SYSTEM AND METHOD FOR GENE EDITING BY USING ENGINEERED CELL

Abstract

A system and method for gene editing by using an engineered cell are provided. The system includes the engineered cell embedded with a synthetic protein receptor and a target cell. The engineered cell contains a CRISPR/CasRx system and a sgRNA gene sequence. The synthetic protein receptor includes an extracellular target cell recognition domain, a native Notch core domain, an intramembranous hydrolyzable polypeptide and effectors. The extracellular target cell recognition domain can recognize antigen molecules on the target cell surface; and the effectors act as transcription factors for CasRx enzyme and sgRNAs. CasRx and gRNA are expressed in the engineered cell for gene editing to edit mRNA of the target cell. In this way, the application range of the engineered cell is expanded, the pertinence and specificity of gene editing are improved, the off-target effect is reduced, the collective non-specific reaction is reduced, and the safety of gene editing is improved.

Claims

1. A system for gene editing on a target cell by using an engineered cell, comprising an engineered cell embedded with a synthetic protein receptor and the target cell, the engineered cell containing a CRISPR/CasRx system and a sgRNA gene sequence, a surface of the target cell containing antigenic molecules; wherein the synthetic protein receptor is a synthetic Notch receptor based on a native Notch receptor and is composed of an extracellular target cell recognition domain, a native Notch core domain, an intramembranous hydrolyzable polypeptide and effectors; the extracellular target cell recognition domain is configured to recognize antigen molecules on the surface of the target cell; the effectors act as transcription factors for a CasRx enzyme and sgRNAs in the CRISPR/CasRx system.

2. The system according to claim 1, wherein the effectors are selected from domains of a tetracycline transcription activator protein or a Cre recombinase.

3. The system according to claim 1, wherein after the extracellular target cell recognition domain of the engineered cell recognizes the antigen molecules on the surface of the target cell, a cleavage of the intramembranous hydrolyzable polypeptide is initiated, the effectors shed into a nucleus, and a synthesis of CasRx and the sgRNAs in the engineered cell is initiated; the CasRx and the sgRNAs synthesized are fused with the target cell, and the CasRx edits target mRNA in the target cell under a guidance of the sgRNAs.

4. The system according to claim 3, wherein the CasRx and the sgRNAs are secreted into a vicinity of the target cell in a form of microvesicles.

5. The system according to claim 1, wherein the target cell refers to microglia, and the sgRNAs are targeting sgRNAs of three cytokine mRNAs IL-1a, TNFa and C1q, and DNA sequences of encoding the sgRNAs of the three cytokine mRNAs IL-1a, TNFa and C1q are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

6. The system according to claim 5, wherein the extracellular target cell recognition domain is CD62L, CD62E or CD62P in a Selectin family.

7. The system according to claim 1, wherein the engineered cell is obtained by introducing the synthetic protein receptor into an eukaryotic cell by DNA recombination, DNA injection, plasmid transfection or viral transfection.

8. The system according to claim 7, wherein the eukaryotic cell is a neural stem cell, a macrophage, an endothelial progenitor cell, a T lymphocyte or a glial cell.

