CN112522316B - Construction method and application of humanized KDR gene modified animal model - Google Patents

Abstract

The application provides a construction method and application of a humanized KDR gene modified animal model, and relates to the technical field of genetic engineering. The humanized KDR gene modified animal model constructed by the application can accelerate the research progress of the fields related to the human KDR gene or protein. Preferably, the application utilizes homologous recombination gene editing technology to replace a mouse-derived Kdr gene with a human-derived KDR gene on a mouse with a sound immune system, and constructs a mouse model capable of interacting with an anti-human-derived KDR antibody.

Description

Construction method and application of humanized KDR gene modified animal model

Technical Field

The application relates to the technical field of genetic engineering, in particular to a construction method and application of a humanized KDR gene modified animal model.

Background

Cancer is a major public health problem facing the world, and the incidence and mortality of human cancers is still rising. With the progress of aging and the increase in population, cancer is expected to be the leading cause of death in the 21 st century, and has become a heavy burden for public medical care.

Angiogenesis is a necessary step in tumorigenesis, the development process: in the process of tumor growth, when the diameter of the tumor is larger than 2mm, the required nutrients and oxygen cannot be obtained only by diffusion in tissues, and angiogenesis is required to provide nutrients and oxygen for cancer cells for growth and amplification of the cancer cells. In 1971, Folkman et al proposed that growth and metastasis of solid tumors depend on angiogenesis, and further proposed an anti-tumor therapy that "starves" tumors by destroying or inhibiting tumor angiogenesis through tumor angiogenesis inhibitors, with tumor angiogenesis as a target. Further research shows that when providing nutrient substances and oxygen for tumor cells, tumor blood vessels can form a microenvironment with hypertonicity, hypoxia, acidity and high pressure due to structural abnormality, so that proliferation, infiltration, survival and function exertion of immune cells are influenced, and the resistance of tumors to chemotherapy, radiotherapy and immunotherapy is caused. Jain et al proposed the theory of normalization of tumor vessels: the anti-angiogenesis medicine is reasonably applied, the tumor blood vessels tend to be normal in structure and function in a specific time window, and finally the anti-tumor effect is improved and the tumor metastasis is inhibited.

A great deal of research shows that factors such as Vascular Endothelial Growth Factor (VEGF) and angiogenin (Ang) are over-expressed in tumor tissues and play an important role in promoting the formation and growth of tumor blood vessels. For these pro-angiogenic factors, a series of drug molecules have been developed, among which, drugs directed to VEGF/VEGFR (vascular endothelial growth factor receptor) signaling pathway are the fastest growing and most successful, and several drugs are on the market, such as bevacizumab directed to VEGF, ramucirumab directed to VEGFR, etc. VEGF is a glycoprotein, and the human VEGF family comprises 6 members of VEGFA, VEGFB and the like, and VEGF can selectively act on VEGFR. VEGFR is a class of tyrosine kinase transmembrane proteins, which are distributed predominantly on the surface of vascular endothelial cells and comprise 3 members of VEGFR1, VEGFR2 (also known as KDR) and VEGFR 3. VEGF binds to the extracellular segment of VEGFR, so that VEGFR conformation is changed to form a dimer, the tyrosine site of the intracellular segment of the dimer is phosphorylated, a downstream signal transduction pathway is activated, and angiogenesis is promoted. Among them, VEGFR2(KDR) is the most important endothelial cell proliferation and differentiation promoting signal transduction protein on vascular endothelial cells, KDR plays an important role in tumor angiogenesis in addition, recent studies show that it also plays an important role in regulating and controlling tumor microenvironment, and KDR antibody can reverse the inhibitory effect of VEGF on T cells. Therefore, the inhibitor aiming at KDR can play a role in resisting tumors by inhibiting angiogenesis and improving the tumor microenvironment and enhancing the immune function of immune cells. At present, aiming at a KDR target point, a large-molecular antibody medicament, namely Ramucirumab (Cyramza, Ramucirumab), is available on the market for the Gift company, and many companies in China are developing the large-molecular antibody medicament aiming at the KDR, such as the target points are distributed in the scientific pharmaceutical industry, the Kangfang organism, the Sansheng pharmacy, the step-size pharmacy and the like; besides macromolecular drugs, several companies in China have already or will come into the market for small-molecule kinase inhibitors of KDR, such as Apatinib of Henry medicine and Sofantinib of Megaku.

In preclinical research of drugs, mice are widely applied to drug effect evaluation, pharmacological toxicological analysis and other researches, and the premise for applying the researches is that drugs acting on human-derived targets can be combined with corresponding targets of the mice, and if tested drugs cannot be combined with mouse-derived targets, the mice cannot be used for drug effect evaluation, pharmacological toxicological evaluation and other evaluations. The contradiction between the drug effect and the pharmacological and toxicological evaluation of animals cannot be caused due to the fact that the developed human-derived target drugs cannot identify target molecules of the animals, and the contradiction is particularly prominent in the macromolecular antibody drugs, because the macromolecular antibody drugs can only identify one target site consisting of a plurality of amino acids, and the difference of 1 amino acid of the target molecules between the animals and the human can cause the drugs not to identify the target sites in the animal bodies, further influence the subsequent drug evaluation, prolong the research and development period and increase the uncertainty. The consistency of amino acid sequences of KDR proteins of human and mouse is only 85%, the consistency of amino acid of extracellular regions is lower and is only 79.9% (the extracellular regions are main action regions of antibody macromolecular drugs aiming at KDR targets), and due to the difference of the amino acid sequences, the marketed Ramomucirumab aiming at the KDR targets cannot identify the Kdr proteins of the mouse, and the drug effect, the toxicology, the safety and the like of the Ramomucirumab by the mouse cannot be utilized for evaluation. In order to reduce the difference between human and mice, the wild type mouse is humanized and transformed, so that the microenvironment of a human body can be well reproduced, and the method can be applied to drug toxicity and safety evaluation while being used for drug effect evaluation. At present, humanized mouse models have been widely used in the field of disease research such as tumor and virus infection.

Nowadays, genetic engineering technology has been developed relatively mature, target molecules are humanized and transformed by means of gene modification, and the problem of target difference between human and animals can be well solved by establishing a humanized animal model to research human diseases: after gene modification, the humanized model animal body can fully or partially express corresponding human protein, imitate the interaction between various molecules when human diseases occur, reappear the characteristics of the human diseases, greatly reduce the difference between the animal model and the human body before clinic, and enable the screening research of the medicine at the whole level of the animal to be possible; meanwhile, the model can evaluate the toxicity and drug metabolism of the drug on a model with a normal immune system, can better fit the clinical drug efficacy, and improves the success probability of drug screening.

Disclosure of Invention

The application aims to provide a construction method and application of a humanized KDR gene modified animal model, so as to accelerate research progress of fields related to human KDR genes or proteins.

In order to achieve the above object, the present application first provides a method for constructing a humanized KDR gene-modified animal cell, comprising:

introducing a human KDR gene into a non-human animal cell, so that the human KDR gene is expressed in the non-human animal cell to generate humanized KDR protein, and simultaneously reducing or eliminating the expression of an endogenous KDR gene in the non-human animal cell.

In some embodiments of the present application, a gene editing technology is used to modify an endogenous KDR gene in a non-human animal cell to obtain a humanized KDR gene;

preferably, the non-human animal is a rodent;

preferably, the rodent is a mouse, and the construction method comprises the following steps: replacing the whole sequence of exons 4 to 14 and a part of the sequence of exon 15 of a mouse Kdr gene with the whole sequence of exons 4 to 14 and a part of the sequence of exon 15 of a human KDR gene;

preferably, the cell is an embryonic stem cell.

It is understood that the sequence of the coding protein corresponding to the entire sequence of exon 4 to exon 14 and a part of the sequence of exon 15 of mouse Kdr gene is the sequence of the extracellular region of KDR protein.

When the non-human animal cell is an embryonic stem cell, a humanized KDR gene cell is obtained by carrying out gene editing on the embryonic stem cell, the humanized KDR gene cell is transferred to a blastocyst of a wild type mouse, and a F0 generation chimeric animal can be obtained after embryo transplantation and development.

In some embodiments of the present application, the humanized KDR gene is selected from at least one of the following group:

(a) the CDS coding sequence of the humanized KDR gene is shown as SEQ ID NO: 6 is shown in the specification;

(b) the mRNA sequence transcribed by the humanized KDR gene is shown as SEQ ID NO: 7 is shown in the specification;

(c) the protein sequence coded by the humanized KDR gene is shown as SEQ ID NO: shown in fig. 8.

In some embodiments of the present application, the construction method specifically includes: providing a human KDR gene homologous recombination vector, introducing the human KDR gene homologous recombination vector into mouse cells, and transferring the mouse cells into a culture solution for culture;

preferably, the human KDR gene homologous recombination vector comprises a 5 'homology arm, a humanized KDR gene coding region, a resistance gene expression box and a 3' homology arm which are sequentially arranged from a 5 'end to a 3' end;

specific sites which can be recognized by recombinase are arranged between the coding region of the humanized KDR gene and the expression frame of the resistance gene and between the expression frame of the resistance gene and the 3' homology arm;

preferably, the specific site is an FRT site.

In some embodiments of the present application, the DNA sequence of the 5' homology arm is as set forth in SEQ ID NO: 9 is shown in the figure;

preferably, the DNA sequence of the 3' homology arm is as set forth in SEQ ID NO: 10 is shown in the figure;

preferably, the DNA sequence of the coding region of the humanized KDR gene is as set forth in SEQ ID NO: shown at 11.

SEQ ID NO: 11 does not include an intron, and a plurality of continuous exons are fused by cutting off the intron, so that the efficiency of homologous recombination is improved while the KDR gene expression is not influenced.

The application also provides a humanized KDR gene modified animal cell which is obtained by the construction method of the humanized KDR gene modified animal cell.

The application also provides a construction method of the humanized KDR gene modified animal model, the humanized KDR gene modified animal cell is injected into a blastocyst, and is transplanted into a surrogate mother body for development through embryo to obtain an F0 animal;

preferably, the blastocyst is a blastocyst of a wild-type animal.

Specifically, the method for screening the animals of the F0 generation can be as follows: and (4) screening through the character of hair color. For example, blastocysts are selected from animals having a first coat color (e.g., black), humanized KDR genetically modified animal cells (embryonic stem cells) are selected from animals having a second coat color (e.g., gray), the obtained F0 generation animals are chimeras, and thus the coat color is a mixed color of the first coat color and the second coat color, and then F0 generation animals having a second coat color ratio of 50% or more are selected for breeding the next generation, because the second coat color ratio of 50% or more of the F0 generation animals has a higher probability that the reproductive system thereof is developed from the humanized KDR genetically modified animal cells.

In some embodiments of the present application, the method for constructing the humanized KDR genetically modified animal model further comprises: mating the F0 generation animals with animals expressing recombinase to obtain F1 generation animals with resistance genes removed;

mating the F1 generation animal with a wild animal, expanding the population, mating the heterozygote animal with the heterozygote animal to obtain a homozygote animal, and establishing a stable humanized KDR gene modified animal strain;

preferably, the recombinase is a Flp recombinase.

It is understood that the cells of the F1 generation animals obtained by mating the F0 generation animals with an animal expressing a recombinase can contain both the humanized KDR gene and the recombinase gene, and the recombinase can recognize specific sites on both sides of the resistance gene, thereby splicing the resistance gene transformed into the genome of the animal to obtain F1 generation animals from which the resistance gene is deleted.

In some embodiments of the present application, the specific site is an FRT site and the recombinase is a Flp recombinase. The Flp recombinase can recognize specific sites on both sides of the resistance gene expression frame, and then the resistance gene expression frame is deleted.

Preferably, the animal expressing the recombinase is an animal expressing the recombinase at an early stage of oocyte/embryo development. This is because the early stage of oocyte/embryo development, i.e., the animals expressing the recombinase after maturation, can ensure that all cells throughout the body contain the recombinase gene, thereby ensuring that germ cells thereof contain the recombinase gene. It is understood that the early stage of embryonic development generally refers to within 72 hours after fertilization of the ovum.

The application also provides a cell or a cell line or a cell culture, the cell or the cell line or the cell culture is derived from a humanized KDR gene modified animal model or filial generation thereof, and the humanized KDR gene modified animal model is obtained by the construction method of the humanized KDR gene modified animal model.

The application also provides a tissue or organ, the tissue or organ is derived from a humanized KDR gene modified animal model or offspring thereof, and the humanized KDR gene modified animal model is obtained by the construction method of the humanized KDR gene modified animal model.

The application also provides a humanized KDR gene modified animal model obtained by the construction method of the humanized KDR gene modified animal model, and application of cells, cell lines or cell cultures, tissues and organs derived from the humanized KDR gene modified animal model in the fields related to the human KDR gene or protein;

optionally, the application includes at least one of human KDR gene function research, human KDR antibody research, drug preparation aiming at a human KDR target site and drug effect research.

The beneficial effect of this application:

the humanized KDR gene modified animal model constructed by the method can accelerate the research progress of the fields related to the human KDR gene or protein, for example, the humanized KDR gene modified animal model is used for replacing human reagents, and an effective model and a powerful tool are provided for preclinical experiments of KDR target drugs.

Preferably, the application utilizes homologous recombination gene editing technology to replace a mouse-derived Kdr gene with a human-derived KDR gene on a mouse with a sound immune system, and constructs a mouse model capable of interacting with an anti-human-derived KDR antibody.

Furthermore, the KDR humanized mouse constructed by the application replaces the extracellular region of mouse Kdr gene with human source sequence, and the intracellular region retains the complete mouse source sequence. The humanized mouse which is successfully manufactured is provided with the human extracellular region, so that a human KDR target drug can be screened, and the mouse intracellular region ensures that the intracellular signal conduction is not influenced, and the external stimulation is faithfully converted into the intracellular behavior.

Drawings

To more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments are briefly described below, and it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope of the present application.

FIG. 1 is a schematic diagram showing the comparison of the murine KDr and human KDR genes.

FIG. 2 is a schematic diagram of the Kdr gene of the humanized mouse after being transformed.

FIG. 3 is a schematic of the targeting protocol for humanizing the mouse Kdr gene.

FIG. 4 is a schematic diagram showing the cleavage result in example 4 of the present application; as for the results of enzyme digestion identification of EcoRV, the theoretical band sizes should be 6404bp, 5108bp, 3181bp, 2704bp, 314bp and 104bp (the bands of 314bp and 104bp have no band display because the product amount is too small); m: 1kb DNA ladder.

FIG. 5 is a schematic diagram showing the results of PCR identification of the targeted ES cell clone in example 5 of the present application. A is an electrophoresis chart of 5' homologous arm homologous recombination identification result; and B is an electrophoresis chart of the 3' homologous arm homologous recombination identification result. The letters above the pictures are the ES cell clone numbers; m: DNA marker (1kb DNA ladder).

FIG. 6 is a schematic diagram showing the results of PCR identification of F1 mouse homologous recombination in example 6 of the present application. A is an electrophoresis chart of 5' homologous arm homologous recombination identification result; and B is an electrophoresis chart of the 3' homologous arm homologous recombination identification result. The upper number is the mouse number; m: DNA marker (1kb DNA ladder).

FIG. 7 is a schematic diagram of the position design of the RT-PCR electrophoresis primer in example 8 of the present application.

FIG. 8 is a graph showing the results of reverse transcription PCR electrophoresis in example 8 of the present application. He: a heterozygote mouse; WT: a wild-type mouse; m: and (5) electrophoresis of the DNA marker. P1/P2 are the primers P1 and P2 shown in FIG. 7, and are used for detecting the expression of murine Kdr mRNA; P3/P4 are primers P3 and P4 shown in FIG. 7, and are used for detecting whether the humanized KDR mRNA is expressed.

FIG. 9 shows the results of enzyme-linked immunosorbent assay (ELISA) for detecting KDR protein expression of mouse of different genotypes in example 9 of the present application. A picture is the detection result of the content of the murine Kdr protein; and the B picture is the detection result of the content of the human KDR protein. WT: a wild-type mouse; and (3) Homo: KDR humanized homozygous mice.

