Chimeric polypeptide, pharmaceutical composition and application

Abstract

The invention provides a chimeric polypeptide, a pharmaceutical composition and application. According to the chimeric antigen, at least one B cell epitope of the carrier protein CRM197 is replaced by the first polypeptide and / or the second polypeptide, humoral immune response of an organism to the carrier protein CRM197 can be reduced, the density of the same epitope in the chimeric antigen can be improved, the humoral immune response of the organism can be better induced, and the chimeric antigen has a good application prospect. The medicine has a good effect on preventing and / or treating the Alzheimer's disease, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology, and particularly relates to a chimeric polypeptide, a pharmaceutical composition, a nucleic acid molecule, a nucleic acid immunotherapy composition and their uses. Background Art

[0002] Alzheimer's disease (AD) is a degenerative disease of the central nervous system that occurs in old age and is the most common cause of dementia. Clinically, it is manifested as follows: (1) insidious onset, continuous and progressive decline in intelligence, with a disease course of 5-10 years; (2) significant episodic memory impairment in the early stage, first recent memory impairment, followed by remote memory impairment, time and place disorientation; (3) cognitive impairment and visuospatial impairment; (4) personality changes and mental disorders may occur; (5) generally no positive neurological signs in the early stage, and pyramidal and extrapyramidal signs such as bradykinesia and muscle rigidity appear in the late stage; (6) atrophy of the hippocampus and entorhinal cortex is found by head MRI. Its main pathological features are senile plaques caused by the accumulation of extracellular β-amyloid (Aβ) and neurofibrillary tangles, and their main core components are Aβ and hyperphosphorylated microtubule-associated protein (Tau protein), respectively. According to epidemiological surveys, with the rapid development of human society, the continuous extension of human lifespan, the intensification of social aging, and the acceleration of the pace of life, the incidence of Alzheimer's disease is increasing year by year, especially in the elderly population. As China gradually enters a severely aging society, the number of Alzheimer's disease patients will continue to climb, and it is expected to exceed 25 million in 2050, becoming the country with the largest number of AD patients in the world, which will bring an extremely heavy burden to the families of patients and society. Therefore, effective prevention and treatment means are urgently needed.

[0003] Regarding Alzheimer's disease, although a variety of Aβ / tau active immunization vaccines have been reported to enter the first or second clinical trial stage, none of them have achieved good results in the crucial clinical phase III efficacy trial. Therefore, the active immunotherapy targeting Aβ / tau still faces many challenges and there are still many problems to be solved. Summary of the Invention

[0004] The inventors of the present application designed a chimeric polypeptide, which can be used as a recombinant chimeric antigen in a pharmaceutical composition and / or a vaccine composition, to separately target Aβ polymers, Tau protein tangles or both simultaneously, which is beneficial to inducing a humoral immune response, delaying and reducing the deposition of Aβ in the brains of AD patients and the aggregation of Tau protein to form neurofibrillary tangles (NFT). While achieving the prevention and / or treatment of Alzheimer's disease, adverse reactions are avoided as much as possible.

[0005] Specifically, in the first aspect, the present invention provides a chimeric polypeptide, which comprises:

[0006] (i) A polypeptide obtained by replacing at least one B-cell epitope in the carrier protein CRM197 with a first polypeptide and / or a second polypeptide; wherein the first polypeptide comprises or consists of the sequence shown in SEQ ID NO:1, and the second polypeptide comprises or consists of the sequence shown in SEQ ID NO:2;

[0007] (ii) A polypeptide obtained by replacing at least one B-cell epitope in the A domain (catalytic domain 1-187) and B domain (transmembrane domain 201-384) of the carrier protein CRM197 with a first polypeptide and / or a second polypeptide, and connecting a third polypeptide to the C-terminus of the B domain; wherein the first polypeptide comprises or consists of the sequence shown in SEQ ID NO:1, the second polypeptide comprises or consists of the sequence shown in SEQ ID NO:2, and the third polypeptide is selected from at least one of the polypeptide formed by sequentially connecting the 12th to 60th amino acids of the hepatitis D virus delta antigen fragment, cholera toxin B subunit, and transferrin.

[0008] The carrier protein CRM197 (Cross reacting material 197, a non-toxic mutant of diphtheria toxin) is a protein molecule containing 535 amino acids, and its amino acid sequence information can be found in the National Center for Biotechnology Information (NCBI) of the United States (http: / / www.ncbi.nlm.nih.gov / ), and its three-dimensional structure can be obtained from the Protein Data Bank (PDB ID: 4AE0, https: / / www.rcsb.org / structure / 4AE0). It has been reported that the carrier protein CRM197 has 19 specific B-cell epitopes, as specifically shown in Figure 1 as follows.

[0009] In the present invention, the first polypeptide is derived from Aβ42, and the second polypeptide is derived from the full-length human Tau protein (Tau protein). The full-length amino acid sequence information of Aβ42 and Tau protein can be found in the National Center for Biotechnology Information of the United States.

[0010] In some embodiments, when replacing the B-cell epitope of the carrier protein CRM197 with the first polypeptide and / or the second polypeptide, when the number of amino acids (M) of the B-cell epitope is less than the number of amino acids (N) of the first polypeptide or the second polypeptide, when replacing the B-cell epitope, it is also necessary to replace N - M amino acids at the N-terminus and / or C-terminus of the B-cell epitope to avoid significantly affecting the length of the chimeric polypeptide. However, the general principle is that the first polypeptide or the second polypeptide needs to "completely cover" the B-cell epitope. For example, when the number of amino acids of the B-cell epitope is 5 and the number of amino acids of the first polypeptide is 8, then when replacing the B-cell epitope, a total of 3 amino acids at the N-terminus and / or C-terminus of the B-cell epitope are also replaced. When the number of amino acids of the B-cell epitope is more than the number of amino acids of the first polypeptide or the second polypeptide, an equal number of amino acid substitutions are directly made to this epitope.

[0011] In some embodiments, by epitope grafting technology, at least one B-cell epitope in the carrier protein CRM197 is replaced with different copies of the first polypeptide and / or the second polypeptide.

[0012] In some embodiments, when replacing the B-cell epitope with the first polypeptide, the number of amino acids of the B-cell epitope is less than or equal to the number of amino acids of the first polypeptide.

[0013] In some embodiments, when replacing the B-cell epitope with the second polypeptide, the number of amino acids of the B-cell epitope is less than or equal to the number of amino acids of the second polypeptide.

[0014] In some embodiments, the first polypeptide is the 3rd to 10th positions of Aβ42, that is 3 EFRHDSGY 10 (SEQ ID NO:1), which is also referred to as A1 herein.

[0015] In some embodiments, the second polypeptide is the 294th to 305th positions of Tau protein, that is 294 KDNIKHVPGGGS 305 (SEQ ID NO:2), which is also referred to as T1 herein.

[0016] In some embodiments, 1 to 19 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the B-cell epitopes are replaced with the first polypeptide and / or the second polypeptide.

[0017] In some embodiments, in (i), at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the B cell epitopes is replaced with the first polypeptide.

[0018] In some specific embodiments, the chimeric polypeptide comprises or consists of any one of the sequences shown in SEQ ID NOs: 3-7.

[0019] In some embodiments, in (i), at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the B cell epitopes is replaced with the second polypeptide.

[0020] In some specific embodiments, the chimeric polypeptide comprises or consists of any one of the sequences shown in SEQ ID NOs: 8-12.

[0021] In some embodiments, in (i), at least 2 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the B cell epitopes are respectively replaced with the first polypeptide and the second polypeptide.

[0022] In some specific embodiments, the chimeric polypeptide comprises or consists of any one of the sequences shown in SEQ ID NOs: 13-17.

[0023] In some embodiments, in (ii), after removing the C domain (receptor domain 387-535) of the carrier protein CRM197, at least 1 of the B cell epitopes is replaced with the first polypeptide, and the third polypeptide is linked.

[0024] In some specific embodiments, the chimeric polypeptide comprises or consists of any one of the sequences shown in SEQ ID NOs: 18-20.

[0025] In some specific embodiments, the third polypeptide is a polypeptide (HDAg) formed by sequentially connecting amino acids 12 to 60 of the hepatitis D virus delta antigen fragment, and the chimeric polypeptide comprises or consists of the sequence shown in SEQ ID NO: 18.

[0026] In some specific embodiments, the third polypeptide is cholera toxin B subunit (CTB), and the chimeric polypeptide comprises or consists of the sequence shown in SEQ ID NO: 19.

[0027] In some specific embodiments, the third polypeptide is Transferrin (TRF), and the chimeric polypeptide comprises or consists of the sequence shown in SEQ ID NO: 20.

[0028] The chimeric polypeptide provided by the present invention can be recombinantly expressed and purified in an expression strain (such as Escherichia coli) according to the protein expression and purification methods known in the art. For example, the protein can be purified by affinity chromatography or by ion exchange chromatography.

[0029] In a second aspect, the present invention provides a pharmaceutical composition comprising the chimeric polypeptide provided by the present invention and at least one adjuvant.

[0030] In some embodiments, the pharmaceutical composition is a vaccine composition.

[0031] In some specific embodiments, the chimeric polypeptide comprises or consists of any one of the sequences shown in SEQ ID NOs: 3-7, and the chimeric polypeptide serves as a recombinant chimeric antigen in the vaccine composition, targeting Aβ42.

[0032] In some specific embodiments, the chimeric polypeptide comprises or consists of any one of the sequences shown in SEQ ID NOs: 8-12, and the chimeric polypeptide serves as a recombinant chimeric antigen in the vaccine composition, targeting Tau protein.

[0033] In some specific embodiments, the chimeric polypeptide comprises or consists of any one of the sequences shown in SEQ ID NOs: 13-17, and the chimeric polypeptide serves as a recombinant chimeric antigen in the vaccine composition, targeting both Aβ42 and Tau protein.

[0034] In some specific embodiments, the chimeric polypeptide comprises or consists of any one of the sequences shown in SEQ ID NOs: 18-20, and the chimeric polypeptide serves as a recombinant chimeric antigen in the vaccine composition, targeting Aβ42.

[0035] In some embodiments, the adjuvant is selected from at least one of aluminum hydroxide, aluminum phosphate, aluminum sulfate, 3D-MPL, QS-21, QS-7, QS-17, QS-18, TQL1055, complete or incomplete Freund's adjuvant, oil-in-water emulsion (such as squalene or peanut oil), CpG, polyglutamic acid, polylysine, AddaVax TM 、MF59.

[0036] In a third aspect, the present invention provides a nucleic acid molecule comprising or consisting of a nucleic acid sequence encoding the chimeric polypeptide provided by the present invention.

[0037] In some embodiments, the sequence of the nucleic acid molecule is as shown in SEQ ID NO: 21-38.

[0038] In a fourth aspect, the present invention provides a nucleic acid immunotherapy composition comprising the nucleic acid molecule provided by the present invention and at least one adjuvant.

[0039] In some embodiments, the adjuvant is selected from aluminum hydroxide, aluminum phosphate, aluminum sulfate, 3D-MPL, QS-21, QS-7, QS-17, QS-18, TQL1055, complete or incomplete Freund's adjuvant, oil-in-water emulsion (such as squalene or peanut oil), CpG, polyglutamic acid, polylysine, AddaVax TM and at least one of MF59.

[0040] In a fifth aspect, the present invention provides the use of the chimeric polypeptide, the pharmaceutical composition, the nucleic acid molecule or the nucleic acid immunotherapy composition in the preparation of a medicament for preventing and / or treating Alzheimer's disease.

[0041] In some embodiments, the medicament is a vaccine.

[0042] In some embodiments, the vaccine is a single-target vaccine targeting Aβ or Tau protein.

[0043] In some embodiments, the vaccine is a dual-target vaccine targeting both Aβ and Tau proteins.

[0044] In a sixth aspect, the present invention provides the chimeric polypeptide, the pharmaceutical composition, the nucleic acid molecule or the nucleic acid immunotherapy composition for preventing and / or treating Alzheimer's disease.

[0045] In a seventh aspect, the present invention further provides a method for preventing and / or treating Alzheimer's disease, which comprises administering an effective amount of the chimeric polypeptide, the pharmaceutical composition, the nucleic acid molecule or the nucleic acid immunotherapy composition to a subject in need thereof.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] 1) By replacing at least one B-cell epitope of the carrier protein CRM197 with the first polypeptide and / or the second polypeptide, the present invention can, on the one hand, reduce the humoral immune response of the body to the carrier protein CRM197, thereby reducing the potential carrier-induced epitope suppression (CIES); on the other hand, it can increase the density of the same epitope in the chimeric antigen, thus better inducing the humoral immune response of the body and having a better effect in preventing and / or treating Alzheimer's disease.

[0048] 2) The chimeric polypeptide provided by the present invention can achieve recombinant expression and purification in an expression strain, with better product homogeneity and without the need for chemical coupling reactions between the polypeptide and the carrier protein.

[0049] 3) The nucleic acid sequence encoding the chimeric polypeptide of the present invention was cloned into a vector and expressed in an expression strain. The purified chimeric polypeptide was used as an antigen and mixed with an adjuvant, and then inoculated into AD transgenic mice to complete the verification of the vaccine antigen. The results showed that after immunization, high levels of antibodies were detected in the AD mice, with obvious improvement in behavioral performance, reduction of β-amyloid plaques in the cortex and hippocampus, reduction of insoluble Aβ40 / 42 in the brain, and reduction of the inflammatory level in the brain, and the vaccine had good safety. Description of the Drawings

[0050] Figure 1 In the figure, A is the basic domain of Aβ42; B is the basic domain of the full-length human Tau protein; C and D are the sequence information of the carrier protein CRM197, where the linear B-cell epitopes are marked in blue;

[0051] Figure 2 is the design process of the chimeric polypeptide in Example 1 of the present invention and the antigen structures of some chimeric polypeptides; among them, A and B are the antigen structures of the chimeric polypeptide DA6c; C and D are the antigen structures of the chimeric polypeptide DT6b; E and F are the antigen structures of the chimeric polypeptide DAT8b;

[0052] Figure 3 is the plasmid construction map shown in Example 2 of the present invention;

[0053] Figure 4 is the chimeric antigen structure after the cDNA encoding the chimeric polypeptide was cloned into the pCold-TFM expression vector in Example 3 of the present invention. Among them, in A, the chimeric antigen includes the DA6c chimeric polypeptide sequence and a TF tag; B the chimeric antigen includes the DT6b chimeric polypeptide sequence and a TF tag; C the chimeric antigen includes the DAT8b chimeric polypeptide sequence and a TF tag;

[0054] Figure 5This is the screening result of the immunogenicity of the chimeric polypeptide in Example 4 of the present invention. Among them, A is the comparison of the anti-Aβ42 antibody levels of the single-target Aβ chimeric polypeptide; B is the comparison of the anti-tau antibody levels of the single-target tau chimeric polypeptide; C is the comparison of the anti-Aβ42 antibody levels of the dual-target chimeric polypeptide; D is the comparison of the anti-tau antibody levels of the dual-target tau chimeric polypeptide. The data are expressed as mean ± S.E.M, and P≤0.05 indicates that the difference is statistically significant;

[0055] Figure 6 This is the image of the Aβ42 sample in Example 4 of the present invention under TEM, scale bar = 200 nm;

[0056] Figure 7 This is Tau pp -RD sample image under TEM, scale bar = 200 nm;

[0057] Figure 8 This is the H&E staining result of the main organs in Example 4 of the present invention, scale bar = 100 μm;

[0058] Figure 9 This is the antibody titer result of APP / PS1 mice after inoculating antigens in Example 4 of the present invention. Among them, A is the Aβ42 antibody titer induced by inoculating TF-DA6c (red line) and TF-DAT8b (green), B is the tau antibody titer produced by inoculating TF-DAT8b. The data are expressed as mean ± S.E.M. (n = 12), and P≤0.05 indicates that the difference is statistically significant;

[0059] Figure 10 This is the behavioral test result of APP / PS1 mice in Example 5 of the present invention. Among them, A is the result of the Y-maze experiment. The data are expressed as mean ± S.E.M. (n = 12), and P≤0.05 indicates that the difference is statistically significant; B is the result of the novel object recognition experiment. The data are expressed as mean ± S.E.M. (n = 12), and P≤0.05 indicates that the difference is statistically significant;

[0060] Figure 11 This shows the result of the Aβ level in the mouse brain tissue in Example 7 of the present invention. Among them, A is the representative immunohistochemical image, scale bar = 100 μm, B is the number of β-amyloid plaques in the cortex area. The data are expressed as mean ± S.E.M. (n = 12), and P≤0.05 indicates that the difference is statistically significant; D is the number of β-amyloid plaques in the hippocampal area. The data are expressed as mean ± S.E.M. (n = 12), and P≤0.05 indicates that the difference is statistically significant;

[0061] Figure 12Shows the test results of the concentration of insoluble Aβ42 / 40 in Example 7 of the present invention. Among them, A is the level of insoluble Aβ42 in the brain tissue, B is the level of insoluble Aβ40 in the brain tissue, the data are expressed as mean ± S.E.M. (n = 12), and P ≤ 0.05 indicates that the difference is statistically significant;

[0062] Figure 13 Shows the levels of inflammatory factors IL-6 (A), IL-1β (B), IFN-γ (C) and TNF-α (D) in the supernatant of the brain tissue extract in Example 7 of the present invention. The data are expressed as mean ± S.E.M. (n = 12), and P ≤ 0.05 indicates that the difference is statistically significant;

[0063] Figures 14 - 16 Respectively show the results of evaluating the humoral immune response by immunizing BALB / c mice with different chimeric polypeptide antigens and using the ELISA method in Examples 8-10. Detailed implementation mode

[0064] The present invention will be further described below through specific examples. The examples described in the present invention are only for the purpose of illustrating the present invention and do not limit the scope of the present invention. Unless otherwise specified, the raw materials and materials used in the following examples are commercially available products, and the methods and conditions used are known methods and conventional conditions in the art.

