CN113150130B - Novel coronavirus monoclonal antibody and application thereof - Google Patents

Novel coronavirus monoclonal antibody and application thereof Download PDF

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CN113150130B
CN113150130B CN202110132581.4A CN202110132581A CN113150130B CN 113150130 B CN113150130 B CN 113150130B CN 202110132581 A CN202110132581 A CN 202110132581A CN 113150130 B CN113150130 B CN 113150130B
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CN113150130A (en
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罗均利
徐义
蔡开妹
孙扬清
陈子华
陈志康
袁伟杰
刘青青
龙敏
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Xiangya Hospital of Central South University
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Xiangya Hospital of Central South University
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Priority to CN202110132581.4A priority Critical patent/CN113150130B/en
Priority to CN202210703223.9A priority patent/CN115109150A/en
Priority to CN202210704198.6A priority patent/CN115141272A/en
Priority to CN202210704192.9A priority patent/CN115141271A/en
Priority to CN202210703241.7A priority patent/CN115109151A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a novel coronavirus monoclonal antibody and application thereof. 15 full-humanized antibodies against SARS-Cov-2S1 or RBD protein are screened, the affinity of the antibodies is between 1.888964539nM/L and 0.060928817nM/L, the IC50 of in vitro pseudovirus neutralization is between 0.0005564 mu g/ml and 2.924 mu g/ml, and the 15 antibodies of the invention have important scientific significance and clinical application prospect for the prevention, treatment and diagnosis of SARS-Cov-2.

Description

Novel coronavirus monoclonal antibody and application thereof
The technical field is as follows:
the invention belongs to the technical field of screening and preparation of monoclonal antibodies, and particularly relates to a novel coronavirus monoclonal antibody and application thereof.
The background art comprises the following steps:
SARS-Cov-2 is a linear single-stranded positive-stranded RNA virus whose structural proteins include spike protein (S), small envelope protein (E), envelope protein (M), and nucleoprotein (N). The S protein is composed of S1 and S2 subunits.
SARS-Cov-2 binds to the membrane protein of Angiotensin-converting enzyme 2 (ACE 2) on the surface of lung epithelial cells via spike protein (S protein) on the surface of virus particles, and ACE2 then undergoes a change in shape structure, leading to the virus entering the cell. Based on a new coronavirus pathogenic mechanism, the neutralizing antibody of the S protein blocks the combination of the S protein and ACE2 on the surface of a cell, thereby blocking the virus from entering the cell.
When SARS-Cov-2 invades the body, it will induce the body to produce corresponding effector B cell and memory B cell, and the effector B cell will produce antibody, which can be used for diagnosing and treating SARS-Cov-2. The blood plasma obtained from SARS-Cov-2 convalescent patient is transfused into SARS-Cov-2 severe patient, and the symptoms can be obviously improved. However, in the case of a huge number of patients, the plasma number of the SARS-Cov-2 convalescent patients is limited, and the monoclonal antibody can be produced in vitro in a large scale by screening the SARS-Cov-2 convalescent patients for effective neutralizing antibody against SARS-Cov-2, and by genetic engineering and protein expression techniques.
The invention content is as follows:
the main purpose of the present invention is to provide a specific monoclonal antibody against SARS-Cov-2 and its application. The mononuclear cell is obtained from the SARS-Cov-2 rehabilitative person to construct phage library, and the specific antibody against SARS-Cov-2 is quickly screened, and the antibody can be used for clinical detection, diagnosis, prevention and treatment of novel coronavirus SARS-Cov-2.
In order to achieve the purpose, the invention is realized by the following technical scheme:
extracting peripheral blood from SARS-Cov-2 patient, obtaining mononuclear cell and extracting total RNA, synthesizing cDNA, using heavy chain variable region and light chain variable region doubling primer to amplify heavy chain variable region and light chain variable region, randomly combining obtained heavy chain variable region and light chain variable region, recombining with phage vector, converting recombined product into XL1-Blue, adding auxiliary phage VCSM13 to obtain titer about 10 13 Size SARS-Cov-2 phage antibody library. And (3) screening an anti-Fab antibody by using the antigen S1 or RBD, and sequencing to obtain a heavy chain variable region sequence and a light chain variable region sequence which can be combined with the S1 or RBD, wherein the heavy chain variable region is assembled with an Fc fragment of IgG1, and the heavy chain and the light chain of the antibody are simultaneously expressed in mammalian cells to express the fully humanized anti-S1 or RBD antibody. Through in vitro affinity determination and pseudovirus neutralization experiment, monoclonal antibody for detecting, diagnosing, preventing or treating SARS-Cov-2 is screened.
The novel coronavirus antibody screened by the invention comprises one or more of XY1, XY2, XY3, XY4, XY5, XY6, XY7, XY8, XY9, XY10, XY11, XY12, XY13, XY14, XY15 and 15 antibodies.
The invention screens the following:
the XY1 antibody heavy chain variable region comprises:
the amino acid sequence of CDRH1 is shown in SEQ ID NO.1;
the amino acid sequence of CDRH2 is shown in SEQ ID NO.2;
the amino acid sequence of CDRH3 is shown in SEQ ID NO.3;
preferably, the amino acid sequence of the heavy chain variable region of the XY1 antibody is shown in SEQ ID NO.4.
The XY1 antibody light chain variable region comprises:
the amino acid sequence of CDRL1 is shown in SEQ ID NO.5;
the amino acid sequence of CDRL2 is shown in SEQ ID NO.6;
the amino acid sequence of CDRL3 is shown in SEQ ID NO.7
Preferably, the amino acid sequence of the variable region of the XY1 antibody light chain is shown in SEQ ID NO.8.
The XY2 antibody heavy chain variable region comprises:
the amino acid sequence of CDRH1 is shown in SEQ ID NO.9;
the amino acid sequence of CDRH2 is shown in SEQ ID NO.10;
the amino acid sequence of CDRH3 is shown in SEQ ID NO.11
Preferably, the amino acid sequence of the heavy chain variable region of the XY2 antibody is shown in SEQ ID NO.12.
The XY2 antibody light chain variable region comprises:
the amino acid sequence of CDRL1 is shown in SEQ ID NO.13;
the amino acid sequence of CDRL2 is shown in SEQ ID NO.14;
the amino acid sequence of CDRL3 is shown in SEQ ID NO.15;
preferably, the amino acid sequence of the variable region of the XY2 antibody light chain is shown in SEQ ID NO.16;
the variable region amino acid sequence of the XY3 antibody heavy chain comprises:
the amino acid sequence of CDRH1 is shown in SEQ ID NO.17;
the amino acid sequence of CDRH2 is shown in SEQ ID NO.18;
the amino acid sequence of CDRH3 is shown in SEQ ID NO.19;
preferably, the amino acid sequence of the heavy chain variable region of the XY3 antibody is shown in SEQ ID NO.20.
The variable region amino acid sequence of the XY3 antibody light chain comprises:
the amino acid sequence of CDRL1 is shown in SEQ ID NO.21;
the amino acid sequence of CDRL2 is shown in SEQ ID NO.22;
the amino acid sequence of CDRL3 is shown in SEQ ID NO.23;
preferably, the amino acid sequence of the variable region of the XY3 antibody light chain is shown in SEQ ID NO.24.
The XY4 antibody heavy chain variable region amino acid comprises:
the amino acid sequence of CDRH1 is shown in SEQ ID NO.25;
the amino acid sequence of CDRH2 is shown in SEQ ID NO.26;
the amino acid sequence of CDRH3 is shown in SEQ ID NO.27;
preferably, the amino acid sequence of the heavy chain variable region of the XY4 antibody is shown in SEQ ID NO.28.
The variable region amino acid of the XY4 antibody light chain comprises:
the amino acid sequence of CDRL1 is shown in SEQ ID NO.29;
the amino acid sequence of CDRL2 is shown in SEQ ID NO.30;
the amino acid sequence of CDRL3 is shown in SEQ ID NO.31;
preferably, the amino acid sequence of the variable region of the XY4 antibody light chain is as shown in SEQ ID NO.32;
the XY5 antibody heavy chain variable region amino acid comprises:
the amino acid sequence of CDRH1 is shown in SEQ ID NO.33;
the amino acid sequence of CDRH2 is shown in SEQ ID NO.34;
the amino acid sequence of CDRH3 is shown in SEQ ID NO.35;
preferably, the amino acid sequence of the heavy chain variable region of the XY5 antibody is shown in SEQ ID NO.36.
The variable region amino acid sequence of the XY5 antibody light chain comprises:
the amino acid sequence of CDRL1 is shown in SEQ ID NO.37;
the amino acid sequence of CDRL2 is shown in SEQ ID NO.38;
the amino acid sequence of CDRL3 is shown in SEQ ID NO.39;
preferably, the amino acid sequence of the variable region of the XY5 antibody light chain is shown in SEQ ID NO.40.
The variable region amino acid sequence of the XY6 antibody heavy chain comprises:
the amino acid sequence of CDRH1 is shown in SEQ ID NO.41;
the amino acid sequence of CDRH2 is shown in SEQ ID NO.42;
the amino acid sequence of CDRH3 is shown in SEQ ID NO.43;
preferably, the amino acid sequence of the heavy chain variable region of the XY6 antibody is shown in SEQ ID NO.44.