FIG. 10 shows the results of antitumor effect of ramucirumab (Cyramza) in example 9 of the present application in wild type mice and humanized mice. A picture is the response result of KDR humanized mouse to Ramoplumumab tumor drug effect; panel B is a comparative analysis of tumor size at day 17 of the dosing endpoint of Panel A; the C picture shows the response result of wild type C57BL/6 mouse to ramucirumab. In the A picture, the number of mice in the Vehicle group is 4, and the number of mice in the Cyramza-15mpk-i.p. group is 5; in the C panel, the number of mice in the Vehicle group and the Cyramza-20mpk-i.p. group is 5.

Detailed Description

Embodiments of the present application will be described in detail below with reference to specific examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The mouse strain, biochemical reagent and experimental instrument used in the embodiment of the application are as follows:

primer synthesis and sequencing services were purchased from bio-engineering (shanghai) gmbh;

EcoRV restriction enzymes were purchased from NEB Inc. under the following commercial designations: R0195S;

a1 kb DNA ladder was purchased from Thermo Fisher Scientific, having the following code: SM 0311;

In-Fusion HD Cloning Kits available from Takara under the accession number: 639650, respectively;

coli DH 5. alpha. competent cells were purchased from TaKaRa, cat # 9057;

c57BL/6, ICR mice were purchased from Shanghai Ling Biotech, Inc.;

the RNA extraction kit is purchased from the general gold company, and the goods number is as follows: DP 424;

the RNA reverse transcription kit is purchased from the general gold company, and the goods number is as follows: AT 311-02;

the real-time quantitative PCR detection kit is purchased from the general gold company, and the goods number is as follows: AQ 131-01;

ELISA kits for murine Kdr protein detection were purchased from R & D SYSTEMS, under the accession numbers: MVR 200B;

ELISA kits for murine protein detection were purchased from R & D SYSTEMS, under the accession number: a DVR 200;

ramucirumab (Cyramza) was purchased from american gifts.

Example 1 sequence design

Both the mouse Kdr gene and the human Kdr gene contain multiple transcripts, and the sequence design of this example is described mainly with one transcript as an example. That is, the mouse KDr Gene (NCBI Gene ID: 16542) from exon 4 to 14 and a part of exon 15 (based on the transcript of NCBI accession No. NM-010612.3 → NP-034742.2, whose mRNA sequence is shown in SEQ ID NO: 1 and the corresponding protein sequence is shown in SEQ ID NO: 2) are replaced with exon 4 to exon 14 and a part of exon 15 of human KDR Gene (Gene ID: 3791) (based on the transcript of NCBI accession No. NM-002253.3 → NP-002244.1, whose mRNA sequence is shown in SEQ ID NO: 3 and the corresponding protein sequence is shown in SEQ ID NO: 4), wherein the comparison of mouse KDr Gene and human KDR Gene is schematically shown in FIG. 1, the finally obtained humanized mouse KDR Gene after modification is schematically shown in FIG. 2, and the humanized mouse KDR Gene DNA sequence (chimeric KDR Gene DNA) is shown in SEQ ID NO: and 5, as follows:

SEQ ID NO: 5 lists only the DNA sequences involved in the engineered part, where the underlined region is flanked by the non-engineered regions, the bold italics are the sequences of the new exons formed by fusing together human exons 4 to 14 and a part of exon 15 with a part of mouse exon 15, and the regular italics are the partial sequences of mouse intron 15.

The CDS region and mRNA sequence of the humanized mouse KDR after being transformed and the protein sequence coded by the CDS region and mRNA sequence are respectively shown as SEQ ID NO: 6. SEQ ID NO: 7 and SEQ ID NO: shown in fig. 8.

Given that there are multiple transcripts for the human and mouse KDR genes, the method of humanized sequence design in this example is equally applicable to the humanized engineering of other transcripts. The mouse Kdr gene transcript described above may be replaced with another transcript.

Example 2 design and construction of recombinant vector PBR322-KDR

Based on the sequence design, the inventors further designed the targeting protocol shown in FIG. 3 and a vector comprising a 5 'homology arm, a human KDR gene fragment, a murine Kdr gene fragment, a resistance gene expression cassette, and a 3' homology arm. Wherein the 5 'homology arm (SEQ ID NO: 9) is the 75972817-75968732 nucleotide of NCBI accession No. NC-000071.6, the 3' homology arm (SEQ ID NO: 10) is the 75955853-75951 nucleotide of NCBI accession No. NC-000071.6, the human KDR gene fragment (SEQ ID NO: 11) is the 705-2501 nucleotide of NCBI accession No. NM-002253.3, and the murine Kdr gene fragment (SEQ ID NO: 12) is the 76116880-76116514 nucleotide of NCBI accession No. NC-000071.7.

The construction process of the vector is as follows: an upstream primer for amplifying 3 homologous recombination fragments (LA, KI and RA) and a downstream primer matched with the upstream primer and related sequences are designed. Wherein, the 5 'homologous arm corresponds to LA fragment, elements such as human KDR gene fragment inserted for humanization correspond to KI fragment, the 3' homologous arm corresponds to RA fragment, and the primer sequence is as follows:

LA(4015bp)：

F：5’-CGCGGTCGACAAGCTcaactactctaccactgagc-3’(SEQID NO：13)

R：5’-GCCATGCTGGTCACTGACAG-3’(SEQ ID NO：14)

KI(4097bp)：

F：5’-AGTGACCAGCATGGCGTCGTGTACATTACTGAGAA-3’(SEQ ID NO：15)

R：5’-CCATTATGTACCTGACTGAT-3’(SEQ ID NO：16)

RA(3948bp)：

F：5’-TCAGGTACATAATGGctccacgtgttctttgtaat-3’(SEQ ID NO：17)

R：5’-TAGAGGATCGGCGCGagaaccaatgacagtggtat-3’(SEQ ID NO:18)

using C57BL/6 mouse genome DNA or BAC library as template to make PCR amplification to obtain LA and RA fragments, using the synthesized human KDR gene fragment, mouse Kdr gene fragment and resistance gene expression frame as template to make PCR amplification to obtain KI fragment (SEQ ID NO: 19, containing human KDR gene fragment, mouse Kdr gene fragment and resistance gene expression frame). The fragment was ligated to the PBR322-MCS plasmid by means of an In-fusion kit to finally obtain the vector PBR 322-KDR.

Example 3 validation of the vector PBR322-KDR

Randomly selecting 5 PBR322-KDR clones, using restriction enzyme EcoRV to perform enzyme digestion verification, and generating fragments with the sizes of 6404bp, 5108bp, 3181bp, 2704bp, 314bp and 104bp by enzyme digestion product electrophoresis. The digestion results are shown in FIG. 4, and the plasmid digestion results are all in line with expectations, which indicates that the plasmid digestion verification result is correct. The plasmid was verified to be correct by sequencing company for subsequent experiments.

Example 4 identification of Positive clones of humanized ES cells with KDR

And (3) carrying out electrotransformation on the constructed PBR322-KDR homologous recombination vector by using mouse embryonic stem cells (ES cells), and screening resistant clones to finally obtain 144 resistant ES cell clones. After the resistance ES cell clone genome DNA is extracted, homologous recombination positive clones are screened in a long-fragment PCR mode, the homologous recombination positive clones are identified by PCR, and a PCR product is sequenced and confirmed.

Cloning and identifying homologous recombination positive ES cells:

and respectively carrying out PCR identification on the cloned genomic DNA of the resistant ES cells by using two pairs of primers, wherein the primer I is positioned outside the 5 'homologous arm 5', the primer IV is positioned outside the 3 'homologous arm 3', and the primer II and the primer III are positioned on the inserted fragment, and the specific sequences are as follows:

5' homologous arm recombination identification primer:

primer I: 5'-AACTTCTTACTCTCCCTTGCTTAT-3' (SEQ ID NO: 20)

And (3) primer II: 5'-TGAAATGGGGTTGGTGAGGATGAC-3' (SEQ ID NO: 21) PCR reaction (20. mu.L) is shown in Table 1:

TABLE 1 PCR reaction System (20. mu.L)

Reaction components	Volume (μ l)
		ddH2O	13.2
GXL PCR Buffer	2
		2.5mMdNTP	2
Primer I(10pmol/μl)	0.5
		Primer II(10pmol/μl)	0.5
GXL DNA Polymerase*	0.8
		Genomic DNA	1
Total	20

The PCR amplification reaction conditions are shown in Table 2:

TABLE 2 PCR amplification reaction conditions

Step # of	Temperature (. degree.C.)	Time	Remarks for note
				1	94	3min	-
2	98	15sec	-
				3	61	15sec	-
4	68	5min	Repeating the steps for 2-432 cycles
				5	68	5min	-
6	12	-	Heat preservation

3' homologous arm recombination identification primer:

and (3) primer III: 5'-GCTGTGGGTGCTGATGAACG-3' (SEQ ID NO: 22)

And (3) primer IV: 5'-CAAAGGGCCACTGACACAAGACTC-3' (SEQ ID NO: 23)

The PCR reaction (20. mu.L) is shown in Table 3:

TABLE 3 PCR reaction System (20. mu.L)

The PCR amplification reaction conditions are shown in Table 4:

TABLE 4 PCR amplification reaction conditions

Step # of	Temperature (. degree.C.)	Time	Remarks for note
				1	94	3min	-
2	98	15sec	-
				3	61	15sec	-
4	68	5min	Repeating the steps for 2-432 cycles
				5	68	5min	-
6	12	-	Heat preservation

If the ES cell targeting vector is correctly recombined, the 5' homologous arm identification primer pair I and II can amplify a PCR product with the length of 6.5kb, and a negative clone cannot amplify a PCR product with the length of 6.5 kb; the 3' homology arm identification primer pair III and IV can amplify PCR bands with the length of 6kb, and the negative clone can only amplify a band with the length of 9.2 kb.

The PCR identification of ES cell clones is schematically shown in FIG. 5, in which the clones numbered C2 and H2 are correctly recombined positive ES cell clones.

Example 5 acquisition of humanized heterozygote mice for KDR

The positive ES cell clone No. C2, H2, after expanding, inject into C57BL/6 mouse blastocyst, inject 160 blastocysts totally, through the embryo transfer, obtain 9 high chimeric male mice totally. Mating the chimera male mouse with the mouse expressing Flp recombinase at the early development stage of oocyte/embryo, and breeding to obtain the KDR humanized F1 generation mouse with the resistance gene removed. Through PCR identification and sequencing confirmation, 4 positive mice are obtained in total, and the numbers are as follows: 7. numbers 9, 13, 14.

Example 6 identification of humanized F1 Generation mice with KDR

The KDR humanized F1 generation mouse genotype identification protocol with homologous recombination positive deletion of resistance gene expression cassette was the same as that of example 4 identification primers and identification conditions for positive cloning of KDR humanized ES cells: homologous recombination positive mice, 5' homologous arm identification primer pairs I and II can amplify PCR products with the length of 6.5kb, and negative mice can not amplify PCR products with the length of 6.5 kb; the 3' homology arm identification primer pair III and IV can amplify a PCR product band with the length of 4.2kb, and a negative mouse cannot amplify a PCR product band with the length of 4.2 kb.

The PCR identification result of F1 mouse is shown in FIG. 6, in which 4 mice numbered 7, 9, 13, 14 are positive for homologous recombination and the expression cassette of the resistance gene is deleted.

The obtained F1 mouse generation was mated with a wild type mouse, respectively, to propagate the population. Meanwhile, the heterozygote mice are selfed to obtain homozygote mice.

Example 7 humanized mouse KDR RNA expression analysis

Selecting a KDR humanized heterozygote mouse and a littermate wild type mouse (6 weeks old) to extract total lung RNA, carrying out PCR after carrying out reverse transcription into cDNA by using a reverse transcription kit, and verifying whether the humanized insert fragment in the humanized mouse is expressed or not.

The position of the primer for detecting the murine Kdr RNA in the experimental design is shown as the position of the P1/P2 primer in figure 7, and the sequence is as follows:

p1: 5'-CCATTGGCGAGACCATTGAAGTGA-3' (SEQ ID NO: 24), and

P2：5’-AGCGCTCATCCAAGGGCAAT-3’(SEQ ID NO：25)

the position of the designed detection primer for detecting the humanized KDR RNA is shown as the position of a P3/P4 primer in figure 7, and the sequence is as follows:

p3: 5'-GTATTGGGGAAAGCATCGAAGTCT-3' (SEQ ID NO: 26), and

P4：5’-AGCGCTCATCCAAGGGCAAT-3’(SEQ ID NO：27)

the PCR reaction system and the reaction conditions of the two pairs of primers P1/P2 and P3/P4 are the same, and 20 mu L of the PCR reaction system and the reaction conditions are as follows: 94 ℃ for 4 min; (94 ℃, 15 sec; 62 ℃, 15 sec; 72 ℃, 45sec, 32 cycles); 72 ℃ for 5 min; keeping the temperature at 12 ℃.

Theoretically, by using a P1/P2 primer pair, a wild mouse and a heterozygote mouse can amplify PCR products of reverse transcription of murine RNA with the size of 414 bp; with the primer pair P3/P4, heterozygote mice can amplify PCR products with the size of 414bp specific to humanized RNA after reverse transcription, and wild mice cannot amplify specific PCR products specific to humanized KDR RNA.

The reverse transcription PCR electrophoresis results (see FIG. 8) show that, consistent with the theoretical situation, the expression of murine Kdr mRNA can be detected in both wild type and heterozygous mice; however, the expression of the humanized KDR mRNA was detectable only in the humanized heterozygote mice, and not in the wild-type mice.

Example 8 analysis of KDR protein level expression in humanized mice

The method comprises the steps of selfing a heterozygote mouse to obtain a homozygote mouse, selecting 3 humanized KDR homozygote mice and 3 same-litter wild-type mice (6-7 weeks old), taking peripheral blood, and detecting the expression of the murine and human KDR proteins in the peripheral blood of the wild-type and humanized homozygote mice by respectively using enzyme-linked immunosorbent assay (ELISA) detection kits aiming at the murine Kdr protein and the human KDR protein. According to the operation method provided by the kit, the result is shown in fig. 9, only a mouse-derived KDR protein signal of a background level can be detected in the humanized KDR homozygote mouse, and a high-level human-derived KDR protein signal can be detected, which indicates that the human-derived KDR protein is actively expressed in the humanized KDR mouse; however, only the expression of the mouse-derived Kdr protein could be detected in the wild-type mouse, and the expression of the human-derived Kdr protein could not be detected.

Example 9 humanized mouse KDR model can be applied to drug efficacy validation for human KDR target

Wild type C57BL/6 mice and KDR humanized homozygote mice were inoculated with B16-F1 melanoma tumor cells, respectively, and each mouse was inoculated with 1x10⁶The amount of cells until the tumor grows to 25-50mm³At that time, the administration of groups was initiated and treatment was given with saline and ramucirumab (Cyramza) marketed for KDR target, respectively, as shown in the table below:

after 6 times of administration treatment, the ramucirumab does not show an anti-tumor effect in a wild mouse tumor-bearing group; the ramucirumab shows obvious anti-tumor effect in KDR humanized homozygote mice, and the average tumor volume of a treatment group is 238.87mm³Is obviously lower than 2150.54mm of the normal saline control group³The difference is statistically significant, which indicates that ramucirumab can effectively inhibit the growth of B16-F1 tumor, and the result is shown in FIG. 10. The result shows that the KDR humanized and transformed mouse model overcomes the defects of a wild mouse and can be applied to drug effect verification aiming at a human KDR target point.

Sequence listing

<110> Shanghai's Square model Biotech Co., Ltd

Shanghai Yushi Biological Technology Co., Ltd.

Guangdong Nanmo Biological Technology Co., Ltd.