[0065] Statistical method: In the following examples, the GraphPad Prism 9 software is used to plot and analyze the data. The Student's T-test is used for the comparison between two groups. When comparing multiple groups, the one-way ANOVA method is used for the test analysis, and the Tukey's post-hoc test is used for the analysis of the differences between groups. P ≤ 0.05 is considered to have a significant difference.

[0066] Example 1

[0067] This example provides the design process of the chimeric polypeptide.

[0068] As Figure 1 shown, the A1 ( 3 EFRHDSGY 10 ) polypeptide is located at the N-terminus of the full-length Aβ42 protein ( Figure 1 A), and the T1 ( 294 KDNIKHVGGGS 305 ) polypeptide is located in the microtubule-binding domain of the Tau protein ( Figure 1B). CRM197 consists of 535 amino acids and is divided into a catalytic domain (1-187), a transmembrane domain (201-384), and a receptor domain (387-535). Its B-cell epitopes are shown in ( Figure 1 C and D). Different copies of A1 and / or T1 were transplanted onto CRM197 through epitope grafting technology, the root mean square deviation (RMSD) values of the backbone atoms were calculated, and the structures of the chimeric polypeptides were shown in two ways, Ribbon and Surface, where the red, blue, and grayish-white markings represent the partial structures of the A1, T1 polypeptides, and carrier protein, respectively ( Figure 2 A-F).

[0069] Example 2

[0070] In this example, a plasmid vector TF-DA6c expressing the chimeric polypeptide TF-DA6c as shown in SEQ ID NO: 6 was prepared, and the expressed recombinant fusion protein was purified. In each example of the present invention, the chimeric polypeptide is sometimes also referred to as a recombinant fusion protein, a fusion protein, a chimeric antigen, or a recombinant antigen.

[0071] 1. Plasmid construction (SEQ ID NO: 3-17)

[0072] The cDNA (SEQ ID NO: 24) encoding the chimeric polypeptide as shown in SEQ ID NO: 6 was cloned after the HRV 3C protease site of the pCold-TFM expression vector, and the resulting plasmid is as Figure 3 shown in A.

[0073] Among them, the pCold-TFM plasmid was modified on the basis of the pCold-TF plasmid (TaKaRa, Japan), and the modification process is described in Cao T, Zhang Z, Liu ZG, et al. High-level expression and purification of the major house dust mite allergen Derp 2 in Escherichia coli. Protein Expr Purif. 2016; 121: 97-102. doi: 10.1016 / j.pep.2016.01.012, and the full text of this article is incorporated herein by reference.

[0074] 2. Plasmid construction (SEQ ID NO: 18-20)

[0075] The cDNA (SEQ ID NO: 36) encoding the chimeric polypeptide shown in SEQ ID NO: 18 was cloned downstream of the HRV 3C protease site of the pET-28(a) expression vector (Novagen), and the resulting plasmid is as Figure 3 shown in B.

[0076] 3. Expression and purification of the recombinant chimeric antigen (the recombinant chimeric antigen is a protein fused with the TF tag; the TF tag is fused and expressed without interacting with the target protein and generally does not affect the function and properties of the target protein)

[0077] (1) Expression of the recombinant chimeric antigen:

[0078] ① After the plasmid transformation was completed and incubated overnight at 37 °C on an LB solid culture dish, single colonies were selected and inoculated into 10 ml of LB liquid medium resistant to ampicillin (100 μg / mL), and cultured overnight at 37 °C on a shaker (220 rpm / min);

[0079] ② The next day, the cultured bacterial solution was inoculated into 1 L of fresh LB liquid medium resistant to ampicillin for scale-up culture (37 °C, 220 rpm / min). When the OD600 of the bacterial solution reached 0.6 - 0.8, the culture was stopped and the bacterial solution was cooled to 16 °C.

[0080] ③ After the bacterial solution was cooled, an IPTG inducer with a final concentration of 0.2 mmol / L was added, and the expression was induced at 16 °C on a shaker for 20 hours (220 rpm / min).

[0081] ④ The induced bacterial solution was centrifuged at 4 °C and 5000 rpm for 20 min, and the supernatant of the culture medium after centrifugation was discarded. The cells were resuspended in 50 mL of phosphate resuspension buffer (prepared in the laboratory; the preparation method is: mix 5.3 ml of 0.2 M NaH2PO4 and 94.7 ml of 0.2 M Na2HPO4, adjust the pH to 8.0, then add 4 ml of 5 M NaCl and 2 ml of 5 M EDTA, place in a graduated cylinder, add deionized water to a volume of 1 L, transfer to a reagent bottle and shake well, store at 4 °C). Resuspend the cell pellet of 1 liter and store at -80 °C for later use.

[0082] ⑤ When in use, take out the resuspended bacterial solution from the -80 °C refrigerator and break it with a high-pressure cell crusher (4 °C, 1000 bar, cycle 3 times) to release the fusion protein. Collect the cell lysate in a high-speed centrifuge tube, and then centrifuge it with a high-speed floor-standing refrigerated centrifuge (4 °C, 20000 rpm, 20 min), and collect the supernatant of the centrifugation.

[0083] ⑥ Take an appropriate amount of the cell lysate and the supernatant of the lysate, and use SDS-PAGE to detect the expression of the target protein.

[0084] (2) Purification of recombinant chimeric antigen:

[0085] Affinity chromatography purification of recombinant chimeric antigen

[0086] ① Equilibrate Ni SepHarose 6 Fast Flow affinity chromatography column with phosphate resuspension buffer for at least one column volume. TM 6 Fast Flow affinity chromatography column.

[0087] ② Load the supernatant of lysed bacteria onto the equilibrated Ni SepHarose 6 Fast Flow affinity chromatography column to allow it to fully contact the nickel resin, and bind on a shaker in a chromatography cold cabinet for 10 minutes. TM 6 Fast Flow affinity chromatography column.

[0088] ③ Wash with 50 ml of phosphate washing buffer for 3 column volumes to remove non-specifically bound miscellaneous proteins.

[0089] ④ Finally, elute the fusion protein specifically bound to the nickel resin with 10 ml of phosphate elution buffer.

[0090] ⑤ Collect the protein eluate after affinity purification through the nickel (Ni) column, concentrate the protein solution with a centrifugal ultrafiltration tube, and place it in a 4 °C refrigerator for gel filtration chromatography separation after concentration.

[0091] ⑥ Take an appropriate amount of the corresponding sample for SDS-PAGE electrophoresis analysis.

[0092] Ion exchange chromatography method for recombinant chimeric antigen

[0093] ① Install the ion exchange chromatography (HiTrap DEAE FF column) on the protein purifier, open the corresponding software for control operations. Rinse pump A with filtered phosphate buffer, and equilibrate the ion chromatography column with the binding buffer at a flow rate of 2.0 ml / min for one column volume. TM DEAE FF column

[0094] ③ After equilibration, filter the concentrated protein sample (less than 5 ml) through a 0.22 μm filter membrane, load it onto the ion chromatography column through the protein purifier, and perform column purification with phosphate buffer at a flow rate of 2.0 ml / min.

[0095] ④ According to the molecular sieve chromatogram, collect the protein peaks at each position for SDS-PAGE detection and analysis.

[0096] ⑤ Concentration of the purified protein product: The final product is desalted into the storage buffer with a HiTrap desalting column, concentrated with an ultrafiltration concentrator tube, and the time and rotation speed are determined according to the protein concentration and size, and stored at -80 °C. TM desalting column

[0097] Example 3

[0098] In this example, chimeric polypeptides shown in SEQ ID NO: 3 - 20 were prepared. Among them, the chimeric polypeptides shown in SEQ ID NO: 3 - 7 were named DA1a, DA4a, DA6b, DA6c, and DA10b in sequence; the chimeric polypeptides shown in SEQ ID NO: 8 - 12 were named DT1a, DT5a, DT5b, DT6b, and DT9a in sequence; the chimeric polypeptides shown in SEQ ID NO: 13 - 17 were named DAT4a, DAT4b, DAT6b, DAT8b, and DAT9a in sequence; the chimeric polypeptide shown in SEQ ID NO: 18 was named D - HA01; the chimeric polypeptide shown in SEQ ID NO: 19 was named D - CA01; the chimeric polypeptide shown in SEQ ID NO: 20 was named D - TA01, and the obtained chimeric polypeptides were purified.

[0099] Among them, the partial sequence structure after the cDNA encoding the chimeric polypeptide of SEQ ID NO: 3 - 17 was cloned into the pCold - TFM expression vector is as Figure 3 shown in A, and the partial sequence structure after the cDNA encoding the chimeric polypeptide of SEQ ID NO: 18 - 20 was cloned into the pET - 28(a) expression vector is as Figure 3 shown in B.

[0100] The steps for preparing and purifying the recombinant chimeric antigen were basically the same as those in Example 2, except for the buffer used in the purification step. Specifically, the buffer used for purifying the recombinant chimeric antigens DA1a, DA4a, DA6b, DA6c, DA10b, D - HA01, D - CA01, and D - TA01 was a phosphate series buffer, and the buffer used for purifying the chimeric polypeptides DT1a, DT5a, DT5b, DT6b, DT9a, DAT4a, DAT4b, DAT6b, DAT8b, and DAT9a was a Tris - hydrochloric acid series buffer (preparation method: measure 20 ml of 1 M Tris - HCl (pH 8.0), 4 ml of 5 M NaCl, 2 ml of 5 M EDTA, place them in a measuring cylinder, add deionized water to a volume of 1 L, transfer to a reagent bottle and shake well, and store at 4°C).

[0101] Example 4

[0102] Immunization protocol for BALB / C mice

[0103] To comparatively study the immunogenicity of different recombinant chimeric antigens, experiments were conducted in BALB / c mice. The experiment included 6 groups, with 6 eight-week-old female BALB / c mice selected in each group. One group was the control group (inoculated with the same dose of CRM197 protein fused with the TF tag as the control group), and the other five groups were the recombinant chimeric antigen groups. The dose of antigen inoculated in each group of mice was 40 μg, and the inoculation method was subcutaneous inoculation in the neck. A total of three immunizations were carried out, with an immunization interval of 2 weeks.

[0104] Immunization protocol for APP / PS1 mice

[0105] Two-month-old female APP / PS1 mice were divided into three groups, with 12 mice in each group. One group of APP / PS1 female mice was inoculated with PBS as the control group, and the other two experimental groups were inoculated with the same dose of TF-DA6c and TF-DT6b respectively; another 12 mice of the same age and sex, C57BL / 6J mice, were selected as wild-type mice for the experiment. The dose of antigen inoculated in each group of mice was 40 μg, and the inoculation method was subcutaneous inoculation in the neck. A total of 5 immunizations were carried out. The interval between the first two immunizations was 2 weeks, and the interval between the last three immunizations was 5 weeks.

[0106] ELISA detection of the level of specific antibodies induced by immunization

[0107] The ELISA method was used to detect the level of specific antibodies against Aβ42 or tau induced in immunized mice. A blank control was set in the experiment, with the PBS group as the control group. A detection OD value greater than 2.1 times that of the negative was considered positive.

[0108] (1) Preparation of antigen stock solution: The synthesized Aβ42 peptide or tau was dissolved in PBS solution (Dalian Meilun Biotechnology Co., Ltd.) to a final concentration of 1 mg / ml, and its stock solution was stored frozen at -80 °C in the refrigerator for later use.

[0109] (2) Coating: The stock solution was diluted with the coating solution to make the final concentration of the antigen 1 μg / ml. 100 μl was added to each well and coated overnight at 4 °C.

[0110] (3) Washing: Discard the coating solution, drain the remaining liquid in the ELISA plate, add 200 μl of PBST to each well, and gently tap the ELISA plate for washing. A total of 5 washes were carried out, with each wash lasting 3 min.

[0111] (4) Blocking: Discard the washing solution, add 200 μl of blocking solution to each well, that is, 3% BSA in PBS solution, and block at 37 °C for 1 h.

[0112] (5) Add the serum primary antibody to be detected: Discard the blocking solution, wash 5 times, and then add 100 μl of the serum to be detected with different dilution gradients (starting from a dilution gradient of 1:2000, diluted 4-fold) to each well. Incubate at 37 °C for 1 h.

[0113] (6) Secondary antibody: Discard the primary antibody in the plate, wash 5 times with PBST, and then add 100 μl of HRP-conjugated mouse secondary antibody (diluted 1:5000) to each well. Incubate at 37 °C for 45 min.

[0114] (7) Color development: Discard the mouse secondary antibody, wash 5 times, and then add 100 μl of TMB color development solution (Beijing Solarbio Science & Technology Co., Ltd.) to each well. Develop color in the dark for 20 - 30 min. After the positive control well shows obvious blue, add 50 μl of 2 M sulfuric acid termination solution to each well to terminate the reaction. Read the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader and record the values.

[0115] Results: As Figure 5 shown, by comparing the levels of specific antibodies produced by BALB / c mice, TF-DA6c is the antigen with the highest level of anti-Aβ42 antibody among the Aβ single-target antigens ( Figure 5 A); TF-DT6b is the antigen with the highest level of anti-tau antibody among the tau single-target antigens ( Figure 5 B); TF-DAT8b is the antigen with the highest levels of anti-Aβ42 ( Figure 5 C) and anti-tau ( Figure 5 D) antibodies among the dual-target antigens;

[0116] In vitro functional detection of antibodies:

[0117] 1. Preparation of Aβ42 monomers

[0118] (l) Take 5 mg of Aβ(1 - 42) polypeptide powder (Aladdin), add 550 μL of hexafluoroisopropanol (HFIP), and dissolve it completely at room temperature in the dark;

[0119] (2) Aliquot the dissolved polypeptide solution into multiple tubes, and place them in a freeze dryer until HFIP completely evaporates;

[0120] (3) After sealing, store them in a -20 °C refrigerator for later use.