The variable region amino acid sequence of the XY6 antibody light chain comprises:
the amino acid sequence of CDRL1 is shown in SEQ ID NO.45;
the amino acid sequence of CDRL2 is shown in SEQ ID NO.46;
the amino acid sequence of CDRL3 is shown in SEQ ID NO.47;
preferably the amino acid sequence of the variable region of the XY6 antibody light chain: see SEQ ID NO.48;
the variable region amino acid sequence of the XY7 antibody heavy chain comprises:
the amino acid sequence of CDRH1 is shown in SEQ ID NO.49;
the amino acid sequence of CDRH2 is shown in SEQ ID NO.50;
the amino acid sequence of CDRH3 is shown in SEQ ID NO.51;
preferably, the amino acid sequence of the heavy chain variable region of the XY7 antibody is shown in SEQ ID NO.52;
the variable region amino acid sequence of the XY7 antibody light chain comprises:
the amino acid sequence of CDRL1 is shown in SEQ ID NO.53;
the amino acid sequence of CDRL2 is shown in SEQ ID NO.54;
the amino acid sequence of CDRL3 is shown in SEQ ID NO.55;
preferably the amino acid sequence of the variable region of the XY7 antibody light chain: see SEQ ID NO.56;
the amino acid sequence of the variable region of the XY8 antibody heavy chain comprises:
the amino acid sequence of CDRH1 is shown in SEQ ID NO.57;
the amino acid sequence of CDRH2 is shown in SEQ ID NO.58;
the amino acid sequence of CDRH3 is shown in SEQ ID NO.59;
preferably the amino acid sequence of the variable region of the XY8 antibody heavy chain: see SEQ ID NO.60;
the XY8 antibody light chain variable region amino acid sequence comprises:
the amino acid sequence of CDRL1 is shown in SEQ ID NO.61;
the amino acid sequence of CDRL2 is shown in SEQ ID NO.62;
the amino acid sequence of CDRL3 is shown in SEQ ID NO.63;
preferably the amino acid sequence of the variable region of the XY8 antibody light chain: see SEQ ID NO.64;
the amino acid sequence of the heavy chain variable region of the XY9 antibody comprises:
the amino acid sequence of CDRH1 is shown in SEQ ID NO.65;
the amino acid sequence of CDRH2 is shown in SEQ ID NO.66;
the amino acid sequence of CDRH3 is shown in SEQ ID NO.67;
preferably the amino acid sequence of the heavy chain variable region of the XY9 antibody: see SEQ ID NO.68;
the variable region amino acid sequence of the XY9 antibody light chain comprises:
the amino acid sequence of CDRL1 is shown in SEQ ID NO.69;
the amino acid sequence of CDRL2 is shown in SEQ ID NO.70;
the amino acid sequence of CDRL3 is shown in SEQ ID NO.71;
preferably the amino acid sequence of the variable region of the XY9 antibody light chain: see SEQ ID NO.72;
the variable heavy chain amino acid sequence of the XY10 antibody comprises
The amino acid sequence of CDRH1 is shown in SEQ ID NO.73;
the amino acid sequence of CDRH2 is shown in SEQ ID NO.74;
the amino acid sequence of CDRH3 is shown in SEQ ID NO.75;
preferably the amino acid sequence of the heavy chain variable region of the XY10 antibody is as shown in SEQ ID NO.76;
the variable region amino acid sequence of the XY10 antibody light chain comprises:
the amino acid sequence of CDRL1 is shown in SEQ ID NO.77;
the amino acid sequence of CDRL2 is shown in SEQ ID NO.78;
the amino acid sequence of CDRL3 is shown in SEQ ID NO.79;
preferably, the amino acid sequence of the variable region of the XY10 antibody light chain is shown in SEQ ID NO.80;
the amino acid sequence of the heavy chain variable region of the XY11 antibody comprises:
the amino acid sequence of CDRH1 is shown in SEQ ID NO.81;
the amino acid sequence of CDRH2 is shown in SEQ ID NO.82;
the amino acid sequence of CDRH3 is shown in SEQ ID NO.83;
preferably, the amino acid sequence of the heavy chain variable region of the XY11 antibody is shown in SEQ ID NO.84;
the variable region amino acid sequence of the XY11 antibody light chain comprises:
the amino acid sequence of CDRL1 is shown in SEQ ID NO.85;
the amino acid sequence of CDRL2 is shown in SEQ ID NO.86;
the amino acid sequence of CDRL3 is shown in SEQ ID NO.87;
preferably, the amino acid sequence of the variable region of the XY11 antibody light chain is shown in SEQ ID NO.88;
the variable region amino acid sequence of the XY12 antibody heavy chain comprises:
the amino acid sequence of CDRH1 is shown in SEQ ID NO.89;
the amino acid sequence of CDRH2 is shown in SEQ ID NO.90;
the amino acid sequence of CDRH3 is shown in SEQ ID NO.91;
preferably the amino acid sequence of the heavy chain variable region of the XY12 antibody is shown in SEQ ID NO.92;
the variable region amino acid sequence of the XY12 antibody light chain comprises:
the amino acid sequence of CDRL1 is shown in SEQ ID NO.93;
the amino acid sequence of CDRL2 is shown in SEQ ID NO.94;
the amino acid sequence of CDRL3 is shown in SEQ ID NO.95;
preferably, the amino acid sequence of the variable region of the XY12 antibody light chain is as shown in SEQ ID NO.96;
the amino acid sequence of the variable region of the XY13 antibody heavy chain comprises:
the amino acid sequence of CDRH1 is shown in SEQ ID NO.97;
the amino acid sequence of CDRH2 is shown in SEQ ID NO.98;
the amino acid sequence of CDRH3 is shown in SEQ ID NO.99;
preferably the amino acid sequence of the variable region of the XY13 antibody heavy chain: see SEQ ID NO.100;
the variable region amino acid sequence of the XY13 antibody light chain comprises:
the amino acid sequence of CDRL1 is shown in SEQ ID NO.101;
the amino acid sequence of CDRL2 is shown in SEQ ID NO.102;
the amino acid sequence of CDRL3 is shown in SEQ ID NO.103;
preferably, the amino acid sequence of the variable region of the XY13 antibody light chain is shown in SEQ ID NO.104;
the variable region amino acid sequence of the XY14 antibody heavy chain comprises:
the amino acid sequence of CDRH1 is shown in SEQ ID NO.105;
the amino acid sequence of CDRH2 is shown in SEQ ID NO.106;
the amino acid sequence of CDRH3 is shown in SEQ ID NO.107;
preferably, the amino acid sequence of the heavy chain variable region of the XY14 antibody is shown in SEQ ID NO.108;
the variable region amino acid sequence of the XY14 antibody light chain comprises:
the amino acid sequence of CDRL1 is shown in SEQ ID NO.109;
the amino acid sequence of CDRL2 is shown in SEQ ID NO.110;
the amino acid sequence of CDRL3 is shown in SEQ ID NO.111;
preferably, the amino acid sequence of the variable region of the XY14 antibody light chain is set forth in SEQ ID NO.112;
the variable region amino acid sequence of the XY15 antibody heavy chain comprises:
the amino acid sequence of CDRH1 is shown in SEQ ID NO.113;
the amino acid sequence of CDRH2 is shown in SEQ ID NO.114;
the amino acid sequence of CDRH3 is shown in SEQ ID NO.115;
preferably the amino acid sequence of the heavy chain variable region of the XY15 antibody is shown in SEQ ID NO.116;
the variable region amino acid sequence of the XY15 antibody light chain comprises:
the amino acid sequence of CDRL1 is shown in SEQ ID NO.117;
the amino acid sequence of CDRL2 is shown in SEQ ID NO.118;
the amino acid sequence of CDRL3 is shown in SEQ ID NO.119;
preferably, the amino acid sequence of the variable region of the XY15 antibody light chain is shown in SEQ ID NO.120.
The antibodies of the invention comprise conventional constant regions in addition to the heavy and light chain variable regions described above.
The novel coronavirus antibody disclosed by the invention preferably comprises one or more of XY1, XY2, XY3, XY4, XY5, XY6, XY7, XY8, XY9, XY10, XY11 and 11 antibodies. Through detection of affinity and in vitro neutralization experiments, the 11 antibodies are found to have more excellent affinity which reaches nM level and have neutralization effect on pseudoviruses; further preferably, the antibody composition comprises a combination of XY4 and XY10 antibodies, and the additive neutralization effect of the two antibodies is very significant.
The novel coronavirus antibody of the invention, preferably at least one of XY1, XY4, XY7 and XY10 antibodies is an antibody for novel coronavirus treatment; the IC50 values of these four antibodies have significant advantages; it is further preferred that the XY4 and XY10 antibody combination is an antibody for use in novel coronavirus therapy. According to the ELISA detection results of different antibody combinations, XY4 is suitable for being used as a detection phase for detecting the antigen; at least one of XY1, XY2, XY3, XY5, XY6, XY7, XY8, XY9, XY12, XY13, XY14, XY15, 12 antibodies is suitable for use as a coating phase antibody in the detection of an antigen.
Further preferred antibody combinations of the coating phase and the detection phase of the invention:
the coating phase antibody XY1 combination detects at least one of phase antibodies XY4, XY7, XY8, XY9, XY10, XY11, preferably detects at least one of phase antibodies XY4, XY10, and more preferably detects phase antibody XY4.
Detecting at least one of phase antibodies XY4, XY9 and XY10 by combining the coating phase antibodies XY2, preferably at least one of phase antibodies XY4 and XY10; further preferably, the phase antibody XY4 is detected.
The coating phase antibody XY3 is combined with at least one of detection phase antibodies XY4 and XY10, preferably detection phase antibody XY4.