Construction method and application of humanized KDR gene animal model

<160> 27

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5921

<212> RNA

<213> mouse (mouse)

<400> 1

gaguccucag gaccccaaga gaguaagcug uguuuccuua gaucgcgcgg accgcuaccc 60

ggcaggacug aaagcccaga cugugucccg cagccgggau aaccuggcug acccgauucc 120

gcggacaccg cugcagccgc ggcuggagcc agggcgccgg ugccccgcgc ucuccccggu 180

cuugcgcugc gggggcgcau accgccucug ugacuucuuu gcgggccagg gacggagaag 240

gagucugugc cugagaacug ggcucugugc ccagcgcgag gugcaggaug gagagcaagg 300

cgcugcuagc ugucgcucug ugguucugcg uggagacccg agccgccucu guggguuugc 360

cuggcgauuu ucuccauccc cccaagcuca gcacacagaa agacauacug acaauuuugg 420

caaauacaac ccuucagauu acuugcaggg gacagcggga ccuggacugg cuuuggccca 480

augcucagcg ugauucugag gaaaggguau uggugacuga augcggcggu ggugacagua 540

ucuucugcaa aacacucacc auucccaggg ugguuggaaa ugauacugga gccuacaagu 600

gcucguaccg ggacgucgac auagccucca cuguuuaugu cuauguucga gauuacagau 660

caccauucau cgccucuguc agugaccagc auggcaucgu guacaucacc gagaacaaga 720

acaaaacugu ggugaucccc ugccgagggu cgauuucaaa ccucaaugug ucucuuugcg 780

cuagguaucc agaaaagaga uuuguuccgg auggaaacag aauuuccugg gacagcgaga 840

uaggcuuuac ucuccccagu uacaugauca gcuaugccgg cauggucuuc ugugaggcaa 900

agaucaauga ugaaaccuau cagucuauca uguacauagu ugugguugua ggauauagga 960

uuuaugaugu gauucugagc cccccgcaug aaauugagcu aucugccgga gaaaaacuug 1020

ucuuaaauug uacagcgaga acagagcuca auguggggcu ugauuucacc uggcacucuc 1080

caccuucaaa gucucaucau aagaagauug uaaaccggga ugugaaaccc uuuccuggga 1140

cuguggcgaa gauguuuuug agcaccuuga caauagaaag ugugaccaag agugaccaag 1200

gggaauacac cuguguagcg uccaguggac ggaugaucaa gagaaauaga acauuugucc 1260

gaguucacac aaagccuuuu auugcuuucg guagugggau gaaaucuuug guggaagcca 1320

cagugggcag ucaaguccga aucccuguga aguaucucag uuacccagcu ccugauauca 1380

aaugguacag aaauggaagg cccauugagu ccaacuacac aaugauuguu ggcgaugaac 1440

ucaccaucau ggaagugacu gaaagagaug caggaaacua cacggucauc cucaccaacc 1500

ccauuucaau ggagaaacag agccacaugg ucucucuggu ugugaauguc ccaccccaga 1560

ucggugagaa agccuugauc ucgccuaugg auuccuacca guaugggacc augcagacau 1620

ugacaugcac agucuacgcc aacccucccc ugcaccacau ccagugguac uggcagcuag 1680

aagaagccug cuccuacaga cccggccaaa caagcccgua ugcuuguaaa gaauggagac 1740

acguggagga uuuccagggg ggaaacaaga ucgaagucac caaaaaccaa uaugcccuga 1800

uugaaggaaa aaacaaaacu guaaguacgc uggucaucca agcugccaac gugucagcgu 1860

uguacaaaug ugaagccauc aacaaagcgg gacgaggaga gagggucauc uccuuccaug 1920

ugaucagggg uccugaaauu acugugcaac cugcugccca gccaacugag caggagagug 1980

ugucccuguu gugcacugca gacagaaaua cguuugagaa ccucacgugg uacaagcuug 2040

gcucacaggc aacaucgguc cacaugggcg aaucacucac accaguuugc aagaacuugg 2100

augcucuuug gaaacugaau ggcaccaugu uuucuaacag cacaaaugac aucuugauug 2160

uggcauuuca gaaugccucu cugcaggacc aaggcgacua uguuugcucu gcucaagaua 2220

agaagaccaa gaaaagacau ugccugguca aacagcucau cauccuagag cgcauggcac 2280

ccaugaucac cggaaaucug gagaaucaga caacaaccau uggcgagacc auugaaguga 2340

cuugcccagc aucuggaaau ccuaccccac acauuacaug guucaaagac aacgagaccc 2400

ugguagaaga uucaggcauu guacugagag augggaaccg gaaccugacu auccgcaggg 2460

ugaggaagga ggauggaggc cucuacaccu gccaggccug caauguccuu ggcugugcaa 2520

gagcggagac gcucuucaua auagaaggug cccaggaaaa gaccaacuug gaagucauua 2580

uccucgucgg cacugcagug auugccaugu ucuucuggcu ccuucuuguc auuguccuac 2640

ggaccguuaa gcgggccaau gaaggggaac ugaagacagg cuacuugucu auugucaugg 2700

auccagauga auugcccuug gaugagcgcu gugaacgcuu gccuuaugau gccagcaagu 2760

gggaauuccc cagggaccgg cugaaacuag gaaaaccucu uggccgcggu gccuucggcc 2820

aagugauuga ggcagacgcu uuuggaauug acaagacagc gacuugcaaa acaguagccg 2880

ucaagauguu gaaagaagga gcaacacaca gcgagcaucg agcccucaug ucugaacuca 2940

agauccucau ccacauuggu caccaucuca auguggugaa ccuccuaggc gccugcacca 3000

agccgggagg gccucucaug gugauugugg aauucugcaa guuuggaaac cuaucaacuu 3060

acuuacgggg caagagaaau gaauuuguuc ccuauaagag caaaggggca cgcuuccgcc 3120

agggcaagga cuacguuggg gagcucuccg uggaucugaa aagacgcuug gacagcauca 3180

ccagcagcca gagcucugcc agcucaggcu uuguugagga gaaaucgcuc agugauguag 3240

aggaagaaga agcuucugaa gaacuguaca aggacuuccu gaccuuggag caucucaucu 3300

guuacagcuu ccaaguggcu aagggcaugg aguucuuggc aucaaggaag uguauccaca 3360

gggaccuggc agcacgaaac auucuccuau cggagaagaa ugugguuaag aucugugacu 3420

ucggcuuggc ccgggacauu uauaaagacc cggauuaugu cagaaaagga gaugcccgac 3480

ucccuuugaa guggauggcc ccggaaacca uuuuugacag aguauacaca auucagagcg 3540

augugugguc uuucggugug uugcucuggg aaauauuuuc cuuaggugcc uccccauacc 3600

cuggggucaa gauugaugaa gaauuuugua ggagauugaa agaaggaacu agaaugcggg 3660

cuccugacua cacuacccca gaaauguacc agaccaugcu ggacugcugg caugaggacc 3720

ccaaccagag acccucguuu ucagaguugg uggagcauuu gggaaaccuc cugcaagcaa 3780

augcgcagca ggauggcaaa gacuauauug uucuuccaau gucagagaca cugagcaugg 3840

aagaggauuc uggacucucc cugccuaccu caccuguuuc cuguauggag gaagaggaag 3900

ugugcgaccc caaauuccau uaugacaaca cagcaggaau cagucauuau cuccagaaca 3960

guaagcgaaa gagccggcca gugaguguaa aaacauuuga agauauccca uuggaggaac 4020

cagaaguaaa agugauccca gaugacagcc agacagacag ugggaugguc cuugcaucag 4080

aagagcugaa aacucuggaa gacaggaaca aauuaucucc aucuuuuggu ggaaugaugc 4140

ccaguaaaag cagggagucu guggccucgg aaggcuccaa ccagaccagu ggcuaccagu 4200

cuggguauca cucagaugac acagacacca ccguguacuc cagcgacgag gcaggacuuu 4260

uaaagauggu ggaugcugca guucacgcug acucagggac cacacugcgc ucaccuccug 4320

uuuaaaugga agugguccug ucccggcucc gcccccaacu ccuggaaauc acgagagagg 4380

ugcugcuuag auuuucaagu guuguucuuu ccaccacccg gaaguagcca cauuugauuu 4440

ucauuuuugg aggagggacc ucagacugca aggagcuugu ccucagggca uuuccagaga 4500

agaugcccau gacccaagaa uguguugacu cuacucucuu uuccauucau uuaaaagucc 4560

uauauaaugu gcccugcugu ggucucacua ccaguuaaag caaaagacuu ucaaacagug 4620

gcucuguccu ccaagaagug gcaacggcac cucugugaaa cuggaucgaa ugggcaaugc 4680

uuuguguguu gaggaugggu gagauguccc agggccgagu cugucuaccu uggaggcuuu 4740

guggaggaug cgggcuauga gccaaguguu aaguguggga uguggacugg gaggaaggaa 4800

ggcgcaagcu cgcucggaga gcgguuggag ccugcagaug cauugugcug gcucuggugg 4860

aggugggcuu guggccuguc aggaaacgca aaggcggccg gcaggguuug guuuuggaag 4920

guuugcgugc ucuucacagu cggguuacag gcgaguuccc uguggcguuu ccuacuccua 4980

augagaguuc cuuccggacu cuuacguguc uccuggccug gccccaggaa ggaaaugaug 5040

cagcuugcuc cuuccucauc ucucaggcug ugccuuaauu cagaacacca aaagagagga 5100

acgucggcag aggcuccuga cggggccgaa gaauugugag aacagaacag aaacucaggg 5160

uuucugcugg guggagaccc acguggcugc ccugguggca gugucugagg guucucuguc 5220

aaguggcggu aaaggcucag gcugguguuc uuccucuauc uccacuccug ucaggccccc 5280

aaguccucag uauuuuagcu uuguggcuuc cugauggcag aaaaaucuua auugguuggu 5340

uugcucucca gauaaucacu agccagauuu cgaaauuacu uuuuagccga gguuaugaua 5400

acaucuacug uauccuuuag aauuuuaacc uauaaaacua ugucuacugg uuucugccug 5460

ugugcuuaug uuaaaaaaaa aaagaaagaa agaaacuguu cuuuucauuu gguaccauag 5520

ugugaagagc ugggagcaau gacuguuaaa caugcuaugg cacaucuauu uauagucugu 5580

uauguagaac aaauguaaua uauuaaaacg uuauauuaua uauaaugaac uuuguacuac 5640

ccaccuuuug uaucaguauu auguaccacu agagagauua caaggcuuuc agcagccgcu 5700

guuguuuugu uaaagacuuu gagaaacucg aaggaauccu uucauggaau augcagcuau 5760

auacccuacc gucucucuca ucucaaacgg aggaggagga ggaggaguca gguauaaugu 5820

gaguguguuc uacguguccu uguucucugu ucuuaggagg aaugauuuca ucaaauguuu 5880

auaugcuuua uaaaccaaua aacguauucu gaguaaagag a 5921

<210> 2

<211> 1345

<212> PRT

<213> mouse (mouse)