[0121] 2. Expression and purification of Tau pp -RD, refer to the article: Ferrari L, Rüdiger SGD. Recombinant production and purification of the human protein Tau. Protein Eng Des Sel. 2018;31(12):447 - 455.

[0122] doi:10.1093 / protein / gzz010. The full text of this article is incorporated herein by reference.

[0123] 3. Preparation of antiserum:

[0124] One day before each immunization of the mice, 10 μL of mouse blood was collected through the tail vein of the mice, dissolved in 990 μL of sterile PBS, and gently mixed. Centrifuge (4 °C, 3000 rpm, 1000 g, 15 min), take the supernatant into another clean 1.5 mL EP tube, and store it at -80 °C for later use to obtain the antiserum.

[0125] 4. Observation of Aβ42 and Tau by transmission electron microscopy (TEM) pp -RD aggregation inhibition experiment

[0126] Aβ42 samples: Aβ42 monomer 5 μM, Aβ42 monomer (5 μM) + anti-Aβ42 antibody (anti-TF-DA6c serum prepared in step 3, antibody titer: 10,000), Aβ42 monomer (5 μM) + anti-Aβ42 antibody (anti-TF-DA6c serum prepared in step 3, antibody titer: 10,000), and Aβ42 monomer (5 μM) + pre-immune serum (anti-serum prepared before the first immunization of the mice, titer: 10,000) were incubated at 37 °C for 120 hours. Tau pp -RD samples: Tau alone pp -RD (1 μg / mL) + sodium heparin (0.25 μg / mL), Tau pp -RD (1 μg / mL) + sodium heparin (0.25 μg / mL) + anti-tau antibody (anti-TF-DT6b serum prepared in step 3, antibody titer: 10,000), Tau pp -RD (1 μg / mL) + sodium heparin (0.25 μg / mL) + anti-tau antibody (anti-TF-DAT8b serum prepared in step 3, antibody titer: 10,000), and Tau pp -RD (1 μg / mL) + sodium heparin (0.25 μg / mL) + pre-immune serum (anti-serum prepared before the first immunization of the mice, antibody titer: 10,000) were incubated at 37 °C for 48 hours. The final volume of the samples was 100 μL. The samples were centrifuged (5 minutes, 10,000 rpm), and a total of 10 μL of the samples was placed on a formvar film and carbon-coated copper grid (400 mesh). The prepared samples were treated with 2% uranyl acetate and imaged using TEM. The experimental results are as Figure 6As shown, the protein states of each experimental group were observed by transmission electron microscopy. When Aβ monomers were co-incubated with TF-DA6c or TF-DAT8b antisera, the electron microscopy results showed no obvious changes compared with freshly prepared monomers. However, the experimental results of co-incubating pre-immune serum with monomers were similar to those observed for incubations containing only Aβ42 monomers, with fibrous tangles appearing in the field of view of the transmission electron microscope. Figure 7 The experimental results showed that TF-DT6b or TF-DAT8b antisera blocked the tangles of Tau pp -RD after 48 hours of incubation, indicating that the anti-tau antibodies in the serum could significantly inhibit the aggregation of Tau pp -RD.

[0127] HE staining

[0128] HE staining was performed on the main organ tissues (heart, liver, spleen, lung, kidney) of mice to observe the morphological changes of cells in each organ after immunization, for evaluating the safety of the vaccine.

[0129] (1) Place the sections in an incubator at 70 °C for 30 min;

[0130] (2) Deparaffinization: Take xylene solution and divide it into three parts, namely xylene I solution, xylene II solution, and xylene III solution. Place the sections in xylene I solution for 15 min → xylene II solution for 15 min → xylene III solution for 15 min;

[0131] Hydration: Take absolute ethanol solution and divide it into two parts, namely absolute ethanol I solution and absolute ethanol II solution. Sequentially place the sections in absolute ethanol I solution for 5 min → absolute ethanol II solution for 5 min → 95% ethanol solution for 3 min → 90% ethanol solution for 3 min → 80% ethanol for 3 min → 70% ethanol for 3 min. Then wash with distilled water for 5 - 10 min;

[0132] (3) Staining and washing: Drop a drop of hematoxylin solution onto the sections and stain at 37 °C for 30 min, then rinse with running water to make the sections turn blue;

[0133] (4) Differentiation and rinsing: Place the sections in 0.5% hydrochloric acid ethanol solution to fade until the sections appear lighter red. Then place the sections in running water to restore blue;

[0134] (5) Dehydration: Place the sections sequentially in 50%, 75%, and 85% ethanol, each for 2 min;

[0135] (6) Counterstaining: Stain the sections with 1% eosin ethanol solution for 1 - 3 min;

[0136] (7) Dehydration: Put the sections into 95% ethanol to remove the excess red color, then soak them in absolute ethanol for 5 min, and finally blot dry the excess ethanol.

[0137] (8) Clearing: Put the sections into absolute ethanol, xylene I, and xylene II for 5 min each.

[0138] (9) Mounting: Mount with neutral balsam and examine under the microscope and take pictures.

[0139] As Figure 8 shown, the changes in various organs of mice were detected, and the safety of the vaccine was investigated from multiple perspectives. HE staining results of heart, liver, spleen, lung, and kidney tissues showed no abnormal pathological manifestations.

[0140] TF-DA6c and TF-DAT8b elicited strong immune responses in APP / PS1 mice

[0141] After vaccination according to the immunization protocol, both antigens elicited high levels of immune responses, and the results were as Figure 9 shown. The results showed that TF-DA6c produced high levels of anti-Aβ42 antibodies in APP / PS1 mice, and TF-DAT8b produced both anti-Aβ42 and tau antibodies simultaneously. When the mice were 4 - 5 months old, a significant decrease in the anti-Aβ42 antibody titer was observed. We speculate that this is mainly because the concentration of Aβ protein in the brain of APP / PS1 mice increases and begins to aggregate around 4 months old, and the antibodies are continuously consumed due to clearing the plaque deposits in the brain, so the antibody titer decreases. After increasing the number of immunizations, the antibody level increases ( Figure 9 A). Since APP / PS1 mice lack tau pathological features, the anti-tau antibody titer was maintained at a relatively high level ( Figure 9 B). This phenomenon indicates that in the clinical application of AD vaccines, dynamically detecting the antibody level in serum and intervening in a timely manner may achieve better preventive or therapeutic effects.

[0142] Example 5

[0143] Behavioral tests

[0144] Y-maze and novel object recognition (NOR) experiments were performed on 9-month-old APP / PS1 mice between 7:00 am and 6:00 pm. Mice needed to adapt to the behavioral room one week before the test, and the same experimenter spent 5 minutes with the experimental subjects every day. Before each mouse was tested, each behavioral test device was cleaned with 75% ethanol.

[0145] (1) Y-maze experiment:

[0146] The experimental apparatus was a horizontal three-arm maze, with each arm being 40 cm in length, 10 cm in width, and 25 cm in height, and labeled as Arm A, Arm B, and Arm C respectively. Mice were acclimated for 1 hour under dim light in the behavioral room before the experiment. During the test, the mice were initially placed at the end of one of the arms. Then, the behavior was recorded with a camera, and the mice were allowed to freely explore the maze for 8 minutes. Consecutively entering all three arms was defined as having made a spontaneous alternation, such as ABC, CAB, or BCA, but not BAB. The spontaneous alternation (%) was calculated as {actual alternation / (total alternation times - 2)} × 100 to evaluate short-term spatial working memory.

[0147] (2) Novel object recognition experiment (NOR):

[0148] Experimental apparatus: The experiment was also conducted in a self-made white hard plastic box without a lid, with a length, width, and height of 40 cm. The objects selected for the experiment were all made of colorless and odorless plastic materials to ensure that they would not attract the abnormal attention of the mice.

[0149] Experimental procedure: The experiment was divided into a training period and a test period, with an interval of 4 h in between. During the training period, two identical objects were placed in the experimental apparatus, the camera was turned on to record, the mouse was placed with its head facing the wall into the apparatus, and the mouse was allowed to freely explore in the apparatus for 10 min. Then, the mouse was taken out. Similarly, after each mouse completed the training, it was wiped thoroughly with 75% ethanol to ensure the consistency of the experiment. 4 h after the mice completed the training, the position of one of the objects in the apparatus was moved to the diagonal, the camera was turned on to record, the mouse was placed with its head facing the wall into the apparatus, and the mouse was allowed to freely explore in the apparatus for 10 min. When a mouse's head was facing an object and was 1 cm away from the object, or when its nose touched the object, the mouse was considered to be exploring the object.

[0150] The novel object recognition index (DI, %) was calculated as {(novel object exploration time - familiar object exploration time) / (novel object exploration time + familiar object exploration time)} × 100 to demonstrate learning ability and memory.

[0151] Results:

[0152] TF-DA6c and TF-DAT8b improved the learning and memory of APP / PS1 mice in behavioral tests

[0153] In the Y-maze experiment, compared with the control group, the wild-type and treatment group mice had a higher spontaneous alternation rate index, with a significant difference ( Figure 10 A). Similarly, the wild-type and treatment group mice also showed a higher recognition index in the NOR experiment, with a difference compared to the control group mice ( Figure 10 B).

[0154] In summary, the above behavioral research results indicate that TF-DA6c and TF-DAT8b alleviate the decline of working memory in APP / PS1 mice.

[0155] Example 7

[0156] Brain homogenate preparation:

[0157] First, the mice were anesthetized and perfused with pre-cooled cerebrospinal fluid (Solarbio Science & Technology Co., Ltd.). The brain was divided into two halves along the sagittal suture. One half was used to prepare the homogenate, and the other half was fixed with 4% paraformaldehyde. The hippocampus and cortex were homogenized separately in radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology) containing 1% protease inhibitor (Boster Biological Technology Co., Ltd.) and centrifuged at 14,000×g for 30 min at 4°C. The supernatant containing soluble Aβ, namely the RIPA soluble fraction, was collected. The precipitate was resuspended in 5 M guanidine hydrochloride (Thermo Fisher Scientific (China) Co., Ltd.) solution (pH 8.0) and centrifuged at 14,000×g for 30 min at 4°C to prepare the supernatant containing insoluble Aβ (referred to as guanidine hydrochloride-soluble Aβ). The total protein of the hippocampal and cortical tissues of each mouse was determined by BCA assay.

[0158] Immunohistochemistry experiment:

[0159] Aβ deposits were identified by anti-human Aβ monoclonal antibody 6E10 (Biolegend) (1:1000). Representative regions of Aβ plaques in the cortex and hippocampal regions were analyzed by ImageJ software.

[0160] Determination of Aβ40 / 42, antibodies and cytokines in the brains of APP / PS1 mice:

[0161] The concentrations of Aβ40 / 42, antibodies and cytokines (IL-6, IL-1β, TNF-α and IFN-γ) in the hippocampus and cortex were measured by ELISA assay.

[0162] Results:

[0163] TF-DA6c and TF-DAT8b can reduce plaque formation and the levels of insoluble Aβ42 / 40 in APP / PS1 mice

[0164] The immunohistochemistry experiment of APP / PS1 mice showed that compared with the control group, a significant reduction in the number of senile plaques in the brains of the treated group mice could be observed ( Figure 11 A). Quantitative statistics of the number of Aβ plaques in the cerebral cortex and hippocampus of mice showed that compared with the control group mice, the treated group was significantly reduced ( Figure 11 B and 11C). A significant decrease in the levels of insoluble Aβ40 and Aβ42 in the cerebral cortex and hippocampus of the treated group mice was also detected (Figure 12 A and 12B).

[0165] TF-DA6c and TF-DAT8b affect inflammatory cytokines in the brain tissue

[0166] As Figure 13 shown, the changes in inflammatory factors in the brains of the TF-DA6c and TF-DAT8b vaccination groups and the control group were compared to explore whether there are safety hazards in the immune vaccine. Compared with the control group, the levels of the four inflammatory factors did not increase and slightly decreased, indicating that the treatment with TF-DA6c and TF-DAT8b does not cause an inflammatory reaction in the brain and can reduce the levels of inflammatory factors in the brain to a certain extent.

[0167] Example 8

[0168] Experiment on the AD vaccine based on the chimeric polypeptide D-HA01:

[0169] The chimeric polypeptide D-HA01 antigen was adsorbed into an equal volume of AS03 adjuvant to immunize BALB / c mice, and the humoral immune response was evaluated by ELISA (as in the method of Example 4).

[0170] Control group mice (Control) (n = 3) were only inoculated with 40 μg of the carrier (the carrier is a protein of HDAg fused with CRM197 (domains A and B), and the expression and purification method of this protein is the same as that of D-HA01 in Example 3) combined with the adjuvant, and experimental group mice (n = 3) were inoculated with the same dose of the chimeric polypeptide (D-HA01). The experimental results showed that: compared with the control group, after inoculation with the chimeric polypeptide, higher levels of antibodies were detected in the mice ([[]] Figure 14 ).

[0171] Example 9

[0172] Experiment on the AD vaccine based on the chimeric polypeptide D-CA01:

[0173] The chimeric polypeptide D-CA01 antigen was adsorbed into an equal volume of AS03 adjuvant to immunize BALB / c mice, and the humoral immune response was evaluated by ELISA (as in the method of Example 4).

[0174] Control group mice (Control) (n = 3) were only inoculated with 40 μg of the carrier (the carrier is a protein of cholera toxin B subunit fused with CRM197 (fragments A and B), and the expression and purification method of this protein is the same as that of D-CA01 in Example 3) combined with the adjuvant, and experimental group mice (n = 3) were inoculated with the same dose of the chimeric polypeptide (D-CA01). The experimental results showed that: compared with the control group, after inoculation with the chimeric polypeptide, higher levels of antibodies were detected in the mice ([[]] Figure 15 ).

[0175] Example 10

[0176] Experiment on the AD vaccine obtained based on the chimeric polypeptide D-TA01:

[0177] The chimeric polypeptide D-TA01 antigen was adsorbed into an equal volume of AS03 adjuvant to immunize BALB / c mice, and the humoral immune response was evaluated by ELISA (as the method in Example 4).

[0178] Control group mice (Control) (n = 3) were only inoculated with 40 μg of the vector (the vector is a protein of transferrin fused with CRM197 (A and B fragments), and the expression and purification method of this protein is the same as that of D-TA01 in Example 3) and the adjuvant, and experimental group mice (n = 3) were inoculated with the same dose of the chimeric polypeptide (D-TA01). The experimental results showed that: compared with the control group, after inoculation with the chimeric polypeptide, higher levels of antibodies were detected in the mice ( Figure 16 ).