The coating phase antibody XY4 is combined with at least one of detection phase antibodies XY2, XY8 and XY10, preferably detection phase antibody XY10;
the coating phase antibody XY5 is combined with at least one of detection phase antibodies XY4 and XY10, preferably detection phase antibody XY4;
the coating phase antibody XY6 is combined with at least one of detection phase antibodies XY4 and XY10, preferably detection phase antibody XY4;
the coating phase antibody XY7 is combined with at least one of detection phase antibodies XY4 and XY10, preferably detection phase antibody XY4;
the coating phase antibody XY8 is combined with at least one of detection phase antibodies XY4 and XY10, preferably detection phase antibody XY4;
coating phase antibody XY9 combined detection phase antibody XY4, XY10 at least one, preferably detection phase antibody XY4;
coating phase antibody XY10 combined detection phase antibody XY4;
the coating phase antibody XY11 is combined with the detection phase antibody XY4.
The novel coronavirus neutralizing antibody of the invention further comprises sequences, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region have 80% of homology with the amino acid sequences of the 15 antibody heavy chain variable regions and the amino acid sequences of the antibody light chain variable regions.
Antibodies of the invention that also include conservative sequence variants of the amino acid sequences of the antibodies are also included within the scope of the invention. Conservative amino acid sequence variants include modifications to the amino acid sequence that do not significantly alter the properties of the neutralizing antibodies of the invention, such as variants resulting from similar amino acid substitutions, deletions of amino acids, or additions as are well known in the art.
The neutralizing antibody of the present invention also includes human and non-human antibodies, and all antibodies having the same function as the above neutralizing antibody or modified and optimized. Further comprising: glycosylation or polyethylene glycol modification, etc.
The novel coronavirus monoclonal antibody further comprises any one of Fab, fab '-SH, fv, scFv and (Fab') 2 fragments, and comprises any one of the combined fragments of the heavy chain variable region and the light chain variable region of the antibody.
Fab refers to the part of an antibody molecule that contains one light chain variable and constant region and one heavy chain variable and constant region that are bound by a disulfide bond.
Fab' -SH refers to a Fab fragment that contains part of the hinge region.
Fv refers to the smallest antibody fragment containing the variable region of the antibody heavy chain, the variable region of the light chain and having all antigen binding sites.
scFv refers to an engineered antibody in which the variable region of the light chain is linked to the variable region of the heavy chain either directly or via a peptide chain.
(Fab ') 2 refers to the dimer of Fab'.
The invention also provides the nucleic acid sequences of the variable regions of the heavy chain and the light chain of the novel coronavirus neutralizing antibody.
The XY1 heavy chain variable region nucleic acid sequence is shown in SEQ ID NO.121;
the XY1 light chain variable region nucleic acid sequence is shown in SEQ ID NO.122;
the XY2 heavy chain variable region nucleic acid sequence is shown in SEQ ID NO.123;
the XY2 light chain variable region nucleic acid sequence is shown in SEQ ID NO.124;
the XY3 heavy chain variable region nucleic acid sequence is shown in SEQ ID NO.125;
the XY3 light chain variable region nucleic acid sequence is shown in SEQ ID NO.126;
the XY4 heavy chain variable region nucleic acid sequence is shown in SEQ ID NO.127;
the XY4 light chain variable region nucleic acid sequence is shown in SEQ ID NO.128;
the XY5 heavy chain variable region nucleic acid sequence is shown in SEQ ID NO.129;
the XY5 light chain variable region nucleic acid sequence is shown in SEQ ID NO.130;
the XY6 heavy chain variable region nucleic acid sequence is shown in SEQ ID NO.131;
the XY6 light chain variable region nucleic acid sequence is shown in SEQ ID NO.132;
the XY7 heavy chain variable region nucleic acid sequence is shown in SEQ ID NO.133;
the XY7 light chain variable region nucleic acid sequence is shown in SEQ ID NO.134;
the XY8 heavy chain variable region nucleic acid sequence is shown in SEQ ID NO.135;
the XY8 light chain variable region nucleic acid sequence is shown in SEQ ID NO.136;
the XY9 heavy chain variable region nucleic acid sequence is shown in SEQ ID NO.137;
the XY9 light chain variable region nucleic acid sequence is shown in SEQ ID NO.138;
the XY10 heavy chain variable region nucleic acid sequence is shown in SEQ ID NO.139;
the XY10 light chain variable region nucleic acid sequence is shown in SEQ ID NO.140;
the XY11 heavy chain variable region nucleic acid sequence is shown in SEQ ID NO.141;
the XY11 light chain variable region nucleic acid sequence is shown in SEQ ID NO.142;
the XY12 heavy chain variable region nucleic acid sequence is shown in SEQ ID NO.143;
the XY12 light chain variable region nucleic acid sequence is shown in SEQ ID NO.144;
the XY13 heavy chain variable region nucleic acid sequence is shown in SEQ ID NO.145;
the XY13 light chain variable region nucleic acid sequence is shown in SEQ ID NO.146;
the XY14 heavy chain variable region nucleic acid sequence is shown in SEQ ID NO.147;
the XY14 light chain variable region nucleic acid sequence is shown in SEQ ID NO.148;
the XY15 heavy chain variable region nucleic acid sequence is shown in SEQ ID NO.149;
the XY15 light chain variable region nucleic acid sequence is shown in SEQ ID NO.150.
The antibody nucleic acid sequences of the present invention comprising variable and constant regions:
the XY1 heavy chain nucleic acid sequence is shown in SEQ ID NO.151;
the XY1 light chain nucleic acid sequence is shown in SEQ ID NO.152;
the XY2 heavy chain nucleic acid sequence is shown in SEQ ID NO.153;
the XY2 light chain nucleic acid sequence is shown in SEQ ID NO.154;
the XY3 heavy chain nucleic acid sequence is shown in SEQ ID NO.155;
the XY3 light chain nucleic acid sequence is shown in SEQ ID NO.156;
the XY4 heavy chain nucleic acid sequence is shown in SEQ ID NO.157;
the XY4 light chain nucleic acid sequence is shown in SEQ ID NO.158;
the XY5 heavy chain nucleic acid sequence is shown in SEQ ID NO.159;
the XY5 light chain nucleic acid sequence is shown in SEQ ID NO.160;
the XY6 heavy chain nucleic acid sequence is shown in SEQ ID NO.161;
the XY6 light chain nucleic acid sequence is shown in SEQ ID NO.162;
the XY7 heavy chain nucleic acid sequence is shown in SEQ ID NO.163;
the XY7 light chain nucleic acid sequence is shown in SEQ ID NO.164;
the XY8 heavy chain nucleic acid sequence is shown in SEQ ID NO.165;
the XY8 light chain nucleic acid sequence is shown in SEQ ID NO.166;
the XY9 heavy chain nucleic acid sequence is shown in SEQ ID NO.167;
the XY9 light chain nucleic acid sequence is shown in SEQ ID NO.168;
the XY10 heavy chain nucleic acid sequence is shown in SEQ ID NO.169;
the XY10 light chain nucleic acid sequence is shown in SEQ ID NO.170;
the XY11 heavy chain nucleic acid sequence is shown in SEQ ID NO.171;
the XY11 light chain nucleic acid sequence is shown in SEQ ID NO.172;
the XY12 heavy chain nucleic acid sequence is shown in SEQ ID NO.173;
the XY12 light chain nucleic acid sequence is shown in SEQ ID NO.174;
the XY13 heavy chain nucleic acid sequence is shown in SEQ ID NO.175;
the XY13 light chain nucleic acid sequence is shown in SEQ ID NO.176;
the XY14 heavy chain nucleic acid sequence is shown in SEQ ID NO.177;
the XY14 light chain nucleic acid sequence is shown in SEQ ID NO.178;
the XY15 heavy chain nucleic acid sequence is shown in SEQ ID NO.179;
the XY15 light chain nucleic acid sequence is shown in SEQ ID NO.180.
Also included are nucleic acid sequences that share at least 80% identity therewith or nucleic acid sequences that contain degenerate codons that express the same amino acids.
The invention also provides a preparation for detecting, preventing or treating the novel coronavirus, which comprises the novel coronavirus neutralizing antibody.
The invention also provides the application of the novel coronavirus neutralizing antibody, which comprises any one or more of the following components;
(1) Used for preparing a novel coronavirus detection preparation;
(2) For the preparation of a formulation for the prevention of a novel coronavirus infection;
(3) Can be used for preparing preparations for treating novel coronavirus infection.
The invention obtains mononuclear cells from a Xinguan rehabilitator, extracts RNA, then reversely transcribes the RNA into cDNA, amplifies heavy chain and light chain of an antibody by combining a heavy chain variable region primer and a light chain variable region primer, and then assembles the heavy chain variable region and the light chain variable region primer on a phage vector to construct a SARS-Cov-2 Fab phage antibody library; using antigen S1 or RBD (RBD is a section of protein in S1 protein and plays a key role in binding with ACE 2), screening anti-Fab antibody, and sequencing to obtain heavy chain variable region and light chain variable region sequences capable of binding with S1 or RBD, wherein the heavy chain variable region is assembled with Fc fragment of IgG1, and the heavy chain and the light chain of the antibody are simultaneously expressed in mammalian cells to express fully humanized anti-RBD or S1 antibody. Through in vitro affinity determination and pseudovirus neutralization test, monoclonal antibody with strong affinity and virus neutralization capacity and capable of being used in SARS-Cov-2 detection, diagnosis, prevention or treatment is screened. Plays an active promoting role in preventing and treating the novel coronavirus.
