the antibody light and heavy chains are directly or indirectly connected in tandem via the coding sequence for a linker polypeptide, regardless of the order. and / or (B) (i) gene coding sequences for the light chain variable region (VL) and heavy chain variable region (VH) of an anti-HIV-1-gp160 (or its cleavage products gp120 and gp41) monoclonal antibody; (ii) gene coding sequences for the light chain variable region (VL) and heavy chain variable region (VH) of a monoclonal antibody that binds to the human CD4 receptor site; (iii) a gene coding sequence for a polypeptide that inhibits fusion of HIV with the CD4+ T cell membrane; The coding sequences for the antibody light chain variable region (VL) and heavy chain variable region (VH) are directly or indirectly connected in tandem via the coding sequence for a linker polypeptide, regardless of the order. The gene sequence construct of claim 1.

7. (i) further comprising a promoter located upstream of the gene coding sequence for a single-chain antibody molecule having the ability to inhibit HIV infection and having no constant region and the gene coding sequence for a polypeptide having the ability to inhibit HIV infection; and / or (ii) further comprising a gene coding sequence for a single-chain antibody molecule having the ability to inhibit HIV infection and having no constant region and a secretory signal peptide coding sequence located upstream of the gene coding sequence for a polypeptide having the ability to inhibit HIV infection; The gene sequence construct of claim 1.

8. a gene coding sequence for a first single-chain antibody molecule having the ability to inhibit HIV infection and having no constant region, a gene coding sequence for a second single-chain antibody molecule having the ability to inhibit HIV infection and having no constant region, and a gene coding sequence for a polypeptide having the ability to inhibit HIV infection; VL2-linker-VH2-linker-VL1-linker-VH1-linker-peptide inhibitor; VL2 and VH2 are variable region fragments of the light chain and heavy chain of a first antibody molecule, respectively; VL1 and VH1 are variable region fragments of the light chain and heavy chain of a second antibody molecule, respectively; linker is a linker polypeptide; and peptide inhibitor is a polypeptide that suppresses HIV infection (e.g., a polypeptide that inhibits the fusion of HIV with the CD4+ T cell membrane). The gene sequence construct of claim 1. (a) the protein sequences of VL2 and VH2 comprise SEQ ID NO:2, or a functional fragment thereof, or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto; (b) the protein sequence of VL1 and VH1 comprises SEQ ID NO: 3, or a functional fragment thereof, or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto; (c) the linker sequence is selected from GGGGS, (GGGGS) 2 , (GGGGS) 3 , (GGGGS) 4 , (GGGGS) 5 , (GGGGS) 6 , and (GGGGS) 7 , or other alternative linker polypeptide sequences; and / or (d) the polypeptide that inhibits fusion of HIV with the CD4+ T cell membrane may be selected from membrane fusion-inhibiting polypeptides P52, C34, T20, etc.; Optionally, The sequence of the membrane fusion-inhibiting polypeptide P52 comprises SEQ ID NO: 5 or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto; The polypeptide sequence of C34 comprises SEQ ID NO: 6 or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto; The polypeptide sequence of T20 comprises SEQ ID NO: 7 or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto; The gene sequence construct of claim 8.

10. A viral vector genome comprising the construct of claim 1.

11. A viral vector system comprising the genome of claim 10, optionally a lentiviral vector system or an adeno-associated viral vector system.

12. A viral particle comprising the genome of the construct of claim 1.

13. A method for producing a virus comprising administering to a subject a virus vector system according to claim 11 or a virus particle according to claim 12, and a pharmaceutically acceptable carrier or diluent, optionally comprising cells that are muscle cells, liver cells, or CD4+ T cells; Pharmaceutical compositions.

14. A pharmaceutical composition for use in inhibiting HIV infection, comprising cells transduced by the viral vector system of claim 11 or the viral particles of claim 12.

15. Use of cells transduced by the viral vector system of claim 11 or the viral particles of claim 12 in the manufacture of a medicament for inhibiting HIV infection.

16. A pharmaceutical composition for use in treating HIV infection in a subject in need thereof, comprising cells transduced by the viral vector system of claim 11 or the viral particle of claim 12, Optionally, the subject comprises an HIV-infected individual with early stage HIV infection, or an HIV-infected individual who has already received cocktail drug therapy, or an HIV-infected individual who is resistant to cocktail drug therapy. The pharmaceutical composition.

17. Use of a cell transduced by the viral vector system of claim 11 or the viral particle of claim 12 in the manufacture of a medicament for treating HIV infection in a subject in need thereof, Optionally, the subject comprises an HIV-infected individual with early stage HIV infection, or an HIV-infected individual who has already received cocktail drug therapy, or an HIV-infected individual who is resistant to cocktail drug therapy. The above use.

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