CN110734498A - fusion protein for relieving immunosuppression and application thereof - Google Patents

fusion protein for relieving immunosuppression and application thereof Download PDF

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Publication number
CN110734498A
CN110734498A CN201910977793.5A CN201910977793A CN110734498A CN 110734498 A CN110734498 A CN 110734498A CN 201910977793 A CN201910977793 A CN 201910977793A CN 110734498 A CN110734498 A CN 110734498A
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郝瑞栋
刘根桃
易桥勇
李彦涛
红丽
吴国详
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Shanghai Keqi Pharmaceutical Technology Co Ltd
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Shanghai Keqi Pharmaceutical Technology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators

Abstract

The invention discloses fusion proteins for relieving immunosuppression and application thereof, and belongs to the technical field of biological medicine, wherein the fusion proteins are in PTA or PTR, in addition, part of the fusion proteins is an inhibitor of PD-1, and part of the fusion proteins is an inhibitor of TGF- β. aiming at an inhibitory microenvironment in the interior of a tumor, the invention designs novel fusion proteins PTA and PTR, wherein the PTA and PTR can simultaneously inhibit PD-1 and TGF- β signal channels, and the fusion proteins which can singly or jointly inhibit the PD-1/PD-L1 signal channel and the TGF- β signal channel are singly or jointly used for treating the solid tumor, so that the fusion proteins which can secrete and inhibit the PD-1/PD-L1 signal channel and the TGF- β signal channel when a CAR-T cell targets the solid tumor have stronger capacity of relieving the immunosuppression microenvironment and treating the tumor.

Description

fusion protein for relieving immunosuppression and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to fusion proteins for relieving immunosuppression and application thereof.
Background
However, the CAR-T therapy has not been greatly developed in the treatment of solid tumors, and important reasons for the less significant efficacy of the CAR-T therapy are that the immune microenvironment of the solid tumor is too complex and has strong capacity of inhibiting the vitality of T cells, so that the T cells cannot effectively proliferate in the solid tumor, and cannot kill tumor tissues after being exhausted.
The clinical results also show that the PD-1 inhibitor has curative effect in various solid tumors, but the effective rate is only about 20 percent, TGF- 1 is a common inhibitory cytokine in solid tumor tissues, the TGF- is involved in the regulation of cells through receptor signaling pathways on the cell surface in an autocrine or paracrine manner, mainly comprises the functions of inhibiting immunity, enabling tumor cells to escape from immune monitoring, TGF-3638 inhibits the generation of interleukin 2, so that the proliferation, differentiation and cytotoxicity of the T cells are inhibited, and finally the growth of the tumors is promoted, the TGF- can inhibit the proliferation of NK cells and the expression of the receptor thereof, so that the TGF-NK-584 toxicity is inhibited, TGF-358 receptor transduction is considered to be directly inhibited by TGF-RII receptor 468, TGF-11, TGF-9 receptor 5, TGF-11 receptor 5, TGF-9-II, TGF-9 receptor 5, TGF-9, TGF-9-II, TGF-9, TGF-9-7, TGF-7, 9, 3, III.
In view of the limited curative effect of CAR-T in solid tumor treatment due to immune microenvironment and the limitation of single-drug use of PD-1 antibody, we develop a novel immunotherapy which integrates CAR-T, PD-1 inhibitor and TGF- β inhibitor, so that CAR-T cells target solid tumors, secrete fusion protein for inhibiting PD-1/PD-L1 signaling pathway and TGF- β signaling pathway, and have stronger capability of relieving immune suppression microenvironment and treating tumors.
Disclosure of Invention
In view of the above technical problems, the present invention aims to provide fusion proteins for relieving immunosuppression and applications thereof, and novel fusion proteins PTA and PTR are designed for tumor internal inhibitory microenvironment, wherein the PTA and PTR can inhibit PD-1 and TGF- β signaling pathways simultaneously, and can be used for treating solid tumors alone or in combination with CAR-T.
The technical purpose of the invention is realized by the following technical scheme:
fusion proteins for use in the resolution of immunosuppression, wherein the fusion proteins are of PTA or PTR and wherein moiety is an inhibitor of PD-1 and moiety is an inhibitor of TGF- β.
Further , the inhibitor of PD-1 is a PD-1 antibody.
Further , the inhibitor of TGF- β is a TGF- β antibody or an extracellular domain fragment of TGFBRII.
Further , the fusion protein is PTA, the part of PTA is PD-1 antibody, the other part is TGF- β antibody, and the amino acid sequence of PTA is SEQ ID NO. 1.
Further , the fusion protein is PTR, the part of PTR is PD-1 antibody, the other part is extracellular region fragment of TGFBRII, and the PTR amino acid sequence is SEQ ID NO. 2.
A DNA sequence encoding the above amino acid sequence.
vectors encoding the DNA sequences described above.
cells stably or transiently expressing the above vector.
The use of the above amino acids, DNA, vectors and cells in the treatment of human tumors.
Compared with the prior art, the invention has the following beneficial effects:
the invention solves the problems of limited curative effect and low PD-1 effective rate of CAR-T cells in solid tumor treatment, designs novel fusion proteins PTA and PTR aiming at inhibitory microenvironment in tumors, wherein the PTA and PTR can simultaneously inhibit PD-1 and TGF- β signal pathways, and can be used for treating solid tumors by singly or jointly using CAR-T, so that the CAR-T cells can secrete fusion proteins inhibiting PD-1/PD-L1 signal pathways and TGF- β signal pathways while targeting solid tumors, and the CAR-T cells have stronger capacity of relieving immune inhibition microenvironment and treating tumors.
Drawings
FIG. 1 is a schematic representation of the molecular structures of CARL6-PTA and CARL 6-PTR;
FIG. 2 is a graph of CAR-T cell positivity;
FIG. 3 is a spectrum of the positive rate of A549 cells TM4SF 1;
FIG. 4 is a graph of the killing ability of different CAR-T cells to target cell A549;
FIG. 5 is a graph of the number of mouse peripheral blood CAR-T cells after reinfusion;
FIG. 6 is a plot of tumor growth in mice after reinfusion.
Detailed Description
The present invention will be described in further detail in with reference to examples and drawings, but the embodiments of the present invention are not limited to these.
As shown in FIGS. 1-6, fusion proteins for relieving immunosuppression are of PTA or PTR, wherein part of the fusion protein is inhibitor of PD-1, and part of the fusion protein is inhibitor of TGF- β preferably, the inhibitor of PD-1 is PD-1 antibody and the inhibitor of TGF- β is TGF- β antibody or extracellular region fragment of TGFBRII.
When the fusion protein is PTA, part of PTA is PD-1 antibody, another part is TGF- β antibody, and PTA amino acid sequence is SEQ ID NO. 1.
When the fusion protein is PTR, the part of PTR is PD-1 antibody, the other part is extracellular region fragment of TGFBRII, and the amino acid sequence of PTR is SEQ ID NO. 2.
kinds of vectors, DNA sequences encoding the above mentioned amino acid sequences, kinds of cells, stable or transient expression of the above mentioned vectors, the use of the amino acid, DNA, vectors and cells in the treatment of human tumors.
Example :
in the embodiment, CAR molecules CARL6-PTA or CARL6-PTR expressed by PTA or PTR are constructed, wherein PTA is a fusion protein formed by linking anti-PD1 scFv and anti-TGF β scFv through a flexible GS connecting peptide, PTR is a fusion protein formed by linking anti-PD1 scFv and sTGFBRII through a flexible GS connecting peptide, and sTGFBRII is an extracellular region sequence of TGRIFBI, and a specific molecular structure schematic diagram is shown in figure 1.
The CARL6-PTR vector construction comprises the steps of firstly obtaining anti-PD1 scFv plasmid and sTGFBRII sequence plasmid through gene synthesis, respectively amplifying PD1 antibody sequence and sTGFBRII sequence through PCR, splicing PD1 antibody sequence and sTGF β RII sequence into through bypass PCR, adding flexible connecting polypeptide in the middle to construct complete anti-PD1 scFv-sTGFBRII sequence (marked as PTR), using ClaI to singly cut a lentivirus vector pHAGE-CARL6 coding CARL6 to obtain a vector fragment with a sticky end, connecting PTR into the single-cut vector through recombinase, reacting for 20 minutes at 50 ℃, then transforming st bl3 competent cells, inoculating single colony to LB for amplification the next day, extracting plasmid, verifying the sequence through enzyme cutting and sequencing, and leaving correct plasmid named pHAGE-6-PTR to carry out the next step experiment under CARL .
The CARL6-PTA vector construction comprises the steps of firstly obtaining an anti-PD1 scFv plasmid and an anti-TGF β scFv sequence plasmid through gene synthesis, respectively amplifying the anti-PD1 scFv sequence and the anti-TGF β scFv sequence through a PCR mode, splicing the anti-PD1 scFv sequence and the anti-TGF β scFv sequence to through a bypass PCR mode, adding a flexible connecting polypeptide in the middle to construct a complete anti-PD 1-anti-TGF β scFv sequence (marked as PTA), singly digesting a lentivirus vector pHAGE-CARL6 encoding CARL6 with ClaI to obtain a vector fragment with a sticky end, connecting the PTA to the single digestion vector with a recombinase, reacting for 20 minutes at 50 ℃, then transforming stbl3 competent cells, inoculating the single colony to LB amplification culture on the next day, extracting and verifying the sequence of the plasmid through enzyme digestion and sequencing, and carrying out a CARL 3884 experiment under the sequence of CARL 6.
Example two:
the self-secreting PTA or PTR CARL6-T cells CARL6-PTA-T or CARL6-PTR-T were constructed by this example, in this particular example we achieved in the following way;
the CAR molecule lentivirus with PTA or PTR is first obtained, in this example the recombinant expression vector of choice is a lentiviral vector further steps are carried out by co-transfecting 293T cells with the plasmids pHAGE-CARL6, pHAGE-CARL6-PTA and pHAGE-CARL6-PTR of example in the presence of the helper packaging plasmid psPAX2 and the VSV-G envelope plasmid pMD2.G to package as lentiviral vectors the specific procedure is that 1.7X 10 to package as lentiviral vectors in days in advance7293T cells were inoculated into T175 flasks, and when the cell confluence reached 70-90%, the above plasmids were co-transfected into 293T cells using lipo-3000, and the medium was changed with 50ml before transfection. Cell supernatants were harvested 48 and 72 hours after transfection as crude lentiviral fluid. Then removing impurities such as cell debris by centrifugation and filtration, and concentrating by ultracentrifugation to obtain lentiviruses with CARL6, CARL6-PTA and CARL 6-PTR.