9. A preparation method of an engineered cell embedded with a synthetic protein receptor, comprising the following steps: 1) a preparation of editable cells preparing and culturing neural stem cells, macrophages, endothelial progenitor cells, T lymphocytes or glial cells, extracting primary cells and carrying out a first amplification; 2) a construction of a lentivirus containing a synthetic protein receptor gene sequence respectively designing forward and reverse specific PCR amplification primers for the synthetic protein receptor gene sequence and a gene editing assembly sequence, and introducing enzyme cleavage sites; carrying out an overlap extension PCR for a second amplification using the synthetic protein receptor sequence and the gene editing assembly sequence as templates, respectively, a gene editing assembly comprising a tetracycline response element TRE sequence, a CasRx transcription sequence, and DNA sequences corresponding to sgRNAs; extracting CDS regions of the synthetic protein receptor sequence and the gene editing assembly sequence from cDNA plasmids or library templates, and linking the CDS regions into a T vector; cutting the CDS regions from the T vector and loading into a lentiviral overexpression plasmid vector; synthesizing a DNA neck-loop structure corresponding to siRNA, and linking into a lentiviral interference plasmid vector after annealing; and preparing a lentiviral shuttle plasmid and an auxiliary packaging vector plasmid of the lentiviral shuttle plasmid; respectively extracting the lentiviral overexpression plasmid vector, the lentiviral interference plasmid vector, and the lentiviral shuttle plasmid, and co-transfecting the lentiviral overexpression plasmid vector, the lentiviral interference plasmid vector, and the lentiviral shuttle plasmid into 293T cells to obtain the lentivirus containing the synthetic protein receptor gene sequence and the gene editing assembly sequence; and 3) a transfection of the lentivirus into eukaryotic cells transfecting the lentivirus into the editable cells prepared in step 1), and simultaneously transfecting fluorescent reporter genes to obtain the engineered cell embedded with the synthetic protein receptor.

10. The preparation method according to claim 9, wherein in step 3), lentivirus-transfected editable cells are amplified, and when the lentivirus-transfected editable cells account for 80 to 90% of a culture flask, an expression of a labeling fluorescent protein is observed, and a marker identification is carried out on a transfected cell population to detect an activation of the engineered cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG. 1 is a schematic diagram showing the basic protein structure of a synthetic protein receptor according to an embodiment of the invention and related lentivirus design.

[0049] FIG. 2 is a schematic diagram of the working principle of the intracellular phase response program activated by synthetic receptors after engineered cells bind and recognize microglia in Example 1 of the invention.

[0050] FIG. 3 is the schematic diagram of the initiated expression of CasRx and three kinds of gRNA genes in the nucleus after engineered cells are activated in Example 1 of the invention.

[0051] FIG. 4 is the schematic diagram of the translation and synthesis of the engineered cell CasRx and three sgRNAs and packaging into a complex in the cell in Example 1 of the invention. The packaged complex will act on adjacent target cells via a paracrine pathway.

[0052] FIGS. 5 to 6 show the expression levels of the synthetic receptor in the engineered cell after transfection of lentiviral vector in Example 1 of the invention.

[0053] FIG. 7 shows the change of the nuclear localization ratio of tag protein over time after the engineered cell recognizes the target cell in vitro in Example 2 of the invention. The nuclear localization ratio reaches its peak around 24h.

[0054] FIG. 8 shows a case where the engineered cell is activated after recognizing the target cell in Example 2 of the invention. After the engineered cell is activated, the Cre enzyme can be rapidly released and localized to the nucleus, thereby initiating the downstream synthesis reaction. Arrows indicate tag proteins and localization in the activated engineered cell.

[0055] FIG. 9 shows a fluorescence image of secreted exosomes after the engineered cell is activated in Example 2 of the invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0056] The invention will now be further described in conjunction with specific embodiments.

[0057] The term “synthetic protein receptor” that appears in the invention is referred to as a synthetic receptor, which is a fusion protein that can specifically recognize target cells. The term “engineered cells” appearing refers to cells obtained by introducing the synthetic receptor into eukaryotic cells by DNA recombination, DNA injection, plasmid transfection or viral transfection.

[0058] According to the invention, the fusion gene is constructed by overlap extension PCR, the synthetic receptor is expressed by lentivirus-transfected cells, and simultaneously the fluorescent reporter gene is transfected, thus obtaining the engineered cell modified by the synthetic receptor. The microglia and the engineered cells are co-cultured in vitro to test whether the engineered cells are activated. Disease models, such as intracerebral hemorrhage, are built in vivo to test the in-vivo activation state of the engineered cells. The state of the engineered cells is analyzed by immunofluorescence staining and flow cytometry, and the engineered cells are delivered into the model mice by tail vein injection to test the effects of the engineered cells.