[0179] Sequence information:

[0180] The first polypeptide A1 (SEQ ID NO: 1):

[0181] EFRHDSGY

[0182] The second polypeptide T1 (SEQ ID NO: 2):

[0183] KDNIKHVPGGGS

[0184] The chimeric polypeptide DA1a (SEQ ID NO: 3): (EFRHDSGY: positions 69-76)

[0185] MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVD EFRHDSGY AG

[0186] GVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALS

[0187] VELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEE

[0188] KAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHH

[0189] NTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRT

[0190] GFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAF

[0191] HRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0192] DA1a CDS sequence (SEQ ID NO:21):

[0193] ATGGGCGCAGACGATGTTGTGGACTCAAGTAAATCATTTGTCATGGAAAACTTCTCCTCATATCACGGCACGAAACCGG

[0194] GCTACGTTGATAGCATTCAGAAAGGTATCCAAAAACCGAAATCTGGCACGCAGGGTAACTACGATGACGATTGGAAAGA

[0195] ATTCTACAGCACCGACAACAAATATGATGCGGCCGGTTACTCAGTCGACGAGTTCCGCCACGATTCCGGTTACGCCGGC

[0196] GGTGTGGTTAAAGTGACGTATCCGGGCCTGACCAAAGTCCTGGCCCTGAAAGTGGATAATGCAGAAACCATCAAAAAA

[0197] GAACTGGGTCTGAGCCTGACGGAACCGCTGATGGAACAGGTTGGCACCGAAGAATTTATCAAACGCTTCGGCGATGGT

[0198] GCCAGTCGTGTCGTGCTGTCCCTGCCGTTCGCAGAAGGTAGCTCTAGTGTGGAATATATTAACAATTGGGAACAAGCGA

[0199] AAGCCCTGTCCGTTGAACTGGAAATCAACTTTGAAACCCGCGGCAAACGTGGTCAGGATGCGATGTATGAATACATGGC

[0200] ACAAGCTTGCGCGGGTAATCGCGTTCGTCGCAGCGTCGGCTCCTCACTGTCTTGTATCAACCTGGACTGGGATGTTATCC

[0201] GTGATAAAACCAAAACGAAAATCGAAAGTCTGAAAGAACATGGCCCGATCAAAAACAAAATGAGCGAATCTCCGAATA

[0202] AAACGGTGTCCGAAGAAAAAGCTAAACAGTATCTGGAAGAATTCCACCAAACCGCACTGGAACATCCGGAACTGTCAG

[0203] AACTGAAAACCGTGACGGGTACCAACCCGGTTTTTGCCGGCGCAAATTACGCAGCTTGGGCTGTGAACGTTGCGCAAG

[0204] TGATTGACTCGGAAACGGCCGATAATCTGGAAAAAACCACGGCGGCCCTGAGTATTCTGCCGGGCATCGGTTCCGTTAT

[0205] GGGTATTGCCGACGGCGCAGTCCATCACAACACCGAAGAAATTGTGGCCCAGTCTATCGCACTGTCGAGCCTGATGGTT

[0206] GCTCAAGCGATTCCGCTGGTTGGCGAACTGGTTGATATCGGCTTTGCAGCTTACAACTTCGTGGAAAGTATTATCAACCT

[0207] GTTTCAGGTTGTCCACAACTCATATAATCGCCCGGCCTACTCGCCGGGTCACAAAACCCAACCGTTCCTGCATGACGGCT

[0208] ACGCGGTTAGCTGGAATACGGTCGAAGATTCTATTATCCGTACCGGCTTTCAGGGTGAATCTGGCCACGACATTAAAATC

[0209] ACGGCTGAAAACACCCCGCTGCCGATTGCAGGTGTTCTGCTGCCGACGATCCCGGGTAAACTGGATGTTAACAAATCAA

[0210] AAACCCATATCTCGGTCAACGGTCGCAAAATTCGTATGCGCTGCCGTGCGATCGACGGCGATGTGACCTTCTGTCGTCCG

[0211] AAAAGCCCGGTCTATGTGGGCAACGGTGTCCATGCTAATCTGCACGTGGCGTTTCATCGCTCTAGTTCCGAAAAAATCCA

[0212] TAGTAACGAAATCTCATCGGATTCCATTGGTGTGCTGGGCTACCAGAAAACCGTGGACCATACCAAAGTGAATAGCAAA

[0213] CTGAGCCTGTTCTTCGAAATCAAATCGTAA

[0214] Chimeric polypeptide DA4a (SEQ ID NO:4): (EFRHDSGY: positions 53 - 60, 222 - 229, 348 - 355, 501 - 508)

[0215] MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEEFRHDSGYDAAGYSVDNENPLSGKAG

[0216] GVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALS

[0217] VELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEFRHDSGYMSESPNKTVSEE

[0218] KAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVH

[0219] HNTEEIVAQSIALSSLMVAQAIPLVEFRHDSGYAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDS

[0220] IIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLH

[0221] VAFHRSSSEKIHEFRHDSGYIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0222] DA4a CDS sequence (SEQ ID NO:22):

[0223] ATGGGCGCAGACGATGTTGTGGACTCAAGTAAATCATTTGTCATGGAAAACTTCTCCTCATATCACGGCACGAAACCG

[0224] GGCTACGTTGATAGCATTCAGAAAGGTATCCAAAAACCGAAATCTGGCACGCAGGGTAACTACGATGACGATTGGAA

[0225] AGAAGAGTTCCGCCACGATTCCGGTTACGATGCGGCCGGTTACTCAGTCGACAACGAAAATCCGCTGTCGGGCAAAG

[0226] CCGGCGGTGTGGTTAAAGTGACGTATCCGGGCCTGACCAAAGTCCTGGCCCTGAAAGTGGATAATGCAGAAACCATC

[0227] AAAAAAGAACTGGGTCTGAGCCTGACGGAACCGCTGATGGAACAGGTTGGCACCGAAGAATTTATCAAACGCTTCGG

[0228] CGATGGTGCCAGTCGTGTCGTGCTGTCCCTGCCGTTCGCAGAAGGTAGCTCTAGTGTGGAATATATTAACAATTGGGA

[0229] ACAAGCGAAAGCCCTGTCCGTTGAACTGGAAATCAACTTTGAAACCCGCGGCAAACGTGGTCAGGATGCGATGTATG

[0230] AATACATGGCACAAGCTTGCGCGGGTAATCGCGTTCGTCGCAGCGTCGGCTCCTCACTGTCTTGTATCAACCTGGACT

[0231] GGGATGTTATCCGTGATAAAACCAAAACGAAAATCGAAAGTCTGAAAGAGTTCCGCCACGATTCCGGTTACATGAGC

[0232] GAATCTCCGAATAAAACGGTGTCCGAAGAAAAAGCTAAACAGTATCTGGAAGAATTCCACCAAACCGCACTGGAACA

[0233] TCCGGAACTGTCAGAACTGAAAACCGTGACGGGTACCAACCCGGTTTTTGCCGGCGCAAATTACGCAGCTTGGGCTGT

[0234] GAACGTTGCGCAAGTGATTGACTCGGAAACGGCCGATAATCTGGAAAAAACCACGGCGGCCCTGAGTATTCTGCCGG

[0235] GCATCGGTTCCGTTATGGGTATTGCCGACGGCGCAGTCCATCACAACACCGAAGAAATTGTGGCCCAGTCTATCGCAC

[0236] TGTCGAGCCTGATGGTTGCTCAAGCGATTCCGCTGGTTGAGTTCCGCCACGATTCCGGTTACGCAGCTTACAACTTCGT

[0237] GGAAAGTATTATCAACCTGTTTCAGGTTGTCCACAACTCATATAATCGCCCGGCCTACTCGCCGGGTCACAAAACCCA

[0238] ACCGTTCCTGCATGACGGCTACGCGGTTAGCTGGAATACGGTCGAAGATTCTATTATCCGTACCGGCTTTCAGGGTGA

[0239] ATCTGGCCACGACATTAAAATCACGGCTGAAAACACCCCGCTGCCGATTGCAGGTGTTCTGCTGCCGACGATCCCGGG

[0240] TAAACTGGATGTTAACAAATCAAAAACCCATATCTCGGTCAACGGTCGCAAAATTCGTATGCGCTGCCGTGCGATCGA

[0241] CGGCGATGTGACCTTCTGTCGTCCGAAAAGCCCGGTCTATGTGGGCAACGGTGTCCATGCTAATCTGCACGTGGCGTTT

[0242] CATCGCTCTAGTTCCGAAAAAATCCATGAGTTCCGCCACGATTCCGGTTACATTGGTGTGCTGGGCTACCAGAAAACC

[0243] GTGGACCATACCAAAGTGAATAGCAAACTGAGCCTGTTCTTCGAAATCAAATCGTAA

[0244] Chimeric polypeptide DA6b (SEQ ID NO:5): (EFRHDSGY: positions 3 - 10, 69 - 76, 139 - 146, 254 - 261, 317 - 324, 409 - 416)

[0245] MGAEFRHDSGYSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDEFRHDSGYAG

[0246] GVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLEFRHDSGYVEYINNWEQAKAL

[0247] SVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSE

[0248] EKAKQYLEEFHQTEFRHDSGYELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIEFRHD

[0249] SGYTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVED

[0250] SIIRTEFRHDSGYDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANL

[0251] HVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0252] DA6b CDS sequence (SEQ ID NO:23):

[0253] ATGGGCGCAGAGTTCCGCCACGATTCCGGTTACTCATTTGTCATGGAAAACTTCTCCTCATATCACGGCACGAAACCG

[0254] GGCTACGTTGATAGCATTCAGAAAGGTATCCAAAAACCGAAATCTGGCACGCAGGGTAACTACGATGACGATTGGAA

[0255] AGAATTCTACAGCACCGACAACAAATATGATGCGGCCGGTTACTCAGTCGACGAGTTCCGCCACGATTCCGGTTACGC

[0256] CGGCGGTGTGGTTAAAGTGACGTATCCGGGCCTGACCAAAGTCCTGGCCCTGAAAGTGGATAATGCAGAAACCATCA

[0257] AAAAAGAACTGGGTCTGAGCCTGACGGAACCGCTGATGGAACAGGTTGGCACCGAAGAATTTATCAAACGCTTCGGC

[0258] GATGGTGCCAGTCGTGTCGTGCTGTCCCTGGAGTTCCGCCACGATTCCGGTTACGTGGAATATATTAACAATTGGGAA

[0259] CAAGCGAAAGCCCTGTCCGTTGAACTGGAAATCAACTTTGAAACCCGCGGCAAACGTGGTCAGGATGCGATGTATGA

[0260] ATACATGGCACAAGCTTGCGCGGGTAATCGCGTTCGTCGCAGCGTCGGCTCCTCACTGTCTTGTATCAACCTGGACTG

[0261] GGATGTTATCCGTGATAAAACCAAAACGAAAATCGAAAGTCTGAAAGAACATGGCCCGATCAAAAACAAAATGAGCG

[0262] AATCTCCGAATAAAACGGTGTCCGAAGAAAAAGCTAAACAGTATCTGGAAGAATTCCACCAAACCGAGTTCCGCCAC

[0263] GATTCCGGTTACGAACTGAAAACCGTGACGGGTACCAACCCGGTTTTTGCCGGCGCAAATTACGCAGCTTGGGCTGTG

[0264] AACGTTGCGCAAGTGATTGACTCGGAAACGGCCGATAATCTGGAAAAAACCACGGCGGCCCTGAGTATTCTGCCGGG

[0265] CATCGGTTCCGTTATGGGTATTGAGTTCCGCCACGATTCCGGTTACACCGAAGAAATTGTGGCCCAGTCTATCGCACTG

[0266] TCGAGCCTGATGGTTGCTCAAGCGATTCCGCTGGTTGGCGAACTGGTTGATATCGGCTTTGCAGCTTACAACTTCGTGG

[0267] AAAGTATTATCAACCTGTTTCAGGTTGTCCACAACTCATATAATCGCCCGGCCTACTCGCCGGGTCACAAAACCCAAC

[0268] CGTTCCTGCATGACGGCTACGCGGTTAGCTGGAATACGGTCGAAGATTCTATTATCCGTACCGAGTTCCGCCACGATTC

[0269] CGGTTACGACATTAAAATCACGGCTGAAAACACCCCGCTGCCGATTGCAGGTGTTCTGCTGCCGACGATCCCGGGTAA

[0270] ACTGGATGTTAACAAATCAAAAACCCATATCTCGGTCAACGGTCGCAAAATTCGTATGCGCTGCCGTGCGATCGACGG

[0271] CGATGTGACCTTCTGTCGTCCGAAAAGCCCGGTCTATGTGGGCAACGGTGTCCATGCTAATCTGCACGTGGCGTTTCAT

[0272] CGCTCTAGTTCCGAAAAAATCCATAGTAACGAAATCTCATCGGATTCCATTGGTGTGCTGGGCTACCAGAAAACCGTG

[0273] GACCATACCAAAGTGAATAGCAAACTGAGCCTGTTCTTCGAAATCAAATCGTAA

[0274] Chimeric polypeptide DA6c (SEQ ID NO:6): (EFRHDSGY: positions 20-27, 104-111, 168-175, 276-283, 348-355, 501-508)

[0275] MGADDVVDSSKSFVMENFSSEFRHDSGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAG

[0276] GVVKVTYPGLTKVLALKVDNAETIKEFRHDSGYEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKAL

[0277] SVELEINFEFRHDSGYDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEE

[0278] KAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGEFRHDSGYNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHH

[0279] NTEEIVAQSIALSSLMVAQAIPLVEFRHDSGYAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSII

[0280] RTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHV

[0281] AFHRSSSEKIHEFRHDSGYIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0282] DA6c CDS sequence (SEQ ID NO:24):

[0283] ATGGGCGCAGACGATGTTGTGGACTCAAGTAAATCATTTGTCATGGAAAACTTCTCCTCAGAGTTCCGCCACGATTCC

[0284] GGTTACGTTGATAGCATTCAGAAAGGTATCCAAAAACCGAAATCTGGCACGCAGGGTAACTACGATGACGATTGGAA

[0285] AGAATTCTACAGCACCGACAACAAATATGATGCGGCCGGTTACTCAGTCGACAACGAAAATCCGCTGTCGGGCAAAG

[0286] CCGGCGGTGTGGTTAAAGTGACGTATCCGGGCCTGACCAAAGTCCTGGCCCTGAAAGTGGATAATGCAGAAACCATC

[0287] AAAGAGTTCCGCCACGATTCCGGTTACGAACCGCTGATGGAACAGGTTGGCACCGAAGAATTTATCAAACGCTTCGGC

[0288] GATGGTGCCAGTCGTGTCGTGCTGTCCCTGCCGTTCGCAGAAGGTAGCTCTAGTGTGGAATATATTAACAATTGGGAA

[0289] CAAGCGAAAGCCCTGTCCGTTGAACTGGAAATCAACTTTGAAACCCGCGGCAAACGTGGTCAGGATGCGATGTATGA

[0290] ATACATGGCACAAGCTTGCGCGGGTAATCGCGTTCGTCGCAGCGTCGGCTCCTCACTGTCTTGTATCAACCTGGACTG

[0291] GGATGTTATCCGTGATAAAACCAAAACGAAAATCGAAAGTCTGAAAGAGTTCCGCCACGATTCCGGTTACATGAGCG

[0292] AATCTCCGAATAAAACGGTGTCCGAAGAAAAAGCTAAACAGTATCTGGAAGAATTCCACCAAACCGCACTGGAACAT

[0293] CCGGAACTGTCAGAACTGAAAACCGTGACGGGTACCAACCCGGTTTTTGCCGGCGCAAATTACGCAGCTTGGGCTGTG

[0294] AACGTTGCGCAAGTGATTGAGTTCCGCCACGATTCCGGTTACGAAAAAACCACGGCGGCCCTGAGTATTCTGCCGGGC

[0295] ATCGGTTCCGTTATGGGTATTGCCGACGGCGCAGTCCATCACAACACCGAAGAAATTGTGGCCCAGTCTATCGCACTG

[0296] TCGAGCCTGATGGTTGCTCAAGCGATTCCGCTGGTTGGCGAACTGGTTGATATCGGCTTTGCAGCTTACAACTTCGTGG

[0297] AAAGTATTATCAACCTGTTTCAGGTTGTCCACAACTCATATAATCGCCCGGCCTACTCGCCGGGTCACAAAACCCAAC

[0298] CGTTCCTGCATGACGGCTACGCGGTTAGCTGGAATACGGTCGAAGATTCTATTATCCGTACCGAGTTCCGCCACGATTC

[0299] CGGTTACGACATTAAAATCACGGCTGAAAACACCCCGCTGCCGATTGCAGGTGTTCTGCTGCCGACGATCCCGGGTAA

[0300] ACTGGATGTTAACAAATCAAAAACCCATATCTCGGTCAACGGTCGCAAAATTCGTATGCGCTGCCGTGCGATCGACGG

[0301] CGATGTGACCTTCTGTCGTCCGAAAAGCCCGGTCTATGTGGGCAACGGTGTCCATGCTAATCTGCACGTGGCGTTTCAT

[0302] CGCTCTAGTTCCGAAAAAATCCATGAGTTCCGCCACGATTCCGGTTACATTGGTGTGCTGGGCTACCAGAAAACCGTG

[0303] GACCATACCAAAGTGAATAGCAAACTGAGCCTGTTCTTCGAAATCAAATCGTAA

[0304] Chimeric polypeptide DA10b (SEQ ID NO:7): (EFRHDSGY: positions 3-10, 20-27, 53-70, 69-76, 104-111, 127-134, 139-146, 168-175, 222-229, 254-261)