Construction of CAR-T cells A CAR-T cell was constructed by coating an antibody for activating T cells at days before construction, adding retronectin, anti-human CD3 and CD8 antibodies to a six-well plate, standing overnight at 4 deg.C, obtaining a blood sample by a conventional blood collection method, obtaining PBMCs by centrifugation with lymphocyte separation, then using STEMCELL T cell sorting kit, sorting and counting T cells from PBMCs, and resuspending the T cells with a medium to a density of 1X 106The culture medium is X-VIVO15 culture medium containing human AB serum and IL-2, taking out a six-well plate coated in days before the culture, washing twice by PBS, adding the resuspended T cells into the coated six-well plate for culture, adding polybrene and corresponding lentivirus (CARL6, CARL6-PTA and CARL6-PTR) after the cells are cultured for 24 hours, mixing uniformly, putting the mixture back into an incubator for culture, centrifuging the cells in the six-well plate after infecting for 24 hours, changing the cells into X-VIVO15 culture medium containing human AB serum and IL-2, and then only needing to supplement the culture medium in the culture process to maintain the cell density at 1X 106The dosage is only required to be one ml. 1ml of T or CAR-T cells were incubated with Fab antibody by the fourth day of culture, washed with saline, centrifuged, and scFv expression was detected by flow cytometry to determine transduction efficiency of CAR-T cells. As shown in fig. 2: the positive rate of CARL6-T is 48.2%, the positive rate of CARL6-PTA-T is 42.4%, the positive rate of CARL6-PTR-T is about 30.1%, and the T cell is an untransduced control cell.
Example three:
this example demonstrates that CARL6, CARL6-PTA-T and CARL6-PTR-T are able to kill target cells efficiently in vitro.
After the CAR-T cells are constructed, we verify that the constructed CARL6-T, CARL6-PTA-T and CARL6-PTR-T can effectively kill tumor cells through a cell killing test. As detected by TM4SF1 antibody staining and flow cytometry, the lung cancer cell line a549 was positive for TM4SF1 as shown in fig. 3. CAR-T cells (CARL6-T, CARL6-PTA-T, CARL6-PTR-T) and T cells and target cells A549 with high expression of positive L6 are respectively arranged in a 96-well plate according to effective target ratios of 1:1, 5:1 and 10:1, and a culture medium blank group and a target cell control group are simultaneously arranged. After the CAR-T cells and the target cells were co-cultured for 16h, the killing efficiency of the CAR-T cells was calculated by measuring the OD450 value by a microplate reader using the method of CCK 8. As shown in FIG. 4, the GAR-T cells obtained by the method have obvious killing advantages compared with the ordinary T cells, and the killing efficiency is very high. When the effective target ratio is 1:1, the killing efficiency can reach about 40 percent, when the effective target ratio is 5:1, the killing efficiency can reach 60-70 percent, when the effective target ratio is 10:1, most target cells can be killed, and the killing efficiency can reach about 90 percent. These results fully demonstrate that we constructed CAR-T cells that are able to kill target cells efficiently, and that the ability of CARL6-PTA-T and CARL6-PTR-T to kill target cells is significantly better than CARL 6-T.
Example four:
we demonstrated by this example that CARL6-PTA-T and CARL6-PTR-T have stronger proliferation and tumor-inhibiting abilities than CARL6-T in vivo.
Before the start of the experiment, NOD-SCID immunodeficient mice of 6 weeks of age were used, and the size of tumor bodies was measured after subcutaneously injecting the lung cancer cell line A549 after to two weeks3Is the tumor size in units. When the tumor body reaches 100-300mm3In size, cultured CAR positive CARL6-PTA-T, CARL6-PTR-T, CARL6-T and T cells were injected into mice via tail vein at 5 × 10E6 cells/mouse. After the cells are back transfused, the proliferation condition of the CAR-T cells in a mouse body is detected by blood sampling of a tail vein of the mouse, the change of the size of a tumor body is measured at the same time, and a tumor change curve is drawn.
The proliferation condition of the mouse in vivo CAR-T is that 20ul of tail venous blood of a mouse is added into 1.5ml of sterilized EP tubes, 20ul of heparin anticoagulant is added in advance in the tubes, 2ul of anti-human CD45 antibody is added into each tube, the tubes are incubated for ten minutes at room temperature in a dark place, 1ml of erythrocyte lysate is added into the tubes for lysis for 5 minutes, cell precipitates are collected by centrifugation, times are washed by physiological saline, and the number of CAR-T cells is detected by flow measurement, the proliferation capacity of CARL6-PTA-T and CARL6-PTR-T is obviously 7 to 8 times higher than that of CARL6-T, the constructed CARL6-PTA-T and CARL6-PTR-T are better than that of common CARL6-T in mice, the tumor treatment effect of the constructed CARL6-PTA-T and the constructed CARL6-PTR-T are determined by measuring the size change of the mice after reinfusion, the constructed CARL6-PTA-T and the constructed CART-468-PTA-T-4642 are obviously reduced in vivo compared with the average tumor size of the constructed CARL-PTA-4624 and PTT-9-468-PTA-T-9-T-468-5-2 and the constructed tumor-T-III of mice.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.
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<120> fusion proteins for relieving immunosuppression and application thereof
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Ala Ala Thr Thr Ala Gly Cys Ala Gly Ala Gly Ala Cys Ala Ala Cys
660 665 670
Thr Cys Cys Ala Ala Gly Ala Ala Cys Ala Cys Cys Cys Thr Gly Thr
675 680 685
Thr Cys Cys Thr Cys Cys Ala Gly Ala Thr Gly Ala Ala Cys Thr Cys
690 695 700
Thr Cys Thr Gly Cys Gly Cys Gly Cys Cys Gly Ala Gly Gly Ala Cys
705 710 715 720
Ala Cys Cys Gly Cys Cys Gly Thr Gly Thr Ala Cys Thr Ala Cys Thr
725 730 735
Gly Cys Gly Cys Thr Ala Cys Ala Ala Ala Cys Gly Ala Cys Gly Ala
740 745 750
Cys Thr Ala Cys Thr Gly Gly Gly Gly Ala Cys Ala Gly Gly Gly Cys
755 760 765
Ala Cys Ala Cys Thr Cys Gly Thr Gly Ala Cys Cys Gly Thr Gly Thr
770 775 780
Cys Thr Ala Gly Cys Gly Ala Ala Ala Cys Gly Gly Thr Ala Cys Thr
785 790 795 800
Cys Ala Cys Gly Cys Ala Gly Thr Cys Thr Cys Cys Ala Gly Gly Thr
805 810 815
Ala Cys Cys Cys Thr Gly Thr Cys Thr Thr Thr Gly Thr Cys Thr Cys
820 825 830
Cys Ala Gly Gly Gly Gly Ala Ala Ala Gly Ala Gly Cys Cys Ala Cys