[0059] In the invention, the preparation of engineered neural stem cells and their application in gene editing are described in detail as examples, and the preparation and application of macrophage engineered cells, endothelial progenitor cells engineered cells, T lymphocyte engineered cells, and glial cell engineered cells are carried out in a similar way.

[0060] A neural stem cell modified by a synthetic receptor is provided in an embodiment. The synthetic receptor is composed of an extracellular segment that recognizes the target cells, the minimal transmembrane core domain of the native Notch of the intramembranous segment and a transcriptional regulator of the intracellular segment which are connected in series. The structure of the synthetic receptor is shown in FIG. 1.

[0061] Example 1 A preparation method of an engineered cell capable of identifying microglia includes the following steps.

1) Preparation of Editable Neural Stem Cells

[0062] Neural stem cells were taken from the embryos of pregnant mice by the following specific steps.

[0063] The mice were sacrificed by cervical dislocation, then quickly soaked in 70% ethanol with a temperature of -20° C. for sterilization for 5 min and then placed in a sterilized dissecting tray with the abdomen upward. The top of the uterus was incised with micro scissors, the uterus was opened, the placenta was incised, and the embryo was taken out and rinsed 3 times with 1% P/S. Live embryos of normal size and shape were selected, transferred to 50 ml centrifuge tubes, and immersed in 4° C. DMEM-HG and 1% P/S.

[0064] Subsequent steps were performed on ice, with microscissors cutting the top of each embryo at the level of the cervical spinal cord and quickly transferring to a tray on ice containing 4° C. DMEM-HG and 1% P/S. The skin was peeled off with micro forceps, then the skull and dura were dissected layer by layer, and the entire cerebral hemisphere was excised. The pia mater and blood vessels were removed from the cerebral hemispheres with a microdissection instrument. The dissected cerebral hemispheres were cut into small pieces with a pair of microscopic scissors on ice. The cut tissue was carefully transferred to a 15 ml centrifuge tube and then centrifuged at 200 xg for 5 min to remove the supernatant. 3 to 5 ml of pre-warmed accutase solution containing 20 units/ml DNase I was then added. After digestion, the supernatant was discarded by centrifugation, and the digestion was repeated 2 to 3 times. During the digestion process, the cell suspension was gently pipetted, and cell pellets were resuspended in 20 ml of fresh serum-free medium, cell viability was counted by trypan blue staining, and finally dissociated cells were diluted to 2×10.sup.5 cells/ml and incubated at 37° C. in the presence of 5% CO.sub.2.

[0065] DMEM/F-12, used as basal medium, was added with 20 ng/ml epidermal growth factor, 20 ng/ml basic fibroblast growth factor, 2% B-27 supplement, 2.5 .Math.g/ml heparin, 1 mML aminoamide, 1% P/S to obtain an expansion medium for neural stem cells.

[0066] The stem cells were cultured in the presence of 5% CO.sub.2 at 37° C., for a period of time until the stem cells grown into neural stem cell spheres with a diameter of 80 .Math.m to 100 .Math.m.

2) Construction of the Lentivirus Containing Synthetic Protein Gene Sequence

[0067] In this embodiment, the CMV synthetic protein receptor was composed of an extracellular recognition structure, CD62E, a transmembrane core domain, and an intracellular domain containing tTA tetracycline transcription activator protein. Its specific amino acid sequence was shown in SEQ ID NO: 4, and its nucleotide sequence was shown in SEQ ID NO: 5.

[0068] Forward and reverse specific PCR amplification primers were designed for the synthetic protein receptor sequence and the gene editing assembly sequence, and enzyme cleavage sites were introduced. Using the synthetic protein receptor sequence and the gene editing assembly sequence as templates, overlap extension PCR was carried out for amplification. The gene editing assembly included a tetracycline response element TRE sequence, a CasRx sequence containing a signal peptide sequence, and the targeting sgRNAs (i.e., IL-1a sgRNA, TNFa sgRNA, and C1q sgRNA) for the three cytokine mRNAs IL-1a, TNFa and C1q, and the DNA sequences of the targeting sgRNAs are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0069] The CDS regions of the synthetic protein receptor gene were extracted from cDNA plasmids or library templates, and linked into a T vector, and the CDS regions were cut from the T vector and loaded into a lentiviral overexpression plasmid vector.