Drawings
FIG. 1 is the antibody heavy chain variable region amplification electrophoresis band;
FIG. 2 shows the amplified electrophoretic band of kappa chain in the light chain variable region of the antibody;
FIG. 3 is an electrophoretic band of the lambda chain amplification of the variable region of the antibody light chain;
FIG. 4 is an electrophoretic band after fusion of the heavy and light chains of an antibody;
FIG. 5 is an electrophoretic band after SfiI enzyme digestion of pC3C plasmid;
FIG. 6 shows the result of colony PCR identification of the product after fusion of pC3C plasmid-linked antibody heavy chain and light chain, which is transformed into XL 1-Blue;
FIG. 7 is the ELISA assay of enriched phage antibody library of round 5;
helper phage (VCMS 13) was added to the control wells, and enriched phage was added to the experimental group in round 5;
FIG. 8 shows the result of PCR identification of monoclonal bacteria solution by adding RBD phage to XL 1-Blue;
FIG. 9 shows the expression of monoclonal antibodies and Coomassie blue staining;
FIG. 10 shows the result of ELISA assay for monoclonal antibodies;
no antibody was added to the control wells, but a secondary anti-human Fab-HRP antibody was added;
FIG. 11 is a monoclonal antibody IC50 assay;
FIG. 12 shows the detection of the additive neutralization effect of antibodies using XY1, XY4, XY7, XY10 combinations;
FIG. 13 is a combination of XY4 as the immobilized antibody and XY12, XY13, XY14, XY15 phage antibodies as the detection antibody to detect S1 protein; no phage antibody was added to the control, but anti-M13-HRP antibody was added;
FIG. 14 shows the detection of S1 protein using XY10 as the immobilized antibody and XY12, XY13, XY14, XY15 phage antibodies as the detection antibody combination. Control No phage antibody was added, but anti-M13-HRP antibody was added.
The specific implementation mode is as follows:
the invention is further described with reference to specific examples, which are not intended to be limiting.
The RBD protein of SARS-CoV-2 used in the examples of the present invention was purchased from Beijing Yi Qiao Shen Biotechnology Co., ltd, and S1 of SARS-Cov-2 was purchased from Beijing Baipu Sess Biotechnology Co., ltd.
The construction method of the phage antibody library of the invention is referred to as follows: dimitrov (ed.), generation and Selection of Rabbit Antibody Libraries by phase Display,2009, therapeutic μ tic Antibodies: methods and Protocols, vol.525, 101-128.
Example 1
1. Construction of phage antibody libraries
Obtaining 18 peripheral blood (blood sample from recovered patient within 1 month of recovery discharge) of SARS-Cov-2 patient using human peripheral blood lymph separation liquid to separate mononuclear cells. Extracting total RNA, mixing 18 parts of RNA in equal amount, carrying out reverse transcription to obtain cDNA, amplifying to obtain heavy chain variable region fragments and light chain variable region fragments by using a heavy chain variable region and light chain variable region doubling primer, randomly splicing the antibody heavy chain variable region fragments and light chain variable region fragments, connecting the antibody heavy chain variable region fragments and light chain variable region fragments with a phage vector, converting XL1-Blue bacteria (purchased from Toshiba Biotech Co., ltd.), and assembling into a phage antibody display library in the presence of a helper phage VSCM13 (purchased from NTCC national type culture Collection). The method specifically comprises the following steps:
1. monocyte isolation
10ml of peripheral blood was collected from 18 SARS-Cov-2 patients and mononuclear cells were isolated using human peripheral blood-lymph separation medium (LTS 1077-1, a tertiary ocean biologicals science and technology, inc.; ltd.).
(1) Blood collection: 10mL of peripheral blood of a blood donor is aseptically extracted, anticoagulant (30 u/mL of heparin) is added, and the mixture is subpackaged in 15mL centrifuge tubes.
(2) A15 mL centrifuge tube containing anticoagulated whole blood was set at 3000rpm for 10min, at acceleration of 7 and deceleration of 6.
(3) The upper plasma layer was aspirated and the lower cells were diluted with an equal volume of PBS.
(4) A50 mL centrifuge tube was first filled with the same amount of separation medium as the blood sample.
(5) Carefully aspirate the blood sample with a pipette onto the surface of the separation medium, 500-1100g (1800 rpm), centrifuge for 10min, accelerate 5, decelerate 4.
(6) After centrifugation, the centrifuge tube is divided into four layers from top to bottom; the second layer is a layer of annular milky white lymphocytes.
(7) The second layer of circular opalescent lymphocyte layer was carefully pipetted into a new 15mL centrifuge tube, 10mL of pbs was added to the resulting centrifuge tube and the cells were mixed.
(8) 1800rpm, centrifugation for 10min, acceleration 5, deceleration 4.
(9) The supernatant was discarded, 5ml of erythrocyte lysate was added to each 15ml centrifuge tube, slightly mixed and allowed to stand at room temperature for 5min.
(10) 1800rpm, 10min centrifugation, 5 acceleration, 4 deceleration.
(11) The supernatant was discarded and 10mL of PBS was aspirated to resuspend the resulting cells.
(12) 1800rpm, 10min centrifugation, 5 acceleration, 4 deceleration.
(13) The supernatant was discarded and precipitated as PBMC.
2. Total RNA extraction
Subjecting the obtained mononuclear cells to a reaction at 5X10 6 Total RNA was extracted by adding 0.75ml of Trizol (Thermo fisher) to each cell, and 18 portions of RNA were mixed in equal amounts each taking 1. Mu.g of RNA.
(1)5X10 6 0.75ml Tr was added to each cellizol。
(2) Mixing up and down for several times, and incubating for 5min on ice to ensure the cells to be fully cracked.
(3) Centrifuge at 12000rpm for 10 minutes at 4 ℃ and transfer the supernatant to a clean centrifuge tube.
(4) 0.2ml of chloroform was added to 1ml of Trizol, and the mixture was mixed well.
(5) Incubate on ice for 2-3min.
(6) Centrifuge at 12000rpm for 15 minutes at 4 ℃.
(7) Transfer the supernatant to a clean centrifuge tube.
(8) Adding isopropanol with the same volume into the supernatant and mixing uniformly.
(9) Incubate at room temperature for 10min, centrifuge at 12000rpm at 4 ℃ for 10-15 min.
(10) The supernatant was discarded and the precipitate was retained.
(11) The precipitate was washed by adding 1ml of 75% ethanol.
(12) Centrifuge at 12000rpm for 5 minutes at 4 ℃.
(13) The supernatant was discarded as much as possible.
(15) Drying at room temperature for 5-10min, and no liquid can be seen on the tube wall.
(16) Add 20-50 μ l of RNAse free water to re-dissolve RNA.
(17) Taking 1. Mu.l of RNA for electrophoresis detection and determining the concentration of the RNA.
(18) The RNA is subpackaged and stored in a refrigerator at the temperature of 80 ℃ below zero to avoid repeated freeze thawing.
3. Synthesis of cDNA
Mu.g of RNA was collected and used in a cDNA synthesis kit (K1612, thermo fisher). (1) cDNA was synthesized by taking 5. Mu.g of RNA.
(2) The 10. Mu.l RNA/primer mix. Mu.re system is shown in Table 1 below, and includes:
TABLE 1
System of ingredients Volume of
RNA 5μg
50μM Oligo(dT) 20 1μl
Hex Random Primer 1μl
10mM dNTP 1μl
DEPC-treated water Total volume 10. Mu.l
(3) Incubate at 65 ℃ for 5min, and place on ice for 1min.
(4) The cDNA Synthesis Mix was prepared for the next reaction by adding additional reagents as in Table 2 below.
TABLE 2
Components of the System 1 reaction
10xRT buffer 2μl
25mM MgCl 2 4μl
0.1M DTT 2μl
RNaseOΜT(40Μ/μl) 1μl
Superscript III RT(200Μ/μl) 1μl
(5) Add 10. Mu.l of cDNA Synthesis Mix to the RNA/primer Mix gently mixed, centrifuge, and react according to the following conditions in Table 3:
TABLE 3
Step1 50℃ 50min
Step2 85℃ 5min
Cooling on ice, and centrifuging briefly
Step3 RNaseH 1μl
Step4 37℃ 20min
(6) The cDNA products were stored in aliquots at-20 ℃.
4. Antibody heavy chain variable region (VH) and light chain variable region (VL) amplification
The synthesized cDNA was diluted 10-fold and used to amplify the templates for the heavy and light chain variable regions. (1) the system was formulated as in Table 4 below, using VH as an example:
TABLE 4
Figure GDA0003056007510000211
Figure GDA0003056007510000221
(2) The reaction conditions are shown in the following table
TABLE 5
Step1 98℃ 15s
Step2 98℃ 10s
Step3 55℃ 5s
Step4 72℃ 5s
Go to Step 2x34
Step5 72℃ 1m 30s
Step6
4℃ Forever
(3) Electrophoresis
Mu.l of the DNA was subjected to 1% agarose gel electrophoresis to determine whether the desired band was amplified and the size was about 400bp (see FIG. 1).
(4) All amplified VH's were mixed in equal amounts, added with 0.1 volume of 3M sodium acetate and 2.2 volumes of ethanol and mixed well, left overnight at-20 ℃.
(5) Centrifugation was carried out at 16000g for 15min at 4 ℃ and the supernatant removed, rinsed with 1ml of 70% ethanol (room temperature), dried at room temperature, redissolved in 200. Mu.l of water and gel-electrophoresed with 1% gelatin.
(6) The 400bp band was excised, recovered using QIAquick Gel Extraction Kit (QIAGEN, 28706), and the concentration was measured.
(7) VL was amplified and recovered according to the steps (1) to (6), and the results are shown in FIG. 2 and FIG. 3.