835 840 845
Cys Cys Thr Cys Thr Cys Cys Thr Gly Cys Ala Gly Gly Gly Cys Cys
850 855 860
Ala Gly Thr Cys Ala Gly Ala Gly Thr Cys Thr Thr Gly Gly Cys Ala
865 870 875 880
Gly Cys Ala Gly Cys Thr Ala Cys Thr Thr Ala Gly Cys Cys Thr Gly
885 890 895
Gly Thr Ala Thr Cys Ala Gly Cys Ala Gly Ala Ala Ala Cys Cys Thr
900 905 910
Gly Gly Thr Cys Ala Gly Gly Cys Thr Cys Cys Cys Ala Gly Gly Cys
915 920 925
Thr Cys Cys Thr Cys Ala Thr Cys Thr Ala Thr Gly Gly Thr Gly Cys
930 935 940
Ala Thr Cys Cys Ala Gly Cys Ala Gly Gly Gly Cys Ala Cys Cys Thr
945 950 955 960
Gly Gly Cys Ala Thr Cys Cys Cys Ala Gly Ala Cys Ala Gly Gly Thr
965 970 975
Thr Cys Ala Gly Thr Gly Gly Cys Ala Gly Thr Gly Gly Gly Thr Cys
980 985 990
Thr Gly Gly Thr Ala Cys Cys Gly Ala Cys Thr Thr Cys Ala Cys Thr
995 1000 1005
Cys Thr Cys Ala Cys Cys Ala Thr Cys Ala Gly Cys Cys Gly Ala Cys
1010 1015 1020
Thr Gly Gly Ala Gly Cys Cys Thr Gly Ala Ala Gly Ala Thr Thr Thr
1025 1030 1035 1040
Thr Gly Cys Ala Gly Thr Thr Thr Ala Thr Thr Ala Cys Thr Gly Thr
1045 1050 1055
Cys Ala Gly Cys Ala Gly Thr Ala Thr Gly Cys Thr Gly Ala Cys Thr
1060 1065 1070
Cys Ala Cys Cys Gly Ala Thr Cys Ala Cys Cys Thr Thr Cys Gly Gly
1075 1080 1085
Cys Cys Ala Ala Gly Gly Gly Ala Cys Ala Cys Gly Ala Cys Thr Gly
1090 1095 1100
Gly Ala Gly Ala Thr Thr Ala Ala Ala Gly Gly Cys Thr Cys Cys Ala
1105 1110 1115 1120
Cys Cys Thr Cys Thr Gly Gly Ala Thr Cys Cys Gly Gly Cys Ala Ala
1125 1130 1135
Gly Cys Cys Cys Gly Gly Ala Thr Cys Thr Gly Gly Cys Gly Ala Gly
1140 1145 1150
Gly Gly Ala Thr Cys Cys Ala Cys Cys Ala Ala Gly Gly Gly Cys Cys
1155 1160 1165
Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Gly Thr Gly Cys Ala
1170 1175 1180
Gly Thr Cys Thr Gly Gly Gly Gly Cys Thr Gly Ala Gly Gly Thr Gly
1185 1190 1195 1200
Ala Ala Gly Ala Ala Gly Cys Cys Thr Gly Gly Gly Thr Cys Cys Thr
1205 1210 1215
Cys Gly Gly Thr Gly Ala Ala Gly Gly Thr Cys Thr Cys Cys Thr Gly
1220 1225 1230
Cys Ala Ala Gly Gly Cys Thr Thr Cys Thr Gly Gly Ala Thr Ala Cys
1235 1240 1245
Ala Cys Cys Thr Thr Cys Ala Gly Thr Ala Gly Cys Ala Ala Thr Gly
1250 1255 1260
Thr Thr Ala Thr Cys Ala Gly Cys Thr Gly Gly Gly Thr Gly Cys Gly
1265 1270 1275 1280
Cys Cys Ala Gly Gly Cys Cys Cys Cys Thr Gly Gly Ala Cys Ala Ala
1285 1290 1295
Gly Gly Gly Cys Thr Cys Gly Ala Gly Thr Gly Gly Ala Thr Gly Gly
1300 1305 1310
Gly Gly Gly Gly Gly Gly Thr Cys Ala Thr Cys Cys Cys Thr Ala Thr
1315 1320 1325
Thr Gly Thr Thr Gly Ala Thr Ala Thr Thr Gly Cys Gly Ala Ala Cys
1330 1335 1340
Thr Ala Cys Gly Cys Ala Cys Ala Gly Ala Gly Ala Thr Thr Cys Ala
1345 1350 1355 1360
Ala Gly Gly Gly Cys Ala Gly Ala Gly Thr Cys Ala Cys Gly Ala Thr
1365 1370 1375
Thr Ala Cys Cys Gly Cys Gly Gly Ala Cys Gly Ala Ala Thr Cys Cys
1380 1385 1390
Ala Cys Thr Ala Gly Thr Ala Cys Ala Ala Cys Thr Thr Ala Cys Ala
1395 1400 1405
Thr Gly Gly Ala Gly Thr Thr Gly Ala Gly Cys Ala Gly Cys Cys Thr
1410 1415 1420
Gly Ala Gly Gly Thr Cys Thr Gly Ala Gly Gly Ala Cys Ala Cys Gly
1425 1430 1435 1440
Gly Cys Cys Gly Thr Gly Thr Ala Thr Thr Ala Cys Thr Gly Thr Gly
1445 1450 1455
Cys Gly Ala Gly Cys Ala Cys Ala Cys Thr Thr Gly Gly Thr Cys Thr
1460 1465 1470
Cys Gly Thr Cys Cys Thr Gly Gly Ala Thr Gly Cys Thr Ala Thr Gly
1475 1480 1485
Gly Ala Cys Thr Ala Cys Thr Gly Gly Gly Gly Thr Cys Ala Gly Gly
1490 1495 1500
Gly Thr Ala Cys Gly Thr Thr Gly Gly Thr Cys Ala Cys Cys Gly Thr
1505 1510 1515 1520
Cys Thr Cys Cys Thr Cys Ala
1525
<210>4
<211>1242
<212>PRT
<213>2 Ambystoma laterale x Ambystoma jeffersonianum
<400>4
Ala Thr Gly Gly Ala Gly Ala Cys Ala Cys Cys Ala Gly Cys Thr Cys
1 5 10 15
Ala Gly Cys Thr Gly Cys Thr Gly Thr Thr Cys Cys Thr Cys Cys Thr
20 25 30
Cys Cys Thr Gly Cys Thr Gly Thr Gly Gly Cys Thr Cys Cys Cys Thr
35 40 45
Gly Ala Cys Ala Cys Ala Ala Cys Cys Gly Gly Cys Gly Ala Gly Ala
50 55 60
Thr Thr Gly Thr Gly Cys Thr Gly Ala Cys Cys Cys Ala Gly Thr Cys
65 70 75 80
Thr Cys Cys Cys Gly Cys Thr Ala Cys Ala Cys Thr Gly Thr Cys Thr
85 90 95
Cys Thr Gly Ala Gly Cys Cys Cys Thr Gly Gly Cys Gly Ala Gly Cys
100 105 110
Gly Cys Gly Cys Cys Ala Cys Cys Cys Thr Gly Thr Cys Thr Thr Gly
115 120 125
Cys Cys Gly Cys Gly Cys Thr Thr Cys Thr Cys AlaGly Thr Cys Thr
130 135 140
Gly Thr Gly Thr Cys Ala Thr Cys Thr Thr Ala Cys Cys Thr Cys Gly
145 150 155 160
Cys Thr Thr Gly Gly Thr Ala Thr Cys Ala Gly Cys Ala Gly Ala Ala
165 170 175
Gly Cys Cys Thr Gly Gly Gly Cys Ala Gly Gly Cys Thr Cys Cys Ala
180 185 190
Cys Gly Cys Cys Thr Gly Cys Thr Cys Ala Thr Thr Thr Ala Cys Gly
195 200 205
Ala Thr Gly Cys Ala Thr Cys Thr Ala Ala Cys Ala Gly Ala Gly Cys
210 215 220
Cys Ala Cys Ala Gly Gly Gly Ala Thr Thr Cys Cys Cys Gly Cys Thr
225 230 235 240
Ala Gly Gly Thr Thr Cys Ala Gly Cys Gly Gly Ala Thr Cys Thr Gly
245 250 255
Gly Gly Thr Cys Cys Gly Gly Cys Ala Cys Cys Gly Ala Cys Thr Thr
260 265 270
Cys Ala Cys Ala Cys Thr Gly Ala Cys Cys Ala Thr Thr Thr Cys Thr
275 280 285
Ala Gly Cys Cys Thr Cys Gly Ala Ala Cys Cys Cys Gly AlaGly Gly
290 295 300
Ala Cys Thr Thr Cys Gly Cys Cys Gly Thr Gly Thr Ala Cys Thr Ala
305 310 315 320
Cys Thr Gly Cys Cys Ala Gly Cys Ala Gly Thr Cys Thr Thr Cys Thr
325 330 335
Ala Ala Cys Thr Gly Gly Cys Cys Thr Ala Gly Ala Ala Cys Ala Thr
340 345 350
Thr Cys Gly Gly Cys Cys Ala Gly Gly Gly Ala Ala Cys Cys Ala Ala
355 360 365
Gly Gly Thr Gly Gly Ala Gly Ala Thr Thr Ala Ala Gly Gly Gly Ala
370 375 380
Gly Gly Ala Gly Gly Cys Gly Gly Gly Thr Cys Cys Gly Gly Cys Gly
385 390 395 400
Gly Ala Gly Gly Cys Gly Gly Ala Ala Gly Cys Gly Gly Ala Gly Gly
405 410 415
Cys Gly Gly Ala Gly Gly Ala Ala Gly Thr Gly Gly Cys Gly Gly Ala
420 425 430
Gly Gly Ala Thr Cys Thr Gly Gly Cys Gly Gly Cys Gly Gly Ala Thr
435 440 445
Cys Thr Cys Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Cys Gly Thr
450 455 460
Gly Gly Ala Gly Thr Cys Cys Gly Gly Ala Gly Gly Cys Gly Gly Ala
465 470 475 480
Gly Thr Gly Gly Thr Gly Cys Ala Gly Cys Cys Thr Gly Gly Ala Cys
485 490 495
Gly Gly Thr Cys Cys Cys Thr Cys Ala Gly Ala Cys Thr Cys Gly Ala
500 505 510
Cys Thr Gly Cys Ala Ala Gly Gly Cys Thr Thr Cys Cys Gly Gly Cys
515 520 525
Ala Thr Cys Ala Cys Ala Thr Thr Cys Thr Cys Thr Ala Ala Cys Thr
530 535 540
Cys Thr Gly Gly Thr Ala Thr Gly Cys Ala Cys Thr Gly Gly Gly Thr
545 550 555 560
Gly Ala Gly Ala Cys Ala Gly Gly Cys Ala Cys Cys Ala Gly Gly Ala
565 570 575
Ala Ala Gly Gly Gly Cys Thr Thr Ala Gly Ala Gly Thr Gly Gly Gly
580 585 590
Thr Gly Gly Cys Thr Gly Thr Gly Ala Thr Thr Thr Gly Gly Thr Ala
595 600 605
Cys Gly Ala Cys Gly Gly Gly Thr Cys Gly Ala Ala Gly Ala Gly Ala
610 615 620