[0070] The DNA neck-loop structure corresponding to the siRNA was synthesized, and after annealing, the lentivirus interference plasmid vector was linked to prepare the lentivirus shuttle plasmid and its auxiliary packaging element vector plasmid. The lentiviral overexpression plasmid vector, the lentiviral interference plasmid vector, and the lentiviral shuttle plasmid were subjected to high-purity endotoxin-free extraction, and then co-transfected 293T cells. 6 h after transfection, the process was replaced with the expansion medium of neural stem cells. After culturing for 24 and 48 hours, the cell supernatants rich in lentiviral particles were collected respectively and then ultracentrifuged to concentrate viruses to obtain lentivirus containing the synthetic receptor sequence, the tetracycline response element TRE, and the CasRx sequence: including signal peptide, U6 promoter, terminator and CasRx sequence, IL-1a sgRNA, TNFa sgRNA, C1q sgRNA genes.

[0071] Specific operation steps were as follows:

[0072] 293T cells were seeded on a 15 cm plate one day in advance, so that 293T cells were in logarithmic growth phase during transfection. The transfection plasmids were mixed together thoroughly in proportion to prepare DNA. The desired trans-IT was placed into DMEM, 2 ml of DMEM per 15 cm plate. The trans-IT was directly added to the medium without contact with the wall of a container. The reagents were vortex-mixed thoroughly and then set still for 10 min.

[0073] 2 ml of trans-IT/DMEM was added to 30 .Math.g of the DNA plasmid mixture. The DNA plasmid mixture was vortex-mixed and then set still at room temperature for 15 min, and in the meanwhile, a 293T cell culture dish was taken, the used culture medium was removed by suction, and fresh complete culture medium was then added. 2 ml of trans-IT/DNA/DMEM mixture was added dropwise to each plate. The medium was then shaken back and forth to mix the resulting mixture gently. The resulting mixture was then incubated in a 37° C. incubator. 48 h after transfection, the supernatants were collected every 12 h and ultracentrifuged at 48960 g for 90 min to concentrate viruses. The bottom pellet was taken by suction and aliquoted and stored at -80° C.

3) Synthetic Receptor-Modified Neural Stem Cells

[0074] 1×10.sup.7-5×10.sup.7 neural stem cells were taken. The used medium was discarded, and 2 to 4 mL of fresh DMEM/F12 was added. 200-300 uL of the virus concentrate obtained in step 2) and Polybrene with a final concentration of 5 .Math.g/ml were then added. The cells were then infected in a 37° C., 5% CO.sub.2 incubator for 12 to16 h. Then, the waste solution was discarded and the cells were transferred to an uncoated culture flask. 20 to 40 mL of fresh DMEM/F12 was then added and the cells were further cultured for amplification in a 37° C., 5% CO.sub.2 incubator for 3 to 5 days. The synthetic receptor-modified neural stem cells were thus obtained by infection.

[0075] Specific operation steps were as follows:

[0076] (1) 18 to 24 h before lentivirus transfection, the neural stem cells were digested with 0.25% trypsin, centrifuged and resuspended in DMEM/F12 medium to make a single cell suspension and the cells were counted. The cell suspension was seeded into a 24-well plate at a density of 1×10.sup.5/well.

[0077] (2) 24 h after the cell seeding, the used culture medium was discarded and replaced with 2 ml of fresh serum-free culture medium containing 5 .Math.g/ml polybrene. The dose of virus suspension required to be added when the MOL value is 10 was calculated and the virus suspension was then added to the medium. The mixture was shaken gently to be mixed evenly, and then incubated in a 37° C., 5% CO.sub.2 incubator.

[0078] (3) Four hours later, 2 ml of fresh culture medium was added.