[0305] MGAEFRHDSGYSFVMENFSSEFRHDSGYVDSIQKGIQKPKSGTQGNYDDDWKEEFRHDSGYDAAGYSVDEFRHDSGYAG

[0306] GVVKVTYPGLTKVLALKVDNAETIKEFRHDSGYEPLMEQVGTEEFIKREFRHDSGYVLSLEFRHDSGYVEYINNWEQAKAL

[0307] SVELEINFEFRHDSGYDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEFRHDSGYMSESPNKTVSE

[0308] EKAKQYLEEFHQTEFRHDSGYELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGA

[0309] VHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVE

[0310] DSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHAN

[0311] LHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0312] DA10b CDS sequence (SEQ ID NO:25):

[0313] ATGGGCGCAGAGTTCCGCCACGATTCCGGTTACTCATTTGTCATGGAAAACTTCTCCTCAGAGTTCCGCCACGATTCCG

[0314] GTTACGTTGATAGCATTCAGAAAGGTATCCAAAAACCGAAATCTGGCACGCAGGGTAACTACGATGACGATTGGAAA

[0315] GAAGAGTTCCGCCACGATTCCGGTTACGATGCGGCCGGTTACTCAGTCGACGAGTTCCGCCACGATTCCGGTTACGCC

[0316] GGCGGTGTGGTTAAAGTGACGTATCCGGGCCTGACCAAAGTCCTGGCCCTGAAAGTGGATAATGCAGAAACCATCAA

[0317] AGAGTTCCGCCACGATTCCGGTTACGAACCGCTGATGGAACAGGTTGGCACCGAAGAATTTATCAAACGCGAGTTCCG

[0318] CCACGATTCCGGTTACGTGCTGTCCCTGGAGTTCCGCCACGATTCCGGTTACGTGGAATATATTAACAATTGGGAACA

[0319] AGCGAAAGCCCTGTCCGTTGAACTGGAAATCAACTTTGAGTTCCGCCACGATTCCGGTTACGATGCGATGTATGAATA

[0320] CATGGCACAAGCTTGCGCGGGTAATCGCGTTCGTCGCAGCGTCGGCTCCTCACTGTCTTGTATCAACCTGGACTGGGA

[0321] TGTTATCCGTGATAAAACCAAAACGAAAATCGAAAGTCTGAAAGAGTTCCGCCACGATTCCGGTTACATGAGCGAATC

[0322] TCCGAATAAAACGGTGTCCGAAGAAAAAGCTAAACAGTATCTGGAAGAATTCCACCAAACCGAGTTCCGCCACGATT

[0323] CCGGTTACGAACTGAAAACCGTGACGGGTACCAACCCGGTTTTTGCCGGCGCAAATTACGCAGCTTGGGCTGTGAACG

[0324] TTGCGCAAGTGATTGACTCGGAAACGGCCGATAATCTGGAAAAAACCACGGCGGCCCTGAGTATTCTGCCGGGCATC

[0325] GGTTCCGTTATGGGTATTGCCGACGGCGCAGTCCATCACAACACCGAAGAAATTGTGGCCCAGTCTATCGCACTGTCG

[0326] AGCCTGATGGTTGCTCAAGCGATTCCGCTGGTTGGCGAACTGGTTGATATCGGCTTTGCAGCTTACAACTTCGTGGAAA

[0327] GTATTATCAACCTGTTTCAGGTTGTCCACAACTCATATAATCGCCCGGCCTACTCGCCGGGTCACAAAACCCAACCGTT

[0328] CCTGCATGACGGCTACGCGGTTAGCTGGAATACGGTCGAAGATTCTATTATCCGTACCGGCTTTCAGGGTGAATCTGG

[0329] CCACGACATTAAAATCACGGCTGAAAACACCCCGCTGCCGATTGCAGGTGTTCTGCTGCCGACGATCCCGGGTAAACT

[0330] GGATGTTAACAAATCAAAAACCCATATCTCGGTCAACGGTCGCAAAATTCGTATGCGCTGCCGTGCGATCGACGGCGA

[0331] TGTGACCTTCTGTCGTCCGAAAAGCCCGGTCTATGTGGGCAACGGTGTCCATGCTAATCTGCACGTGGCGTTTCATCGC

[0332] TCTAGTTCCGAAAAAATCCATAGTAACGAAATCTCATCGGATTCCATTGGTGTGCTGGGCTACCAGAAAACCGTGGAC

[0333] CATACCAAAGTGAATAGCAAACTGAGCCTGTTCTTCGAAATCAAATCGTAA

[0334] Chimeric polypeptide DT1a (SEQ ID NO:8): (KDNIKHVPGGGS: positions 16 - 27)

[0335] MGADDVVDSSKSFVMEKDNIKHVPGGGSVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAG

[0336] GVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALS

[0337] VELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEE

[0338] KAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVH

[0339] HNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSI

[0340] IRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLH

[0341] VAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0342] DT1a CDS sequence (SEQ ID NO:26):

[0343] ATGGGCGCAGACGATGTTGTGGACTCAAGTAAATCATTTGTCATGGAAAAAGATAATATTAAACATGTTCCGGGTGGC

[0344] GGCAGTGTTGATAGCATTCAGAAAGGTATCCAAAAACCGAAATCTGGCACGCAGGGTAACTACGATGACGATTGGAA

[0345] AGAATTCTACAGCACCGACAACAAATATGATGCGGCCGGTTACTCAGTCGACAACGAAAATCCGCTGTCGGGCAAAG

[0346] CCGGCGGTGTGGTTAAAGTGACGTATCCGGGCCTGACCAAAGTCCTGGCCCTGAAAGTGGATAATGCAGAAACCATC

[0347] AAAAAAGAACTGGGTCTGAGCCTGACGGAACCGCTGATGGAACAGGTTGGCACCGAAGAATTTATCAAACGCTTCGG

[0348] CGATGGTGCCAGTCGTGTCGTGCTGTCCCTGCCGTTCGCAGAAGGTAGCTCTAGTGTGGAATATATTAACAATTGGGA

[0349] ACAAGCGAAAGCCCTGTCCGTTGAACTGGAAATCAACTTTGAAACCCGCGGCAAACGTGGTCAGGATGCGATGTATG

[0350] AATACATGGCACAAGCTTGCGCGGGTAATCGCGTTCGTCGCAGCGTCGGCTCCTCACTGTCTTGTATCAACCTGGACT

[0351] GGGATGTTATCCGTGATAAAACCAAAACGAAAATCGAAAGTCTGAAAGAACATGGCCCGATCAAAAACAAAATGAGC

[0352] GAATCTCCGAATAAAACGGTGTCCGAAGAAAAAGCTAAACAGTATCTGGAAGAATTCCACCAAACCGCACTGGAACA

[0353] TCCGGAACTGTCAGAACTGAAAACCGTGACGGGTACCAACCCGGTTTTTGCCGGCGCAAATTACGCAGCTTGGGCTGT

[0354] GAACGTTGCGCAAGTGATTGACTCGGAAACGGCCGATAATCTGGAAAAAACCACGGCGGCCCTGAGTATTCTGCCGG

[0355] GCATCGGTTCCGTTATGGGTATTGCCGACGGCGCAGTCCATCACAACACCGAAGAAATTGTGGCCCAGTCTATCGCAC

[0356] TGTCGAGCCTGATGGTTGCTCAAGCGATTCCGCTGGTTGGCGAACTGGTTGATATCGGCTTTGCAGCTTACAACTTCGT

[0357] GGAAAGTATTATCAACCTGTTTCAGGTTGTCCACAACTCATATAATCGCCCGGCCTACTCGCCGGGTCACAAAACCCA

[0358] ACCGTTCCTGCATGACGGCTACGCGGTTAGCTGGAATACGGTCGAAGATTCTATTATCCGTACCGGCTTTCAGGGTGA

[0359] ATCTGGCCACGACATTAAAATCACGGCTGAAAACACCCCGCTGCCGATTGCAGGTGTTCTGCTGCCGACGATCCCGGG

[0360] TAAACTGGATGTTAACAAATCAAAAACCCATATCTCGGTCAACGGTCGCAAAATTCGTATGCGCTGCCGTGCGATCGA

[0361] CGGCGATGTGACCTTCTGTCGTCCGAAAAGCCCGGTCTATGTGGGCAACGGTGTCCATGCTAATCTGCACGTGGCGTTT

[0362] CATCGCTCTAGTTCCGAAAAAATCCATAGTAACGAAATCTCATCGGATTCCATTGGTGTGCTGGGCTACCAGAAAACC

[0363] GTGGACCATACCAAAGTGAATAGCAAACTGAGCCTGTTCTTCGAAATCAAATCGTAA

[0364] Chimeric polypeptide DT5a (SEQ ID NO:9): (KDNIKHVPGGGS: positions 6 - 17, 66 - 77, 222 - 233, 314 - 325, 409 - 420)

[0365] MGADDVKDNIKHVPGGGSSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYKDNIKHVPGGGSG

[0366] GVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALS

[0367] VELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKKDNIKHVPGGGSPNKTVSEE

[0368] KAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVKDNIKHVPG

[0369] GGSEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSI

[0370] IRTKDNIKHVPGGGSTAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANL

[0371] HVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0372] DT5a CDS sequence (SEQ ID NO:27):

[0373] ATGGGCGCAGACGATGTTAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTTCCTCATATCACGGCACGAAACCG

[0374] GGCTACGTTGATAGCATTCAGAAAGGTATCCAAAAACCGAAATCTGGCACGCAGGGTAACTACGATGACGATTGGAA

[0375] AGAATTCTACAGCACCGACAACAAATATGATGCGGCCGGTTACAAAGATAATATTAAACATGTTCCGGGTGGCGGCA

[0376] GTGGCGGTGTGGTTAAAGTGACGTATCCGGGCCTGACCAAAGTCCTGGCCCTGAAAGTGGATAATGCAGAAACCATC

[0377] AAAAAAGAACTGGGTCTGAGCCTGACGGAACCGCTGATGGAACAGGTTGGCACCGAAGAATTTATCAAACGCTTCGG

[0378] CGATGGTGCCAGTCGTGTCGTGCTGTCCCTGCCGTTCGCAGAAGGTAGCTCTAGTGTGGAATATATTAACAATTGGGA

[0379] ACAAGCGAAAGCCCTGTCCGTTGAACTGGAAATCAACTTTGAAACCCGCGGCAAACGTGGTCAGGATGCGATGTATG

[0380] AATACATGGCACAAGCTTGCGCGGGTAATCGCGTTCGTCGCAGCGTCGGCTCCTCACTGTCTTGTATCAACCTGGACT

[0381] GGGATGTTATCCGTGATAAAACCAAAACGAAAATCGAAAGTCTGAAAAAAGATAATATTAAACATGTTCCGGGTGGC

[0382] GGCAGTCCGAATAAAACGGTGTCCGAAGAAAAAGCTAAACAGTATCTGGAAGAATTCCACCAAACCGCACTGGAACA

[0383] TCCGGAACTGTCAGAACTGAAAACCGTGACGGGTACCAACCCGGTTTTTGCCGGCGCAAATTACGCAGCTTGGGCTGT

[0384] GAACGTTGCGCAAGTGATTGACTCGGAAACGGCCGATAATCTGGAAAAAACCACGGCGGCCCTGAGTATTCTGCCGG

[0385] GCATCGGTTCCGTTAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTGAAGAAATTGTGGCCCAGTCTATCGCAC

[0386] TGTCGAGCCTGATGGTTGCTCAAGCGATTCCGCTGGTTGGCGAACTGGTTGATATCGGCTTTGCAGCTTACAACTTCGT

[0387] GGAAAGTATTATCAACCTGTTTCAGGTTGTCCACAACTCATATAATCGCCCGGCCTACTCGCCGGGTCACAAAACCCA

[0388] ACCGTTCCTGCATGACGGCTACGCGGTTAGCTGGAATACGGTCGAAGATTCTATTATCCGTACCAAAGATAATATTAA

[0389] ACATGTTCCGGGTGGCGGCAGTACGGCTGAAAACACCCCGCTGCCGATTGCAGGTGTTCTGCTGCCGACGATCCCGGG

[0390] TAAACTGGATGTTAACAAATCAAAAACCCATATCTCGGTCAACGGTCGCAAAATTCGTATGCGCTGCCGTGCGATCGA

[0391] CGGCGATGTGACCTTCTGTCGTCCGAAAAGCCCGGTCTATGTGGGCAACGGTGTCCATGCTAATCTGCACGTGGCGTTT

[0392] CATCGCTCTAGTTCCGAAAAAATCCATAGTAACGAAATCTCATCGGATTCCATTGGTGTGCTGGGCTACCAGAAAACC

[0393] GTGGACCATACCAAAGTGAATAGCAAACTGAGCCTGTTCTTCGAAATCAAATCGTAA

[0394] Chimeric polypeptide DT5b (SEQ ID NO:10): (KDNIKHVPGGGS: positions 16 - 27, 103 - 114, 270 - 281, 409 - 420, 497 - 508)

[0395] MGADDVVDSSKSFVMEKDNIKHVPGGGSVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAG

[0396] GVVKVTYPGLTKVLALKVDNAETIKDNIKHVPGGGSMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKAL

[0397] SVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSE

[0398] EKAKQYLEEFHQTALEHPELSELKTVTGTKDNIKHVPGGGSAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVH

[0399] HNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSI

[0400] IRTKDNIKHVPGGGSTAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANL

[0401] HVAFHRSSSKDNIKHVPGGGSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0402] CDS sequence of DT5a (SEQ ID NO:28):