Thr Ala Cys Thr Ala Cys Gly Cys Cys Gly Ala Cys Ala Gly Cys Gly
625 630 635 640
Thr Gly Ala Ala Gly Gly Gly Ala Cys Gly Gly Thr Thr Cys Ala Cys
645 650 655
Ala Ala Thr Thr Ala Gly Cys Ala Gly Ala Gly Ala Cys Ala Ala Cys
660 665 670
Thr Cys Cys Ala Ala Gly Ala Ala Cys Ala Cys Cys Cys Thr Gly Thr
675 680 685
Thr Cys Cys Thr Cys Cys Ala Gly Ala Thr Gly Ala Ala Cys Thr Cys
690 695 700
Thr Cys Thr Gly Cys Gly Cys Gly Cys Cys Gly Ala Gly Gly Ala Cys
705 710 715 720
Ala Cys Cys Gly Cys Cys Gly Thr Gly Thr Ala Cys Thr Ala Cys Thr
725 730 735
Gly Cys Gly Cys Thr Ala Cys Ala Ala Ala Cys Gly Ala Cys Gly Ala
740 745 750
Cys Thr Ala Cys Thr Gly Gly Gly Gly Ala Cys Ala Gly Gly Gly Cys
755 760 765
Ala Cys Ala Cys Thr Cys Gly Thr Gly Ala Cys Cys Gly Thr Gly Thr
770 775 780
Cys Thr Ala Gly Cys Gly Gly Cys Gly Gly Cys Gly Gly Cys Thr Cys
785 790 795 800
Gly Gly Gly Cys Gly Gly Cys Gly Gly Gly Thr Cys Cys Gly Gly Cys
805 810 815
Gly Gly Ala Gly Gly Ala Gly Gly Thr Thr Cys Gly Ala Cys Gly Ala
820 825 830
Thr Cys Cys Cys Ala Cys Cys Gly Cys Ala Cys Gly Thr Thr Cys Ala
835 840 845
Gly Ala Ala Gly Thr Cys Gly Gly Ala Thr Gly Thr Gly Gly Ala Ala
850 855 860
Ala Thr Gly Gly Ala Gly Gly Cys Cys Cys Ala Gly Ala Ala Ala Gly
865 870 875 880
Ala Thr Gly Ala Ala Ala Thr Cys Ala Thr Cys Thr Gly Cys Cys Cys
885 890 895
Cys Ala Gly Cys Thr Gly Thr Ala Ala Thr Ala Gly Gly Ala Cys Thr
900 905 910
Gly Cys Cys Cys Ala Thr Cys Cys Ala Cys Thr Gly Ala Gly Ala Cys
915 920 925
Ala Thr Ala Thr Thr Ala Ala Thr Ala Ala Cys Gly Ala Cys Ala Thr
930 935 940
Gly Ala Thr Ala Gly Thr Cys Ala Cys Thr Gly Ala Cys Ala Ala Cys
945 950 955 960
Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly Thr Cys Ala Ala Gly Thr
965 970 975
Thr Thr Cys Cys Ala Cys Ala Ala Cys Thr Gly Thr Gly Thr Ala Ala
980 985 990
Ala Thr Thr Thr Thr Gly Thr Gly Ala Thr Gly Thr Gly Ala Gly Ala
995 1000 1005
Thr Thr Thr Thr Cys Cys Ala Cys Cys Thr Gly Thr Gly Ala Cys Ala
1010 1015 1020
Ala Cys Cys Ala Gly Ala Ala Ala Thr Cys Cys Thr Gly Cys Ala Thr
1025 1030 1035 1040
Gly Ala Gly Cys Ala Ala Cys Thr Gly Cys Ala Gly Cys Ala Thr Cys
1045 1050 1055
Ala Cys Cys Thr Cys Cys Ala Thr Cys Thr Gly Thr Gly Ala Gly Ala
1060 1065 1070
Ala Gly Cys Cys Ala Cys Ala Gly Gly Ala Ala Gly Thr Cys Thr Gly
1075 1080 1085
Thr Gly Thr Gly Gly Cys Thr Gly Thr Ala Thr Gly Gly Ala Gly Ala
1090 1095 1100
Ala Ala Gly Ala Ala Thr Gly Ala Cys Gly Ala Gly Ala Ala Cys Ala
1105 1110 1115 1120
Thr Ala Ala Cys Ala Cys Thr Ala Gly Ala Gly Ala Cys Ala Gly Thr
1125 1130 1135
Thr Thr Gly Cys Cys Ala Thr Gly Ala Cys Cys Cys Cys Ala Ala Gly
1140 1145 1150
Cys Thr Cys Cys Cys Cys Thr Ala Cys Cys Ala Thr Gly Ala Cys Thr
1155 1160 1165
Thr Thr Ala Thr Thr Cys Thr Gly Gly Ala Ala Gly Ala Thr Gly Cys
1170 1175 1180
Thr Gly Cys Thr Thr Cys Thr Cys Cys Ala Ala Ala Gly Thr Gly Cys
1185 1190 1195 1200
Ala Thr Thr Ala Thr Gly Ala Ala Gly Gly Ala Ala Ala Ala Ala Ala
1205 1210 1215
Ala Ala Ala Ala Gly Cys Cys Thr Gly Gly Thr Gly Ala Gly Ala Cys
1220 1225 1230
Thr Thr Thr Cys Thr Thr Cys Thr Ala Ala
1235 1240
<210>5
<211>916
<212>PRT
<213>2 Ambystoma laterale x Ambystoma jeffersonianum
<400>5
Ala Gly Cys Thr Ala Gly Cys Cys Ala Ala Ala Thr Thr Gly Thr Thr
1 5 10 15
Cys Thr Cys Thr Cys Cys Cys Ala Gly Thr Cys Thr Cys Cys Ala Gly
20 25 30
Cys Ala Ala Thr Cys Cys Thr Gly Thr Cys Thr Gly Cys Ala Thr Cys
35 40 45
Thr Cys Cys Ala Gly Gly Gly Gly Ala Gly Ala Ala Gly Gly Thr Cys
50 55 60
Ala Cys Ala Thr Thr Gly Ala Cys Thr Thr Gly Cys Ala Gly Gly Gly
65 70 75 80
Cys Cys Ala Gly Cys Thr Cys Ala Ala Gly Thr Gly Thr Ala Ala Gly
85 90 95
Thr Thr Thr Cys Ala Thr Gly Ala Ala Cys Thr Gly Gly Thr Ala Cys
100 105 110
Cys Ala Gly Cys Ala Gly Ala Ala Gly Cys Cys Ala Gly Gly Ala Thr
115 120 125
Cys Cys Thr Cys Cys Cys Cys Cys Ala Ala Ala Cys Cys Cys Thr Gly
130 135 140
Gly Ala Thr Thr Thr Ala Thr Gly Cys Cys Ala Cys Ala Thr Cys Cys
145 150 155 160
Ala Ala Thr Thr Thr Gly Gly Cys Thr Thr Cys Thr Gly Gly Ala Gly
165 170 175
Thr Cys Cys Cys Thr Gly Gly Thr Cys Gly Cys Thr Thr Cys Ala Gly
180 185 190
Thr Gly Gly Cys Ala Gly Thr Gly Gly Gly Thr Cys Thr Gly Gly Gly
195 200 205
Ala Cys Cys Thr Cys Thr Thr Ala Cys Thr Cys Thr Cys Thr Cys Gly
210 215 220
Cys Ala Ala Thr Cys Ala Gly Cys Ala Gly Ala Gly Thr Gly Gly Ala
225 230 235 240
Gly Gly Cys Thr Gly Ala Ala Gly Ala Thr Gly Cys Thr Gly Cys Cys
245 250 255
Ala Cys Thr Thr Ala Thr Thr Ala Cys Thr Gly Cys Cys Ala Gly Cys
260 265 270
Ala Gly Thr Gly Gly Ala Ala Thr Ala Gly Thr Ala Ala Cys Cys Cys
275 280 285
Ala Cys Thr Cys Ala Cys Gly Thr Thr Cys Gly Gly Thr Gly Cys Thr
290 295 300
Gly Gly Gly Ala Cys Cys Ala Ala Gly Cys Thr Gly Gly Ala Gly Cys
305 310 315 320
Thr Gly Ala Ala Ala Cys Gly Ala Gly Gly Thr Gly Gly Thr Gly Gly
325 330 335
Thr Gly Gly Thr Ala Gly Cys Gly Gly Cys Gly Gly Cys Gly Gly Cys
340 345 350
Gly Gly Cys Thr Cys Thr Gly Gly Thr Gly Gly Thr Gly Gly Cys Gly
355 360 365
Gly Ala Thr Cys Cys Cys Ala Gly Ala Thr Cys Cys Ala Gly Thr Thr
370 375 380
Gly Gly Thr Gly Cys Ala Gly Thr Cys Thr Gly Gly Ala Cys Cys Thr
385 390 395 400
Gly Ala Gly Cys Thr Gly Ala Ala Gly Ala Ala Gly Cys Cys Thr Gly
405 410 415
Gly Ala Gly Ala Gly Ala Cys Ala Gly Thr Cys Ala Ala Gly Ala Thr
420 425 430
Cys Thr Cys Cys Thr Gly Cys Ala Ala Gly Gly Cys Thr Thr Cys Thr
435 440 445
Gly Gly Gly Thr Ala Thr Ala Cys Cys Thr Thr Cys Ala Cys Ala Ala
450 455 460
Ala Cys Thr Ala Thr Gly Gly Ala Ala Thr Gly Ala Ala Cys Thr Gly
465 470 475 480
Gly Gly Thr Gly Ala Ala Gly Cys Ala Gly Gly Cys Thr Cys Cys Ala
485 490 495
Gly Gly Ala Ala Ala Gly Gly Gly Thr Thr Thr Ala Ala Ala Gly Thr
500 505 510
Gly Gly Ala Thr Gly Gly Gly Cys Thr Gly Gly Ala Thr Ala Ala Ala
515 520 525
Cys Ala Cys Cys Thr Ala Cys Ala Cys Thr Gly Gly Ala Cys Ala Gly
530 535 540
Cys Cys Ala Ala Cys Ala Thr Ala Thr Gly Cys Thr Gly Ala Thr Gly
545 550 555 560
Ala Cys Thr Thr Cys Ala Ala Gly Gly Gly Ala Cys Gly Gly Thr Thr
565 570 575
Thr Gly Cys Cys Thr Thr Cys Thr Cys Thr Thr Thr Gly Gly Ala Ala
580 585 590
Ala Cys Cys Thr Cys Thr Gly Cys Cys Thr Ala Cys Ala Cys Thr Gly
595 600 605
Cys Cys Thr Ala Thr Thr Thr Gly Cys Ala Gly Ala Thr Cys Ala Ala
610 615 620
Cys Ala Ala Cys Cys Thr Cys Ala Ala Ala Ala Ala Thr Gly Ala Gly
625 630 635 640
Gly Ala Cys Ala Thr Gly Gly Cys Thr Ala Cys Ala Thr Ala Thr Thr
645 650 655
Thr Cys Thr Gly Thr Gly Cys Ala Ala Gly Ala Thr Thr Thr Ala Gly
660 665 670
Cys Thr Ala Thr Gly Gly Thr Ala Ala Cys Thr Cys Ala Cys Gly Thr
675 680 685
Thr Ala Cys Gly Cys Thr Gly Ala Cys Thr Ala Cys Thr Gly Gly Gly
690 695 700
Gly Cys Cys Ala Ala Gly Gly Cys Ala Cys Cys Ala Cys Thr Cys Thr