<400> 2

Met Glu Ser Lys Ala Leu Leu Ala Val Ala Leu Trp Phe Cys Val Glu

1 5 10 15

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

20 25 30

Lys Leu Ser Thr Gln Lys Asp Ile Leu Thr Ile Leu Ala Asn Thr Thr

35 40 45

Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro

50 55 60

Asn Ala Gln Arg Asp Ser Glu Glu Arg Val Leu Val Thr Glu Cys Gly

65 70 75 80

Gly Gly Asp Ser Ile Phe Cys Lys Thr Leu Thr Ile Pro Arg Val Val

85 90 95

Gly Asn Asp Thr Gly Ala Tyr Lys Cys Ser Tyr Arg Asp Val Asp Ile

100 105 110

Ala Ser Thr Val Tyr Val Tyr Val Arg Asp Tyr Arg Ser Pro Phe Ile

115 120 125

Ala Ser Val Ser Asp Gln His Gly Ile Val Tyr Ile Thr Glu Asn Lys

130 135 140

Asn Lys Thr Val Val Ile Pro Cys Arg Gly Ser Ile Ser Asn Leu Asn

145 150 155 160

Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly

165 170 175

Asn Arg Ile Ser Trp Asp Ser Glu Ile Gly Phe Thr Leu Pro Ser Tyr

180 185 190

Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp

195 200 205

Glu Thr Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg

210 215 220

Ile Tyr Asp Val Ile Leu Ser Pro Pro His Glu Ile Glu Leu Ser Ala

225 230 235 240

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

245 250 255

Gly Leu Asp Phe Thr Trp His Ser Pro Pro Ser Lys Ser His His Lys

260 265 270

Lys Ile Val Asn Arg Asp Val Lys Pro Phe Pro Gly Thr Val Ala Lys

275 280 285

Met Phe Leu Ser Thr Leu Thr Ile Glu Ser Val Thr Lys Ser Asp Gln

290 295 300

Gly Glu Tyr Thr Cys Val Ala Ser Ser Gly Arg Met Ile Lys Arg Asn

305 310 315 320

Arg Thr Phe Val Arg Val His Thr Lys Pro Phe Ile Ala Phe Gly Ser

325 330 335

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

340 345 350

Pro Val Lys Tyr Leu Ser Tyr Pro Ala Pro Asp Ile Lys Trp Tyr Arg

355 360 365

Asn Gly Arg Pro Ile Glu Ser Asn Tyr Thr Met Ile Val Gly Asp Glu

370 375 380

Leu Thr Ile Met Glu Val Thr Glu Arg Asp Ala Gly Asn Tyr Thr Val

385 390 395 400

Ile Leu Thr Asn Pro Ile Ser Met Glu Lys Gln Ser His Met Val Ser

405 410 415

Leu Val Val Asn Val Pro Pro Gln Ile Gly Glu Lys Ala Leu Ile Ser

420 425 430

Pro Met Asp Ser Tyr Gln Tyr Gly Thr Met Gln Thr Leu Thr Cys Thr

435 440 445

Val Tyr Ala Asn Pro Pro Leu His His Ile Gln Trp Tyr Trp Gln Leu

450 455 460

Glu Glu Ala Cys Ser Tyr Arg Pro Gly Gln Thr Ser Pro Tyr Ala Cys

465 470 475 480

Lys Glu Trp Arg His Val Glu Asp Phe Gln Gly Gly Asn Lys Ile Glu

485 490 495

Val Thr Lys Asn Gln Tyr Ala Leu Ile Glu Gly Lys Asn Lys Thr Val

500 505 510

Ser Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr Lys Cys

515 520 525

Glu Ala Ile Asn Lys Ala Gly Arg Gly Glu Arg Val Ile Ser Phe His

530 535 540

Val Ile Arg Gly Pro Glu Ile Thr Val Gln Pro Ala Ala Gln Pro Thr

545 550 555 560

Glu Gln Glu Ser Val Ser Leu Leu Cys Thr Ala Asp Arg Asn Thr Phe

565 570 575

Glu Asn Leu Thr Trp Tyr Lys Leu Gly Ser Gln Ala Thr Ser Val His

580 585 590

Met Gly Glu Ser Leu Thr Pro Val Cys Lys Asn Leu Asp Ala Leu Trp

595 600 605

Lys Leu Asn Gly Thr Met Phe Ser Asn Ser Thr Asn Asp Ile Leu Ile

610 615 620

Val Ala Phe Gln Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr Val Cys

625 630 635 640

Ser Ala Gln Asp Lys Lys Thr Lys Lys Arg His Cys Leu Val Lys Gln

645 650 655

Leu Ile Ile Leu Glu Arg Met Ala Pro Met Ile Thr Gly Asn Leu Glu

660 665 670

Asn Gln Thr Thr Thr Ile Gly Glu Thr Ile Glu Val Thr Cys Pro Ala

675 680 685

Ser Gly Asn Pro Thr Pro His Ile Thr Trp Phe Lys Asp Asn Glu Thr

690 695 700

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

705 710 715 720

Thr Ile Arg Arg Val Arg Lys Glu Asp Gly Gly Leu Tyr Thr Cys Gln

725 730 735

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

740 745 750

Glu Gly Ala Gln Glu Lys Thr Asn Leu Glu Val Ile Ile Leu Val Gly

755 760 765

Thr Ala Val Ile Ala Met Phe Phe Trp Leu Leu Leu Val Ile Val Leu

770 775 780

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

785 790 795 800

Ser Ile Val Met Asp Pro Asp Glu Leu Pro Leu Asp Glu Arg Cys Glu

805 810 815

Arg Leu Pro Tyr Asp Ala Ser Lys Trp Glu Phe Pro Arg Asp Arg Leu

820 825 830

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

835 840 845

Ala Asp Ala Phe Gly Ile Asp Lys Thr Ala Thr Cys Lys Thr Val Ala

850 855 860

Val Lys Met Leu Lys Glu Gly Ala Thr His Ser Glu His Arg Ala Leu

865 870 875 880

Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly His His Leu Asn Val

885 890 895

Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gly Gly Pro Leu Met Val

900 905 910

Ile Val Glu Phe Cys Lys Phe Gly Asn Leu Ser Thr Tyr Leu Arg Gly

915 920 925

Lys Arg Asn Glu Phe Val Pro Tyr Lys Ser Lys Gly Ala Arg Phe Arg

930 935 940

Gln Gly Lys Asp Tyr Val Gly Glu Leu Ser Val Asp Leu Lys Arg Arg

945 950 955 960

Leu Asp Ser Ile Thr Ser Ser Gln Ser Ser Ala Ser Ser Gly Phe Val

965 970 975

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

980 985 990

Leu Tyr Lys Asp Phe Leu Thr Leu Glu His Leu Ile Cys Tyr Ser Phe

995 1000 1005

Gln Val Ala Lys Gly Met Glu Phe Leu Ala Ser Arg Lys Cys Ile His

1010 1015 1020

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

1025 1030 1035 1040

Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asp Pro Asp

1045 1050 1055

Tyr Val Arg Lys Gly Asp Ala Arg Leu Pro Leu Lys Trp Met Ala Pro

1060 1065 1070

Glu Thr Ile Phe Asp Arg Val Tyr Thr Ile Gln Ser Asp Val Trp Ser

1075 1080 1085

Phe Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Ala Ser Pro Tyr

1090 1095 1100

Pro Gly Val Lys Ile Asp Glu Glu Phe Cys Arg Arg Leu Lys Glu Gly

1105 1110 1115 1120

Thr Arg Met Arg Ala Pro Asp Tyr Thr Thr Pro Glu Met Tyr Gln Thr

1125 1130 1135

Met Leu Asp Cys Trp His Glu Asp Pro Asn Gln Arg Pro Ser Phe Ser

1140 1145 1150

Glu Leu Val Glu His Leu Gly Asn Leu Leu Gln Ala Asn Ala Gln Gln

1155 1160 1165

Asp Gly Lys Asp Tyr Ile Val Leu Pro Met Ser Glu Thr Leu Ser Met

1170 1175 1180

Glu Glu Asp Ser Gly Leu Ser Leu Pro Thr Ser Pro Val Ser Cys Met

1185 1190 1195 1200

Glu Glu Glu Glu Val Cys Asp Pro Lys Phe His Tyr Asp Asn Thr Ala

1205 1210 1215

Gly Ile Ser His Tyr Leu Gln Asn Ser Lys Arg Lys Ser Arg Pro Val

1220 1225 1230

Ser Val Lys Thr Phe Glu Asp Ile Pro Leu Glu Glu Pro Glu Val Lys

1235 1240 1245

Val Ile Pro Asp Asp Ser Gln Thr Asp Ser Gly Met Val Leu Ala Ser

1250 1255 1260

Glu Glu Leu Lys Thr Leu Glu Asp Arg Asn Lys Leu Ser Pro Ser Phe

1265 1270 1275 1280

Gly Gly Met Met Pro Ser Lys Ser Arg Glu Ser Val Ala Ser Glu Gly

1285 1290 1295

Ser Asn Gln Thr Ser Gly Tyr Gln Ser Gly Tyr His Ser Asp Asp Thr

1300 1305 1310

Asp Thr Thr Val Tyr Ser Ser Asp Glu Ala Gly Leu Leu Lys Met Val

1315 1320 1325

Asp Ala Ala Val His Ala Asp Ser Gly Thr Thr Leu Arg Ser Pro Pro

1330 1335 1340

Val

1345

<210> 3

<211> 5849

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 3

acugaguccc gggaccccgg gagagcgguc aauguguggu cgcugcguuu ccucugccug 60

cgccgggcau cacuugcgcg ccgcagaaag uccgucuggc agccuggaua uccucuccua 120

ccggcacccg cagacgcccc ugcagccgcg gucggcgccc gggcucccua gcccugugcg 180

cucaacuguc cugcgcugcg gggugccgcg aguuccaccu ccgcgccucc uucucuagac 240

aggcgcuggg agaaagaacc ggcucccgag uucugggcau uucgcccggc ucgaggugca 300

ggaugcagag caaggugcug cuggccgucg cccuguggcu cugcguggag acccgggccg 360

ccucuguggg uuugccuagu guuucucuug aucugcccag gcucagcaua caaaaagaca 420

uacuuacaau uaaggcuaau acaacucuuc aaauuacuug caggggacag agggacuugg 480

acuggcuuug gcccaauaau cagaguggca gugagcaaag gguggaggug acugagugca 540

gcgauggccu cuucuguaag acacucacaa uuccaaaagu gaucggaaau gacacuggag 600

ccuacaagug cuucuaccgg gaaacugacu uggccucggu cauuuauguc uauguucaag 660

auuacagauc uccauuuauu gcuucuguua gugaccaaca uggagucgug uacauuacug 720

agaacaaaaa caaaacugug gugauuccau gucucggguc cauuucaaau cucaacgugu 780

cacuuugugc aagauaccca gaaaagagau uuguuccuga ugguaacaga auuuccuggg 840

acagcaagaa gggcuuuacu auucccagcu acaugaucag cuaugcuggc auggucuucu 900

gugaagcaaa aauuaaugau gaaaguuacc agucuauuau guacauaguu gucguuguag 960

gguauaggau uuaugaugug guucugaguc cgucucaugg aauugaacua ucuguuggag 1020

aaaagcuugu cuuaaauugu acagcaagaa cugaacuaaa uguggggauu gacuucaacu 1080

gggaauaccc uucuucgaag caucagcaua agaaacuugu aaaccgagac cuaaaaaccc 1140

agucugggag ugagaugaag aaauuuuuga gcaccuuaac uauagauggu guaacccgga 1200

gugaccaagg auuguacacc ugugcagcau ccagugggcu gaugaccaag aagaacagca 1260

cauuugucag gguccaugaa aaaccuuuug uugcuuuugg aaguggcaug gaaucucugg 1320

uggaagccac ggugggggag cgugucagaa ucccugcgaa guaccuuggu uacccacccc 1380

cagaaauaaa augguauaaa aauggaauac cccuugaguc caaucacaca auuaaagcgg 1440

ggcauguacu gacgauuaug gaagugagug aaagagacac aggaaauuac acugucaucc 1500

uuaccaaucc cauuucaaag gagaagcaga gccauguggu cucucugguu guguaugucc 1560

caccccagau uggugagaaa ucucuaaucu cuccugugga uuccuaccag uacggcacca 1620

cucaaacgcu gacauguacg gucuaugcca uuccuccccc gcaucacauc cacugguauu 1680

ggcaguugga ggaagagugc gccaacgagc ccagccaagc ugucucagug acaaacccau 1740

acccuuguga agaauggaga aguguggagg acuuccaggg aggaaauaaa auugaaguua 1800

auaaaaauca auuugcucua auugaaggaa aaaacaaaac uguaaguacc cuuguuaucc 1860

aagcggcaaa ugugucagcu uuguacaaau gugaagcggu caacaaaguc gggagaggag 1920

agagggugau cuccuuccac gugaccaggg guccugaaau uacuuugcaa ccugacaugc 1980

agcccacuga gcaggagagc gugucuuugu ggugcacugc agacagaucu acguuugaga 2040

accucacaug guacaagcuu ggcccacagc cucugccaau ccauguggga gaguugccca 2100

caccuguuug caagaacuug gauacucuuu ggaaauugaa ugccaccaug uucucuaaua 2160

gcacaaauga cauuuugauc auggagcuua agaaugcauc cuugcaggac caaggagacu 2220

augucugccu ugcucaagac aggaagacca agaaaagaca uugcgugguc aggcagcuca 2280

caguccuaga gcguguggca cccacgauca caggaaaccu ggagaaucag acgacaagua 2340

uuggggaaag caucgaaguc ucaugcacgg caucugggaa ucccccucca cagaucaugu 2400

gguuuaaaga uaaugagacc cuuguagaag acucaggcau uguauugaag gaugggaacc 2460

ggaaccucac uauccgcaga gugaggaagg aggacgaagg ccucuacacc ugccaggcau 2520

gcaguguucu uggcugugca aaaguggagg cauuuuucau aauagaaggu gcccaggaaa 2580

agacgaacuu ggaaaucauu auucuaguag gcacggcggu gauugccaug uucuucuggc 2640

uacuucuugu caucauccua cggaccguua agcgggccaa uggaggggaa cugaagacag 2700

gcuacuuguc caucgucaug gauccagaug aacucccauu ggaugaacau ugugaacgac 2760

ugccuuauga ugccagcaaa ugggaauucc ccagagaccg gcugaagcua gguaagccuc 2820

uuggccgugg ugccuuuggc caagugauug aagcagaugc cuuuggaauu gacaagacag 2880

caacuugcag gacaguagca gucaaaaugu ugaaagaagg agcaacacac agugagcauc 2940

gagcucucau gucugaacuc aagauccuca uucauauugg ucaccaucuc aaugugguca 3000

accuucuagg ugccuguacc aagccaggag ggccacucau ggugauugug gaauucugca 3060

aauuuggaaa ccuguccacu uaccugagga gcaagagaaa ugaauuuguc cccuacaaga 3120

ccaaaggggc acgauuccgu caagggaaag acuacguugg agcaaucccu guggaucuga 3180

aacggcgcuu ggacagcauc accaguagcc agagcucagc cagcucugga uuuguggagg 3240

agaagucccu cagugaugua gaagaagagg aagcuccuga agaucuguau aaggacuucc 3300

ugaccuugga gcaucucauc uguuacagcu uccaaguggc uaagggcaug gaguucuugg 3360

caucgcgaaa guguauccac agggaccugg cggcacgaaa uauccucuua ucggagaaga 3420

acgugguuaa aaucugugac uuuggcuugg cccgggauau uuauaaagau ccagauuaug 3480

ucagaaaagg agaugcucgc cucccuuuga aauggauggc cccagaaaca auuuuugaca 3540

gaguguacac aauccagagu gacgucuggu cuuuuggugu uuugcugugg gaaauauuuu 3600

ccuuaggugc uucuccauau ccugggguaa agauugauga agaauuuugu aggcgauuga 3660

aagaaggaac uagaaugagg gccccugauu auacuacacc agaaauguac cagaccaugc 3720

uggacugcug gcacggggag cccagucaga gacccacguu uucagaguug guggaacauu 3780

ugggaaaucu cuugcaagcu aaugcucagc aggauggcaa agacuacauu guucuuccga 3840

uaucagagac uuugagcaug gaagaggauu cuggacucuc ucugccuacc ucaccuguuu 3900

ccuguaugga ggaggaggaa guaugugacc ccaaauucca uuaugacaac acagcaggaa 3960

ucagucagua ucugcagaac aguaagcgaa agagccggcc ugugagugua aaaacauuug 4020

aagauauccc guuagaagaa ccagaaguaa aaguaauccc agaugacaac cagacggaca 4080

gugguauggu ucuugccuca gaagagcuga aaacuuugga agacagaacc aaauuaucuc 4140

caucuuuugg uggaauggug cccagcaaaa gcagggaguc uguggcaucu gaaggcucaa 4200

accagacaag cggcuaccag uccggauauc acuccgauga cacagacacc accguguacu 4260

ccagugagga agcagaacuu uuaaagcuga uagagauugg agugcaaacc gguagcacag 4320

cccagauucu ccagccugac ucggggacca cacugagcuc uccuccuguu uaaaaggaag 4380

cauccacacc cccaacuccu ggacaucaca ugagaggugc ugcucagauu uucaaguguu 4440

guucuuucca ccagcaggaa guagccgcau uugauuuuca uuucgacaac agaaaaagga 4500

ccucggacug cagggagcca gucuucuagg cauauccugg aagaggcuug ugacccaaga 4560

augugucugu gucuucuccc aguguugacc ugauccucuu uuucauucau uuaaaaagca 4620

uuuaucaugc ccccugcugc gggucucacc auggguuuag aacaaagacg uucaagaaau 4680

ggccccaucc ucaaagaagu agcaguaccu ggggagcuga cacuucugua aaacuagaag 4740

auaaaccagg caauguaagu guucgaggug uugaagaugg gaaggauuug cagggcugag 4800

ucuauccaag aggcuuuguu uaggacgugg gucccaagcc aagccuuaag uguggaauuc 4860

ggauugauag aaaggaagac uaacguuacc uugcuuugga gaguacugga gccugcaaau 4920

gcauuguguu ugcucuggug gaggugggca uggggucugu ucugaaaugu aaaggguuca 4980

gacgggguuu cugguuuuag aagguugcgu guucuucgag uugggcuaaa guagaguucg 5040

uugugcuguu ucugacuccu aaugagaguu ccuuccagac cguuacgugu cuccuggcca 5100

agccccagga aggaaaugau gcagcucugg cuccuugucu cccaggcuga uccuuuauuc 5160

agaauaccac aaagaaagga cauucagcuc aaggcucccu gccguguuga agaguucuga 5220

cugcacaaac cagcuucugg uuucuucugg aaugaauacc cucauaucug uccugaugug 5280

auaugucuga gacugaaugc gggagguuca augugaagcu guguguggug ucaaaguuuc 5340

aggaaggauu uuacccuuuu guucuucccc cuguccccaa cccacucuca ccccgcaacc 5400

caucaguauu uuaguuauuu ggccucuacu ccaguaaacc ugauuggguu uguucacucu 5460

cugaaugauu auuagccaga cuucaaaauu auuuuauagc ccaaauuaua acaucuauug 5520

uauuauuuag acuuuuaaca uauagagcua uuucuacuga uuuuugcccu uguucugucc 5580

uuuuuuucaa aaaagaaaau guguuuuuug uuugguacca uagugugaaa ugcugggaac 5640

aaugacuaua agacaugcua uggcacauau auuuauaguc uguuuaugua gaaacaaaug 5700

uaauauauua aagccuuaua uauaaugaac uuuguacuau ucacauuuug uaucaguauu 5760

auguagcaua acaaagguca uaaugcuuuc agcaauugau gucauuuuau uaaagaacau 5820

ugaaaaacuu gaaaaaaaaa aaaaaaaaa 5849

<210> 4

<211> 1356

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 4

Met Gln Ser Lys Val Leu Leu Ala Val Ala Leu Trp Leu Cys Val Glu

1 5 10 15

Thr Arg Ala Ala Ser Val Gly Leu Pro Ser Val Ser Leu Asp Leu Pro

20 25 30

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

35 40 45

Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro

50 55 60

Asn Asn Gln Ser Gly Ser Glu Gln Arg Val Glu Val Thr Glu Cys Ser

65 70 75 80

Asp Gly Leu Phe Cys Lys Thr Leu Thr Ile Pro Lys Val Ile Gly Asn

85 90 95

Asp Thr Gly Ala Tyr Lys Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser

100 105 110