(8) The nucleic acid concentration after purification of VH and VL was adjusted to a final concentration of 100 ng/. Mu.l and stored at-20 ℃.
5. Amplification of Ckappa-pelB
(1) Amplification of Ckappa-pelB and CL-pelB Using vectors pC kappa and pCL (vectors from add gene) (100 ng/. Mu.l) as templates
For an example of amplification of C.kappa. -pelB, see Table 6.
TABLE 6
pCκ 10μl
HCK (sequence see the aforementioned references) 60μl
Pelb (sequence see the aforementioned references) 60μl
ddH 2 O 370μl
PrimerSTAR 500μl
Total 1000μl
(2) The reaction conditions are shown in Table 7
TABLE 7
Figure GDA0003056007510000231
Figure GDA0003056007510000241
(3) 10. Mu.l of the product was electrophoretically detected to give a band of about 400bp in size.
(4) All amplified C.kappa. -pelB were mixed, added with 0.1 volume of 3M sodium acetate and 2.2 volumes of ethanol, and mixed well, left overnight at-20 ℃.
(5) Centrifugation was carried out at 16000g for 15min at 4 ℃ and the supernatant removed, rinsed with 1ml of 70% ethanol (room temperature), dried at room temperature, redissolved in 200. Mu.l of water and subjected to gel electrophoresis using 1% gel.
(6) The 400bp band was excised, recovered using QIAquick Gel Extraction Kit (QIAGEN, 28706), and the concentration was measured.
(7) The purified C.kappa. -pelB was diluted to a final concentration of 100 ng/. Mu.l and stored at-20 ℃.
(8) CL-pelB was amplified and the template pC κ was replaced with pCL by performing the procedures according to steps (1) to (7).
6. human Vkappa/human Ckappa/CL/human VH or human V λ The example of a Human CL/Human VH fusion is a Human Vkappa/Human Ckappa/CL/Human VH fusion
(1) Mixing different amounts of VL, VH, ck-pelB according to Table 8 below
VL concentration 100 ng/. Mu.l, VH concentration 100 ng/. Mu.l, ckappa-pelB concentration 100 ng/. Mu.l
TABLE 8
VL 10μl
VH 10μl
Ck-pelB 10μl
Primerstar Mix 500μl
H 2 O 470μl
Total 1000μl
(2) The reaction conditions are shown in Table 9
TABLE 9
Step1 98℃ 15s
Step2 98℃ 10s
Step3
50℃ 5s
Step4 72℃ 5s
Step5 Go to Step 2x10
Step6 72℃ 1m 30s
Step7
4℃ Forever
(3) Amplification of human VL/human Ck/human VH, see Table 10, table 10
Figure GDA0003056007510000251
Figure GDA0003056007510000261
(4) The amplification conditions are shown in Table 11
TABLE 11
Step1 98℃ 15s
Step2 98℃ 10s
Step3 55℃ 5s
Step4 72℃ 5s(Go to Step2 x 39)
Step5 72℃ 1m 30s
Step6
4℃ Forever
(5) Electrophoretic detection
Taking 10 mu l of PCR product, detecting by electrophoresis, and fusing the target gene band with the size of 1.2Kb.
(the results are shown in FIG. 4)
(6) All amplified human VL/human Ck/human VH were mixed with 0.1 volume of 3M sodium acetate and 2.2 volumes of ethanol and mixed together overnight at-20 ℃.
(7) Centrifugation was carried out at 16000g for 15min at 4 ℃ and the supernatant removed, rinsed with 1ml of 70% ethanol (room temperature), dried at room temperature, redissolved in 200. Mu.l of water and subjected to gel electrophoresis using 1% gel.
(8) The 1.2Kb sized band was excised, recovered using QIAquick Gel Extraction Kit (QIAGEN, 28706), and the concentration was measured.
(9) Human VL/h. Mu.man Ck/Human VH purified nucleic acid was diluted to a final concentration of 150 ng/. Mu.l and stored at-20 ℃.
(10) The fusion of human V.lamda/human CL/human VH was performed according to steps (1) - (9).
7. SfiI enzyme digestion of human V kappa/human Ckappa/CL/human VH and human V λ /human CL/human VH
Taking the example of a human V kappa/human Ckappa/CL/human VH cleavage
(1) The enzyme digestion system is shown in Table 12
TABLE 12
human Vκ/human Cκ/CL/human VH(150ng/μl) 200μl
10Xbuffer 30μl
H 2 O 60μl
SfiI 40u/μl 10μl
(2) Water bath at 50 ℃ and enzyme digestion reaction for 3h.
(3) The Gel was electrophoresed on a 1% Gel, and a 1.2kb band was excised and collected using QIAquick Gel Extraction Kit (QIAGEN, 28706).
(4) The human VL/human Ck/human VH fragments were recovered after detection of SfiI cleavage, adjusted to a concentration of 50 ng/. Mu.l and stored at-20 ℃.
(5) The Human V.lamda/Human CL/Human VH was cleaved and the procedure was as in steps (1) to (4).
8. SfiI restriction enzyme pC3C (vector purchased from add gene)
(1) The enzyme digestion system is shown in Table 13
Watch 13
Figure GDA0003056007510000271
Figure GDA0003056007510000281
(2) Water bath at 50 ℃ and enzyme digestion reaction for 3h.
(3) Electrophoresis using a 1% gel will cut two bands, a 3.5Kb and a 1.2Kb sized fragment. (see FIG. 5 for results)
(4) A3.5 Kb vector backbone was recovered using the QIAquick Gel Extraction Kit (QIAGEN, 28706).
(5) After the enzyme digestion of Sfi, the concentration of the recovered carrier skeleton is adjusted to 100 ng/mul.
9. Connecting pC3C (SfiI) and Human VL/Human Ck/Human VH (SfiI) and Human V λ /human CL/human VH
Taking pC3C (SfiI) and human VL/human Ck/human VH (SfiI) as examples (1) the ligation system is shown in Table 14
TABLE 14
Figure GDA0003056007510000282
The control group is shown in Table 15
Watch 15
Figure GDA0003056007510000283
Figure GDA0003056007510000291
(2) The reaction was carried out at 16 ℃ overnight.
(3) All ligation reactions were combined and 1/10 volume of 3M sodium acetate solution was added.
(4) 2.2 times volume of precooled absolute ethyl alcohol is added, mixed evenly up and down, and precipitated at-20 ℃ overnight.
(5) 16000g, centrifuge at 4 deg.C for 30min, carefully discard the supernatant.
(6) Adding 70% ethanol, gently cleaning the precipitate, centrifuging at 16000g and 4 deg.C for 5min, and discarding the supernatant.
(7) Drying at room temperature.
(8) Dissolution with appropriate amount of water, typically 1 ligation reaction with 1. Mu.l volume of ddH 2 Dissolving O completely, and storing at-20 deg.C.
(9) The ligation of pC3C (SfiI) and Human V.lamda/Human CL/Human VH was performed according to the procedures (1) to (8).
10. Conversion of ligation product to XL1-Blue
(1) XL1-Blue shock-receptive ice is placed for 10min for dissolution.
(2) And adding 19 mu l of the precipitated and concentrated ligation product into a 1.5ml centrifuge tube, carrying out ice bath, adding 300 mu l of XL1-Blue shock competence, mixing uniformly, quickly transferring into a 2mm shock cup, and standing on ice for 1min.
(3) Electric shock condition: 2.5KV,4ms
(4) 5ml (1ml +2ml) SOC culture medium is rapidly added after the electric conversion is finished, and the SOC culture medium is transferred to a 50ml conical centrifuge tube and cultured for 1h at 37 ℃ and 250 rpm. Mu.l of the bacterial liquid is added into 198 mu.l of LB, and meanwhile, the bacterial liquid is evenly coated on an LB plate containing 100 mu g/mu.l of carbenicillin, and the bacterial liquid is placed at 37 ℃ for overnight culture and used for calculating the transformation efficiency and the colony PCR identification positive rate. The PCR results are shown in FIG. 6.
(5) 10ml of SB medium, 3. Mu.l of 100. Mu.g/. Mu.l carbenicillin, and 30. Mu.l of 5. Mu.g/. Mu.l tetracycline were added. Incubated at 37 ℃ and 250rpm for 1h.
(6) Add 4.5. Mu.l of 100. Mu.g/. Mu.l carbenicillin, continue at 37 ℃,250rpm, incubate for 1-4h.
(7) The cultured bacteria were transferred to a 500ml flask and added with 84ml of SB medium, 42.5. Mu.l of 100. Mu.g/. Mu.l carbenicillin, 170. Mu.l of 5. Mu.g/. Mu.l tetracycline, and 1ml of VCMS13 helper phage (10 ml) 11 -10 12 pfu/ml)，37℃，275rpm，90min。
(8) Mu.l of 50. Mu.g/. Mu.l kanamycin was added thereto, and cultured at 37 ℃ and 275rpm overnight.
(9) 3000g, centrifuged at 4 ℃ for 15min.
(10) Precipitated phages
The supernatant (200 ml) was transferred to a 500ml clean centrifuge tube, and 8g of PEG-8000 and 6g of NaCl were added, and the mixture was placed at 37 ℃ and 300rpm for 5min to promote dissolution. Standing on ice for 30min-1h.15000g, centrifuge at 4 ℃ for 15min. The supernatant was discarded, the flask was placed upside down on filter paper and allowed to dry for 10min, and excess liquid was carefully removed. The phage were resuspended in 2ml TBS containing 1% BSA (pipetter mix up and down), 16000g, centrifuged at 4 ℃ for 5min, and the supernatant was passed through a 0.22 μm filter (Millipore) and transferred to a 2ml centrifuge tube. The product can be directly stored on ice in a short time, or added with 2% sodium azide of 0.01 times of volume for storage at 4 ℃, and added with 1 time of volume of glycerol for long-time storage at-20 ℃.