705 710 715 720
Cys Ala Cys Ala Gly Thr Cys Thr Cys Cys Thr Cys Ala Gly Gly Ala
725 730 735
Thr Cys Cys Thr Thr Cys Thr Gly Gly Thr Thr Ala Cys Cys Cys Ala
740 745 750
Thr Ala Gly Gly Ala Thr Gly Thr Gly Cys Ala Gly Cys Cys Thr Thr
755 760 765
Thr Gly Thr Thr Gly Thr Ala Gly Thr Cys Thr Gly Cys Ala Thr Thr
770 775 780
Thr Thr Gly Gly Gly Ala Thr Gly Cys Ala Thr Ala Cys Thr Thr Ala
785 790 795 800
Thr Thr Thr Gly Thr Thr Gly Gly Cys Thr Thr Ala Cys Ala Ala Ala
805 810 815
Ala Ala Ala Gly Ala Ala Gly Thr Ala Thr Thr Cys Ala Thr Cys Cys
820 825 830
Ala Gly Thr Gly Thr Gly Cys Ala Cys Gly Ala Cys Cys Cys Thr Ala
835 840 845
Ala Cys Gly Gly Thr Gly Ala Ala Thr Ala Cys Ala Thr Gly Thr Thr
850 855 860
Cys Ala Thr Gly Ala Gly Ala Gly Cys Ala Gly Thr Gly Ala Ala Cys
865 870 875 880
Ala Cys Ala Gly Cys Cys Ala Ala Ala Ala Ala Ala Thr Cys Thr Ala
885 890 895
Gly Ala Cys Thr Cys Ala Cys Ala Gly Ala Thr Gly Thr Gly Ala Cys
900 905 910
Cys Cys Thr Ala
915
<210>6
<211>2545
<212>PRT
<213>2 Ambystoma laterale x Ambystoma jeffersonianum
<400>6
Ala Gly Cys Thr AlaGly Cys Cys Ala Ala Ala Thr Thr Gly Thr Thr
1 5 10 15
Cys Thr Cys Thr Cys Cys Cys Ala Gly Thr Cys Thr Cys Cys Ala Gly
20 25 30
Cys Ala Ala Thr Cys Cys Thr Gly Thr Cys Thr Gly Cys Ala Thr Cys
35 40 45
Thr Cys Cys Ala Gly Gly Gly Gly Ala Gly Ala Ala Gly Gly Thr Cys
50 55 60
Ala Cys Ala Thr Thr Gly Ala Cys Thr Thr Gly Cys Ala Gly Gly Gly
65 70 75 80
Cys Cys Ala Gly Cys Thr Cys Ala Ala Gly Thr Gly Thr Ala Ala Gly
85 90 95
Thr Thr Thr Cys Ala Thr Gly Ala Ala Cys Thr Gly Gly Thr Ala Cys
100 105 110
Cys Ala Gly Cys Ala Gly Ala Ala Gly Cys Cys Ala Gly Gly Ala Thr
115 120 125
Cys Cys Thr Cys Cys Cys Cys Cys Ala Ala Ala Cys Cys Cys Thr Gly
130 135 140
Gly Ala Thr Thr Thr Ala Thr Gly Cys Cys Ala Cys Ala Thr Cys Cys
145 150 155 160
Ala Ala Thr Thr Thr Gly Gly Cys Thr Thr Cys Thr Gly Gly Ala Gly
165 170 175
Thr Cys Cys Cys Thr Gly Gly Thr Cys Gly Cys Thr Thr Cys Ala Gly
180 185 190
Thr Gly Gly Cys Ala Gly Thr Gly Gly Gly Thr Cys Thr Gly Gly Gly
195 200 205
Ala Cys Cys Thr Cys Thr Thr Ala Cys Thr Cys Thr Cys Thr Cys Gly
210 215 220
Cys Ala Ala Thr Cys Ala Gly Cys Ala Gly Ala Gly Thr Gly Gly Ala
225 230 235 240
Gly Gly Cys Thr Gly Ala Ala Gly Ala Thr Gly Cys Thr Gly Cys Cys
245 250 255
Ala Cys Thr Thr Ala Thr Thr Ala Cys Thr Gly Cys Cys Ala Gly Cys
260 265 270
Ala Gly Thr Gly Gly Ala Ala Thr Ala Gly Thr Ala Ala Cys Cys Cys
275 280 285
Ala Cys Thr Cys Ala Cys Gly Thr Thr Cys Gly Gly Thr Gly Cys Thr
290 295 300
Gly Gly Gly Ala Cys Cys Ala Ala Gly Cys Thr Gly Gly Ala Gly Cys
305 310 315 320
Thr Gly Ala Ala Ala Cys Gly Ala Gly Gly Thr Gly Gly Thr Gly Gly
325 330 335
Thr Gly Gly Thr Ala Gly Cys Gly Gly Cys Gly Gly Cys Gly Gly Cys
340 345 350
Gly Gly Cys Thr Cys Thr Gly Gly Thr Gly Gly Thr Gly Gly Cys Gly
355 360 365
Gly Ala Thr Cys Cys Cys Ala Gly Ala Thr Cys Cys Ala Gly Thr Thr
370 375 380
Gly Gly Thr Gly Cys Ala Gly Thr Cys Thr Gly Gly Ala Cys Cys Thr
385 390 395 400
Gly Ala Gly Cys Thr Gly Ala Ala Gly Ala Ala Gly Cys Cys Thr Gly
405 410 415
Gly Ala Gly Ala Gly Ala Cys Ala Gly Thr Cys Ala Ala Gly Ala Thr
420 425 430
Cys Thr Cys Cys Thr Gly Cys Ala Ala Gly Gly Cys Thr Thr Cys Thr
435 440 445
Gly Gly Gly Thr Ala Thr Ala Cys Cys Thr Thr Cys Ala Cys Ala Ala
450 455 460
Ala Cys Thr Ala Thr Gly Gly Ala Ala Thr Gly Ala Ala Cys Thr Gly
465 470 475 480
Gly Gly Thr Gly Ala Ala Gly Cys Ala Gly Gly Cys Thr Cys Cys Ala
485 490 495
Gly Gly Ala Ala Ala Gly Gly Gly Thr Thr Thr Ala Ala Ala Gly Thr
500 505 510
Gly Gly Ala Thr Gly Gly Gly Cys Thr Gly Gly Ala Thr Ala Ala Ala
515 520 525
Cys Ala Cys Cys Thr Ala Cys Ala Cys Thr Gly Gly Ala Cys Ala Gly
530 535 540
Cys Cys Ala Ala Cys Ala Thr Ala Thr Gly Cys Thr Gly Ala Thr Gly
545 550 555 560
Ala Cys Thr Thr Cys Ala Ala Gly Gly Gly Ala Cys Gly Gly Thr Thr
565 570 575
Thr Gly Cys Cys Thr Thr Cys Thr Cys Thr Thr Thr Gly Gly Ala Ala
580 585 590
Ala Cys Cys Thr Cys Thr Gly Cys Cys Thr Ala Cys Ala Cys Thr Gly
595 600 605
Cys Cys Thr Ala Thr Thr Thr Gly Cys Ala Gly Ala Thr Cys Ala Ala
610 615 620
Cys Ala Ala Cys Cys Thr Cys Ala Ala Ala Ala Ala Thr Gly Ala Gly
625 630 635 640
Gly Ala Cys Ala Thr Gly Gly Cys Thr Ala Cys Ala Thr Ala Thr Thr
645 650 655
Thr Cys Thr Gly Thr Gly Cys Ala Ala Gly Ala Thr Thr Thr Ala Gly
660 665 670
Cys Thr Ala Thr Gly Gly Thr Ala Ala Cys Thr Cys Ala Cys Gly Thr
675 680 685
Thr Ala Cys Gly Cys Thr Gly Ala Cys Thr Ala Cys Thr Gly Gly Gly
690 695 700
Gly Cys Cys Ala Ala Gly Gly Cys Ala Cys Cys Ala Cys Thr Cys Thr
705 710 715 720
Cys Ala Cys Ala Gly Thr Cys Thr Cys Cys Thr Cys Ala Gly Gly Ala
725 730 735
Thr Cys Cys Thr Thr Cys Thr Gly Gly Thr Thr Ala Cys Cys Cys Ala
740 745 750
Thr Ala Gly Gly Ala Thr Gly Thr Gly Cys Ala Gly Cys Cys Thr Thr
755 760 765
Thr Gly Thr Thr Gly Thr Ala Gly Thr Cys Thr Gly Cys Ala Thr Thr
770 775 780
Thr Thr Gly Gly Gly Ala Thr Gly Cys Ala Thr Ala Cys Thr Thr Ala
785 790 795 800
Thr Thr Thr Gly Thr Thr Gly Gly Cys Thr Thr Ala Cys Ala Ala Ala
805 810 815
Ala Ala Ala Gly Ala Ala Gly Thr Ala Thr Thr Cys Ala Thr Cys Cys
820 825 830
Ala Gly Thr Gly Thr Gly Cys Ala Cys Gly Ala Cys Cys Cys Thr Ala
835 840 845
Ala Cys Gly Gly Thr Gly Ala Ala Thr Ala Cys Ala Thr Gly Thr Thr
850 855 860
Cys Ala Thr Gly Ala Gly Ala Gly Cys Ala Gly Thr Gly Ala Ala Cys
865 870 875 880
Ala Cys Ala Gly Cys Cys Ala Ala Ala Ala Ala Ala Thr Cys Thr Ala
885 890 895
Gly Ala Cys Thr Cys Ala Cys Ala Gly Ala Thr Gly Thr Gly Ala Cys
900 905 910
Cys Cys Thr Ala Thr Cys Thr Ala Gly Ala Gly Ala Gly Gly Gly Cys
915 920 925
Ala Gly Ala Gly Gly Ala Ala Gly Thr Cys Thr Thr Cys Thr Ala Ala
930 935 940
Cys Ala Thr Gly Cys Gly Gly Thr Gly Ala Cys Gly Thr Gly Gly Ala
945 950 955 960
Gly Gly Ala Gly Ala Ala Thr Cys Cys Cys Gly Gly Cys Cys Cys Thr
965 970 975
Ala Thr Gly Gly Ala Gly Ala Cys Ala Cys Cys Ala Gly Cys Thr Cys
980 985 990
Ala Gly Cys Thr Gly Cys Thr Gly Thr Thr Cys Cys Thr Cys Cys Thr
995 1000 1005
Cys Cys Thr Gly Cys Thr Gly Thr Gly Gly Cys Thr Cys Cys Cys Thr
1010 1015 1020
Gly Ala Cys Ala Cys Ala Ala Cys Cys Gly Gly Cys Gly Ala Gly Ala
1025 1030 1035 1040
Thr Thr Gly Thr Gly Cys Thr Gly Ala Cys Cys Cys Ala Gly Thr Cys
1045 1050 1055
Thr Cys Cys Cys Gly Cys Thr Ala Cys Ala Cys Thr Gly Thr Cys Thr
1060 1065 1070
Cys Thr Gly Ala Gly Cys Cys Cys Thr Gly Gly Cys Gly Ala Gly Cys
1075 1080 1085
Gly Cys Gly Cys Cys Ala Cys Cys Cys Thr Gly Thr Cys Thr Thr Gly
1090 1095 1100
Cys Cys Gly Cys Gly Cys Thr Thr Cys Thr Cys Ala Gly Thr Cys Thr
1105 1110 1115 1120
Gly Thr Gly Thr Cys Ala Thr Cys Thr Thr Ala Cys Cys Thr Cys Gly