Val Ile Tyr Val Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser

115 120 125

Val Ser Asp Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys

130 135 140

Thr Val Val Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser

145 150 155 160

Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg

165 170 175

Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile

180 185 190

Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser

195 200 205

Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr

210 215 220

Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu

225 230 235 240

Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile

245 250 255

Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu

260 265 270

Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe

275 280 285

Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu

290 295 300

Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr

305 310 315 320

Phe Val Arg Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met

325 330 335

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

340 345 350

Lys Tyr Leu Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly

355 360 365

Ile Pro Leu Glu Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr

370 375 380

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

385 390 395 400

Thr Asn Pro Ile Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val

405 410 415

Val Tyr Val Pro Pro Gln Ile Gly Glu Lys Ser Leu Ile Ser Pro Val

420 425 430

Asp Ser Tyr Gln Tyr Gly Thr Thr Gln Thr Leu Thr Cys Thr Val Tyr

435 440 445

Ala Ile Pro Pro Pro His His Ile His Trp Tyr Trp Gln Leu Glu Glu

450 455 460

Glu Cys Ala Asn Glu Pro Ser Gln Ala Val Ser Val Thr Asn Pro Tyr

465 470 475 480

Pro Cys Glu Glu Trp Arg Ser Val Glu Asp Phe Gln Gly Gly Asn Lys

485 490 495

Ile Glu Val Asn Lys Asn Gln Phe Ala Leu Ile Glu Gly Lys Asn Lys

500 505 510

Thr Val Ser Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr

515 520 525

Lys Cys Glu Ala Val Asn Lys Val Gly Arg Gly Glu Arg Val Ile Ser

530 535 540

Phe His Val Thr Arg Gly Pro Glu Ile Thr Leu Gln Pro Asp Met Gln

545 550 555 560

Pro Thr Glu Gln Glu Ser Val Ser Leu Trp Cys Thr Ala Asp Arg Ser

565 570 575

Thr Phe Glu Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gln Pro Leu Pro

580 585 590

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

595 600 605

Leu Trp Lys Leu Asn Ala Thr Met Phe Ser Asn Ser Thr Asn Asp Ile

610 615 620

Leu Ile Met Glu Leu Lys Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr

625 630 635 640

Val Cys Leu Ala Gln Asp Arg Lys Thr Lys Lys Arg His Cys Val Val

645 650 655

Arg Gln Leu Thr Val Leu Glu Arg Val Ala Pro Thr Ile Thr Gly Asn

660 665 670

Leu Glu Asn Gln Thr Thr Ser Ile Gly Glu Ser Ile Glu Val Ser Cys

675 680 685

Thr Ala Ser Gly Asn Pro Pro Pro Gln Ile Met Trp Phe Lys Asp Asn

690 695 700

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

705 710 715 720

Asn Leu Thr Ile Arg Arg Val Arg Lys Glu Asp Glu Gly Leu Tyr Thr

725 730 735

Cys Gln Ala Cys Ser Val Leu Gly Cys Ala Lys Val Glu Ala Phe Phe

740 745 750

Ile Ile Glu Gly Ala Gln Glu Lys Thr Asn Leu Glu Ile Ile Ile Leu

755 760 765

Val Gly Thr Ala Val Ile Ala Met Phe Phe Trp Leu Leu Leu Val Ile

770 775 780

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

785 790 795 800

Tyr Leu Ser Ile Val Met Asp Pro Asp Glu Leu Pro Leu Asp Glu His

805 810 815

Cys Glu Arg Leu Pro Tyr Asp Ala Ser Lys Trp Glu Phe Pro Arg Asp

820 825 830

Arg Leu Lys Leu Gly Lys Pro Leu Gly Arg Gly Ala Phe Gly Gln Val

835 840 845

Ile Glu Ala Asp Ala Phe Gly Ile Asp Lys Thr Ala Thr Cys Arg Thr

850 855 860

Val Ala Val Lys Met Leu Lys Glu Gly Ala Thr His Ser Glu His Arg

865 870 875 880

Ala Leu Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly His His Leu

885 890 895

Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gly Gly Pro Leu

900 905 910

Met Val Ile Val Glu Phe Cys Lys Phe Gly Asn Leu Ser Thr Tyr Leu

915 920 925

Arg Ser Lys Arg Asn Glu Phe Val Pro Tyr Lys Thr Lys Gly Ala Arg

930 935 940

Phe Arg Gln Gly Lys Asp Tyr Val Gly Ala Ile Pro Val Asp Leu Lys

945 950 955 960

Arg Arg Leu Asp Ser Ile Thr Ser Ser Gln Ser Ser Ala Ser Ser Gly

965 970 975

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

980 985 990

Glu Asp Leu Tyr Lys Asp Phe Leu Thr Leu Glu His Leu Ile Cys Tyr

995 1000 1005

Ser Phe Gln Val Ala Lys Gly Met Glu Phe Leu Ala Ser Arg Lys Cys

1010 1015 1020

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

1025 1030 1035 1040

Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asp

1045 1050 1055

Pro Asp Tyr Val Arg Lys Gly Asp Ala Arg Leu Pro Leu Lys Trp Met

1060 1065 1070

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

1075 1080 1085

Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Ala Ser

1090 1095 1100

Pro Tyr Pro Gly Val Lys Ile Asp Glu Glu Phe Cys Arg Arg Leu Lys

1105 1110 1115 1120

Glu Gly Thr Arg Met Arg Ala Pro Asp Tyr Thr Thr Pro Glu Met Tyr

1125 1130 1135

Gln Thr Met Leu Asp Cys Trp His Gly Glu Pro Ser Gln Arg Pro Thr

1140 1145 1150

Phe Ser Glu Leu Val Glu His Leu Gly Asn Leu Leu Gln Ala Asn Ala

1155 1160 1165

Gln Gln Asp Gly Lys Asp Tyr Ile Val Leu Pro Ile Ser Glu Thr Leu

1170 1175 1180

Ser Met Glu Glu Asp Ser Gly Leu Ser Leu Pro Thr Ser Pro Val Ser

1185 1190 1195 1200

Cys Met Glu Glu Glu Glu Val Cys Asp Pro Lys Phe His Tyr Asp Asn

1205 1210 1215

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

1220 1225 1230

Pro Val Ser Val Lys Thr Phe Glu Asp Ile Pro Leu Glu Glu Pro Glu

1235 1240 1245

Val Lys Val Ile Pro Asp Asp Asn Gln Thr Asp Ser Gly Met Val Leu

1250 1255 1260

Ala Ser Glu Glu Leu Lys Thr Leu Glu Asp Arg Thr Lys Leu Ser Pro

1265 1270 1275 1280

Ser Phe Gly Gly Met Val Pro Ser Lys Ser Arg Glu Ser Val Ala Ser

1285 1290 1295

Glu Gly Ser Asn Gln Thr Ser Gly Tyr Gln Ser Gly Tyr His Ser Asp

1300 1305 1310

Asp Thr Asp Thr Thr Val Tyr Ser Ser Glu Glu Ala Glu Leu Leu Lys

1315 1320 1325

Leu Ile Glu Ile Gly Val Gln Thr Gly Ser Thr Ala Gln Ile Leu Gln

1330 1335 1340

Pro Asp Ser Gly Thr Thr Leu Ser Ser Pro Pro Val

1345 1350 1355

<210> 5

<211> 2601

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

agttcatgga cccaaagact acttaacttt cctgctaata attcactagg agaattccct 60

cttcttctag attacagatc accattcatc gcctctgtca gtgaccagca tggcgtcgtg 120

tacattactg agaacaaaaa caaaactgtg gtgattccat gtctcgggtc catttcaaat 180

ctcaacgtgt cactttgtgc aagataccca gaaaagagat ttgttcctga tggtaacaga 240

atttcctggg acagcaagaa gggctttact attcccagct acatgatcag ctatgctggc 300

atggtcttct gtgaagcaaa aattaatgat gaaagttacc agtctattat gtacatagtt 360

gtcgttgtag ggtataggat ttatgatgtg gttctgagtc cgtctcatgg aattgaacta 420

tctgttggag aaaagcttgt cttaaattgt acagcaagaa ctgaactaaa tgtggggatt 480

gacttcaact gggaataccc ttcttcgaag catcagcata agaaacttgt aaaccgagac 540

ctaaaaaccc agtctgggag tgagatgaag aaatttttga gcaccttaac tatagatggt 600

gtaacccgga gtgaccaagg attgtacacc tgtgcagcat ccagtgggct gatgaccaag 660

aagaacagca catttgtcag ggtccatgaa aaaccttttg ttgcttttgg aagtggcatg 720

gaatctctgg tggaagccac ggtgggggag cgtgtcagaa tccctgcgaa gtaccttggt 780

tacccacccc cagaaataaa atggtataaa aatggaatac cccttgagtc caatcacaca 840

attaaagcgg ggcatgtact gacgattatg gaagtgagtg aaagagacac aggaaattac 900

actgtcatcc ttaccaatcc catttcaaag gagaagcaga gccatgtggt ctctctggtt 960

gtgtatgtcc caccccagat tggtgagaaa tctctaatct ctcctgtgga ttcctaccag 1020

tacggcacca ctcaaacgct gacatgtacg gtctatgcca ttcctccccc gcatcacatc 1080

cactggtatt ggcagttgga ggaagagtgc gccaacgagc ccagccaagc tgtctcagtg 1140

acaaacccat acccttgtga agaatggaga agtgtggagg acttccaggg aggaaataaa 1200

attgaagtta ataaaaatca atttgctcta attgaaggaa aaaacaaaac tgtaagtacc 1260

cttgttatcc aagcggcaaa tgtgtcagct ttgtacaaat gtgaagcggt caacaaagtc 1320

gggagaggag agagggtgat ctccttccac gtgaccaggg gtcctgaaat tactttgcaa 1380

cctgacatgc agcccactga gcaggagagc gtgtctttgt ggtgcactgc agacagatct 1440

acgtttgaga acctcacatg gtacaagctt ggcccacagc ctctgccaat ccatgtggga 1500

gagttgccca cacctgtttg caagaacttg gatactcttt ggaaattgaa tgccaccatg 1560

ttctctaata gcacaaatga cattttgatc atggagctta agaatgcatc cttgcaggac 1620

caaggagact atgtctgcct tgctcaagac aggaagacca agaaaagaca ttgcgtggtc 1680

aggcagctca cagtcctaga gcgtgtggca cccacgatca caggaaacct ggagaatcag 1740

acgacaagta ttggggaaag catcgaagtc tcatgcacgg catctgggaa tccccctcca 1800

cagatcatgt ggtttaaaga taatgagacc cttgtagaag actcaggcat tgtattgaag 1860

gatgggaacc ggaacctcac tatccgcaga gtgaggaagg aggacgaagg cctctacacc 1920

tgccaggcct gcaatgtcct tggctgtgca agagcggaga cgctcttcat aatagaaggt 1980

cagtgtgatg tcataggctc atcagatggt ctctggcaat tcgatcgcat tggacatagc 2040

tgcacatact ttgagtagga tgccttgtgg gtgactcatg gtgacactgg tggttgttaa 2100

gcctcgtctc tgagatgttc atttaagtgt cacacaagtg tacatagcct gaaggcaatt 2160

ttacacaata tttttcagtg tacctacctt ttttgactgg gatctgtcac atgaaattag 2220

gggtcggtgc tcagaaagtt ttagatctta ttcgtctgtg aggtctgagt tacggtttgg 2280

cgcggctgtg ggtgctgatg aacggtcatc ctcaccaacc ccatttcata agcttgatat 2340

cgaattccga agttcctatt ctctagaaag tataggaact tcatcagtca ggtacataat 2400

ggctccacgt gttctttgta atctatcttc aatggctctg cgtcctttta accagaaagc 2460

aaaattagca cgcgcttcag tctgtaccat attttctgtc cacgtagtca tcactgtgag 2520

aaaactattt gccatgagta agaaatgcct ttctgttccc gattctcact ttgtttgaat 2580

ggttaactca cagaaatgac a 2601

<210> 6

<211> 4050

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atggagagca aggcgctgct agctgtcgct ctgtggttct gcgtggagac ccgagccgcc 60

tctgtgggtt tgcctggcga ttttctccat ccccccaagc tcagcacaca gaaagacata 120

ctgacaattt tggcaaatac aacccttcag attacttgca ggggacagcg ggacctggac 180

tggctttggc ccaatgctca gcgtgattct gaggaaaggg tattggtgac tgaatgcggc 240

ggtggtgaca gtatcttctg caaaacactc accattccca gggtggttgg aaatgatact 300

ggagcctaca agtgctcgta ccgggacgtc gacatagcct ccactgttta tgtctatgtt 360

cgagattaca gatctccatt tattgcttct gttagtgacc aacatggagt cgtgtacatt 420

actgagaaca aaaacaaaac tgtggtgatt ccatgtctcg ggtccatttc aaatctcaac 480

gtgtcacttt gtgcaagata cccagaaaag agatttgttc ctgatggtaa cagaatttcc 540

tgggacagca agaagggctt tactattccc agctacatga tcagctatgc tggcatggtc 600

ttctgtgaag caaaaattaa tgatgaaagt taccagtcta ttatgtacat agttgtcgtt 660

gtagggtata ggatttatga tgtggttctg agtccgtctc atggaattga actatctgtt 720

ggagaaaagc ttgtcttaaa ttgtacagca agaactgaac taaatgtggg gattgacttc 780

aactgggaat acccttcttc gaagcatcag cataagaaac ttgtaaaccg agacctaaaa 840

acccagtctg ggagtgagat gaagaaattt ttgagcacct taactataga tggtgtaacc 900

cggagtgacc aaggattgta cacctgtgca gcatccagtg ggctgatgac caagaagaac 960

agcacatttg tcagggtcca tgaaaaacct tttgttgctt ttggaagtgg catggaatct 1020

ctggtggaag ccacggtggg ggagcgtgtc agaatccctg cgaagtacct tggttaccca 1080

cccccagaaa taaaatggta taaaaatgga ataccccttg agtccaatca cacaattaaa 1140

gcggggcatg tactgacgat tatggaagtg agtgaaagag acacaggaaa ttacactgtc 1200

atccttacca atcccatttc aaaggagaag cagagccatg tggtctctct ggttgtgtat 1260

gtcccacccc agattggtga gaaatctcta atctctcctg tggattccta ccagtacggc 1320

accactcaaa cgctgacatg tacggtctat gccattcctc ccccgcatca catccactgg 1380

tattggcagt tggaggaaga gtgcgccaac gagcccagcc aagctgtctc agtgacaaac 1440

ccataccctt gtgaagaatg gagaagtgtg gaggacttcc agggaggaaa taaaattgaa 1500

gttaataaaa atcaatttgc tctaattgaa ggaaaaaaca aaactgtaag tacccttgtt 1560

atccaagcgg caaatgtgtc agctttgtac aaatgtgaag cggtcaacaa agtcgggaga 1620

ggagagaggg tgatctcctt ccacgtgacc aggggtcctg aaattacttt gcaacctgac 1680

atgcagccca ctgagcagga gagcgtgtct ttgtggtgca ctgcagacag atctacgttt 1740

gagaacctca catggtacaa gcttggccca cagcctctgc caatccatgt gggagagttg 1800

cccacacctg tttgcaagaa cttggatact ctttggaaat tgaatgccac catgttctct 1860

aatagcacaa atgacatttt gatcatggag cttaagaatg catccttgca ggaccaagga 1920

gactatgtct gccttgctca agacaggaag accaagaaaa gacattgcgt ggtcaggcag 1980

ctcacagtcc tagagcgtgt ggcacccacg atcacaggaa acctggagaa tcagacgaca 2040

agtattgggg aaagcatcga agtctcatgc acggcatctg ggaatccccc tccacagatc 2100

atgtggttta aagataatga gacccttgta gaagactcag gcattgtatt gaaggatggg 2160

aaccggaacc tcactatccg cagagtgagg aaggaggacg aaggcctcta cacctgccag 2220

gcctgcaatg tccttggctg tgcaagagcg gagacgctct tcataataga aggtgcccag 2280

gaaaagacca acttggaagt cattatcctc gtcggcactg cagtgattgc catgttcttc 2340

tggctccttc ttgtcattgt cctacggacc gttaagcggg ccaatgaagg ggaactgaag 2400

acaggctact tgtctattgt catggatcca gatgaattgc ccttggatga gcgctgtgaa 2460

cgcttgcctt atgatgccag caagtgggaa ttccccaggg accggctgaa actaggaaaa 2520

cctcttggcc gcggtgcctt cggccaagtg attgaggcag acgcttttgg aattgacaag 2580

acagcgactt gcaaaacagt agccgtcaag atgttgaaag aaggagcaac acacagcgag 2640

catcgagccc tcatgtctga actcaagatc ctcatccaca ttggtcacca tctcaatgtg 2700

gtgaacctcc taggcgcctg caccaagccg ggagggcctc tcatggtgat tgtggaattc 2760

tgcaagtttg gaaacctatc aacttactta cggggcaaga gaaatgaatt tgttccctat 2820

aagagcaaag gggcacgctt ccgccagggc aaggactacg ttggggagct ctccgtggat 2880

ctgaaaagac gcttggacag catcaccagc agccagagct ctgccagctc aggctttgtt 2940

gaggagaaat cgctcagtga tgtagaggaa gaagaagctt ctgaagaact gtacaaggac 3000

ttcctgacct tggagcatct catctgttac agcttccaag tggctaaggg catggagttc 3060

ttggcatcaa ggaagtgtat ccacagggac ctggcagcac gaaacattct cctatcggag 3120

aagaatgtgg ttaagatctg tgacttcggc ttggcccggg acatttataa agacccggat 3180

tatgtcagaa aaggagatgc ccgactccct ttgaagtgga tggccccgga aaccattttt 3240

gacagagtat acacaattca gagcgatgtg tggtctttcg gtgtgttgct ctgggaaata 3300

ttttccttag gtgcctcccc ataccctggg gtcaagattg atgaagaatt ttgtaggaga 3360

ttgaaagaag gaactagaat gcgggctcct gactacacta ccccagaaat gtaccagacc 3420

atgctggact gctggcatga ggaccccaac cagagaccct cgttttcaga gttggtggag 3480

catttgggaa acctcctgca agcaaatgcg cagcaggatg gcaaagacta tattgttctt 3540

ccaatgtcag agacactgag catggaagag gattctggac tctccctgcc tacctcacct 3600

gtttcctgta tggaggaaga ggaagtgtgc gaccccaaat tccattatga caacacagca 3660

ggaatcagtc attatctcca gaacagtaag cgaaagagcc ggccagtgag tgtaaaaaca 3720

tttgaagata tcccattgga ggaaccagaa gtaaaagtga tcccagatga cagccagaca 3780

gacagtggga tggtccttgc atcagaagag ctgaaaactc tggaagacag gaacaaatta 3840

tctccatctt ttggtggaat gatgcccagt aaaagcaggg agtctgtggc ctcggaaggc 3900

tccaaccaga ccagtggcta ccagtctggg tatcactcag atgacacaga caccaccgtg 3960

tactccagcg acgaggcagg acttttaaag atggtggatg ctgcagttca cgctgactca 4020

gggaccacac tgcgctcacc tcctgtttaa 4050

<210> 7

<211> 5933

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gaguccucag gaccccaaga gaguaagcug uguuuccuua gaucgcgcgg accgcuaccc 60