2. Phage antibody library screening RBD phage antibody screening is exemplified
RBD-specific antibodies were identified by selecting single clones after 5 rounds of screening using RBD protein-coated 96-well plates.
1. Antigen coating
(1) RBD was added to a carbonate buffer at a concentration of 2. Mu.g/ml,
(2) Adding 50 mu l RBD into each well of a corning high protein adsorption enzyme-linked immunosorbent assay 96 pore plate, and standing overnight at 4 ℃;
(3) Discarding supernatant, adding 5% skimmed milk powder 200 μ l/well, standing at 37 deg.C for 1 hr;
(4) The supernatant was discarded and washed 5 times with 0.05% TBST.
2. RBD antibody screening
(1) Diluting the phage antibody library with 2% skimmed milk powder by 10 times, taking 2RBD antigen-coated 96-well micropores, adding 100 μ l diluted phage, and standing at 37 deg.C for 1h.
(2) Discard the supernatant, wash 5 times with 0.05% TBST, add 100. Mu.l 100mM glycine, stand at 37 ℃ for 15min, blow-beat continuously, add 9. Mu.l 1M Tris.
(3) And (3) adding the phage eluted in the step (2) into 2ml of XL1-Blue bacteria, and standing for 15min at room temperature.
(4) 6ml of SB medium was added, 1.6. Mu.l of 100. Mu.g/. Mu.l carbenicillin and 12. Mu.g of 5. Mu.g/. Mu.l tetracycline were added, and the mixture was incubated at 37 ℃ and 250rpm for 1 hour.
(5) Adding 2.4 μ l of carbenicillin 100 μ g/μ l, and culturing for 1 hr;
(6) Add 1ml of helper phage VCSM13 (10) 11 -10 12 pfu/ml), transferred to a 500ml flask, added with 91ml SB medium, added with 46. Mu.l of 100. Mu.g/. Mu.l carbenicillin, 184. Mu.l of 5. Mu.g/. Mu.l tetracycline, incubated at 37 ℃ for 1.5h at 250rpm, added with 140. Mu.l of 50. Mu.g/. Mu.l kanamycin, incubated at 37 ℃ overnight at 250 rpm.
(7) And (3) concentrating the phage antibody: centrifuging the bacterial liquid of the previous step to remove bacteria, and collecting supernatant (100 ml); 4g of PEG-8000 and 3g of NaCl were added thereto, and the mixture was heated at 37 ℃ and 300rpm for 5min to promote dissolution. Standing on ice for 30min-1h.15000g, centrifuge at 4 ℃ for 15min. The supernatant was discarded, the flask was placed upside down on filter paper and allowed to dry for 10min, and excess liquid was carefully removed. 2ml of TBS containing 1% BSA was used to resuspend the phage (pipetted up and down), 16000g, centrifuged at 4 ℃ for 5min, and the supernatant was passed through a 0.22 μm filter (Millipore) and transferred to a 2ml centrifuge tube. Can be directly stored on ice in a short time, or added with 0.01 time of 2 percent sodium azide for 4 ℃ and added with 1 time of glycerol for long-time storage for-20 ℃.
(8) And (5) taking the phage in the step (7), and performing 5 rounds of screening according to the steps (1) to (7).
(9) ELISA identification of five rounds of screened antibody libraries:
1) ELISA 96-well plate, each well coated with 100ng of RBD;
2) Adding 100 μ l phage (diluted 10 times with 2% skimmed milk powder) and incubating at 37 deg.C for 1h;
3) Washing 5 times with 0.05% TBST;
4) Adding 100 μ l of anti-M13-HRP antibody (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., ltd.) diluted 2000 times with 5% skimmed milk powder, and incubating at 37 deg.C for 1h;
5) Washing 5 times with 0.05% TBST;
6) Adding 100 μ l TMB, and developing in dark for 5min;
7) Add 50. Mu.l of 1M H 2 SO 4 The reaction was terminated.
8) Absorbance was measured at 450nm using a microplate reader.
The results are shown in FIG. 7.
3. Identification of RBD monoclonal antibody
(1) Taking the RBD phage screened in the 5 th round, diluting the RBD phage by 10- 7 Adding 1 μ l into 200 μ l XL1-Blue bacteria, standing at room temperature for 15min;
(2) Coating all the bacteria in the step (1) on an LB individual culture medium containing 100 mu g/ml carbenicillin, and culturing at 37 ℃ overnight;
(3) 200 monoclonals are selected and added into 6ml of liquid LB culture medium containing 10 mu g/ml tetracycline and 100 mu g/ml carbenicillin, and cultured at 37 ℃ and 250rpm for XY4 hours, a small amount of bacterial liquid is taken for PCR identification of the bacterial liquid, and the clone with the band size of 1200bp can be selected and amplified (the result is shown in figure 8).
The colony PCR amplification system is shown in Table 16:
TABLE 16
Bacterial liquid 0.5μl
VHSEQ (sequences see the aforementioned references) 0.2μl
vlseq (sequences see the aforementioned references) 0.2μl
ddH 2 O 4.1μl
PrimerSTAR 5μl
Total 10μl
Colony PCR reaction conditions are shown in table 17:
TABLE 17
Figure GDA0003056007510000331
Figure GDA0003056007510000341
(4) Uniformly dividing the positive clone bacterial liquid obtained in the step (3) into 2 parts, and adding one part into the mixture3 μ l of helper phage VCSM13 (10) 11 -10 12 pfu/ml), left at room temperature for 20min, 2.1. Mu.l of 50. Mu.g/. Mu.l kanamycin was added, and the other aliquot was incubated overnight at 37 ℃ and 250rpm without any substance;
(5) Centrifuging the bacteria added with the helper phage at 4000rpm for 10min, transferring the supernatant into a clean centrifuge tube, diluting the supernatant by 10 times, adding 100 mu l of the diluted supernatant into an enzyme-linked immunosorbent assay (ELISA) plate coated with RBD, incubating at 37 ℃ for 1h, washing 5 times with 0.05% TBST, adding 2000 times diluted antibody (purchased from Beijing Yinqiao Hibiscus biotechnology, inc.), incubating at 37 ℃ for 1h, washing 5 times with 0.05% TBST, adding 100 mu l TMB, developing for 2min, adding 50 mu l of 1M H, and adding 2 SO 4 The reaction is stopped, and the absorbance is measured at 450nm by using a microplate reader, and positive clones with the absorbance 2.1 times higher than that of the control are obtained.
(6) And (5) selecting the positive clones in the step (5), extracting plasmids of the bacteria without adding the helper phage, and sequencing to obtain sequences of the heavy chain variable region and the light chain variable region.
4. S1 phage antibody screening was performed according to steps 1-3.
3. Expression and activity identification of fully humanized antibody
1. Construction of fully humanized vector
(1) Designing corresponding primers according to the sequence of the heavy chain variable region or the light chain variable region of the obtained antibody, respectively amplifying, adding secretion signal peptides at the N ends of the heavy chain and the light chain, and adding an Fc fragment of IgG1 at the C end of the heavy chain;
(2) The heavy chain variable region and the light chain variable region were homologously recombined into pcDNA3.4 vector, respectively. (pcDNA3.4 vector was purchased from Wuhan \28156
2. Expression of fully humanized antibodies
(1) Extracting corresponding heavy chain expression vector and light chain expression vector respectively by using an endotoxin removal medium extraction kit (purchased from OMEGA);
(2) When the fusion level of 293T cells (purchased from Wuhan Punuoist Life technologies, inc.) reached 80%, transfection was performed using PEI;
(3) 12h after transfection, the medium is replaced by serum-free protein expression cultureBase, 37 ℃,5% CO 2 Culturing for 7 days;
(4) The antibody was purified using protein A/G packing. The results are shown in FIG. 9.
3. Identification of fully humanized antibody Activity
Exemplified by RBD antibody Activity
(1) ELISA identification
The obtained antibody was adjusted to a concentration of 1mg/ml, diluted 2000-fold with 5% skim milk powder, added to an enzyme-linked immunosorbent assay well coated with RBD antigen, incubated at 37 ℃ for 1H, washed 5 times with 0.05% TBST, diluted 2000-fold anti-human Fab-HRP secondary antibody (purchased from Beijing Solebao Biotech Co., ltd.) with 5% skim milk powder, incubated at 37 ℃ for 1H, washed 5 times with 0.05% TBST, added with 100. Mu.l TMB, developed for 2min, and added with 50. Mu.l 1M H 2 SO 4 The reaction was terminated and absorbance was measured at 450nm using a microplate reader. The results are shown in FIG. 10.
(2) Fully humanized antibody pseudovirus neutralization assay
1) Mixing 200 TCID50 pseudoviruses expressing SARS-Cov-2S protein with a series of antibodies with different dilution concentrations in equal amount, adding the mixture into a 96-well cell culture plate with the total volume of 100 mu l, incubating the mixture at 37 ℃ for 1h, and adding 100 mu l 293T cells containing 20000 over-expressed ACE 2; control without antibody, equivalent pseudovirus was mixed with ACE2 overexpressing 293T; mixing background equivalent pseudoviruses with 293T cells; 5% CO at 37 ℃ 2 After standing for 48h, the fluorescence intensity was measured. Inhibition efficiency = (experimental group-background)/(control-background) × 100%.
The results are shown in FIG. 11.