1125 1130 1135
Cys Thr Thr Gly Gly Thr Ala Thr Cys Ala Gly Cys Ala Gly Ala Ala
1140 1145 1150
Gly Cys Cys Thr Gly Gly Gly Cys Ala Gly Gly Cys Thr Cys Cys Ala
1155 1160 1165
Cys Gly Cys Cys Thr Gly Cys Thr Cys Ala Thr Thr Thr Ala Cys Gly
1170 1175 1180
Ala Thr Gly Cys Ala Thr Cys Thr Ala Ala Cys Ala Gly Ala Gly Cys
1185 1190 1195 1200
Cys Ala Cys Ala Gly Gly Gly Ala Thr Thr Cys Cys Cys Gly Cys Thr
1205 1210 1215
Ala Gly Gly Thr Thr Cys Ala Gly Cys Gly Gly Ala Thr Cys Thr Gly
1220 1225 1230
Gly Gly Thr Cys Cys Gly Gly Cys Ala Cys Cys Gly Ala Cys Thr Thr
1235 1240 1245
Cys Ala Cys Ala Cys Thr Gly Ala Cys Cys Ala Thr Thr Thr Cys Thr
1250 1255 1260
Ala Gly Cys Cys Thr Cys Gly Ala Ala Cys Cys Cys Gly Ala Gly Gly
1265 1270 1275 1280
Ala Cys Thr Thr Cys Gly Cys Cys Gly Thr Gly Thr Ala Cys Thr Ala
1285 1290 1295
Cys Thr Gly Cys Cys Ala Gly Cys Ala Gly Thr Cys Thr Thr Cys Thr
1300 1305 1310
Ala Ala Cys Thr Gly Gly Cys Cys Thr Ala Gly Ala Ala Cys Ala Thr
1315 1320 1325
Thr Cys Gly Gly Cys Cys Ala Gly Gly Gly Ala Ala Cys Cys Ala Ala
1330 1335 1340
Gly Gly Thr Gly Gly Ala Gly Ala Thr Thr Ala Ala Gly Gly Gly Ala
1345 1350 1355 1360
Gly Gly Ala Gly Gly Cys Gly Gly Gly Thr Cys Cys Gly Gly Cys Gly
1365 1370 1375
Gly Ala Gly Gly Cys Gly Gly Ala Ala Gly Cys Gly Gly Ala Gly Gly
1380 1385 1390
Cys Gly Gly Ala Gly Gly Ala Ala Gly Thr Gly Gly Cys Gly Gly Ala
1395 1400 1405
Gly Gly Ala Thr Cys Thr Gly Gly Cys Gly Gly Cys Gly Gly Ala Thr
1410 1415 1420
Cys Thr Cys Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Cys Gly Thr
1425 1430 1435 1440
Gly Gly Ala Gly Thr Cys Cys Gly Gly Ala Gly Gly Cys Gly Gly Ala
1445 1450 1455
Gly Thr Gly Gly Thr Gly Cys Ala Gly Cys Cys Thr Gly Gly Ala Cys
1460 1465 1470
Gly Gly Thr Cys Cys Cys Thr Cys Ala Gly Ala Cys Thr Cys Gly Ala
1475 1480 1485
Cys Thr Gly Cys Ala Ala Gly Gly Cys Thr Thr Cys Cys Gly Gly Cys
1490 1495 1500
Ala Thr Cys Ala Cys Ala Thr Thr Cys Thr Cys Thr Ala Ala Cys Thr
1505 1510 1515 1520
Cys Thr Gly Gly Thr Ala Thr Gly Cys Ala Cys Thr Gly Gly Gly Thr
1525 1530 1535
Gly Ala Gly Ala Cys Ala Gly Gly Cys Ala Cys Cys Ala Gly Gly Ala
1540 1545 1550
Ala Ala Gly Gly Gly Cys Thr Thr Ala Gly Ala Gly Thr Gly Gly Gly
1555 1560 1565
Thr Gly Gly Cys Thr Gly Thr Gly Ala Thr Thr Thr Gly Gly Thr Ala
1570 1575 1580
Cys Gly Ala Cys Gly Gly Gly Thr Cys Gly Ala Ala Gly Ala Gly Ala
1585 1590 1595 1600
Thr Ala Cys Thr Ala Cys Gly Cys Cys Gly Ala Cys Ala Gly Cys Gly
1605 1610 1615
Thr Gly Ala Ala Gly Gly Gly Ala Cys Gly Gly Thr Thr Cys Ala Cys
1620 1625 1630
Ala Ala Thr Thr Ala Gly Cys Ala Gly Ala Gly Ala Cys Ala Ala Cys
1635 1640 1645
Thr Cys Cys Ala Ala Gly Ala Ala Cys Ala Cys Cys Cys Thr Gly Thr
1650 1655 1660
Thr Cys Cys Thr Cys Cys Ala Gly Ala Thr Gly Ala Ala Cys Thr Cys
1665 1670 1675 1680
Thr Cys Thr Gly Cys Gly Cys Gly Cys Cys Gly Ala Gly Gly Ala Cys
1685 1690 1695
Ala Cys Cys Gly Cys Cys Gly Thr Gly Thr Ala Cys Thr Ala Cys Thr
1700 1705 1710
Gly Cys Gly Cys Thr Ala Cys Ala Ala Ala Cys Gly Ala Cys Gly Ala
1715 1720 1725
Cys Thr Ala Cys Thr Gly Gly Gly Gly Ala Cys Ala Gly Gly Gly Cys
1730 1735 1740
Ala Cys Ala Cys Thr Cys Gly Thr Gly Ala Cys Cys Gly Thr Gly Thr
1745 1750 1755 1760
Cys Thr Ala Gly Cys Gly Gly Cys Gly Gly Cys Gly Gly Cys Thr Cys
1765 1770 1775
Gly Gly Gly Cys Gly Gly Cys Gly Gly Gly Thr Cys Cys Gly Gly Cys
1780 1785 1790
Gly Gly Ala Gly Gly Ala Gly Gly Thr Thr Cys Gly Gly Ala Ala Ala
1795 1800 1805
Cys Gly Gly Thr Ala Cys Thr Cys Ala Cys Gly Cys Ala Gly Thr Cys
1810 1815 1820
Thr Cys Cys Ala Gly Gly Thr Ala Cys Cys Cys Thr Gly Thr Cys Thr
1825 1830 1835 1840
Thr Thr Gly Thr Cys Thr Cys Cys Ala Gly Gly Gly Gly Ala Ala Ala
1845 1850 1855
Gly Ala Gly Cys Cys Ala Cys Cys Cys Thr Cys Thr Cys Cys Thr Gly
1860 1865 1870
Cys Ala Gly Gly Gly Cys Cys Ala Gly Thr Cys Ala Gly Ala Gly Thr
1875 1880 1885
Cys Thr Thr Gly Gly Cys Ala Gly Cys Ala Gly Cys Thr Ala Cys Thr
1890 1895 1900
Thr Ala Gly Cys Cys Thr Gly Gly Thr Ala Thr Cys Ala Gly Cys Ala
1905 1910 1915 1920
Gly Ala Ala Ala Cys Cys Thr Gly Gly Thr Cys Ala Gly Gly CysThr
1925 1930 1935
Cys Cys Cys Ala Gly Gly Cys Thr Cys Cys Thr Cys Ala Thr Cys Thr
1940 1945 1950
Ala Thr Gly Gly Thr Gly Cys Ala Thr Cys Cys Ala Gly Cys Ala Gly
1955 1960 1965
Gly Gly Cys Ala Cys Cys Thr Gly Gly Cys Ala Thr Cys Cys Cys Ala
1970 1975 1980
Gly Ala Cys Ala Gly Gly Thr Thr Cys Ala Gly Thr Gly Gly Cys Ala
1985 1990 1995 2000
Gly Thr Gly Gly Gly Thr Cys Thr Gly Gly Thr Ala Cys Cys Gly Ala
2005 2010 2015
Cys Thr Thr Cys Ala Cys Thr Cys Thr Cys Ala Cys Cys Ala Thr Cys
2020 2025 2030
Ala Gly Cys Cys Gly Ala Cys Thr Gly Gly Ala Gly Cys Cys Thr Gly
2035 2040 2045
Ala Ala Gly Ala Thr Thr Thr Thr Gly Cys Ala Gly Thr Thr Thr Ala
2050 2055 2060
Thr Thr Ala Cys Thr Gly Thr Cys Ala Gly Cys Ala Gly Thr Ala Thr
2065 2070 2075 2080
Gly Cys Thr Gly Ala Cys Thr Cys Ala Cys Cys Gly Ala Thr Cys Ala
2085 2090 2095
Cys Cys Thr Thr Cys Gly Gly Cys Cys Ala Ala Gly Gly Gly Ala Cys
2100 2105 2110
Ala Cys Gly Ala Cys Thr Gly Gly Ala Gly Ala Thr Thr Ala Ala Ala
2115 2120 2125
Gly Gly Cys Thr Cys Cys Ala Cys Cys Thr Cys Thr Gly Gly Ala Thr
2130 2135 2140
Cys Cys Gly Gly Cys Ala Ala Gly Cys Cys Cys Gly Gly Ala Thr Cys
2145 2150 2155 2160
Thr Gly Gly Cys Gly Ala Gly Gly Gly Ala Thr Cys Cys Ala Cys Cys
2165 2170 2175
Ala Ala Gly Gly Gly Cys Cys Ala Gly Gly Thr Gly Cys Ala Gly Cys
2180 2185 2190
Thr Gly Gly Thr Gly Cys Ala Gly Thr Cys Thr Gly Gly Gly Gly Cys
2195 2200 2205
Thr Gly Ala Gly Gly Thr Gly Ala Ala Gly Ala Ala Gly Cys Cys Thr
2210 2215 2220
Gly Gly Gly Thr Cys Cys Thr Cys Gly Gly Thr Gly Ala Ala Gly Gly
2225 2230 2235 2240
Thr Cys Thr Cys Cys Thr Gly Cys Ala Ala Gly Gly Cys ThrThr Cys
2245 2250 2255
Thr Gly Gly Ala Thr Ala Cys Ala Cys Cys Thr Thr Cys Ala Gly Thr
2260 2265 2270
Ala Gly Cys Ala Ala Thr Gly Thr Thr Ala Thr Cys Ala Gly Cys Thr
2275 2280 2285
Gly Gly Gly Thr Gly Cys Gly Cys Cys Ala Gly Gly Cys Cys Cys Cys
2290 2295 2300
Thr Gly Gly Ala Cys Ala Ala Gly Gly Gly Cys Thr Cys Gly Ala Gly
2305 2310 2315 2320
Thr Gly Gly Ala Thr Gly Gly Gly Gly Gly Gly Gly Gly Thr Cys Ala
2325 2330 2335
Thr Cys Cys Cys Thr Ala Thr Thr Gly Thr Thr Gly Ala Thr Ala Thr
2340 2345 2350
Thr Gly Cys Gly Ala Ala Cys Thr Ala Cys Gly Cys Ala Cys Ala Gly
2355 2360 2365
Ala Gly Ala Thr Thr Cys Ala Ala Gly Gly Gly Cys Ala Gly Ala Gly
2370 2375 2380
Thr Cys Ala Cys Gly Ala Thr Thr Ala Cys Cys Gly Cys Gly Gly Ala
2385 2390 2395 2400
Cys Gly Ala Ala Thr Cys Cys Ala Cys Thr Ala Gly Thr Ala Cys Ala
2405 2410 2415
Ala Cys Thr Thr Ala Cys Ala Thr Gly Gly Ala Gly Thr Thr Gly Ala
2420 2425 2430