ggcaggacug aaagcccaga cugugucccg cagccgggau aaccuggcug acccgauucc 120

gcggacaccg cugcagccgc ggcuggagcc agggcgccgg ugccccgcgc ucuccccggu 180

cuugcgcugc gggggcgcau accgccucug ugacuucuuu gcgggccagg gacggagaag 240

gagucugugc cugagaacug ggcucugugc ccagcgcgag gugcaggaug gagagcaagg 300

cgcugcuagc ugucgcucug ugguucugcg uggagacccg agccgccucu guggguuugc 360

cuggcgauuu ucuccauccc cccaagcuca gcacacagaa agacauacug acaauuuugg 420

caaauacaac ccuucagauu acuugcaggg gacagcggga ccuggacugg cuuuggccca 480

augcucagcg ugauucugag gaaaggguau uggugacuga augcggcggu ggugacagua 540

ucuucugcaa aacacucacc auucccaggg ugguuggaaa ugauacugga gccuacaagu 600

gcucguaccg ggacgucgac auagccucca cuguuuaugu cuauguucga gauuacagau 660

caccauucau cgccucuguc agugaccagc auggcgucgu guacauuacu gagaacaaaa 720

acaaaacugu ggugauucca ugucucgggu ccauuucaaa ucucaacgug ucacuuugug 780

caagauaccc agaaaagaga uuuguuccug augguaacag aauuuccugg gacagcaaga 840

agggcuuuac uauucccagc uacaugauca gcuaugcugg cauggucuuc ugugaagcaa 900

aaauuaauga ugaaaguuac cagucuauua uguacauagu ugucguugua ggguauagga 960

uuuaugaugu gguucugagu ccgucucaug gaauugaacu aucuguugga gaaaagcuug 1020

ucuuaaauug uacagcaaga acugaacuaa auguggggau ugacuucaac ugggaauacc 1080

cuucuucgaa gcaucagcau aagaaacuug uaaaccgaga ccuaaaaacc cagucuggga 1140

gugagaugaa gaaauuuuug agcaccuuaa cuauagaugg uguaacccgg agugaccaag 1200

gauuguacac cugugcagca uccagugggc ugaugaccaa gaagaacagc acauuuguca 1260

ggguccauga aaaaccuuuu guugcuuuug gaaguggcau ggaaucucug guggaagcca 1320

cgguggggga gcgugucaga aucccugcga aguaccuugg uuacccaccc ccagaaauaa 1380

aaugguauaa aaauggaaua ccccuugagu ccaaucacac aauuaaagcg gggcauguac 1440

ugacgauuau ggaagugagu gaaagagaca caggaaauua cacugucauc cuuaccaauc 1500

ccauuucaaa ggagaagcag agccaugugg ucucucuggu uguguauguc ccaccccaga 1560

uuggugagaa aucucuaauc ucuccugugg auuccuacca guacggcacc acucaaacgc 1620

ugacauguac ggucuaugcc auuccucccc cgcaucacau ccacugguau uggcaguugg 1680

aggaagagug cgccaacgag cccagccaag cugucucagu gacaaaccca uacccuugug 1740

aagaauggag aaguguggag gacuuccagg gaggaaauaa aauugaaguu aauaaaaauc 1800

aauuugcucu aauugaagga aaaaacaaaa cuguaaguac ccuuguuauc caagcggcaa 1860

augugucagc uuuguacaaa ugugaagcgg ucaacaaagu cgggagagga gagaggguga 1920

ucuccuucca cgugaccagg gguccugaaa uuacuuugca accugacaug cagcccacug 1980

agcaggagag cgugucuuug uggugcacug cagacagauc uacguuugag aaccucacau 2040

gguacaagcu uggcccacag ccucugccaa uccauguggg agaguugccc acaccuguuu 2100

gcaagaacuu ggauacucuu uggaaauuga augccaccau guucucuaau agcacaaaug 2160

acauuuugau cauggagcuu aagaaugcau ccuugcagga ccaaggagac uaugucugcc 2220

uugcucaaga caggaagacc aagaaaagac auugcguggu caggcagcuc acaguccuag 2280

agcguguggc acccacgauc acaggaaacc uggagaauca gacgacaagu auuggggaaa 2340

gcaucgaagu cucaugcacg gcaucuggga aucccccucc acagaucaug ugguuuaaag 2400

auaaugagac ccuuguagaa gacucaggca uuguauugaa ggaugggaac cggaaccuca 2460

cuauccgcag agugaggaag gaggacgaag gccucuacac cugccaggcc ugcaaugucc 2520

uuggcugugc aagagcggag acgcucuuca uaauagaagg ugcccaggaa aagaccaacu 2580

uggaagucau uauccucguc ggcacugcag ugauugccau guucuucugg cuccuucuug 2640

ucauuguccu acggaccguu aagcgggcca augaagggga acugaagaca ggcuacuugu 2700

cuauugucau ggauccagau gaauugcccu uggaugagcg cugugaacgc uugccuuaug 2760

augccagcaa gugggaauuc cccagggacc ggcugaaacu aggaaaaccu cuuggccgcg 2820

gugccuucgg ccaagugauu gaggcagacg cuuuuggaau ugacaagaca gcgacuugca 2880

aaacaguagc cgucaagaug uugaaagaag gagcaacaca cagcgagcau cgagcccuca 2940

ugucugaacu caagauccuc auccacauug gucaccaucu caauguggug aaccuccuag 3000

gcgccugcac caagccggga gggccucuca uggugauugu ggaauucugc aaguuuggaa 3060

accuaucaac uuacuuacgg ggcaagagaa augaauuugu ucccuauaag agcaaagggg 3120

cacgcuuccg ccagggcaag gacuacguug gggagcucuc cguggaucug aaaagacgcu 3180

uggacagcau caccagcagc cagagcucug ccagcucagg cuuuguugag gagaaaucgc 3240

ucagugaugu agaggaagaa gaagcuucug aagaacugua caaggacuuc cugaccuugg 3300

agcaucucau cuguuacagc uuccaagugg cuaagggcau ggaguucuug gcaucaagga 3360

aguguaucca cagggaccug gcagcacgaa acauucuccu aucggagaag aaugugguua 3420

agaucuguga cuucggcuug gcccgggaca uuuauaaaga cccggauuau gucagaaaag 3480

gagaugcccg acucccuuug aaguggaugg ccccggaaac cauuuuugac agaguauaca 3540

caauucagag cgaugugugg ucuuucggug uguugcucug ggaaauauuu uccuuaggug 3600

ccuccccaua cccugggguc aagauugaug aagaauuuug uaggagauug aaagaaggaa 3660

cuagaaugcg ggcuccugac uacacuaccc cagaaaugua ccagaccaug cuggacugcu 3720

ggcaugagga ccccaaccag agacccucgu uuucagaguu gguggagcau uugggaaacc 3780

uccugcaagc aaaugcgcag caggauggca aagacuauau uguucuucca augucagaga 3840

cacugagcau ggaagaggau ucuggacucu cccugccuac cucaccuguu uccuguaugg 3900

aggaagagga agugugcgac cccaaauucc auuaugacaa cacagcagga aucagucauu 3960

aucuccagaa caguaagcga aagagccggc cagugagugu aaaaacauuu gaagauaucc 4020

cauuggagga accagaagua aaagugaucc cagaugacag ccagacagac agugggaugg 4080

uccuugcauc agaagagcug aaaacucugg aagacaggaa caaauuaucu ccaucuuuug 4140

guggaaugau gcccaguaaa agcagggagu cuguggccuc ggaaggcucc aaccagacca 4200

guggcuacca gucuggguau cacucagaug acacagacac caccguguac uccagcgacg 4260

aggcaggacu uuuaaagaug guggaugcug caguucacgc ugacucaggg accacacugc 4320

gcucaccucc uguuuaaaug gaaguggucc ugucccggcu ccgcccccaa cuccuggaaa 4380

ucacgagaga ggugcugcuu agauuuucaa guguuguucu uuccaccacc cggaaguagc 4440

cacauuugau uuucauuuuu ggaggaggga ccucagacug caaggagcuu guccucaggg 4500

cauuuccaga gaagaugccc augacccaag aauguguuga cucuacucuc uuuuccauuc 4560

auuuaaaagu ccuauauaau gugcccugcu guggucucac uaccaguuaa agcaaaagac 4620

uuucaaacag uggcucuguc cuccaagaag uggcaacggc accucuguga aacuggaucg 4680

aaugggcaau gcuuugugug uugaggaugg gugagauguc ccagggccga gucugucuac 4740

cuuggaggcu uuguggagga ugcgggcuau gagccaagug uuaagugugg gauguggacu 4800

gggaggaagg aaggcgcaag cucgcucgga gagcgguugg agccugcaga ugcauugugc 4860

uggcucuggu ggaggugggc uuguggccug ucaggaaacg caaaggcggc cggcaggguu 4920

ugguuuugga agguuugcgu gcucuucaca gucggguuac aggcgaguuc ccuguggcgu 4980

uuccuacucc uaaugagagu uccuuccgga cucuuacgug ucuccuggcc uggccccagg 5040

aaggaaauga ugcagcuugc uccuuccuca ucucucaggc ugugccuuaa uucagaacac 5100

caaaagagag gaacgucggc agaggcuccu gacggggccg aagaauugug agaacagaac 5160

agaaacucag gguuucugcu ggguggagac ccacguggcu gcccuggugg cagugucuga 5220

ggguucucug ucaaguggcg guaaaggcuc aggcuggugu ucuuccucua ucuccacucc 5280

ugucaggccc ccaaguccuc aguauuuuag cuuuguggcu uccugauggc agaaaaaucu 5340

uaauugguug guuugcucuc cagauaauca cuagccagau uucgaaauua cuuuuuagcc 5400

gagguuauga uaacaucuac uguauccuuu agaauuuuaa ccuauaaaac uaugucuacu 5460

gguuucugcc ugugugcuua uguuaaaaaa aaaaagaaag aaagaaacug uucuuuucau 5520

uugguaccau agugugaaga gcugggagca augacuguua aacaugcuau ggcacaucua 5580

uuuauagucu guuauguaga acaaauguaa uauauuaaaa cguuauauua uauauaauga 5640

acuuuguacu acccaccuuu uguaucagua uuauguacca cuagagagau uacaaggcuu 5700

ucagcagccg cuguuguuuu guuaaagacu uugagaaacu cgaaggaauc cuuucaugga 5760

auaugcagcu auauacccua ccgucucucu caucucaaac ggaggaggag gaggaggagu 5820

cagguauaau gugagugugu ucuacguguc cuuguucucu guucuuagga ggaaugauuu 5880

caucaaaugu uuauaugcuu uauaaaccaa uaaacguauu cugaguaaag aga 5933

<210> 8

<211> 1349

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Glu Ser Lys Ala Leu Leu Ala Val Ala Leu Trp Phe Cys Val Glu