[0079] (4) The cells were further incubated for 24 h and the used culture medium was then replaced with fresh virus-free complete medium.

[0080] (5) Three to four days after transfection, puromycin was added into complete medium, with the final concentration of puromycin being 5 ug/ml, to screen stably transfected cell lines to obtain the synthetic receptor-modified neural stem cells.

[0081] The synthetic receptor-modified neural stem cells can specifically recognize the target cell, initiate the expression of intracellular CasRx and gRNA, and then perform gene editing at the mRNA level for the target cells. Their working principle is shown in FIGS. 2 to 4. In the constructed engineered cells, the synthetic receptors are distributed on the cell membrane, and the synthetic receptors span the entire cell membrane, of which the outer segment of the cell membrane is the recognition domain, which can bind to the CD68 protein, a molecular marker on the surface of microglia, thus endowing the engineered cells with the ability to specifically recognize microglia. CD62E on the synthetic receptor binds to CD68, resulting in the adhesion of engineered cells to activated microglia. The minimal transmembrane core domain of native Notch of the hydrolyzable peptide segment of the synthetic receptor is exposed due to mechanical pulling. After the hydrolyzable peptide segment is hydrolyzed, the connection between the effector and the intramembranous segment is destroyed, and the effectors shed from the cell membrane, enter the nucleus, and dactivate the downstream response elements and targeted genes, thus achieving the specific response of the synthetic receptor.

[0082] The neural stem cells were transfected with the constructed lentivirus to obtain the engineered cells containing the synthetic protein receptors, and the expression levels of the synthetic receptors in the engineered cells after transfection with lentiviral vectors are shown in FIGS. 5 to 6, where 1 represents the empty vector group, 2 represents the control group, and 3 represents the synthetic receptor group.

[0083] The expression of the synthetic receptors in engineered cells was verified respectively in terms of transcription and translation levels. The qPCR results (see FIG. 5) showed that the empty vector group or the control group contained almost no or a very small amount of synthetic receptor mRNA, and the Western blot results (see FIG. 6) showed that the neural stem cells in the natural state did not express synthetic receptors, and the engineered cells (the synthetic receptor group) detected synthetic receptors in the form of proteins with a high expression level.

Example 2 Co-Culture of the Engineered Cells and Activated Microglia

1. Culture of Microglia

[0084] The microglia cell lines of BV-2 mice and the mononuclear macrophage leukemia cells of Raw264.7 mice were selected as culture objects, DMEM/F12+10% FBS was used as a complete medium, and during the culture process, the activation of microglia due to excessive pipetting in the case of passage was avoided. The activated microglia were incubated for 12 h with medium containing 1 ug/m1 LPS. After activation, microglia positive for the surface antigen CD68 were sorted by flow cytometry for co-culture.

2. Transfection and Co-Culture

[0085] The lentivirus containing the synthetic receptor sequence, tetracycline response element TRE and CasRx transcription sequence, IL-1a sgRNA, TNFa sgRNA, and C1q sgRNA genes, obtained in Example 1, was transfected into the neural stem cells. When the synthetic receptor binds to microglia, the tetracycline transcription activator protein tTA is detached from the cell and enters the nucleus, where it binds to the tetracycline response element TRE, thereby initiating the expression of CasRx, IL-1a sgRNA, TNFa sgRNA and C1q sgRNA.

[0086] The digested microglia and engineered cells were adjusted to a cell density of about 1×10.sup.6 with DMEM/F12 complete medium, and the microglia and the engineered cells were mixed at a ratio of 1:1, and added to a petri dish with a diameter of 6 cm. The activation of the engineered cells was detected, and after 24 hours of co-culture, the activation and the concentrations of CasRx and IL-1a sgRNA, TNFa sgRNA, and C1q sgRNA in the medium were detected.