Gly Cys Ala Gly Cys Cys Thr Gly Ala Gly Gly Thr Cys Thr Gly Ala
2435 2440 2445
Gly Gly Ala Cys Ala Cys Gly Gly Cys Cys Gly Thr Gly Thr Ala Thr
2450 2455 2460
Thr Ala Cys Thr Gly Thr Gly Cys Gly Ala Gly Cys Ala Cys Ala Cys
2465 2470 2475 2480
Thr Thr Gly Gly Thr Cys Thr Cys Gly Thr Cys Cys Thr Gly Gly Ala
2485 2490 2495
Thr Gly Cys Thr Ala Thr Gly Gly Ala Cys Thr Ala Cys Thr Gly Gly
2500 2505 2510
Gly Gly Thr Cys Ala Gly Gly Gly Thr Ala Cys Gly Thr Thr Gly Gly
2515 2520 2525
Thr Cys Ala Cys Cys Gly Thr Cys Thr Cys Cys Thr Cys Ala Thr Ala
2530 2535 2540
Ala
2545
<210>7
<211>2218
<212>PRT
<213>2 Ambystoma laterale x Ambystoma jeffersonianum
<400>7
Ala Gly Cys Thr Ala Gly Cys Cys Ala Ala Ala Thr Thr Gly Thr Thr
1 5 10 15
Cys Thr Cys Thr Cys Cys Cys Ala Gly Thr Cys Thr Cys Cys Ala Gly
20 25 30
Cys Ala Ala Thr Cys Cys Thr Gly Thr Cys Thr Gly Cys Ala Thr Cys
35 40 45
Thr Cys Cys Ala Gly Gly Gly Gly Ala Gly Ala Ala Gly Gly Thr Cys
50 55 60
Ala Cys Ala Thr Thr Gly Ala Cys Thr Thr Gly Cys Ala Gly Gly Gly
65 70 75 80
Cys Cys Ala Gly Cys Thr Cys Ala Ala Gly Thr Gly Thr Ala Ala Gly
85 90 95
Thr Thr Thr Cys Ala Thr Gly Ala Ala Cys Thr Gly Gly Thr Ala Cys
100 105 110
Cys Ala Gly Cys Ala Gly Ala Ala Gly Cys Cys Ala Gly Gly Ala Thr
115 120 125
Cys Cys Thr Cys Cys Cys Cys Cys Ala Ala Ala Cys Cys Cys Thr Gly
130 135 140
Gly Ala Thr Thr Thr Ala Thr Gly Cys Cys Ala Cys Ala Thr Cys Cys
145 150 155 160
Ala Ala Thr Thr Thr Gly Gly Cys Thr Thr Cys Thr Gly Gly Ala Gly
165 170 175
Thr Cys Cys Cys Thr Gly Gly Thr Cys Gly Cys Thr Thr Cys Ala Gly
180 185 190
Thr Gly Gly Cys Ala Gly Thr Gly Gly Gly Thr Cys Thr Gly Gly Gly
195 200 205
Ala Cys Cys Thr Cys Thr Thr Ala Cys Thr Cys Thr Cys Thr Cys Gly
210 215 220
Cys Ala Ala Thr Cys Ala Gly Cys Ala Gly Ala Gly Thr Gly Gly Ala
225 230 235 240
Gly Gly Cys Thr Gly Ala Ala Gly Ala Thr Gly Cys Thr Gly Cys Cys
245 250 255
Ala Cys Thr Thr Ala Thr Thr Ala Cys Thr Gly Cys Cys Ala Gly Cys
260 265 270
Ala Gly Thr Gly Gly Ala Ala Thr Ala Gly Thr Ala Ala Cys Cys Cys
275 280 285
Ala Cys Thr Cys Ala Cys Gly Thr Thr Cys Gly Gly Thr Gly Cys Thr
290 295 300
Gly Gly Gly Ala Cys Cys Ala Ala Gly Cys Thr Gly Gly Ala Gly Cys
305 310 315 320
Thr Gly Ala Ala Ala Cys Gly Ala Gly Gly Thr Gly Gly Thr Gly Gly
325 330 335
Thr Gly Gly Thr Ala Gly Cys Gly Gly Cys Gly Gly Cys Gly Gly Cys
340 345 350
Gly Gly Cys Thr Cys Thr Gly Gly Thr Gly Gly Thr Gly Gly Cys Gly
355 360 365
Gly Ala Thr Cys Cys Cys Ala Gly Ala Thr Cys Cys Ala Gly Thr Thr
370 375 380
Gly Gly Thr Gly Cys Ala Gly Thr Cys Thr Gly Gly Ala Cys Cys Thr
385 390 395 400
Gly Ala Gly Cys Thr Gly Ala Ala Gly Ala Ala Gly Cys Cys Thr Gly
405 410 415
Gly Ala Gly Ala Gly Ala Cys Ala Gly Thr Cys Ala Ala Gly Ala Thr
420 425 430
Cys Thr Cys Cys Thr Gly Cys Ala Ala Gly Gly Cys Thr Thr Cys Thr
435 440 445
Gly Gly Gly Thr Ala Thr Ala Cys Cys Thr Thr Cys Ala Cys Ala Ala
450 455 460
Ala Cys Thr Ala Thr Gly Gly Ala Ala Thr Gly Ala Ala Cys Thr Gly
465 470 475 480
Gly Gly Thr Gly Ala Ala Gly Cys Ala Gly Gly Cys Thr Cys Cys Ala
485 490 495
Gly Gly Ala Ala Ala Gly Gly Gly Thr Thr Thr Ala Ala Ala Gly Thr
500 505 510
Gly Gly Ala Thr Gly Gly Gly Cys Thr Gly Gly Ala Thr Ala Ala Ala
515 520 525
Cys Ala Cys Cys Thr Ala Cys Ala Cys Thr Gly Gly Ala Cys Ala Gly
530 535 540
Cys Cys Ala Ala Cys Ala Thr Ala Thr Gly Cys Thr Gly Ala Thr Gly
545 550 555 560
Ala Cys Thr Thr Cys Ala Ala Gly Gly Gly Ala Cys Gly Gly Thr Thr
565 570 575
Thr Gly Cys Cys Thr Thr Cys Thr Cys Thr Thr Thr Gly Gly Ala Ala
580 585 590
Ala Cys Cys Thr Cys Thr Gly Cys Cys Thr Ala Cys Ala Cys Thr Gly
595 600 605
Cys Cys Thr Ala Thr Thr Thr Gly Cys Ala Gly Ala Thr Cys Ala Ala
610 615 620
Cys Ala Ala Cys Cys Thr Cys Ala Ala Ala Ala Ala Thr Gly Ala Gly
625 630 635 640
Gly Ala Cys Ala Thr Gly Gly Cys Thr Ala Cys Ala Thr Ala Thr Thr
645 650 655
Thr Cys Thr Gly Thr Gly Cys Ala Ala Gly Ala Thr Thr Thr Ala Gly
660 665 670
Cys Thr Ala Thr Gly Gly Thr Ala Ala Cys Thr Cys Ala Cys Gly Thr
675 680 685
Thr Ala Cys Gly Cys Thr Gly Ala Cys Thr Ala Cys Thr Gly Gly Gly
690 695 700
Gly Cys Cys Ala Ala Gly Gly Cys Ala Cys Cys Ala Cys Thr Cys Thr
705 710 715 720
Cys Ala Cys Ala Gly Thr Cys Thr Cys Cys Thr Cys Ala Gly Gly Ala
725 730 735
Thr Cys Cys Thr Thr Cys Thr Gly Gly Thr Thr Ala Cys Cys Cys Ala
740 745 750
Thr Ala Gly Gly Ala Thr Gly Thr Gly Cys Ala Gly Cys Cys Thr Thr
755 760 765
Thr Gly Thr Thr Gly Thr Ala Gly Thr Cys Thr Gly Cys Ala Thr Thr
770 775 780
Thr Thr Gly Gly Gly Ala Thr Gly Cys Ala Thr Ala Cys Thr Thr Ala
785 790 795 800
Thr Thr Thr Gly Thr Thr Gly Gly Cys Thr Thr Ala Cys Ala Ala Ala
805 810 815
Ala Ala Ala Gly Ala Ala Gly Thr Ala Thr Thr Cys Ala Thr Cys Cys
820 825 830
Ala Gly Thr Gly Thr Gly Cys Ala Cys Gly Ala Cys Cys Cys Thr Ala
835 840 845
Ala Cys Gly Gly Thr Gly Ala Ala Thr Ala Cys Ala Thr Gly Thr Thr
850 855 860
Cys Ala Thr Gly Ala Gly Ala Gly Cys Ala Gly Thr Gly Ala Ala Cys
865 870 875 880
Ala Cys Ala Gly Cys Cys Ala Ala Ala Ala Ala Ala Thr Cys Thr Ala
885 890 895
Gly Ala Cys Thr Cys Ala Cys Ala Gly Ala Thr Gly Thr Gly Ala Cys
900 905 910
Cys Cys Thr Ala Thr Cys Thr Ala Gly Ala Gly Ala Gly Gly Gly Cys
915 920 925
Ala Gly Ala Gly Gly Ala Ala Gly Thr Cys Thr Thr Cys Thr Ala Ala
930 935 940
Cys Ala Thr Gly Cys Gly Gly Thr Gly Ala Cys Gly Thr Gly Gly Ala
945 950 955 960
Gly Gly Ala Gly Ala Ala Thr Cys Cys Cys Gly Gly Cys Cys Cys Thr
965 970 975
Ala Thr Gly Gly Ala Gly Ala Cys Ala Cys Cys Ala Gly Cys Thr Cys
980 985 990
Ala Gly Cys Thr Gly Cys Thr Gly Thr Thr Cys Cys Thr Cys Cys Thr
995 1000 1005
Cys Cys Thr Gly Cys Thr Gly Thr Gly Gly Cys Thr Cys Cys Cys Thr
1010 1015 1020
Gly Ala Cys Ala Cys Ala Ala Cys Cys Gly Gly Cys Gly Ala Gly Ala
1025 1030 1035 1040
Thr Thr Gly Thr Gly Cys Thr Gly Ala Cys Cys Cys Ala Gly Thr Cys
1045 1050 1055
Thr Cys Cys Cys Gly Cys Thr Ala Cys Ala Cys Thr Gly Thr Cys Thr
1060 1065 1070
Cys Thr Gly Ala Gly Cys Cys Cys Thr Gly Gly Cys Gly Ala Gly Cys
1075 1080 1085
Gly Cys Gly Cys Cys Ala Cys Cys Cys Thr Gly Thr Cys Thr Thr Gly
1090 1095 1100
Cys Cys Gly Cys Gly Cys Thr Thr Cys Thr Cys Ala Gly Thr Cys Thr
1105 1110 1115 1120
Gly Thr Gly Thr Cys Ala Thr Cys Thr Thr Ala Cys Cys Thr Cys Gly
1125 1130 1135
Cys Thr Thr Gly Gly Thr Ala Thr Cys Ala Gly Cys Ala Gly Ala Ala
1140 1145 1150
Gly Cys Cys Thr Gly Gly Gly Cys Ala Gly Gly Cys Thr Cys Cys Ala
1155 1160 1165
Cys Gly Cys Cys Thr Gly Cys Thr Cys Ala Thr Thr Thr Ala Cys Gly
1170 1175 1180
Ala Thr Gly Cys Ala Thr Cys Thr Ala Ala Cys Ala Gly Ala Gly Cys
1185 1190 1195 1200
Cys Ala Cys Ala Gly Gly Gly Ala Thr Thr Cys Cys Cys Gly Cys Thr