1 5 10 15

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

20 25 30

Lys Leu Ser Thr Gln Lys Asp Ile Leu Thr Ile Leu Ala Asn Thr Thr

35 40 45

Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro

50 55 60

Asn Ala Gln Arg Asp Ser Glu Glu Arg Val Leu Val Thr Glu Cys Gly

65 70 75 80

Gly Gly Asp Ser Ile Phe Cys Lys Thr Leu Thr Ile Pro Arg Val Val

85 90 95

Gly Asn Asp Thr Gly Ala Tyr Lys Cys Ser Tyr Arg Asp Val Asp Ile

100 105 110

Ala Ser Thr Val Tyr Val Tyr Val Arg Asp Tyr Arg Ser Pro Phe Ile

115 120 125

Ala Ser Val Ser Asp Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys

130 135 140

Asn Lys Thr Val Val Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn

145 150 155 160

Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly

165 170 175

Asn Arg Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr

180 185 190

Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp

195 200 205

Glu Ser Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg

210 215 220

Ile Tyr Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val

225 230 235 240

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

245 250 255

Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys

260 265 270

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

275 280 285

Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln

290 295 300

Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn

305 310 315 320

Ser Thr Phe Val Arg Val His Glu Lys Pro Phe Val Ala Phe Gly Ser

325 330 335

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

340 345 350

Pro Ala Lys Tyr Leu Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys

355 360 365

Asn Gly Ile Pro Leu Glu Ser Asn His Thr Ile Lys Ala Gly His Val

370 375 380

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

385 390 395 400

Ile Leu Thr Asn Pro Ile Ser Lys Glu Lys Gln Ser His Val Val Ser

405 410 415

Leu Val Val Tyr Val Pro Pro Gln Ile Gly Glu Lys Ser Leu Ile Ser

420 425 430

Pro Val Asp Ser Tyr Gln Tyr Gly Thr Thr Gln Thr Leu Thr Cys Thr

435 440 445

Val Tyr Ala Ile Pro Pro Pro His His Ile His Trp Tyr Trp Gln Leu

450 455 460

Glu Glu Glu Cys Ala Asn Glu Pro Ser Gln Ala Val Ser Val Thr Asn

465 470 475 480

Pro Tyr Pro Cys Glu Glu Trp Arg Ser Val Glu Asp Phe Gln Gly Gly

485 490 495

Asn Lys Ile Glu Val Asn Lys Asn Gln Phe Ala Leu Ile Glu Gly Lys

500 505 510

Asn Lys Thr Val Ser Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala

515 520 525

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

530 535 540

Ile Ser Phe His Val Thr Arg Gly Pro Glu Ile Thr Leu Gln Pro Asp

545 550 555 560

Met Gln Pro Thr Glu Gln Glu Ser Val Ser Leu Trp Cys Thr Ala Asp

565 570 575

Arg Ser Thr Phe Glu Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gln Pro

580 585 590

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

595 600 605

Asp Thr Leu Trp Lys Leu Asn Ala Thr Met Phe Ser Asn Ser Thr Asn

610 615 620

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

625 630 635 640

Asp Tyr Val Cys Leu Ala Gln Asp Arg Lys Thr Lys Lys Arg His Cys

645 650 655

Val Val Arg Gln Leu Thr Val Leu Glu Arg Val Ala Pro Thr Ile Thr

660 665 670

Gly Asn Leu Glu Asn Gln Thr Thr Ser Ile Gly Glu Ser Ile Glu Val

675 680 685

Ser Cys Thr Ala Ser Gly Asn Pro Pro Pro Gln Ile Met Trp Phe Lys

690 695 700

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

705 710 715 720

Asn Arg Asn Leu Thr Ile Arg Arg Val Arg Lys Glu Asp Glu Gly Leu

725 730 735

Tyr Thr Cys Gln Ala Cys Asn Val Leu Gly Cys Ala Arg Ala Glu Thr

740 745 750

Leu Phe Ile Ile Glu Gly Ala Gln Glu Lys Thr Asn Leu Glu Val Ile

755 760 765

Ile Leu Val Gly Thr Ala Val Ile Ala Met Phe Phe Trp Leu Leu Leu

770 775 780

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

785 790 795 800

Thr Gly Tyr Leu Ser Ile Val Met Asp Pro Asp Glu Leu Pro Leu Asp

805 810 815

Glu Arg Cys Glu Arg Leu Pro Tyr Asp Ala Ser Lys Trp Glu Phe Pro

820 825 830

Arg Asp Arg Leu Lys Leu Gly Lys Pro Leu Gly Arg Gly Ala Phe Gly

835 840 845

Gln Val Ile Glu Ala Asp Ala Phe Gly Ile Asp Lys Thr Ala Thr Cys

850 855 860

Lys Thr Val Ala Val Lys Met Leu Lys Glu Gly Ala Thr His Ser Glu

865 870 875 880

His Arg Ala Leu Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly His

885 890 895

His Leu Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gly Gly

900 905 910

Pro Leu Met Val Ile Val Glu Phe Cys Lys Phe Gly Asn Leu Ser Thr

915 920 925

Tyr Leu Arg Gly Lys Arg Asn Glu Phe Val Pro Tyr Lys Ser Lys Gly

930 935 940

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

945 950 955 960

Leu Lys Arg Arg Leu Asp Ser Ile Thr Ser Ser Gln Ser Ser Ala Ser

965 970 975

Ser Gly Phe Val Glu Glu Lys Ser Leu Ser Asp Val Glu Glu Glu Glu

980 985 990

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

995 1000 1005

Cys Tyr Ser Phe Gln Val Ala Lys Gly Met Glu Phe Leu Ala Ser Arg

1010 1015 1020

Lys Cys Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu

1025 1030 1035 1040

Lys Asn Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr

1045 1050 1055

Lys Asp Pro Asp Tyr Val Arg Lys Gly Asp Ala Arg Leu Pro Leu Lys

1060 1065 1070

Trp Met Ala Pro Glu Thr Ile Phe Asp Arg Val Tyr Thr Ile Gln Ser

1075 1080 1085

Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly

1090 1095 1100

Ala Ser Pro Tyr Pro Gly Val Lys Ile Asp Glu Glu Phe Cys Arg Arg

1105 1110 1115 1120

Leu Lys Glu Gly Thr Arg Met Arg Ala Pro Asp Tyr Thr Thr Pro Glu

1125 1130 1135

Met Tyr Gln Thr Met Leu Asp Cys Trp His Glu Asp Pro Asn Gln Arg

1140 1145 1150

Pro Ser Phe Ser Glu Leu Val Glu His Leu Gly Asn Leu Leu Gln Ala

1155 1160 1165

Asn Ala Gln Gln Asp Gly Lys Asp Tyr Ile Val Leu Pro Met Ser Glu

1170 1175 1180

Thr Leu Ser Met Glu Glu Asp Ser Gly Leu Ser Leu Pro Thr Ser Pro

1185 1190 1195 1200

Val Ser Cys Met Glu Glu Glu Glu Val Cys Asp Pro Lys Phe His Tyr

1205 1210 1215

Asp Asn Thr Ala Gly Ile Ser His Tyr Leu Gln Asn Ser Lys Arg Lys

1220 1225 1230

Ser Arg Pro Val Ser Val Lys Thr Phe Glu Asp Ile Pro Leu Glu Glu

1235 1240 1245

Pro Glu Val Lys Val Ile Pro Asp Asp Ser Gln Thr Asp Ser Gly Met

1250 1255 1260

Val Leu Ala Ser Glu Glu Leu Lys Thr Leu Glu Asp Arg Asn Lys Leu

1265 1270 1275 1280

Ser Pro Ser Phe Gly Gly Met Met Pro Ser Lys Ser Arg Glu Ser Val

1285 1290 1295

Ala Ser Glu Gly Ser Asn Gln Thr Ser Gly Tyr Gln Ser Gly Tyr His

1300 1305 1310

Ser Asp Asp Thr Asp Thr Thr Val Tyr Ser Ser Asp Glu Ala Gly Leu

1315 1320 1325

Leu Lys Met Val Asp Ala Ala Val His Ala Asp Ser Gly Thr Thr Leu

1330 1335 1340

Arg Ser Pro Pro Val

1345

<210> 9

<211> 4000

<212> DNA

<213> mouse (mouse)

<400> 9

caactactct accactgagc gtgatatcct tggtccctta aaagttatcc tctgtcctta 60

ataatgctta gcaatcatat ttgcttaaaa tatttattga atgactgcat gaatgaatga 120

atgaatgagc taacagaaaa ctcatgacca tgtgggtgat ttccgaaaca gagtgtgaga 180

tctttggtgg catgtccttg tagactgtct gccaccagta tctatcatct tgaaggtgac 240

tattgagtag tttatatgca tgtgaaaaac caaaccttct attctcttac tcatagcctc 300

tcttaatcat agccctgtgg catggagtgt accattgata tcttcctgga atactttttc 360

aggggacagc gggacctgga ctggctttgg cccaatgctc agcgtgattc tgaggaaagg 420

gtattggtga ctgaatgcgg cggtggtgac agtatcttct gcaaaacact caccattccc 480

agggtggttg gaaatgatac tggagcctac aagtgctcgt accgggacgt cgacatagcc 540

tccactgttt atgtctatgt tcgaggtaag tggtgggcca gaatgtactt ccccgtctgc 600

ttccaatgtc gtaaatgcaa tagagtcaag aagagttgag tctagtaaga ggcttgttgg 660

aaaagatgtt tgcccctata aatctcggag cttctctgcc aggctccctt agggaaggac 720

tcaccagaag tctgggtttt ggttggagct gcacgtctct tctctggcta ggtcagccct 780

gcttcctttt ctaccttgct ctgtgattcc ttgggacccg acaggagcta tggagacgcc 840

taagatgtct tctgctcttt gagactctgg gatctttgca aatgaggagg cttggaggtg 900

aataatagag aggtttctgt ggtgagatag gttatctgga gcagggagga ggtttttaca 960

ggaatgcata gggaaatagc caaggactct agaggctgtt tttttttttt tcttttttct 1020

ttctccctcc cccataagat ctttcctttt cacattaccc ctttattaaa ataaattcac 1080

atacccatgt gcaaagcaca acactgtctt gtctttgctt gtgtgaactg agagggcttg 1140

ggaccaaccc cagggagata tgaacagtct tcatggacat agcaagtatc ttattttgag 1200

gtgctggcta gctgcttggg cttccttcat agactttccc tttcagggct aaagatctag 1260

gactgattca caggacacct ttagtttcat gtcaaaggaa cttagctttt gtcttacata 1320

aaaaagttag ttttgtgatg taatcaaggt ttgtaacttt acatccatat aataaaaact 1380

acagtgtgac atgtactcca tatgtagggg atgaatagga tctcttttac attctaaaaa 1440

ttgcacacca ccttttcttg tgatcactgc ctgtaacaac atctgcttca caggaatcta 1500

aagatttctt tgtcactctt ttttttcttt ctttctttct ttttgtgaaa gagagttttg 1560

catgtatgta tgtgtagttt gtgcgtgcat gctatctgtg aaggtcagaa gtgggcattg 1620

gatcccctgg acctgcagtt acagagccac cacgtgggtg ctgggcactg aacccaggtc 1680

ctctgcagga gcagccagtg cttccatctg ctaggccatc tctccagccc tgagttgcat 1740

tatatttgac acaggcatac aatgttgtat ttctttcaga gaaggatgga tctatacaag 1800

tattttattt attcatctgt atgtacccct acgtctccgg gaatttgaga cgggtgaaaa 1860

tgactttaag gttttttatt tcctgaaagc cactcaccag gcaggaaggt tctttaccgg 1920

ggcaccaacc acccttgcat atctacactt ccattcagta gggtcagtgt atggaagacc 1980

agacgccata tccaaatatc ccatgatgct cttgagcttc tcagtggttg tctattcttt 2040

ttgctccggg taagccaagt ataccacaga gttctgtgcc tcccctcctc accctgcaga 2100

gctgagggag gcaaggctca gtcagacctt ggagaatgcc tggctccttt tgatctcgca 2160

gtcttgggag acgtggctct ctcagaagat tgcaaggatt tcctgctctc agcctgtctg 2220

gaaatgcttc aagatgagct tctcctccag tatcttgata cttccttctc cccgagtcag 2280

gaaacctgga gacatgagaa aagtcatttc gtggcttaca ataaatgttt tattttggga 2340

ggatgcgtgg ttgtttggta ttcttttgtt tacttcccct ttagggttac tgaaatagct 2400

tacggcatag ctttagtctt aagtcaagag gtgaatttag cttctcaagg cttgctcatg 2460

aactattcca ttaccatgtc catggaggag aggcaggagc aacaagcact ggtttccagc 2520

actgtctgct tgtatccttg gtgcctctgg atataccata aaattctgct cctgcctcag 2580

cttaggtttc agagttgagc tattcaagag tttgctcatg aaatttaaca acaggaaaaa 2640

aagcattgaa tgtagtattc ccagtggata ggtctttagc gtcccctctt acttcctcag 2700

tcctctgacc catctagcgg tggatggagc caggaagctg gccttcgctg tgcaggataa 2760

tggtgatggc atgtggggag tgaagtttgc tgcattgctc tgctttccag ctgggaagga 2820

aatcaggttt ctgctgttga aaggaattag ttactcttct tccctttggt ttcattaact 2880

ctttcttcac ctctgaaaag gcttgatcag atgtgtgggg gttaggaggt gaggtgttat 2940

ctctgggatt tcaagtagtg gctccagacc atgtgttctg tctctagggg atgaagttac 3000

tcccagacat gtattaaaac tgtatgaatg atgtctgaaa gatatcaaaa cttctctggc 3060

tttcatctca atctttggaa ttgtgttttt cctaatactt attgtcatag atatgaataa 3120

gtgctaaatc ttgaactaaa tgtagtggct atgaaacatt actttataaa gactcttatc 3180

tacagtcctc acagacttaa gattattata aaaaccattc ttctgaatta ctgaatcatc 3240

tgaaataaaa aacaatcaaa tcattgttat tttctcttag tttaaagatc ctattttttt 3300

ttttttagtc atttacatta ctttcctaat ggaaaaccat gaacttttat catcataaaa 3360

ctcagagttt aagtgtatgg tcttaaatga aatcttttat tttatgtaaa aataatctat 3420

taggccgggc ggtggtggca caagccttta atcccagcac ttgggagaca gaggcagacg 3480

aatctctgag ttcgaggaca gcctggtcta cagagtgagt tccaggacag ccagggctat 3540

acagagaaag cctgtctcaa aaaacaaaca aacaaacaaa caaacaaaca aaaaaacaaa 3600

aaaatctatt aaagcatgga ctattaaaat agattctgcc tggtaatatt ccatgtagaa 3660

gaagctaagt tattttgttg gaatctttct tcactttctc tctgtctgtc tgtctgtgtc 3720

tctctgtctc tctgtttctc tgtctgtctg tttctgtctc tgtgtgtatg tgtttctctg 3780

ccctcctcga tcacatcttt agagccagat agctatgtgc agactagcct gtggatgtcc 3840

gcattcatgc aagttgtcaa tgatagccat tctgtcattc aattagagtt catggaccca 3900

aagactactt aactttcctg ctaataattc actaggagaa ttccctcttc ttctagatta 3960

cagatcacca ttcatcgcct ctgtcagtga ccagcatggc 4000

<210> 10

<211> 3933

<212> DNA

<213> mouse (mouse)