[0087] FIG. 7 shows, from left to right, the image of tag antibody, fusion of tag antibody and nucleus, CD68 staining, and fusion of tag antibody to EGFP and CD68. The rightmost column is an enlarged image of the white box in the fourth column. It can be seen that when the engineered cells are cultured alone, there is no activation of the CD68 molecule, and the tag antibody representing the intracellular segment of the synthetic receptor is localized on the cell membrane and will not enter the nucleus. When the engineered cells were co-cultured with BV2 microglia or Raw264.7 macrophages, the CD68 molecules on the surface of the latter two cells activated the engineered cells, and the tag antibody appeared nuclear localization, indicating that the intracellular segments of part of the synthetic receptors entered the nucleus. The engineered cells can thus recognize activated microglia and activate the intracellular domain into the nucleus.

[0088] In FIG. 8, N2A represents the single culture of engineered cells, and BV2 and Raw264.7 respectively represent the co-culture of BV2 microglia or Raw264.7 macrophages with engineered cells. By quantifying the change of the nuclear localization ratio of the tag protein of the engineered cell over time, the results show that when the engineered cells are cultured alone, there is only a small amount of nuclear localization of the tag antibody, and it hardly changes with time, which may represent non-specific activation. Under the co-culture conditions, the nuclear localization ratio of the tag antibody increases significantly after activation, and gradually increases over time, indicating that the activated Cre enzyme can be rapidly released and localized to the nucleus within 6h, thereby initiating the downstream synthesis reaction. In addition, this process reaches its peak around 24h.

[0089] The function of synthesizing and secreting CasRx and sgRNA by the engineered cells was tracked using exosome fluorescent dyes. The results are shown in FIG. 9. The results show that the engineered cells can secrete CasRx and sgRNAs outside the cells in the form of exosomes.