[0403] ATGGGCGCAGACGATGTTGTGGACTCAAGTAAATCATTTGTCATGGAAAAAGATAATATTAAACATGTTCCGGGTGGC

[0404] GGCAGTGTTGATAGCATTCAGAAAGGTATCCAAAAACCGAAATCTGGCACGCAGGGTAACTACGATGACGATTGGAA

[0405] AGAATTCTACAGCACCGACAACAAATATGATGCGGCCGGTTACTCAGTCGACAACGAAAATCCGCTGTCGGGCAAAG

[0406] CCGGCGGTGTGGTTAAAGTGACGTATCCGGGCCTGACCAAAGTCCTGGCCCTGAAAGTGGATAATGCAGAAACCATC

[0407] AAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTATGGAACAGGTTGGCACCGAAGAATTTATCAAACGCTTCGGC

[0408] GATGGTGCCAGTCGTGTCGTGCTGTCCCTGCCGTTCGCAGAAGGTAGCTCTAGTGTGGAATATATTAACAATTGGGAA

[0409] CAAGCGAAAGCCCTGTCCGTTGAACTGGAAATCAACTTTGAAACCCGCGGCAAACGTGGTCAGGATGCGATGTATGA

[0410] ATACATGGCACAAGCTTGCGCGGGTAATCGCGTTCGTCGCAGCGTCGGCTCCTCACTGTCTTGTATCAACCTGGACTG

[0411] GGATGTTATCCGTGATAAAACCAAAACGAAAATCGAAAGTCTGAAAGAACATGGCCCGATCAAAAACAAAATGAGCG

[0412] AATCTCCGAATAAAACGGTGTCCGAAGAAAAAGCTAAACAGTATCTGGAAGAATTCCACCAAACCGCACTGGAACAT

[0413] CCGGAACTGTCAGAACTGAAAACCGTGACGGGTACCAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTGCTGT

[0414] GAACGTTGCGCAAGTGATTGACTCGGAAACGGCCGATAATCTGGAAAAAACCACGGCGGCCCTGAGTATTCTGCCGG

[0415] GCATCGGTTCCGTTATGGGTATTGCCGACGGCGCAGTCCATCACAACACCGAAGAAATTGTGGCCCAGTCTATCGCAC

[0416] TGTCGAGCCTGATGGTTGCTCAAGCGATTCCGCTGGTTGGCGAACTGGTTGATATCGGCTTTGCAGCTTACAACTTCGT

[0417] GGAAAGTATTATCAACCTGTTTCAGGTTGTCCACAACTCATATAATCGCCCGGCCTACTCGCCGGGTCACAAAACCCA

[0418] ACCGTTCCTGCATGACGGCTACGCGGTTAGCTGGAATACGGTCGAAGATTCTATTATCCGTACCAAAGATAATATTAA

[0419] ACATGTTCCGGGTGGCGGCAGTACGGCTGAAAACACCCCGCTGCCGATTGCAGGTGTTCTGCTGCCGACGATCCCGGG

[0420] TAAACTGGATGTTAACAAATCAAAAACCCATATCTCGGTCAACGGTCGCAAAATTCGTATGCGCTGCCGTGCGATCGA

[0421] CGGCGATGTGACCTTCTGTCGTCCGAAAAGCCCGGTCTATGTGGGCAACGGTGTCCATGCTAATCTGCACGTGGCGTTT

[0422] CATCGCTCTAGTTCCAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTATTGGTGTGCTGGGCTACCAGAAAACC

[0423] GTGGACCATACCAAAGTGAATAGCAAACTGAGCCTGTTCTTCGAAATCAAATCGTAA

[0424] Chimeric polypeptide DT6b (SEQ ID NO:11): (KDNIKHVPGGGS: positions 6 - 17, 103 - 114, 222 - 233, 270 - 281, 381 - 392, 497 - 508)

[0425] MGADDVKDNIKHVPGGGSSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAG

[0426] GVVKVTYPGLTKVLALKVDNAETIKDNIKHVPGGGSMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKAL

[0427] SVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKKDNIKHVPGGGSPNKTVSE

[0428] EKAKQYLEEFHQTALEHPELSELKTVTGTKDNIKHVPGGGSAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVH

[0429] HNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYKDNIKHVPGGGSGYAVSWNTVEDSI

[0430] IRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLH

[0431] VAFHRSSSKDNIKHVPGGGSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0432] DT6b CDS sequence (SEQ ID NO:29):

[0433] ATGGGCGCAGACGATGTTAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTTCCTCATATCACGGCACGAAACCG

[0434] GGCTACGTTGATAGCATTCAGAAAGGTATCCAAAAACCGAAATCTGGCACGCAGGGTAACTACGATGACGATTGGAA

[0435] AGAATTCTACAGCACCGACAACAAATATGATGCGGCCGGTTACTCAGTCGACAACGAAAATCCGCTGTCGGGCAAAG

[0436] CCGGCGGTGTGGTTAAAGTGACGTATCCGGGCCTGACCAAAGTCCTGGCCCTGAAAGTGGATAATGCAGAAACCATC

[0437] AAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTATGGAACAGGTTGGCACCGAAGAATTTATCAAACGCTTCGGC

[0438] GATGGTGCCAGTCGTGTCGTGCTGTCCCTGCCGTTCGCAGAAGGTAGCTCTAGTGTGGAATATATTAACAATTGGGAA

[0439] CAAGCGAAAGCCCTGTCCGTTGAACTGGAAATCAACTTTGAAACCCGCGGCAAACGTGGTCAGGATGCGATGTATGA

[0440] ATACATGGCACAAGCTTGCGCGGGTAATCGCGTTCGTCGCAGCGTCGGCTCCTCACTGTCTTGTATCAACCTGGACTG

[0441] GGATGTTATCCGTGATAAAACCAAAACGAAAATCGAAAGTCTGAAAAAAGATAATATTAAACATGTTCCGGGTGGCG

[0442] GCAGTCCGAATAAAACGGTGTCCGAAGAAAAAGCTAAACAGTATCTGGAAGAATTCCACCAAACCGCACTGGAACAT

[0443] CCGGAACTGTCAGAACTGAAAACCGTGACGGGTACCAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTGCTGT

[0444] GAACGTTGCGCAAGTGATTGACTCGGAAACGGCCGATAATCTGGAAAAAACCACGGCGGCCCTGAGTATTCTGCCGG

[0445] GCATCGGTTCCGTTATGGGTATTGCCGACGGCGCAGTCCATCACAACACCGAAGAAATTGTGGCCCAGTCTATCGCAC

[0446] TGTCGAGCCTGATGGTTGCTCAAGCGATTCCGCTGGTTGGCGAACTGGTTGATATCGGCTTTGCAGCTTACAACTTCGT

[0447] GGAAAGTATTATCAACCTGTTTCAGGTTGTCCACAACTCATATAATCGCCCGGCCTACAAAGATAATATTAAACATGTT

[0448] CCGGGTGGCGGCAGTGGCTACGCGGTTAGCTGGAATACGGTCGAAGATTCTATTATCCGTACCGGCTTTCAGGGTGAA

[0449] TCTGGCCACGACATTAAAATCACGGCTGAAAACACCCCGCTGCCGATTGCAGGTGTTCTGCTGCCGACGATCCCGGGT

[0450] AAACTGGATGTTAACAAATCAAAAACCCATATCTCGGTCAACGGTCGCAAAATTCGTATGCGCTGCCGTGCGATCGAC

[0451] GGCGATGTGACCTTCTGTCGTCCGAAAAGCCCGGTCTATGTGGGCAACGGTGTCCATGCTAATCTGCACGTGGCGTTTC

[0452] ATCGCTCTAGTTCCAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTATTGGTGTGCTGGGCTACCAGAAAACCG

[0453] TGGACCATACCAAAGTGAATAGCAAACTGAGCCTGTTCTTCGAAATCAAATCGTAA

[0454] Chimeric polypeptide DT9a (SEQ ID NO: 12): (KDNIKHVPGGGS: positions 16 - 27, 66 - 77, 128 - 139, 164 - 175, 249 - 260, 286 - 297, 344 - 355, 409 - 420, 497 - 508)

[0455] MGADDVVDSSKSFVMEKDNIKHVPGGGSVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYKDNIKHVPGGGSG

[0456] GVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFKDNIKHVPGGGSFAEGSSSVEYINNWEQAKALS

[0457] VELKDNIKHVPGGGSDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEE

[0458] KAKQYLEKDNIKHVPGGGSSELKTVTGTNPVFAGANYAAWAVNVKDNIKHVPGGGSEKTTAALSILPGIGSVMGIADGAV

[0459] HHNTEEIVAQSIALSSLMVAQAKDNIKHVPGGGSAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVE

[0460] DSIIRTKDNIKHVPGGGSTAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHA

[0461] NLHVAFHRSSSKDNIKHVPGGGSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0462] CDS sequence of DT9a (SEQ ID NO:30):

[0463] ATGGGCGCAGACGATGTTGTGGACTCAAGTAAATCATTTGTCATGGAAAAAGATAATATTAAACATGTTCCGGGTGGC

[0464] GGCAGTGTTGATAGCATTCAGAAAGGTATCCAAAAACCGAAATCTGGCACGCAGGGTAACTACGATGACGATTGGAA

[0465] AGAATTCTACAGCACCGACAACAAATATGATGCGGCCGGTTACAAAGATAATATTAAACATGTTCCGGGTGGCGGCA

[0466] GTGGCGGTGTGGTTAAAGTGACGTATCCGGGCCTGACCAAAGTCCTGGCCCTGAAAGTGGATAATGCAGAAACCATC

[0467] AAAAAAGAACTGGGTCTGAGCCTGACGGAACCGCTGATGGAACAGGTTGGCACCGAAGAATTTATCAAACGCTTCAA

[0468] AGATAATATTAAACATGTTCCGGGTGGCGGCAGTTTCGCAGAAGGTAGCTCTAGTGTGGAATATATTAACAATTGGGA

[0469] ACAAGCGAAAGCCCTGTCCGTTGAACTGAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTGATGCGATGTATGA

[0470] ATACATGGCACAAGCTTGCGCGGGTAATCGCGTTCGTCGCAGCGTCGGCTCCTCACTGTCTTGTATCAACCTGGACTG

[0471] GGATGTTATCCGTGATAAAACCAAAACGAAAATCGAAAGTCTGAAAGAACATGGCCCGATCAAAAACAAAATGAGCG

[0472] AATCTCCGAATAAAACGGTGTCCGAAGAAAAAGCTAAACAGTATCTGGAAAAAGATAATATTAAACATGTTCCGGGT

[0473] GGCGGCAGTTCAGAACTGAAAACCGTGACGGGTACCAACCCGGTTTTTGCCGGCGCAAATTACGCAGCTTGGGCTGTG

[0474] AACGTTAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTGAAAAAACCACGGCGGCCCTGAGTATTCTGCCGGG

[0475] CATCGGTTCCGTTATGGGTATTGCCGACGGCGCAGTCCATCACAACACCGAAGAAATTGTGGCCCAGTCTATCGCACT

[0476] GTCGAGCCTGATGGTTGCTCAAGCGAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTGCAGCTTACAACTTCGT

[0477] GGAAAGTATTATCAACCTGTTTCAGGTTGTCCACAACTCATATAATCGCCCGGCCTACTCGCCGGGTCACAAAACCCA

[0478] ACCGTTCCTGCATGACGGCTACGCGGTTAGCTGGAATACGGTCGAAGATTCTATTATCCGTACCAAAGATAATATTAA

[0479] ACATGTTCCGGGTGGCGGCAGTACGGCTGAAAACACCCCGCTGCCGATTGCAGGTGTTCTGCTGCCGACGATCCCGGG

[0480] TAAACTGGATGTTAACAAATCAAAAACCCATATCTCGGTCAACGGTCGCAAAATTCGTATGCGCTGCCGTGCGATCGA

[0481] CGGCGATGTGACCTTCTGTCGTCCGAAAAGCCCGGTCTATGTGGGCAACGGTGTCCATGCTAATCTGCACGTGGCGTTT

[0482] CATCGCTCTAGTTCCAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTATTGGTGTGCTGGGCTACCAGAAAACC

[0483] GTGGACCATACCAAAGTGAATAGCAAACTGAGCCTGTTCTTCGAAATCAAATCGTAA

[0484] Chimeric polypeptide DAT4a (SEQ ID NO:13): (EFRHDSGY: positions 139 - 146, 501 - 508; KDNIKHVPGGGS: positions 6 - 17, 314 - 325)

[0485] MGADDVKDNIKHVPGGGSSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAG

[0486] GVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLEFRHDSGYVEYINNWEQAKAL

[0487] SVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSE

[0488] EKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVKDNIKHVP

[0489] GGGSEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVED

[0490] SIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANL

[0491] HVAFHRSSSEKIHEFRHDSGYIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0492] DAT4a CDS sequence (SEQ ID NO:31):

[0493] ATGGGCGCAGACGATGTTAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTTCCTCATATCACGGCACGAAACCG

[0494] GGCTACGTTGATAGCATTCAGAAAGGTATCCAAAAACCGAAATCTGGCACGCAGGGTAACTACGATGACGATTGGAA

[0495] AGAATTCTACAGCACCGACAACAAATATGATGCGGCCGGTTACTCAGTCGACAACGAAAATCCGCTGTCGGGCAAAG

[0496] CCGGCGGTGTGGTTAAAGTGACGTATCCGGGCCTGACCAAAGTCCTGGCCCTGAAAGTGGATAATGCAGAAACCATC

[0497] AAAAAAGAACTGGGTCTGAGCCTGACGGAACCGCTGATGGAACAGGTTGGCACCGAAGAATTTATCAAACGCTTCGG

[0498] CGATGGTGCCAGTCGTGTCGTGCTGTCCCTGGAGTTCCGCCACGATTCCGGTTACGTGGAATATATTAACAATTGGGA

[0499] ACAAGCGAAAGCCCTGTCCGTTGAACTGGAAATCAACTTTGAAACCCGCGGCAAACGTGGTCAGGATGCGATGTATG

[0500] AATACATGGCACAAGCTTGCGCGGGTAATCGCGTTCGTCGCAGCGTCGGCTCCTCACTGTCTTGTATCAACCTGGACT

[0501] GGGATGTTATCCGTGATAAAACCAAAACGAAAATCGAAAGTCTGAAAGAACATGGCCCGATCAAAAACAAAATGAGC

[0502] GAATCTCCGAATAAAACGGTGTCCGAAGAAAAAGCTAAACAGTATCTGGAAGAATTCCACCAAACCGCACTGGAACA

[0503] TCCGGAACTGTCAGAACTGAAAACCGTGACGGGTACCAACCCGGTTTTTGCCGGCGCAAATTACGCAGCTTGGGCTGT

[0504] GAACGTTGCGCAAGTGATTGACTCGGAAACGGCCGATAATCTGGAAAAAACCACGGCGGCCCTGAGTATTCTGCCGG

[0505] GCATCGGTTCCGTTAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTGAAGAAATTGTGGCCCAGTCTATCGCAC

[0506] TGTCGAGCCTGATGGTTGCTCAAGCGATTCCGCTGGTTGGCGAACTGGTTGATATCGGCTTTGCAGCTTACAACTTCGT

[0507] GGAAAGTATTATCAACCTGTTTCAGGTTGTCCACAACTCATATAATCGCCCGGCCTACTCGCCGGGTCACAAAACCCA

[0508] ACCGTTCCTGCATGACGGCTACGCGGTTAGCTGGAATACGGTCGAAGATTCTATTATCCGTACCGGCTTTCAGGGTGA

[0509] ATCTGGCCACGACATTAAAATCACGGCTGAAAACACCCCGCTGCCGATTGCAGGTGTTCTGCTGCCGACGATCCCGGG

[0510] TAAACTGGATGTTAACAAATCAAAAACCCATATCTCGGTCAACGGTCGCAAAATTCGTATGCGCTGCCGTGCGATCGA

[0511] CGGCGATGTGACCTTCTGTCGTCCGAAAAGCCCGGTCTATGTGGGCAACGGTGTCCATGCTAATCTGCACGTGGCGTTT

[0512] CATCGCTCTAGTTCCGAAAAAATCCATGAGTTCCGCCACGATTCCGGTTACATTGGTGTGCTGGGCTACCAGAAAACC

[0513] GTGGACCATACCAAAGTGAATAGCAAACTGAGCCTGTTCTTCGAAATCAAATCGTAA

[0514] Chimeric polypeptide DAT4b (SEQ ID NO:14): (EFRHDSGY: positions 3-10, 317-324; KDNIKHVPGGGS: positions 138-149, 497-508)

[0515] MGAEFRHDSGYSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAG

[0516] GVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSKDNIKHVPGGGSINNWEQAKAL

[0517] SVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSE

[0518] EKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIEFRHD

[0519] SGYTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVED

[0520] SIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANL

[0521] HVAFHRSSSKDNIKHVPGGGSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0522] DAT4b CDS sequence (SEQ ID NO:32):