2) The combination of fully humanized antibodies XY1, XY4, XY7 and XY10 is used for detecting the antibody superposition neutralization effect as follows:
200 TCID50 pseudoviruses expressing SARS-Cov-2S protein are mixed with a series of antibodies with different dilution concentrations in equal volume (single antibody concentration is 0.009766 mu g/ml; when two antibodies are combined, the equal volume is mixed, the total concentration is 0.009766 mu g/ml), the total volume is 100 mu l, the pseudoviruses are added into a 96-well cell culture plate, the culture plate is incubated at 37 ℃ for 1h, and 100 mu l of 293T cells containing 20000 over-expressed ACE2 are added; control without antibody, equivalent pseudovirus was mixed with ACE2 overexpressing 293T; background of the inventionMixing the same amount of pseudovirus and 293T cells; 37 ℃ and 5% CO 2 After standing for 48h, the fluorescence intensity was measured. Inhibition efficiency = (experimental group-background)/(control-background) × 100%.
The results are shown in FIG. 12.
(3) The S1 protein is detected by using XY4 as a fixed antibody and XY12, XY13, XY14 and XY15 as detection antibody combinations as follows:
the detection is carried out by adopting an ELISA sandwich method, a monoclonal fully humanized antibody XY4 is coated on an ELISA plate, SARS-Cov-2 antigen S1 is added, XY12, XY13, XY14 and XY15 phage antibodies are added, different combinations are carried out, an antibody with HRP mark anti-M13 is added, the detection color development is carried out, the darker the color is, the stronger the SARS-Cov-2 antigen capture capability is, and meanwhile, the pair of antibodies is suitable for the detection of SARS-Cov antigen.
The specific operation is as follows:
1) ELISA 96-well plates coated with 100ng of XY4 fully humanized antibody per well;
2) Add 100. Mu.l S1 (10 pg/ml), incubate 1h at 37 ℃;
3) Washing 5 times with 0.05% TBST;
4) Adding phage antibody diluted by 10 times with 2% skimmed milk powder, and incubating at 37 deg.C for 1h;
5) Washing 5 times with 0.05% TBST;
6) Adding 2000-fold diluted anti-M13-HRP antibody (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., ltd.) with 5% skimmed milk powder, and incubating at 37 deg.C for 1h;
7) Washing 5 times with 0.05% TBST;
8) Adding 100 μ l TMB, and developing in dark for 5min;
9) Add 50. Mu.l of 1M H 2 SO 4 The reaction was terminated.
10 Absorbance at 450nm using a microplate reader.
The results are shown in FIG. 13.
(4) The S1 protein is detected by using a fully humanized XY10 as an immobilized antibody and XY12, XY13, XY14 and XY15 as detection antibody combinations as follows:
the detection is carried out by adopting an ELISA sandwich method, a monoclonal fully humanized antibody XY10 is coated on an ELISA plate, SARS-Cov-2 antigen S1 is added, XY12, XY13, XY14 and XY15 phage antibodies are added, different combinations are carried out, an antibody with HRP mark anti-M13 is added, the detection color development is carried out, the darker the color is, the stronger the SARS-Cov-2 antigen capture capability is, and meanwhile, the pair of antibodies is suitable for the detection of SARS-Cov antigen.
The specific operation is as follows:
1) ELISA 96-well plates coated with 100ng of XY10 fully humanized antibody per well;
2) Adding 100. Mu.l S1 (10 pg/ml), incubating at 37 ℃ for 1h;
3) Washing 5 times with 0.05% TBST;
4) Adding phage antibody diluted by 10 times with 2% skimmed milk powder, and incubating at 37 deg.C for 1h;
5) Washing 5 times with 0.05% TBST;
6) Adding anti-M13-HRP antibody (purchased from Beijing Yinqiao Shenzhou Biotechnology Co., ltd.) diluted 2000 times with 5% skimmed milk powder, and incubating at 37 deg.C for 1h;
7) Washing 5 times with 0.05% TBST;
8) Adding 100 μ l TMB, and developing in dark for 5min;
9) Add 50. Mu.l of 1M H 2 SO 4 The reaction was terminated.
10 Absorbance at 450nm using a microplate reader.
The results are shown in FIG. 14.
(5) Fully humanized antibody affinity assay
Measurement of antibody affinity the assay was performed using BIAcore 8k (BIAcore, cytiva) and antibody diluted to a concentration of 20. Mu.g/ml injected at a flow rate of 10. Mu.l/min for a period of 20s, giving a response of 1000R μm. Binding affinity to SARS-CoV-2RBD protein was measured at a flow rate of 50. Mu.L/min and an injection time of 2 minutes. The affinity results are shown in Table 18.
TABLE 18 antibody affinity assay
Figure GDA0003056007510000381
Figure GDA0003056007510000391
(6) Antibody combination detection of RBD of SARS-Cov-2
The detection is carried out by adopting an ELISA sandwich method, monoclonal fully-humanized antibodies XY1, XY2, XY3, XY4, XY5, XY6, XY7, XY8, XY9, XY10 and XY11 coat an ELISA plate, SARS-Cov-2 antigen RBD is added, phage antibodies XY1, XY2, XY3, XY4, XY5, XY6, XY7, XY8, XY9, XY10 and XY11 are added for different combinations, then an antibody with HRP-marked anti-M13 is added, the color development is detected, the darker the color indicates that the capturing capability of the SARS-Cov-2 antigen is stronger, and the pair of antibodies is suitable for detecting SARS-Cov antigen. The results are shown in Table 19.
The specific operation is as follows:
1) ELISA 96-well plates coated with 100ng of fully humanized antibody per well;
2) Add 100. Mu.l antigen (10 pg/ml), incubate 1h at 37 ℃;
3) Washing 5 times with 0.05% TBST;
4) Adding phage antibody diluted by 10 times with 2% skimmed milk powder, and incubating at 37 deg.C for 1h;
5) Washing 5 times with 0.05% TBST;
6) Adding 2000-fold diluted anti-M13-HRP antibody (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., ltd.) with 5% skimmed milk powder, and incubating at 37 deg.C for 1h;
7) Adding 100 μ l TMB, and developing in dark for 5min;
8) Add 50. Mu.l of 1M H 2 SO 4 The reaction was terminated.
9) Absorbance was measured at 450nm using a microplate reader.
10 Detection of S1 antigen was performed according to steps (1) to (9).
In the following table 19, the column is a monoclonal fully humanized antibody, the column is a phage antibody, and the heavy chain variable region and the light chain variable region of the phage antibody and the full antibody are the same, but the same antibody has different forms, and the combination of underlined data in the following table shows more excellent effects.
TABLE 19 detection of RBD proteins by cross-combination of different monoclonal antibodies
XY1 XY2 XY3 XY4 XY5 XY6 XY7 XY8 XY9 XY10 XY11 Control of
XY1 0.8848 0.7326 3.497333 0.7735 0.475067 1.022033 1.0165 1.800633 3.002033 1.066633 0.07912
XY2 0.6635 0.7934 3.561467 0.551133 0.2924 0.631633 0.847033 1.0656 2.019467 0.958733 0.05423
XY3 0.3361 0.59015 3.0677 0.33515 0.17445 0.369 0.4125 0.5762 1.27265 0.64975 0.06881
XY4 0.8688 1.1878 0.90005 0.60735 0.3471 0.9918 1.03855 0.83135 2.4142 0.90515 0.05987
XY5 0.56505 0.79005 0.7313 2.83085 0.24635 0.69735 0.52915 0.7721 1.7483 0.6084 0.07762
XY6 0.42205 0.55845 0.54215 2.7278 0.4206 0.5086 0.48525 0.7785 1.3035 0.702 0.06931
XY7 0.398 0.73475 0.9844 3.1126 0.4679 0.1885 0.41515 0.92155 1.0476 0.6443 0.05542
XY8 0.37835 0.46405 0.5455 3.0873 0.50125 0.24975 0.4681 0.7979 1.71195 0.68565 0.08339
XY9 0.5695 0.5438 0.4639 2.3447 0.3912 0.206 0.6099 0.45535 1.32155 0.52985 0.06871
XY10 0.7053 0.75605 0.75415 3.2243 0.47525 0.17805 0.54015 0.5249 0.45935 0.5754 0.05324
XY11 0.27535 0.4699 0.4894 2.7212 0.27635 0.13865 0.3255 0.4718 0.69185 0.92985 0.04996
The column is the coating antibody, the row is the detection antibody, and the blank is not added with the phage antibody, but with the helper phage VCSM13 and the anti-M13-HRP antibody.
Through the series of activity identification, the monoclonal fully-humanized antibodies XY1, XY2, XY3, XY4, XY5, XY6, XY7, XY8, XY9, XY10, XY11, XY12, XY13, XY14, XY15, 15 novel coronavirus antibodies are obtained. The amino acid and nucleic acid sequences of the heavy chain and the light chain can be seen in a sequence table.