Sequence Listing

[0090] <110> Huashan Hospital, Fudan University ZHU Jianhong

[0091] <120> SYSTEM AND METHOD FOR GENE EDITING BY USING ENGINEERED CELL

[0092] <130> 2111189

[0093] < 160> 5

[0094] <170> SIPOSequenceListing 1 .0

[0095] <210> 1

[0096] <211> 29

[0097] <212> DNA

[0098] <213> Artificial Sequence

[0099] <400> 1

TABLE-US-00001 catgaagtga gccatagctt gcatcatag 29

[0100] <210> 2

[0101] <211> 29

[0102] <212> DNA

[0103] <213> Artificial Sequence

[0104] <400> 2

TABLE-US-00002 tttgctacga cgtgggctac aggcttgtc 29

[0105] <210> 3

[0106] <211> 29

[0107] <212> DNA

[0108] <213> Artificial Sequence

[0109] <400> 3

TABLE-US-00003 aggaccttgt caaagataac cacgttgcc 29

[0110] <210> 4

[0111] <211> 612

[0112] <212> PRT

[0113] <213> Artificial Sequence

[0114] <400> 4

TABLE-US-00004 Met Asn Ala Ser Arg Phe Leu Ser Ala Leu Val Phe Val Leu Leu Ala 1               5                   10                  15 Glu Glu Ser Thr Ala Trp Tyr Tyr Asn Ala Ser Ser Glu Leu Met Thr             20                  25                  30 Tyr Asp Glu Ala Ser Ala Tyr Cys Gln Arg Asp Tyr Thr His Leu Val         35                  40                  45 Ala Ile Gln Asn Lys Glu Glu Ile Asn Tyr Leu Asn Ser Asn Leu Lys     50                  55                  60 His Ser Pro Ser Tyr Tyr Trp Ile Gly Ile Arg Lys Val Asn Asn Val 65                  70                  75                  80 Trp Ile Trp Val Gly Thr Gly Lys Pro Leu Thr Glu Glu Ala Gln Asn                 85                  90                  95 Trp Ala Pro Gly Glu Pro Asn Asn Lys Gln Arg Asn Glu Asp Cys Val             100                 105                 110 Glu Ile Tyr Ile Gln Arg Thr Lys Asp Ser Gly Met Trp Asn Asp Glu         115                 120                 125 Arg Cys Asn Lys Lys Lys Leu Ala Leu Cys Tyr Thr Ala Ser Cys Thr     130                 135                 140 Asn Ala Ser Cys Ser Gly His Gly Glu Cys Ile Glu Thr Ile Asn Ser 145                 150                 155                 160 Tyr Thr Cys Lys Cys His Pro Gly Phe Leu Gly Pro Asn Cys Glu Gln                 165                 170                 175 Ala Val Thr Cys Lys Pro Gln Glu His Pro Asp Tyr Gly Ser Leu Asn             180                 185                 190 Cys Ser His Pro Phe Gly Pro Phe Ser Tyr Asn Ser Ser Cys Ser Phe         195                 200                 205 Gly Cys Lys Arg Gly Tyr Leu Pro Ser Ser Met Glu Thr Thr Val Arg     210                 215                 220 Cys Thr Ser Ser Gly Glu Trp Ser Ala Pro Ala Pro Ala Cys His Val 225                 230                 235                 240 Val Glu Cys Glu Ala Leu Thr His Pro Ala His Gly Ile Arg Lys Cys                 245                 250                 255 Ser Ser Asn Pro Gly Ser Tyr Pro Trp Asn Thr Thr Cys Thr Phe Asp             260                 265                 270 Cys Val Glu Gly Tyr Arg Arg Val Gly Ala Gln Asn Leu Gln Cys Thr         275                 280                 285 Ser Ser Gly Ile Trp Asp Asn Glu Thr Pro Ser Cys Lys Ala Val Thr     290                 295                 300 Cys Asp Ala Ile Pro Gln Pro Gln Asn Gly Phe Val Ser Cys Ser His 305                 310                 315                 320 Ser Thr Ala Gly Glu Leu Ala Phe Lys Ser Ser Cys Asn Phe Thr Cys                 325                 330                 335 Glu Gln Ser Phe Thr Leu Gln Gly Pro Ala Gln Val Glu Cys Ser Ala             340                 345                 350 Gln Gly Gln Trp Thr Pro Gln Ile Pro Val Cys Lys Ala Val Gln Cys         355                 360                 365 Glu Ala Leu Ser Ala Pro Gln Gln Gly Asn Met Lys Cys Leu Pro Ser     370                 375                 380 Ala Ser Gly Pro Phe Gln Asn Gly Ser Ser Cys Glu Phe Ser Cys Glu 385                 390                 395                 400 Glu Gly Phe Glu Leu Lys Gly Ser Arg Arg Leu Gln Cys Gly Pro Arg                 405                 410                 415 Gly Glu Trp Asp Ser Lys Lys Pro Thr Cys Ser Ala Val Lys Cys Asp             420                 425                 430 Asp Val Pro Arg Pro Gln Asn Gly Val Met Glu Cys Ala His Ala Thr         435                 440                 445 Thr Gly Glu Phe Thr Tyr Lys Ser Ser Cys Ala Phe Gln Cys Asn Glu     450                 455                 460 Gly Phe Ser Leu His Gly Ser Ala Gln Leu Glu Cys Thr Ser Gln Gly 465                 470                 475                 480 Lys Trp Thr Gln Glu Val Pro Ser Cys Gln Val Val Gln Cys Pro Ser                 485                 490                 495 Leu Asp Val Pro Gly Lys Met Asn Met Ser Cys Ser Gly Thr Ala Val             500                 505                 510 Phe Gly Thr Val Cys Glu Phe Thr Cys Pro Asp Asp Trp Thr Leu Asn         515                 520                 525 Gly Ser Ala Val Leu Thr Cys Gly Ala Thr Gly Arg Trp Ser Gly Met     530                 535                 540 Pro Pro Thr Cys Glu Ala Pro Val Ser Pro Thr Arg Pro Leu Val Val 545                 550                 555                 560 Ala Leu Ser Ala Ala Gly Thr Ser Leu Leu Thr Ser Ser Ser Leu Leu                 565                 570                 575 Tyr Leu Leu Met Arg Tyr Phe Arg Lys Lys Ala Lys Lys Phe Val Pro             580                 585                 590 Ala Ser Ser Cys Gln Ser Leu Gln Ser Phe Glu Asn Tyr His Val Pro         595                 600                 605 Ser Tyr Asn Val     610