[0523] ATGGGCGCAGAGTTCCGCCACGATTCCGGTTACTCATTTGTCATGGAAAACTTCTCCTCATATCACGGCACGAAACCG

[0524] GGCTACGTTGATAGCATTCAGAAAGGTATCCAAAAACCGAAATCTGGCACGCAGGGTAACTACGATGACGATTGGAA

[0525] AGAATTCTACAGCACCGACAACAAATATGATGCGGCCGGTTACTCAGTCGACAACGAAAATCCGCTGTCGGGCAAAG

[0526] CCGGCGGTGTGGTTAAAGTGACGTATCCGGGCCTGACCAAAGTCCTGGCCCTGAAAGTGGATAATGCAGAAACCATC

[0527] AAAAAAGAACTGGGTCTGAGCCTGACGGAACCGCTGATGGAACAGGTTGGCACCGAAGAATTTATCAAACGCTTCGG

[0528] CGATGGTGCCAGTCGTGTCGTGCTGTCCAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTATTAACAATTGGGA

[0529] ACAAGCGAAAGCCCTGTCCGTTGAACTGGAAATCAACTTTGAAACCCGCGGCAAACGTGGTCAGGATGCGATGTATG

[0530] AATACATGGCACAAGCTTGCGCGGGTAATCGCGTTCGTCGCAGCGTCGGCTCCTCACTGTCTTGTATCAACCTGGACT

[0531] GGGATGTTATCCGTGATAAAACCAAAACGAAAATCGAAAGTCTGAAAGAACATGGCCCGATCAAAAACAAAATGAGC

[0532] GAATCTCCGAATAAAACGGTGTCCGAAGAAAAAGCTAAACAGTATCTGGAAGAATTCCACCAAACCGCACTGGAACA

[0533] TCCGGAACTGTCAGAACTGAAAACCGTGACGGGTACCAACCCGGTTTTTGCCGGCGCAAATTACGCAGCTTGGGCTGT

[0534] GAACGTTGCGCAAGTGATTGACTCGGAAACGGCCGATAATCTGGAAAAAACCACGGCGGCCCTGAGTATTCTGCCGG

[0535] GCATCGGTTCCGTTATGGGTATTGAGTTCCGCCACGATTCCGGTTACACCGAAGAAATTGTGGCCCAGTCTATCGCACT

[0536] GTCGAGCCTGATGGTTGCTCAAGCGATTCCGCTGGTTGGCGAACTGGTTGATATCGGCTTTGCAGCTTACAACTTCGTG

[0537] GAAAGTATTATCAACCTGTTTCAGGTTGTCCACAACTCATATAATCGCCCGGCCTACTCGCCGGGTCACAAAACCCAA

[0538] CCGTTCCTGCATGACGGCTACGCGGTTAGCTGGAATACGGTCGAAGATTCTATTATCCGTACCGGCTTTCAGGGTGAAT

[0539] CTGGCCACGACATTAAAATCACGGCTGAAAACACCCCGCTGCCGATTGCAGGTGTTCTGCTGCCGACGATCCCGGGTA

[0540] AACTGGATGTTAACAAATCAAAAACCCATATCTCGGTCAACGGTCGCAAAATTCGTATGCGCTGCCGTGCGATCGACG

[0541] GCGATGTGACCTTCTGTCGTCCGAAAAGCCCGGTCTATGTGGGCAACGGTGTCCATGCTAATCTGCACGTGGCGTTTCA

[0542] TCGCTCTAGTTCCAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTATTGGTGTGCTGGGCTACCAGAAAACCGT

[0543] GGACCATACCAAAGTGAATAGCAAACTGAGCCTGTTCTTCGAAATCAAATCGTAA

[0544] Chimeric polypeptide DAT6b (SEQ ID NO: 15): (EFRHDSGY: positions 69 - 76, 290 - 297, 5901 - 508; KDNIKHVPGGGS: positions 6 - 17, 164 - 175, 409 - 420)

[0545] MGADDVKDNIKHVPGGGSSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDEFRHDSGYAG

[0546] GVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALS

[0547] VELKDNIKHVPGGGSDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEE

[0548] KAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIEFRHDSGYEKTTAALSILPGIGSVMGIADGAVH

[0549] HNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSI

[0550] IRTKDNIKHVPGGGSTAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANL

[0551] HVAFHRSSSEKIHEFRHDSGYIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0552] CDS sequence of DAT6b (SEQ ID NO:33):

[0553] ATGGGCGCAGAGTTCCGCCACGATTCCGGTTACTCATTTGTCATGGAAAACTTCTCCTCATATCACGGCACGAAACCG

[0554] GGCTACGTTGATAGCATTCAGAAAGGTATCCAAAAACCGAAATCTGGCACGCAGGGTAACTACGATGACGATTGGAA

[0555] AGAATTCTACAGCACCGACAACAAATATGATGCGGCCGGTTACAAAGATAATATTAAACATGTTCCGGGTGGCGGCA

[0556] GTGGCGGTGTGGTTAAAGTGACGTATCCGGGCCTGACCAAAGTCCTGGCCCTGAAAGTGGATAATGCAGAAACCATC

[0557] AAAAAAGAACTGGGTCTGAGCCTGACGGAACCGCTGATGGAACAGGTTGGCACCGAAGAATTTATCAAACGCTTCGG

[0558] CGATGGTGCCAGTCGTGTCGTGCTGTCCCTGCCGTTCGCAGAAGGTAGCTCTAGTGTGGAATATATTAACAATTGGGA

[0559] ACAAGCGAAAGCCCTGTCCGTTGAACTGGAAATCAACTTTGAGTTCCGCCACGATTCCGGTTACGATGCGATGTATGA

[0560] ATACATGGCACAAGCTTGCGCGGGTAATCGCGTTCGTCGCAGCGTCGGCTCCTCACTGTCTTGTATCAACCTGGACTG

[0561] GGATGTTATCCGTGATAAAACCAAAACGAAAATCGAAAGTCTGAAAGAACATGGCCCGATCAAAAACAAAATGAGCG

[0562] AATCTCCGAATAAAACGGTGTCCGAAGAAAAAGCTAAACAGTATCTGGAAGAATTCCACCAAACCGCACTGGAACAT

[0563] CCGGAACTGTCAGAACTGAAAACCGTGACGGGTACCAACCCGGTTTTTGCCGGCGCAAATTACGCAGCTTGGGCTGTG

[0564] AACGTTAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTGAAAAAACCACGGCGGCCCTGAGTATTCTGCCGGG

[0565] CATCGGTTCCGTTATGGGTATTGCCGACGGCGCAGTCCATCACAACACCGAAGAAATTGTGGCCCAGTCTATCGCACT

[0566] GTCGAGCCTGATGGTTGCTCAAGCGATTCCGCTGGTTGGCGAACTGGTTGATATCGGCTTTGCAGCTTACAACTTCGTG

[0567] GAAAGTATTATCAACCTGTTTCAGGTTGTCCACAACTCATATAATCGCCCGGCCTACTCGCCGGGTCACAAAACCCAA

[0568] CCGTTCCTGCATGACGGCTACGCGGTTAGCTGGAATACGGTCGAAGATTCTATTATCCGTACCGAGTTCCGCCACGATT

[0569] CCGGTTACGACATTAAAATCACGGCTGAAAACACCCCGCTGCCGATTGCAGGTGTTCTGCTGCCGACGATCCCGGGTA

[0570] AACTGGATGTTAACAAATCAAAAACCCATATCTCGGTCAACGGTCGCAAAATTCGTATGCGCTGCCGTGCGATCGACG

[0571] GCGATGTGACCTTCTGTCGTCCGAAAAGCCCGGTCTATGTGGGCAACGGTGTCCATGCTAATCTGCACGTGGCGTTTCA

[0572] TCGCTCTAGTTCCAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTATTGGTGTGCTGGGCTACCAGAAAACCGT

[0573] GGACCATACCAAAGTGAATAGCAAACTGAGCCTGTTCTTCGAAATCAAATCGTAA

[0574] Chimeric polypeptide DAT8b (SEQ ID NO:16): (EFRHDSGY: positions 3 - 10, 127 - 130, 276 - 273, 409 - 416; KDNIKHVPGGGS: positions 49 - 60, 222 - 233, 344 - 355, 497 - 508)

[0575] MGAEFRHDSGYSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDKDNIKHVPGGGSDAAGYSVDNENPLSGKAG

[0576] GVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKREFRHDSGYVLSLPFAEGSSSVEYINNWEQAKALS

[0577] VELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKKDNIKHVPGGGSPNKTVSEE

[0578] KAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGEFRHDSGYNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHH

[0579] NTEEIVAQSIALSSLMVAQAKDNIKHVPGGGSAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSI

[0580] IRTEFRHDSGYDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLH

[0581] VAFHRSSSKDNIKHVPGGGSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0582] DAT8b CDS sequence (SEQ ID NO:34):

[0583] ATGGGCGCAGAGTTCCGCCACGATTCCGGTTACTCATTTGTCATGGAAAACTTCTCCTCATATCACGGCACGAAACCG

[0584] GGCTACGTTGATAGCATTCAGAAAGGTATCCAAAAACCGAAATCTGGCACGCAGGGTAACTACGATGACAAAGATAA

[0585] TATTAAACATGTTCCGGGTGGCGGCAGTGATGCGGCCGGTTACTCAGTCGACAACGAAAATCCGCTGTCGGGCAAAGC

[0586] CGGCGGTGTGGTTAAAGTGACGTATCCGGGCCTGACCAAAGTCCTGGCCCTGAAAGTGGATAATGCAGAAACCATCA

[0587] AAAAAGAACTGGGTCTGAGCCTGACGGAACCGCTGATGGAACAGGTTGGCACCGAAGAATTTATCAAACGCGAGTTC

[0588] CGCCACGATTCCGGTTACGTGCTGTCCCTGCCGTTCGCAGAAGGTAGCTCTAGTGTGGAATATATTAACAATTGGGAA

[0589] CAAGCGAAAGCCCTGTCCGTTGAACTGGAAATCAACTTTGAAACCCGCGGCAAACGTGGTCAGGATGCGATGTATGA

[0590] ATACATGGCACAAGCTTGCGCGGGTAATCGCGTTCGTCGCAGCGTCGGCTCCTCACTGTCTTGTATCAACCTGGACTG

[0591] GGATGTTATCCGTGATAAAACCAAAACGAAAATCGAAAGTCTGAAAAAAGATAATATTAAACATGTTCCGGGTGGCG

[0592] GCAGTCCGAATAAAACGGTGTCCGAAGAAAAAGCTAAACAGTATCTGGAAGAATTCCACCAAACCGCACTGGAACAT

[0593] CCGGAACTGTCAGAACTGAAAACCGTGACGGGTACCAACCCGGTTTTTGCCGGCGAGTTCCGCCACGATTCCGGTTAC

[0594] AACGTTGCGCAAGTGATTGACTCGGAAACGGCCGATAATCTGGAAAAAACCACGGCGGCCCTGAGTATTCTGCCGGG

[0595] CATCGGTTCCGTTATGGGTATTGCCGACGGCGCAGTCCATCACAACACCGAAGAAATTGTGGCCCAGTCTATCGCACT

[0596] GTCGAGCCTGATGGTTGCTCAAGCGAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTGCAGCTTACAACTTCGT

[0597] GGAAAGTATTATCAACCTGTTTCAGGTTGTCCACAACTCATATAATCGCCCGGCCTACTCGCCGGGTCACAAAACCCA

[0598] ACCGTTCCTGCATGACGGCTACGCGGTTAGCTGGAATACGGTCGAAGATTCTATTATCCGTACCGAGTTCCGCCACGA

[0599] TTCCGGTTACGACATTAAAATCACGGCTGAAAACACCCCGCTGCCGATTGCAGGTGTTCTGCTGCCGACGATCCCGGG

[0600] TAAACTGGATGTTAACAAATCAAAAACCCATATCTCGGTCAACGGTCGCAAAATTCGTATGCGCTGCCGTGCGATCGA

[0601] CGGCGATGTGACCTTCTGTCGTCCGAAAAGCCCGGTCTATGTGGGCAACGGTGTCCATGCTAATCTGCACGTGGCGTTT

[0602] CATCGCTCTAGTTCCAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTATTGGTGTGCTGGGCTACCAGAAAACC

[0603] GTGGACCATACCAAAGTGAATAGCAAACTGAGCCTGTTCTTCGAAATCAAATCGTAA

[0604] Chimeric polypeptide DAT9a (SEQ ID NO:17): (EFRHDSGY: positions 3 - 10, 104 - 111, 222 - 229, 317 - 324, 501 - 508; KDNIKHVPGGGS: positions 49 - 60, 138 - 149, 270 - 281, 409 - 420)

[0605] MGAEFRHDSGYSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDKDNIKHVPGGGSDAAGYSVDNENPLSGKAG

[0606] GVVKVTYPGLTKVLALKVDNAETIKEFRHDSGYEPLMEQVGTEEFIKRFGDGASRVVLSKDNIKHVPGGGSINNWEQAKA

[0607] LSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEFRHDSGYMSESPNKTVS

[0608] EEKAKQYLEEFHQTALEHPELSELKTVTGTKDNIKHVPGGGSAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIEFRHD

[0609] SGYTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVED

[0610] SIIRTKDNIKHVPGGGSTAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHAN

[0611] LHVAFHRSSSEKIHEFRHDSGYIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0612] DAT9a CDS sequence (SEQ ID NO:35):

[0613] ATGGGCGCAGAGTTCCGCCACGATTCCGGTTACTCATTTGTCATGGAAAACTTCTCCTCATATCACGGCACGAAACCG

[0614] GGCTACGTTGATAGCATTCAGAAAGGTATCCAAAAACCGAAATCTGGCACGCAGGGTAACTACGATGACAAAGATAA

[0615] TATTAAACATGTTCCGGGTGGCGGCAGTGATGCGGCCGGTTACTCAGTCGACAACGAAAATCCGCTGTCGGGCAAAGC

[0616] CGGCGGTGTGGTTAAAGTGACGTATCCGGGCCTGACCAAAGTCCTGGCCCTGAAAGTGGATAATGCAGAAACCATCA

[0617] AAGAGTTCCGCCACGATTCCGGTTACGAACCGCTGATGGAACAGGTTGGCACCGAAGAATTTATCAAACGCTTCGGCG

[0618] ATGGTGCCAGTCGTGTCGTGCTGTCCAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTATTAACAATTGGGAAC

[0619] AAGCGAAAGCCCTGTCCGTTGAACTGGAAATCAACTTTGAAACCCGCGGCAAACGTGGTCAGGATGCGATGTATGAA

[0620] TACATGGCACAAGCTTGCGCGGGTAATCGCGTTCGTCGCAGCGTCGGCTCCTCACTGTCTTGTATCAACCTGGACTGG

[0621] GATGTTATCCGTGATAAAACCAAAACGAAAATCGAAAGTCTGAAAGAGTTCCGCCACGATTCCGGTTACATGAGCGA

[0622] ATCTCCGAATAAAACGGTGTCCGAAGAAAAAGCTAAACAGTATCTGGAAGAATTCCACCAAACCGCACTGGAACATC

[0623] CGGAACTGTCAGAACTGAAAACCGTGACGGGTACCAAAGATAATATTAAACATGTTCCGGGTGGCGGCAGTGCTGTG

[0624] AACGTTGCGCAAGTGATTGACTCGGAAACGGCCGATAATCTGGAAAAAACCACGGCGGCCCTGAGTATTCTGCCGGG

[0625] CATCGGTTCCGTTATGGGTATTGAGTTCCGCCACGATTCCGGTTACACCGAAGAAATTGTGGCCCAGTCTATCGCACTG

[0626] TCGAGCCTGATGGTTGCTCAAGCGATTCCGCTGGTTGGCGAACTGGTTGATATCGGCTTTGCAGCTTACAACTTCGTGG

[0627] AAAGTATTATCAACCTGTTTCAGGTTGTCCACAACTCATATAATCGCCCGGCCTACTCGCCGGGTCACAAAACCCAAC

[0628] CGTTCCTGCATGACGGCTACGCGGTTAGCTGGAATACGGTCGAAGATTCTATTATCCGTACCAAAGATAATATTAAAC

[0629] ATGTTCCGGGTGGCGGCAGTACGGCTGAAAACACCCCGCTGCCGATTGCAGGTGTTCTGCTGCCGACGATCCCGGGTA

[0630] AACTGGATGTTAACAAATCAAAAACCCATATCTCGGTCAACGGTCGCAAAATTCGTATGCGCTGCCGTGCGATCGACG

[0631] GCGATGTGACCTTCTGTCGTCCGAAAAGCCCGGTCTATGTGGGCAACGGTGTCCATGCTAATCTGCACGTGGCGTTTCA

[0632] TCGCTCTAGTTCCGAAAAAATCCATGAGTTCCGCCACGATTCCGGTTACATTGGTGTGCTGGGCTACCAGAAAACCGT

[0633] GGACCATACCAAAGTGAATAGCAAACTGAGCCTGTTCTTCGAAATCAAATCGTAA

[0634] Chimeric polypeptide D-HA01 (SEQ ID NO:18): (EFRHDSGY: positions 69-76, 290-297)