<400> 10

ctccacgtgt tctttgtaat ctatcttcaa tggctctgcg tccttttaac cagaaagcaa 60

aattagcacg cgcttcagtc tgtaccatat tttctgtcca cgtagtcatc actgtgagaa 120

aactatttgc catgagtaag aaatgccttt ctgttcccga ttctcacttt gtttgaatgg 180

ttaactcaca gaaatgacac ttcaatatat acctggggaa aaatagattt ggtgttgggc 240

attgctattc aactagtgac aaggatcaaa ggttcaggat tttccccaga gcagagacaa 300

gattcactca taaacaatag atgattaata ggtgattgct tttgtgatat ggacaatccg 360

gggttataat tgaaatatga aaggaaactt cgtttcccct ggctgtgcgt gtgcgagaga 420

gtgtgtgtgt gtatgagtgt gttttgtgtg tgtgtgtgtg tatgggtgtg ttatgtgtgt 480

gtgtgtgtat gatcgatatg aaccctaacc tttgtttttc agtcagtatt ctgtgtcaga 540

acatttcatc ttcagaagag cgttcctgga tcttgttttt gtaatacact aagggaacaa 600

tgtgactcta ttgtgtgagt gaaggagtgg tgtgcaaggg ggtgactcag ggtggttaga 660

gaggtaggtt tggcagaatt ctaacatctc caagtctaaa tgttggcccc tttcatctgc 720

ccaactcacc atgtccagtg ctgcatctag agccactagc atgctctcaa atctctaaga 780

tggttaggat tctataaact gcacctgcaa ctgaagatgc caaagtgcat ggtagttctg 840

tcactaccaa agtggttcat ggcttggtgc caaatccagg gaggatgcac ccgccagttc 900

tctctggtac tctgcctcca cccactagcc atctgcatta ggttatcatg cctatgtggg 960

cacagtggca tagatactat ggagacagga aaggggtggg ggggagcgag agagagagag 1020

agagagagag agagagagag agagagagag agagagagag agagaggaag agaggaagag 1080

aggaagagag gaagagagga agagagagga gagggtagac attgaatgct tctacagaag 1140

ttcagctgtc agatgttaat atgagactat tcacacataa actgattgag accattgaat 1200

ctttgacctt tcttctgtct cttacatgag taggctttta agcctttaca tacaggtaga 1260

accattactt ctctggaaac atcagagaaa ccaaatccac ttacagatgc actcagagct 1320

cactcccaac tggtaactct tattttgcca actaattaat tgcttgtctt ctggcttctg 1380

gcttcaagcc tcccagcttg aggctcattt caactgacac tgcacagtgt gagtggagag 1440

actgagaccc taagtgataa ttgttttctc ttaatcacct ggtgctaagc atttggtgct 1500

gcgagcctcc tgcccgtatc tctccacttg gaatgtgttc tggagagcaa cctacagtct 1560

tacttgctag cctttccctt gctccctcca cacataaggc ctttatcttg gcgctctctg 1620

tctctgcctg tctctctgtg tgagtctctg tctgtctgtc tatctgtcta tctctgtgtt 1680

cgtctttctg tctgcctgcc tgcctgcctg cctgtctgtc tgtctgtctg tctgtctgtc 1740

tctccctctc tttatttctg tgtctctccc tcacctatcc ttcccttctc tgtcttcctc 1800

tcttttctcc tctttcaggc ttctgccaca tagggacaag gtggtcagct caatttggac 1860

aaatccagta taacccttga acagaccacc aagttcctgt atctcacaga caagtagctt 1920

aaccttttaa gccttatttc ttcatctgaa aactgaaggt tactgaaaaa ataaagaagg 1980

gcgctatatt taaagtgaag ggcgctatat ttaaagtgac cagtgcatag taggtgtgtg 2040

gtcacaagta gctgtctgcg caaagagctt tacttatctc cttgtgtaga ttcctaaaca 2100

atcaattttc ccatgccaca taagcaaaga tgttgactat catcacaaaa tcagactcaa 2160

gtacccatga atagaagtat attttttcct tagagttctc tcaccagaca catccctata 2220

atgtatctct tttttaaaat atttttatta catattttcc tcaattacat ttccaatgct 2280

atctccaaag tcccccatac cctccccccc ttccctaccc agccattccc atttttttgg 2340

ccctggcatt cccctgtact ggggcatata cagtttgcgt gtccaatggg cctctctttc 2400

cagtgatggc cgactaggcc atcttttgat acatatgcag ctagagtcaa gagctccggg 2460

gtactggtta gttcataatg ttgttccacc tatagggttg cagatccctt tagctccttg 2520

ggaactttct ctagctcctc ctttgggagc cctgtgatcc atccaatagc tgactgagca 2580

tccacttctg tgtttgctag gccctggcat agtctcacaa gagacagcta catctgggtc 2640

ctttcgataa aatcttgcta tataatgtat ctcttaagac acgtgtaagc acctgtggtt 2700

cattgctgga tgtcttcatg agtggtgtgt ttctgttgtt ctttaaaagt atgaactcta 2760

tccagcttaa ggagctattg gtccctagga atcttaaaag tgggtgacat gaggtcctgc 2820

cttttctccc ttcatgtcac ccactttgtc accttctacc ataggctatg aatttccagc 2880

tgttgatcac tcaaacatgt cttccaggtg cccaggaaaa gaccaacttg gaagtcatta 2940

tcctcgtcgg cactgcagtg attgccatgt tcttctggct ccttcttgtc attgtcctac 3000

ggaccgttaa gcgggtaaca acataatttc cctcctgtcc cttgtgtctt ggtttttgtg 3060

attaatggaa gctgactggg tttctttcag cggcttcttc ccattgttat tggctcaatg 3120

ggcacatttt tacctcaata caataacatt cttgtccatt ttctttgggt ggactgtggg 3180

cattaattga tgggagccac cagaggggtg aaaggtttgg actgtccact gtaattaaga 3240

tttagaaacc ttattctgaa ctcttttttg gaaactgaag tggcactaaa ggcaggatta 3300

taatagcagc gctctcaaac accatgagtt ttattggaaa atgagattat catgaatgag 3360

gcccttatta aacaacaata tgtatacaaa acaacaacca aacaagaggc cgtgtgtgta 3420

tgacggggag gatttatgct ttccaggcca atgaagggga actgaagaca ggctacttgt 3480

ctattgtcat ggatccagat gaattgccct tggatgagcg ctgtgaacgc ttgccttatg 3540

atgccagcaa gtgggaattc cccagggacc ggctgaaact aggtgagttg tcaactgcta 3600

ttaacttgat ataagttttt acccgctcat ctggctctct gttaagacaa tgacagatct 3660

ggtctattta gatgatgtat tctgatttat aaatattaat tttatctctt gactttgggt 3720

aatcatccat ttagctttct agtagtaagg agcctgtgca accatccaaa gcagggcatt 3780

ttgaaaagca aatggaaaaa ccagaacaaa ggaggaaagt gctctgtggg tctaactggg 3840

atgcaccatc ctcttgacca attggaatct tcatatgcct tttgacagtg tgacaatcaa 3900

agtgtatttt gtaataccac tgtcattggt tct 3933

<210> 11

<211> 1797

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 11

gtcgtgtaca ttactgagaa caaaaacaaa actgtggtga ttccatgtct cgggtccatt 60

tcaaatctca acgtgtcact ttgtgcaaga tacccagaaa agagatttgt tcctgatggt 120

aacagaattt cctgggacag caagaagggc tttactattc ccagctacat gatcagctat 180

gctggcatgg tcttctgtga agcaaaaatt aatgatgaaa gttaccagtc tattatgtac 240

atagttgtcg ttgtagggta taggatttat gatgtggttc tgagtccgtc tcatggaatt 300

gaactatctg ttggagaaaa gcttgtctta aattgtacag caagaactga actaaatgtg 360

gggattgact tcaactggga atacccttct tcgaagcatc agcataagaa acttgtaaac 420

cgagacctaa aaacccagtc tgggagtgag atgaagaaat ttttgagcac cttaactata 480

gatggtgtaa cccggagtga ccaaggattg tacacctgtg cagcatccag tgggctgatg 540

accaagaaga acagcacatt tgtcagggtc catgaaaaac cttttgttgc ttttggaagt 600

ggcatggaat ctctggtgga agccacggtg ggggagcgtg tcagaatccc tgcgaagtac 660

cttggttacc cacccccaga aataaaatgg tataaaaatg gaatacccct tgagtccaat 720

cacacaatta aagcggggca tgtactgacg attatggaag tgagtgaaag agacacagga 780

aattacactg tcatccttac caatcccatt tcaaaggaga agcagagcca tgtggtctct 840

ctggttgtgt atgtcccacc ccagattggt gagaaatctc taatctctcc tgtggattcc 900

taccagtacg gcaccactca aacgctgaca tgtacggtct atgccattcc tcccccgcat 960

cacatccact ggtattggca gttggaggaa gagtgcgcca acgagcccag ccaagctgtc 1020

tcagtgacaa acccataccc ttgtgaagaa tggagaagtg tggaggactt ccagggagga 1080

aataaaattg aagttaataa aaatcaattt gctctaattg aaggaaaaaa caaaactgta 1140

agtacccttg ttatccaagc ggcaaatgtg tcagctttgt acaaatgtga agcggtcaac 1200

aaagtcggga gaggagagag ggtgatctcc ttccacgtga ccaggggtcc tgaaattact 1260

ttgcaacctg acatgcagcc cactgagcag gagagcgtgt ctttgtggtg cactgcagac 1320

agatctacgt ttgagaacct cacatggtac aagcttggcc cacagcctct gccaatccat 1380

gtgggagagt tgcccacacc tgtttgcaag aacttggata ctctttggaa attgaatgcc 1440

accatgttct ctaatagcac aaatgacatt ttgatcatgg agcttaagaa tgcatccttg 1500

caggaccaag gagactatgt ctgccttgct caagacagga agaccaagaa aagacattgc 1560

gtggtcaggc agctcacagt cctagagcgt gtggcaccca cgatcacagg aaacctggag 1620

aatcagacga caagtattgg ggaaagcatc gaagtctcat gcacggcatc tgggaatccc 1680

cctccacaga tcatgtggtt taaagataat gagacccttg tagaagactc aggcattgta 1740

ttgaaggatg ggaaccggaa cctcactatc cgcagagtga ggaaggagga cgaaggc 1797

<210> 12

<211> 367

<212> DNA

<213> mouse (mouse)

<400> 12

ctctacacct gccaggcctg caatgtcctt ggctgtgcaa gagcggagac gctcttcata 60

atagaaggtc agtgtgatgt cataggctca tcagatggtc tctggcaatt cgatcgcatt 120

ggacatagct gcacatactt tgagtaggat gccttgtggg tgactcatgg tgacactggt 180

ggttgttaag cctcgtctct gagatgttca tttaagtgtc acacaagtgt acatagcctg 240

aaggcaattt tacacaatat ttttcagtgt acctaccttt tttgactggg atctgtcaca 300

tgaaattagg ggtcggtgct cagaaagttt tagatcttat tcgtctgtga ggtctgagtt 360

acggttt 367

<210> 13

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cgcggtcgac aagctcaact actctaccac tgagc 35

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gccatgctgg tcactgacag 20

<210> 15

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

agtgaccagc atggcgtcgt gtacattact gagaa 35

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ccattatgta cctgactgat 20

<210> 17

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tcaggtacat aatggctcca cgtgttcttt gtaat 35

<210> 18

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tagaggatcg gcgcgagaac caatgacagt ggtat 35

<210> 19

<211> 4082

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gtcgtgtaca ttactgagaa caaaaacaaa actgtggtga ttccatgtct cgggtccatt 60

tcaaatctca acgtgtcact ttgtgcaaga tacccagaaa agagatttgt tcctgatggt 120

aacagaattt cctgggacag caagaagggc tttactattc ccagctacat gatcagctat 180

gctggcatgg tcttctgtga agcaaaaatt aatgatgaaa gttaccagtc tattatgtac 240

atagttgtcg ttgtagggta taggatttat gatgtggttc tgagtccgtc tcatggaatt 300

gaactatctg ttggagaaaa gcttgtctta aattgtacag caagaactga actaaatgtg 360

gggattgact tcaactggga atacccttct tcgaagcatc agcataagaa acttgtaaac 420

cgagacctaa aaacccagtc tgggagtgag atgaagaaat ttttgagcac cttaactata 480

gatggtgtaa cccggagtga ccaaggattg tacacctgtg cagcatccag tgggctgatg 540

accaagaaga acagcacatt tgtcagggtc catgaaaaac cttttgttgc ttttggaagt 600

ggcatggaat ctctggtgga agccacggtg ggggagcgtg tcagaatccc tgcgaagtac 660

cttggttacc cacccccaga aataaaatgg tataaaaatg gaatacccct tgagtccaat 720

cacacaatta aagcggggca tgtactgacg attatggaag tgagtgaaag agacacagga 780

aattacactg tcatccttac caatcccatt tcaaaggaga agcagagcca tgtggtctct 840

ctggttgtgt atgtcccacc ccagattggt gagaaatctc taatctctcc tgtggattcc 900

taccagtacg gcaccactca aacgctgaca tgtacggtct atgccattcc tcccccgcat 960

cacatccact ggtattggca gttggaggaa gagtgcgcca acgagcccag ccaagctgtc 1020

tcagtgacaa acccataccc ttgtgaagaa tggagaagtg tggaggactt ccagggagga 1080

aataaaattg aagttaataa aaatcaattt gctctaattg aaggaaaaaa caaaactgta 1140

agtacccttg ttatccaagc ggcaaatgtg tcagctttgt acaaatgtga agcggtcaac 1200

aaagtcggga gaggagagag ggtgatctcc ttccacgtga ccaggggtcc tgaaattact 1260

ttgcaacctg acatgcagcc cactgagcag gagagcgtgt ctttgtggtg cactgcagac 1320

agatctacgt ttgagaacct cacatggtac aagcttggcc cacagcctct gccaatccat 1380

gtgggagagt tgcccacacc tgtttgcaag aacttggata ctctttggaa attgaatgcc 1440

accatgttct ctaatagcac aaatgacatt ttgatcatgg agcttaagaa tgcatccttg 1500

caggaccaag gagactatgt ctgccttgct caagacagga agaccaagaa aagacattgc 1560

gtggtcaggc agctcacagt cctagagcgt gtggcaccca cgatcacagg aaacctggag 1620

aatcagacga caagtattgg ggaaagcatc gaagtctcat gcacggcatc tgggaatccc 1680

cctccacaga tcatgtggtt taaagataat gagacccttg tagaagactc aggcattgta 1740

ttgaaggatg ggaaccggaa cctcactatc cgcagagtga ggaaggagga cgaaggcctc 1800

tacacctgcc aggcctgcaa tgtccttggc tgtgcaagag cggagacgct cttcataata 1860

gaaggtcagt gtgatgtcat aggctcatca gatggtctct ggcaattcga tcgcattgga 1920

catagctgca catactttga gtaggatgcc ttgtgggtga ctcatggtga cactggtggt 1980

tgttaagcct cgtctctgag atgttcattt aagtgtcaca caagtgtaca tagcctgaag 2040

gcaattttac acaatatttt tcagtgtacc tacctttttt gactgggatc tgtcacatga 2100

aattaggggt cggtgctcag aaagttttag atcttattcg tctgtgaggt ctgagttacg 2160

gtttggcgcg gctgtgggtg ctgatgaacg gtcatcctca ccaaccccat ttcataagct 2220

tgatatcgaa ttccgaagtt cctattctct agaaagtata ggaacttcag gtctgaagag 2280

gagtttacgt ccagccaagc tagcttggct gcaggtcgtc gaaattctac cgggtagggg 2340

aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc 2400

gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg 2460

ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc 2520

cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta 2580

gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg 2640

gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg 2700

ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc 2760

cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc 2820

cgggcctttc gacctgcagc ctgttgacaa ttaatcatcg gcatagtata tcggcatagt 2880

ataatacgac aaggtgagga actaaaccat gggatcggcc attgaacaag atggattgca 2940

cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 3000

aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt 3060

tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc 3120

gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 3180

aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 3240

tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 3300

ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 3360

ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 3420

cgaactgttc gccaggctca aggcgcgcat gcccgacggc gatgatctcg tcgtgaccca 3480

tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 3540

ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 3600

tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 3660

tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gaggggatca 3720

attctctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt 3780

ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta 3840

ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg 3900

ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc 3960

ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctcgacta gagcttgcgg 4020

aacccttcga agttcctatt ctctagaaag tataggaact tcatcagtca ggtacataat 4080

gg 4082

<210> 20

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

aacttcttac tctcccttgc ttat 24

<210> 21

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

tgaaatgggg ttggtgagga tgac 24

<210> 22

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gctgtgggtg ctgatgaacg 20

<210> 23

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

caaagggcca ctgacacaag actc 24

<210> 24

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ccattggcga gaccattgaa gtga 24

<210> 25

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

agcgctcatc caagggcaat 20

<210> 26

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

gtattgggga aagcatcgaa gtct 24

<210> 27

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

agcgctcatc caagggcaat 20

Claims (8)

1. A construction method of a humanized KDR gene modified animal cell is characterized by comprising the following steps:

transforming an endogenous KDR gene in the mouse stem cells by using a gene editing technology to obtain a humanized KDR gene;

the construction method comprises the following steps: the whole sequence of the No. 4 to No. 14 exons and a part of the sequence of the No. 15 exon of the mouse Kdr gene have the mRNA sequence shown as SEQ ID NO: 1, the sequence of the mRNA is shown as SEQ ID NO: 3 is shown in the specification;

the construction method specifically comprises the following steps: providing a human KDR gene homologous recombination vector, introducing the human KDR gene homologous recombination vector into mouse cells, and transferring the mouse cells into a culture solution for culture;

the human KDR gene homologous recombination vector comprises a 5 'homology arm, a humanized KDR gene coding region, a resistance gene expression frame and a 3' homology arm which are sequentially arranged from a 5 'end to a 3' end;

specific sites which can be recognized by recombinase are arranged between the coding region of the humanized KDR gene and the expression frame of the resistance gene and between the expression frame of the resistance gene and the 3' homology arm;

the specific site is an FRT site.

2. The method for constructing a humanized KDR genetically modified animal cell according to claim 1, wherein the humanized KDR gene is selected from at least one of the following groups:

(a) the CDS coding sequence of the humanized KDR gene is shown as SEQ ID NO: 6 is shown in the specification;

(b) the mRNA sequence transcribed by the humanized KDR gene is shown as SEQ ID NO: 7 is shown in the specification;

(c) the protein sequence coded by the humanized KDR gene is shown as SEQ ID NO: shown in fig. 8.

3. The method for constructing a humanized KDR genetically engineered animal cell of claim 1, wherein the DNA sequence of the 5' homology arm is as set forth in SEQ ID NO: 9 is shown in the figure;

the DNA sequence of the 3' homology arm is shown as SEQ ID NO: 10 is shown in the figure;

the DNA sequence of the coding region of the humanized KDR gene is shown as SEQ ID NO: shown at 11.

4. A humanized KDR genetically modified animal cell obtained by the method for constructing a humanized KDR genetically modified animal cell according to any one of claims 1 to 3.

5. A method for constructing a humanized KDR gene modified animal model, which is characterized in that the humanized KDR gene modified animal cell of claim 4 is injected into blastocyst, and is transplanted into a surrogate mother body for development through embryo to obtain an animal of generation F0;

the blastocyst is a wild animal blastocyst.

6. The method of constructing a humanized KDR genetically engineered animal model of claim 5, further comprising: mating the F0 generation animals with animals expressing recombinase to obtain F1 generation animals with resistance genes removed;

mating the F1 generation animal with a wild animal, expanding the population, mating the heterozygote animal with the heterozygote animal to obtain a homozygote animal, and establishing a stable humanized KDR gene modified animal strain;

the recombinase is a Flp recombinase.

7. A cell or cell line or cell culture derived from a humanized KDR genetically engineered animal model obtained by the method of constructing a humanized KDR genetically engineered animal model according to any one of claims 5 to 6, or progeny thereof.

8. A tissue or organ derived from a humanized KDR genetically engineered animal model obtained by the method for constructing a humanized KDR genetically engineered animal model according to any one of claims 5 to 6, or a progeny thereof.

Images