[0115] <210> 5

[0116] <211> 1836

[0117] <212> DNA

[0118] <213> Artificial Sequence

[0119] <400> 5

TABLE-US-00005 atgaatgcct cgcgctttct ctctgctctt gtttttgttc tcctcgctgg agagagcaca 60 gcttggtact acaatgcctc cagtgagctc atgacgtatg atgaagccag tgcatactgt 120 cagcgggact acacacatct ggtggcaatt cagaacaagg aagagatcaa ctaccttaac 180 tccaatctga aacattcacc gagttactac tggattggaa tcagaaaagt caataacgta 240 tggatctggg tggggacggg gaagcctctg acagaggaag ctcagaactg ggctccaggt 300 gaaccaaaca acaaacaaag aaatgaggac tgtgtagaga tttacatcca acgaaccaaa 360 gactcgggca tgtggaatga cgagagatgt aacaaaaaga agctggctct gtgctacaca 420 gcttcgtgta ccaatgcatc ctgcagtggt catggtgaat gcatagagac catcaatagt 480 tacacctgca agtgccaccc tggcttcctg ggacccaact gtgagcaagc tgtgacttgc 540 aaaccacagg aacaccctga ctatggaagc ctgaactgct cccacccgtt cggccccttc 600 agctataatt cctcctgctc ctttggctgt aaaaggggct acctgcccag cagcatggag 660 accaccgtgc ggtgtacgtc ctctggagag tggagtgcgc ctgctccagc ctgccatgtg 720 gttgaatgtg aagctttgac ccaccctgcc cacggtatca ggaaatgttc ctcaaatcct 780 gggagctacc catggaacac gacatgcacg tttgactgtg tggaagggta caggcgagtt 840 ggagctcaga atctacagtg tacctcatct ggcatctggg ataacgagac gccatcatgc 900 aaagctgtga cctgtgacgc catccctcag cctcagaatg gctttgtgag ctgcagccac 960 tcaacagctg gagaacttgc gtttaagtca tcctgtaact tcacctgtga gcagagtttc 1020 acgttgcagg ggccagcgca ggttgaatgc agcgcacaag ggcagtggac accacaaatc 1080 ccagtctgca aagctgtcca gtgtgaagcc ttatctgcgc cacagcaggg caacatgaaa 1140 tgtcttccca gtgcttctgg acctttccaa aatgggtcca gttgtgagtt ctcctgcgaa 1200 gaaggatttg aactgaaggg atcaagaaga cttcagtgtg gtccaagagg ggaatgggat 1260 agcaagaagc ccacgtgttc agctgtgaaa tgtgatgatg tccctcggcc ccagaatggc 1320 gtcatggagt gtgctcatgc tactactgga gaattcacct acaagtcctc atgtgccttt 1380 caatgcaatg agggctttag cttgcatggc tcagctcaac ttgagtgcac atctcaggga 1440 aagtggaccc aggaagtccc ctcctgccaa gtggtacaat gtccaagcct tgacgtcccg 1500 ggaaagatga acatgagctg cagcggaaca gcagttttcg gcacagtgtg tgagtttaca 1560 tgtcctgatg attggacact caatggatct gcagttctga cgtgtggtgc cacgggacgc 1620 tggtctggga tgccgcctac ctgtgaagcc ccagtcagcc ccacccgtcc cttggtagtt 1680 gcactttctg cggcaggaac ctcactcctg acatcgtcct cattgctcta cttgttgatg 1740 agatactttc ggaagaaagc aaagaaattt gttcctgcta gcagctgcca aagccttcaa 1800 tcgtttgaaa actaccatgt gccttcttac aacgtc 1836