[0635] MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDEFRHDSGYAG

[0636] GVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALS

[0637] VELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEE

[0638] KAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIEFRHDSGYEKTTAALSILPGIGSVMGIADGAVH

[0639] HNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKGREDILEQWVSGRKKLEELE

[0640] RDLRKLKKKIKKLEEDNPWLGNIKGIIGK

[0641] D-HA01 CDS sequence (SEQ ID NO:36):

[0642] ATGGGCGCAGACGATGTTGTGGACTCAAGTAAATCATTTGTCATGGAAAACTTCTCCTCATATCACGGCACGAAACCGG

[0643] GCTACGTTGATAGCATTCAGAAAGGTATCCAAAAACCGAAATCTGGCACGCAGGGTAACTACGATGACGATTGGAAAGA

[0644] ATTCTACAGCACCGACAACAAATATGATGCGGCCGGTTACTCAGTCGACGAGTTCCGCCACGATTCCGGTTACGCCGGC

[0645] GGTGTGGTTAAAGTGACGTATCCGGGCCTGACCAAAGTCCTGGCCCTGAAAGTGGATAATGCAGAAACCATCAAAAAA

[0646] GAACTGGGTCTGAGCCTGACGGAACCGCTGATGGAACAGGTTGGCACCGAAGAATTTATCAAACGCTTCGGCGATGGT

[0647] GCCAGTCGTGTCGTGCTGTCCCTGCCGTTCGCAGAAGGTAGCTCTAGTGTGGAATATATTAACAATTGGGAACAAGCGA

[0648] AAGCCCTGTCCGTTGAACTGGAAATCAACTTTGAAACCCGCGGCAAACGTGGTCAGGATGCGATGTATGAATACATGGC

[0649] ACAAGCTTGCGCGGGTAATCGCGTTCGTCGCAGCGTCGGCTCCTCACTGTCTTGTATCAACCTGGACTGGGATGTTATCC

[0650] GTGATAAAACCAAAACGAAAATCGAAAGTCTGAAAGAACATGGCCCGATCAAAAACAAAATGAGCGAATCTCCGAATA

[0651] AAACGGTGTCCGAAGAAAAAGCTAAACAGTATCTGGAAGAATTCCACCAAACCGCACTGGAACATCCGGAACTGTCAG

[0652] AACTGAAAACCGTGACGGGTACCAACCCGGTTTTTGCCGGCGCAAATTACGCAGCTTGGGCTGTGAACGTTGCGCAAG

[0653] TGATTGAGTTCCGCCACGATTCCGGTTACGAAAAAACCACGGCGGCCCTGAGTATTCTGCCGGGCATCGGTTCCGTTATG

[0654] GGTATTGCCGACGGCGCAGTCCATCACAACACCGAAGAAATTGTGGCCCAGTCTATCGCACTGTCGAGCCTGATGGTTG

[0655] CTCAAGCGATTCCGCTGGTTGGCGAACTGGTTGATATCGGCTTTGCAGCTTACAACTTCGTGGAAAGTATTATCAACCTG

[0656] TTTCAGGTTGTCCACAACTCATATAATCGCCCGGCCTACTCGCCGGGTCACAAAGGGAGGGAAGACATCCTCGAGCAGT

[0657] GGGTGAGCGGAAGAAAGAAGTTAGAGGAACTCGAGAGAGACCTCCGGAAGCTCAAGAAGAAAATCAAGAAACTAGA

[0658] GGAAGACAATCCCTGGCTGGGAAACATCAAAGGAATAATCGGAAAGTAA

[0659] Chimeric polypeptide D-CA01 (SEQ ID NO:19): (EFRHDSGY: positions 69-76, 290-297)

[0660] MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDEFRHDSGYAG

[0661] GVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALS

[0662] VELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEE

[0663] KAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIEFRHDSGYEKTTAALSILPGIGSVMGIADGAVH

[0664] HNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTPQNITDLCAEYHNTQIHTLN

[0665] DKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAND-CA01 CDS sequence (SEQ ID NO:37):

[0666] ATGGGCGCAGACGATGTTGTGGACTCAAGTAAATCATTTGTCATGGAAAACTTCTCCTCATATCACGGCACGAAACCGG

[0667] GCTACGTTGATAGCATTCAGAAAGGTATCCAAAAACCGAAATCTGGCACGCAGGGTAACTACGATGACGATTGGAAAGA

[0668] ATTCTACAGCACCGACAACAAATATGATGCGGCCGGTTACTCAGTCGACGAGTTCCGCCACGATTCCGGTTACGCCGGC

[0669] GGTGTGGTTAAAGTGACGTATCCGGGCCTGACCAAAGTCCTGGCCCTGAAAGTGGATAATGCAGAAACCATCAAAAAA

[0670] GAACTGGGTCTGAGCCTGACGGAACCGCTGATGGAACAGGTTGGCACCGAAGAATTTATCAAACGCTTCGGCGATGGT

[0671] GCCAGTCGTGTCGTGCTGTCCCTGCCGTTCGCAGAAGGTAGCTCTAGTGTGGAATATATTAACAATTGGGAACAAGCGA

[0672] AAGCCCTGTCCGTTGAACTGGAAATCAACTTTGAAACCCGCGGCAAACGTGGTCAGGATGCGATGTATGAATACATGGC

[0673] ACAAGCTTGCGCGGGTAATCGCGTTCGTCGCAGCGTCGGCTCCTCACTGTCTTGTATCAACCTGGACTGGGATGTTATCC

[0674] GTGATAAAACCAAAACGAAAATCGAAAGTCTGAAAGAACATGGCCCGATCAAAAACAAAATGAGCGAATCTCCGAATA

[0675] AAACGGTGTCCGAAGAAAAAGCTAAACAGTATCTGGAAGAATTCCACCAAACCGCACTGGAACATCCGGAACTGTCAG

[0676] AACTGAAAACCGTGACGGGTACCAACCCGGTTTTTGCCGGCGCAAATTACGCAGCTTGGGCTGTGAACGTTGCGCAAG

[0677] TGATTGAGTTCCGCCACGATTCCGGTTACGAAAAAACCACGGCGGCCCTGAGTATTCTGCCGGGCATCGGTTCCGTTATG

[0678] GGTATTGCCGACGGCGCAGTCCATCACAACACCGAAGAAATTGTGGCCCAGTCTATCGCACTGTCGAGCCTGATGGTTG

[0679] CTCAAGCGATTCCGCTGGTTGGCGAACTGGTTGATATCGGCTTTGCAGCTTACAACTTCGTGGAAAGTATTATCAACCTG

[0680] TTTCAGGTTGTCCACAACTCATATAATCGCCCGGCCTACTCGCCGGGTCACAAAACACCTCAAAATATTACTGATTTGTG

[0681] TGCAGAATACCACAACACACAAATACATACGCTAAATGATAAGATATTTTCGTATACAGAATCTCTAGCTGGAAAAAGAG

[0682] AGATGGCTATCATTACTTTTAAGAATGGTGCAACTTTTCAAGTAGAAGTACCAGGTAGTCAACATATAGATTCACAAAAA

[0683] AAAGCGATTGAAAGGATGAAGGATACCCTGAGGATTGCATATCTTACTGAAGCTAAAGTCGAAAAGTTATGTGTATGGA

[0684] ATAATAAAACGCCTCATGCGATTGCCGCAATTAGTATGGCAAATTAA

[0685] Chimeric polypeptide D-TA01 (SEQ ID NO:20): (EFRHDSGY: positions 69 - 76, 290 - 297)

[0686] MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDEFRHDSGYAG

[0687] GVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALS

[0688] VELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEE

[0689] KAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIEFRHDSGYEKTTAALSILPGIGSVMGIADGAVH

[0690] HNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKMGSSHHHHHHSQDPMLSKD

[0691] IIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQ

[0692] KAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS

[0693] D-T01 CDS sequence (SEQ ID NO:38):

[0694] ATGGGCGCAGACGATGTTGTGGACTCAAGTAAATCATTTGTCATGGAAAACTTCTCCTCATATCACGGCACGAAACCGG

[0695] GCTACGTTGATAGCATTCAGAAAGGTATCCAAAAACCGAAATCTGGCACGCAGGGTAACTACGATGACGATTGGAAAGA

[0696] ATTCTACAGCACCGACAACAAATATGATGCGGCCGGTTACTCAGTCGACGAGTTCCGCCACGATTCCGGTTACGCCGGC

[0697] GGTGTGGTTAAAGTGACGTATCCGGGCCTGACCAAAGTCCTGGCCCTGAAAGTGGATAATGCAGAAACCATCAAAAAA

[0698] GAACTGGGTCTGAGCCTGACGGAACCGCTGATGGAACAGGTTGGCACCGAAGAATTTATCAAACGCTTCGGCGATGGT

[0699] GCCAGTCGTGTCGTGCTGTCCCTGCCGTTCGCAGAAGGTAGCTCTAGTGTGGAATATATTAACAATTGGGAACAAGCGA

[0700] AAGCCCTGTCCGTTGAACTGGAAATCAACTTTGAAACCCGCGGCAAACGTGGTCAGGATGCGATGTATGAATACATGGC

[0701] ACAAGCTTGCGCGGGTAATCGCGTTCGTCGCAGCGTCGGCTCCTCACTGTCTTGTATCAACCTGGACTGGGATGTTATCC

[0702] GTGATAAAACCAAAACGAAAATCGAAAGTCTGAAAGAACATGGCCCGATCAAAAACAAAATGAGCGAATCTCCGAATA

[0703] AAACGGTGTCCGAAGAAAAAGCTAAACAGTATCTGGAAGAATTCCACCAAACCGCACTGGAACATCCGGAACTGTCAG

[0704] AACTGAAAACCGTGACGGGTACCAACCCGGTTTTTGCCGGCGCAAATTACGCAGCTTGGGCTGTGAACGTTGCGCAAG

[0705] TGATTGAGTTCCGCCACGATTCCGGTTACGAAAAAACCACGGCGGCCCTGAGTATTCTGCCGGGCATCGGTTCCGTTATG

[0706] GGTATTGCCGACGGCGCAGTCCATCACAACACCGAAGAAATTGTGGCCCAGTCTATCGCACTGTCGAGCCTGATGGTTG

[0707] CTCAAGCGATTCCGCTGGTTGGCGAACTGGTTGATATCGGCTTTGCAGCTTACAACTTCGTGGAAAGTATTATCAACCTG

[0708] TTTCAGGTTGTCCACAACTCATATAATCGCCCGGCCTACTCGCCGGGTCACAAAGAGTCTCAGGTCAGACAACAGTTCT

[0709] CAAAAGACATCGAAAAGTTGCTAAACGAACAAGTGAATAAGGAAATGCAGTCTTCCAACTTGTATATGAGCATGAGTTC

[0710] ATGGTGCTATACCCATAGCTTAGATGGCGCGGGGCTTTTCTTGTTTGACCATGCGGCTGAAGAATACGAGCATGCTAAAA

[0711] AGCTTATTATCTTCTTGAATGAAAACAATGTGCCTGTGCAATTGACCAGCATCAGCGCGCCTGAGCATAAGTTTGAAGGT

[0712] TTGACTCAAATTTTCCAAAAAGCCTATGAACATGAGCAACACATCAGCGAGTCTATTAACAATATCGTAGATCACGCCAT

[0713] AAAAAGCAAAGATCATGCGACTTTCAATTTCTTGCAATGGTATGTGGCTGAACAGCATGAAGAAGAAGTGCTTTTCAAG

[0714] GATATTTTGGATAAAATTGAGTTGATTGGTAATGAAAACCATGGCTTGTATTTAGCCGATCAGTATGTCAAAGGGATCGCT

[0715] AAAAGCAGGAAATCTTAA

Claims

1. A chimeric polypeptide, characterized in that, The chimeric polypeptide comprises: (i) a polypeptide obtained by replacing at least one of the B cell epitopes of the carrier protein CRM197 with a first polypeptide and / or a second polypeptide; wherein, the first polypeptide comprises or consists of the sequence shown in SEQ ID NO:1, and the second polypeptide comprises or consists of the sequence shown in SEQ ID NO:2; (ii) a polypeptide obtained by replacing at least one of the B cell epitopes in the A domain and the B domain of the carrier protein CRM197 with a first polypeptide and / or a second polypeptide, and connecting a third polypeptide to the C-terminus of the B domain; wherein, the first polypeptide comprises or consists of the sequence shown in SEQ ID NO:1, the second polypeptide comprises or consists of the sequence shown in SEQ ID NO:2, and the third polypeptide is selected from at least one of the polypeptides formed by sequentially connecting amino acids at positions 12 to 60 of the hepatitis D virus delta antigen fragment, cholera toxin B subunit, and transferrin.

2. The chimeric polypeptide according to claim 1, wherein Replace 1 to 19 of the B cell epitopes with the first polypeptide and / or the second polypeptide.

3. The chimeric polypeptide according to claim 1 or 2, characterized in that, In (i), replace at least 1 of the B cell epitopes with the first polypeptide; Preferably, the chimeric polypeptide comprises or consists of any one of the sequences shown in SEQ ID NO:3-7.

4. The chimeric polypeptide according to claim 1 or 2, characterized in that, In (i), replace at least 1 of the B cell epitopes with the second polypeptide; Preferably, the chimeric polypeptide comprises or consists of any one of the sequences shown in SEQ ID NO:8-12.

5. The chimeric polypeptide according to claim 1 or 2, characterized in that In (i), replace at least 2 of the B cell epitopes with the first polypeptide and the second polypeptide respectively; Preferably, the chimeric polypeptide comprises or consists of any one of the sequences shown in SEQ ID NO:13-17.

6. The chimeric polypeptide according to claim 1 or 2, characterized in that, In (ii), replace at least 1 of the B cell epitopes with the first polypeptide; Preferably, the chimeric polypeptide comprises or consists of any one of the sequences shown in SEQ ID NO:18-20.

7. A pharmaceutical composition, characterized in that, Comprising the chimeric polypeptide according to any one of claims 1-6 and at least one adjuvant.

8. A nucleic acid molecule, characterized in that, Comprising or consisting of a nucleic acid sequence encoding the chimeric polypeptide according to any one of claims 1-6; Preferably, the sequence of the nucleic acid molecule is as shown in SEQ ID NO:21-38.

9. A nucleic acid immunotherapy composition, characterized in that Comprising the nucleic acid molecule according to claim 8 and at least one adjuvant.

10. Use of the chimeric polypeptide according to any one of claims 1-6, the pharmaceutical composition according to claim 7, the nucleic acid molecule according to claim 8, or the nucleic acid immunotherapy composition according to claim 9 in the preparation of a drug for preventing and / or treating Alzheimer's disease; Preferably, the drug is a vaccine.

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