1205 1210 1215
Ala Gly Gly Thr Thr Cys Ala Gly Cys Gly Gly Ala Thr Cys Thr Gly
1220 1225 1230
Gly Gly Thr Cys Cys Gly Gly Cys Ala Cys Cys Gly Ala Cys Thr Thr
1235 1240 1245
Cys Ala Cys Ala Cys Thr Gly Ala Cys Cys Ala Thr Thr Thr Cys Thr
1250 1255 1260
Ala Gly Cys Cys Thr Cys Gly Ala Ala Cys Cys Cys Gly Ala Gly Gly
1265 1270 1275 1280
Ala Cys Thr Thr Cys Gly Cys Cys Gly Thr Gly Thr Ala Cys Thr Ala
1285 1290 1295
Cys Thr Gly Cys Cys Ala Gly Cys Ala Gly Thr Cys Thr Thr Cys Thr
1300 1305 1310
Ala Ala Cys Thr Gly Gly Cys Cys Thr Ala Gly Ala Ala Cys Ala Thr
1315 1320 1325
Thr Cys Gly Gly Cys Cys Ala Gly Gly Gly Ala Ala Cys Cys Ala Ala
1330 1335 1340
Gly Gly Thr Gly Gly Ala Gly Ala Thr Thr Ala Ala Gly Gly Gly Ala
1345 1350 1355 1360
Gly Gly Ala Gly Gly Cys Gly Gly Gly Thr Cys Cys Gly Gly Cys Gly
1365 1370 1375
Gly Ala Gly Gly Cys Gly Gly Ala Ala Gly Cys Gly Gly Ala Gly Gly
1380 1385 1390
Cys Gly Gly Ala Gly Gly Ala Ala Gly Thr Gly Gly Cys Gly Gly Ala
1395 1400 1405
Gly Gly Ala Thr Cys Thr Gly Gly Cys Gly Gly Cys Gly Gly Ala Thr
1410 1415 1420
Cys Thr Cys Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Cys Gly Thr
1425 1430 1435 1440
Gly Gly Ala Gly Thr Cys Cys Gly Gly Ala Gly Gly Cys Gly Gly Ala
1445 1450 1455
Gly Thr Gly Gly Thr Gly Cys Ala Gly Cys Cys Thr Gly Gly Ala Cys
1460 1465 1470
Gly Gly Thr Cys Cys Cys Thr Cys Ala Gly Ala Cys Thr Cys Gly Ala
1475 1480 1485
Cys Thr Gly Cys Ala Ala Gly Gly Cys Thr Thr Cys Cys Gly Gly Cys
1490 1495 1500
Ala Thr Cys Ala Cys Ala Thr Thr Cys Thr Cys Thr Ala Ala Cys Thr
1505 1510 1515 1520
Cys Thr Gly Gly Thr Ala Thr Gly Cys Ala Cys Thr Gly Gly Gly Thr
1525 1530 1535
Gly Ala Gly Ala Cys Ala Gly Gly Cys Ala Cys Cys Ala Gly Gly Ala
1540 1545 1550
Ala Ala Gly Gly Gly Cys Thr Thr Ala Gly Ala Gly Thr Gly Gly Gly
1555 1560 1565
Thr Gly Gly Cys Thr Gly Thr Gly Ala Thr Thr Thr Gly Gly Thr Ala
1570 1575 1580
Cys Gly Ala Cys Gly Gly Gly Thr Cys Gly Ala Ala Gly Ala Gly Ala
1585 1590 1595 1600
Thr Ala Cys Thr Ala Cys Gly Cys Cys Gly Ala Cys Ala Gly Cys Gly
1605 1610 1615
Thr Gly Ala Ala Gly Gly Gly Ala Cys Gly Gly Thr Thr Cys Ala Cys
1620 1625 1630
Ala Ala Thr Thr Ala Gly Cys Ala Gly Ala Gly Ala Cys Ala Ala Cys
1635 1640 1645
Thr Cys Cys Ala Ala Gly Ala Ala Cys Ala Cys Cys Cys Thr Gly Thr
1650 1655 1660
Thr Cys Cys Thr Cys Cys Ala Gly Ala Thr Gly Ala Ala Cys Thr Cys
1665 1670 1675 1680
Thr Cys Thr Gly Cys Gly Cys Gly Cys Cys Gly Ala Gly Gly Ala Cys
1685 1690 1695
Ala Cys Cys Gly Cys Cys Gly Thr Gly Thr Ala Cys Thr Ala Cys Thr
1700 1705 1710
Gly Cys Gly Cys Thr Ala Cys Ala Ala Ala Cys Gly Ala Cys Gly Ala
1715 1720 1725
Cys Thr Ala Cys Thr Gly Gly Gly Gly Ala Cys Ala Gly Gly Gly Cys
1730 1735 1740
Ala Cys Ala Cys Thr Cys Gly Thr Gly Ala Cys Cys Gly Thr Gly Thr
1745 1750 1755 1760
Cys Thr Ala Gly Cys Gly Gly Cys Gly Gly Cys Gly Gly Cys Thr Cys
1765 1770 1775
Gly Gly Gly Cys Gly Gly Cys Gly Gly Gly Thr Cys Cys Gly Gly Cys
1780 1785 1790
Gly Gly Ala Gly Gly Ala Gly Gly Thr Thr Cys Gly Ala Cys Gly Ala
1795 1800 1805
Thr Cys Cys Cys Ala Cys Cys Gly Cys Ala Cys Gly Thr Thr Cys Ala
1810 1815 1820
Gly Ala Ala Gly Thr Cys Gly Gly Ala Thr Gly Thr Gly Gly Ala Ala
1825 1830 1835 1840
Ala Thr Gly Gly Ala Gly Gly Cys Cys Cys Ala Gly Ala Ala Ala Gly
1845 1850 1855
Ala Thr Gly Ala Ala Ala Thr Cys Ala Thr Cys Thr Gly Cys Cys Cys
1860 1865 1870
Cys Ala Gly Cys Thr Gly Thr Ala Ala Thr Ala Gly Gly Ala Cys Thr
1875 1880 1885
Gly Cys Cys Cys Ala Thr Cys Cys Ala Cys Thr Gly Ala Gly Ala Cys
1890 1895 1900
Ala Thr Ala Thr Thr Ala Ala Thr Ala Ala Cys Gly Ala Cys Ala Thr
1905 1910 1915 1920
Gly Ala Thr Ala Gly Thr Cys Ala Cys Thr Gly Ala Cys Ala Ala Cys
1925 1930 1935
Ala Ala Cys Gly Gly Thr Gly Cys Ala Gly Thr Cys Ala Ala Gly Thr
1940 1945 1950
Thr Thr Cys Cys Ala Cys Ala Ala Cys Thr Gly Thr Gly Thr Ala Ala
1955 1960 1965
Ala Thr Thr Thr Thr Gly Thr Gly Ala Thr Gly Thr Gly Ala Gly Ala
1970 1975 1980
Thr Thr Thr Thr Cys Cys Ala Cys Cys Thr Gly Thr Gly Ala Cys Ala
1985 1990 1995 2000
Ala Cys Cys Ala Gly Ala Ala Ala Thr Cys Cys Thr Gly Cys Ala Thr
2005 2010 2015
Gly Ala Gly Cys Ala Ala Cys Thr Gly Cys Ala Gly Cys Ala Thr Cys
2020 2025 2030
Ala Cys Cys Thr Cys Cys Ala Thr Cys Thr Gly Thr Gly Ala Gly Ala
2035 2040 2045
Ala Gly Cys Cys Ala Cys Ala Gly Gly Ala Ala Gly Thr Cys Thr Gly
2050 2055 2060
Thr Gly Thr Gly Gly Cys Thr Gly Thr Ala Thr Gly Gly Ala Gly Ala
2065 2070 2075 2080
Ala Ala Gly Ala Ala Thr Gly Ala Cys Gly Ala Gly Ala Ala Cys Ala
2085 2090 2095
Thr Ala Ala Cys Ala Cys Thr Ala Gly Ala Gly Ala Cys Ala Gly Thr
2100 2105 2110
Thr Thr Gly Cys Cys Ala Thr Gly Ala Cys Cys Cys Cys Ala Ala Gly
2115 2120 2125
Cys Thr Cys Cys Cys Cys Thr Ala Cys Cys Ala Thr Gly Ala Cys Thr
2130 2135 2140
Thr Thr Ala Thr Thr Cys Thr Gly Gly Ala Ala Gly Ala Thr Gly Cys
2145 2150 2155 2160
Thr Gly Cys Thr Thr Cys Thr Cys Cys Ala Ala Ala Gly Thr Gly Cys
2165 2170 2175
Ala Thr Thr Ala Thr Gly Ala Ala Gly Gly Ala Ala Ala Ala Ala Ala
2180 2185 2190
Ala Ala Ala Ala Gly Cys Cys Thr Gly Gly Thr Gly Ala Gly Ala Cys
2195 2200 2205
Thr Thr Thr Cys Thr Thr Cys Thr Ala Ala
2210 2215

Claims (9)

1, fusion protein for relieving immunosuppression, wherein the fusion protein is in PTA or PTR, the fusion protein has part as inhibitor of PD-1 and part as inhibitor of TGF- β.
2. fusion protein for use in the resolution of immunosuppression according to claim 1, wherein the inhibitor of PD-1 is a PD-1 antibody.
3. fusion protein for use in relieving immunosuppression according to claim 2, wherein the inhibitor of TGF- β is TGF- β antibody or extracellular region fragment of TGFBRII.
4. The fusion protein for relieving immunosuppression according to claim 3, wherein the fusion protein is PTA, the part of PTA is PD-1 antibody, the other part is TGF- β antibody, and the amino acid sequence of PTA is SEQ ID No. 1.
5. fusion protein for relieving immunosuppression according to claim 3, wherein the fusion protein is PTR, part of the PTR is PD-1 antibody, part is extracellular region fragment of TGFBRII, and the PTR amino acid sequence is SEQ ID NO. 2.
6. A DNA sequence encoding the amino acid sequence of any of claims 1-5.
A vector of 7, , encoding the DNA sequence of claim 6.
8, cells stably or transiently expressing the vector of claim 7.
9. The amino acid, DNA, vector and cell of any of claims 1-8 for use in the treatment of a human tumor.
CN201910977793.5A 2019-10-15 2019-10-15 fusion protein for relieving immunosuppression and application thereof Pending CN110734498A (en)

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