Sequence listing
<110> Hunan ya Hospital of Zhongnan university
<120> novel coronavirus monoclonal antibody and application thereof
<160> 180
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 1
Glu Asp Thr Phe Thr Ser His Tyr
1 5
<210> 2
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 2
Ile Asn Pro Thr Gly Gly Ser Ile
1 5
<210> 3
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 3
Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val
1 5 10 15
<210> 4
<211> 127
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 4
Gly Ser Thr Gly Asp Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
1 5 10 15
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp
20 25 30
Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln
35 40 45
Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser
50 55 60
Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser
65 70 75 80
Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
85 90 95
Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala
100 105 110
Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 5
<211> 6
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 5
Gln Gly Ile Arg Asn Ser
1 5
<210> 6
<211> 3
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 6
Asp Ala Ser
1
<210> 7
<211> 9
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 7
Gln His Tyr Phe Gly Thr Pro Leu Thr
1 5
<210> 8
<211> 113
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 8
Gly Ser Thr Gly Asp Ala Glu Ile Val Met Thr Gln Ser Pro Ser Ser
1 5 10 15
Leu Ser Ala Ser Glu Gly Asp Arg Val Ile Ile Thr Cys Arg Ala Ser
20 25 30
Gln Gly Ile Arg Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
35 40 45
Ala Pro Lys Leu Leu Leu Tyr Asp Ala Ser Lys Leu Glu Ser Gly Val
50 55 60
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr
65 70 75 80
Ile Asp Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His
85 90 95
Tyr Phe Gly Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
100 105 110
Lys
<210> 9
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 9
Glu Asp Thr Phe Thr Ser His Tyr
1 5
<210> 10
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 10
Ile Asn Pro Thr Gly Gly Ser Ile
1 5
<210> 11
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 11
Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val
1 5 10 15
<210> 12
<211> 127
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 12
Gly Ser Thr Gly Asp Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val
1 5 10 15
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp
20 25 30
Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln
35 40 45
Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser
50 55 60
Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser
65 70 75 80
Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
85 90 95
Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala
100 105 110
Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 13
<211> 6
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 13
Gln Thr Ile Ser Lys Tyr
1 5
<210> 14
<211> 3
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 14
Glu Ala Ser
1
<210> 15
<211> 9
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 15
Gln Gln Ser Tyr Ser Ser Arg Phe Thr
1 5
<210> 16
<211> 113
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 16
Gly Ser Thr Gly Asp Ala Ala Ile Arg Leu Thr Gln Ser Pro Ser Ser
1 5 10 15
Leu Ser Ala Ser Val Gly Asp Thr Val Thr Ile Thr Cys Arg Ala Ser
20 25 30
Gln Thr Ile Ser Lys Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Glu
35 40 45
Ala Pro Lys Leu Leu Ile Ser Glu Ala Ser Thr Phe Gln Gly Gly Val
50 55 60
Ser Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Tyr Ser Leu Gln Pro Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln
85 90 95
Ser Tyr Ser Ser Arg Phe Thr Phe Gly Pro Gly Thr Lys Val Glu Ile
100 105 110
Lys
<210> 17
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 17
Glu Asp Thr Phe Thr Ser His Tyr
1 5
<210> 18
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 18
Ile Asn Pro Thr Gly Gly Ser Ile
1 5
<210> 19
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 19
Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val
1 5 10 15
<210> 20
<211> 127
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 20
Gly Ser Thr Gly Asp Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val
1 5 10 15
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp
20 25 30
Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln
35 40 45
Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser
50 55 60
Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser
65 70 75 80
Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
85 90 95
Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala
100 105 110
Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 21
<211> 6
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 21
Gln Gly Ile Ser Ser Trp
1 5
<210> 22
<211> 3
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 22
Ala Ala Ser
1
<210> 23
<211> 9
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 23
Gln Gln Ala Asn Ser Phe Pro Leu Thr
1 5
<210> 24
<211> 113
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 24
Gly Ser Thr Gly Asp Ala Ala Ile Gln Met Thr Gln Ser Pro Ser Ser
1 5 10 15
Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
20 25 30
Gln Gly Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
35 40 45
Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
50 55 60
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
85 90 95
Ala Asn Ser Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 25
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 25
Gly Gly Thr Phe Ser Ser Ile Ala
1 5
<210> 26
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 26
Ile Ile Pro Ile Phe Gly Thr Ala
1 5
<210> 27
<211> 14
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 27
Ala Arg Asp Val Ile Glu Ala Thr Ile Tyr Gly Met Asp Val
1 5 10
<210> 28
<211> 126
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 28
Gly Ser Thr Gly Asp Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
1 5 10 15
Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly
20 25 30
Thr Phe Ser Ser Ile Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln
35 40 45
Gly Leu Ala Trp Met Gly Lys Ile Ile Pro Ile Phe Gly Thr Ala Asn
50 55 60
Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Ala Asp Glu Ser
65 70 75 80
Thr Asn Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
85 90 95
Ala Val Tyr Tyr Cys Ala Arg Asp Val Ile Glu Ala Thr Ile Tyr Gly
100 105 110
Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 29
<211> 6
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 29
Gln Ser Ile Ser Thr Tyr
1 5
<210> 30
<211> 3
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 30
Gly Ala Ser
1
<210> 31
<211> 9
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 31
Gln Gln Ser Tyr Ser Ala Pro Tyr Thr
1 5
<210> 32
<211> 113
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 32
Gly Ser Thr Gly Asp Ala Asp Ile Val Met Thr Gln Ser Pro Ser Ser
1 5 10 15
Leu Pro Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Thr Ser
20 25 30
Gln Ser Ile Ser Thr Tyr Val Asn Trp Tyr Gln Gln Lys Ser Gly Asn
35 40 45
Ala Pro Glu Leu Leu Met Tyr Gly Ala Ser Ile Leu Gln Ser Gly Val
50 55 60
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
85 90 95
Ser Tyr Ser Ala Pro Tyr Thr Phe Ala Gln Gly Thr Lys Leu Glu Ile
100 105 110
Arg
<210> 33
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 33
Glu Asp Thr Phe Thr Ser His Tyr
1 5
<210> 34
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 34
Ile Asn Pro Thr Gly Gly Ser Ile
1 5
<210> 35
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 35
Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val
1 5 10 15
<210> 36
<211> 127
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 36
Gly Ser Thr Gly Asp Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
1 5 10 15
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp
20 25 30
Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln
35 40 45
Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser
50 55 60
Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser
65 70 75 80
Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
85 90 95
Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala
100 105 110
Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 37
<211> 6
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 37
Gln Gly Val Ser Asn Tyr
1 5
<210> 38
<211> 3
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 38
Ala Ala Ser
1
<210> 39
<211> 9
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 39
Gln His Tyr Asp Ser Pro Pro Tyr Thr
1 5
<210> 40
<211> 113
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 40
Gly Ser Thr Gly Asp Ala Val Ile Trp Met Thr Gln Ser Pro Ser Ser
1 5 10 15
Leu Ser Ala Ser Met Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
20 25 30
Gln Gly Val Ser Asn Tyr Leu Ala Trp Tyr Gln His Lys Pro Gly Lys
35 40 45
Ala Pro Glu Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val
50 55 60
Pro Ser Arg Phe Ser Ala Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His
85 90 95
Tyr Asp Ser Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Val
100 105 110
Lys
<210> 41
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 41
Glu Asp Thr Phe Thr Ser His Tyr
1 5
<210> 42
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 42
Ile Asn Pro Thr Gly Gly Ser Ile
1 5
<210> 43
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 43
Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val
1 5 10 15
<210> 44
<211> 127
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 44
Gly Ser Thr Gly Asp Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
1 5 10 15
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp
20 25 30
Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln
35 40 45
Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser
50 55 60
Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser
65 70 75 80
Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
85 90 95
Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala
100 105 110
Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 45
<211> 6
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 45
Ala Leu Ala Lys His Phe
1 5
<210> 46
<211> 3
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 46
Lys Asp Thr
1
<210> 47
<211> 9
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 47
Gln Ser Pro Asp Thr Thr Gly Arg Ile
1 5
<210> 48
<211> 117
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 48
Gly Ser Thr Gly Asp Ala Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val
1 5 10 15
Ser Val Ser Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala
20 25 30
Leu Ala Lys His Phe Gly His Trp Tyr Gln Gln Arg Pro Gly Gln Ala
35 40 45
Pro Val Leu Val Ile Tyr Lys Asp Thr Glu Arg Pro Leu Gly Ile Pro
50 55 60
Glu Arg Phe Ser Gly Ser Ser Ser Gly Ala Thr Val Thr Leu Thr Ile
65 70 75 80
Ser Ala Val Glu Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Pro
85 90 95
Asp Thr Thr Gly Arg Ile Phe Gly Gly Gly Thr Lys Val Thr Val Leu
100 105 110
Gly Gln Pro Lys Ala
115
<210> 49
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 49
Glu Asp Thr Phe Thr Ser His Tyr
1 5
<210> 50
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 50
Ile Asn Pro Thr Gly Gly Ser Ile
1 5
<210> 51
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 51
Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val
1 5 10 15
<210> 52
<211> 127
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 52
Gly Ser Thr Gly Asp Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
1 5 10 15
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp
20 25 30
Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln
35 40 45
Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser
50 55 60
Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser
65 70 75 80
Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
85 90 95
Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala
100 105 110
Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 53
<211> 6
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 53
Gln Gly Ile Ser Ser Trp
1 5
<210> 54
<211> 3
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 54
Ala Ala Ser
1
<210> 55
<211> 9
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 55
Gln Gln Ser Tyr Ser Ile Pro Arg Thr
1 5
<210> 56
<211> 113
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 56
Gly Ser Thr Gly Asp Ala Ala Ile Arg Leu Thr Gln Ser Pro Ser Ser
1 5 10 15
Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
20 25 30
Gln Gly Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
35 40 45
Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
50 55 60
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
85 90 95
Ser Tyr Ser Ile Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 57
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 57
Glu Asp Thr Phe Thr Ser His Tyr
1 5
<210> 58
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 58
Ile Asn Pro Thr Gly Gly Ser Ile
1 5
<210> 59
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 59
Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val
1 5 10 15
<210> 60
<211> 122
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 60
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp Thr Phe Thr Ser His
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val Trp
100 105 110
Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 61
<211> 6
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 61
Gln Asp Ile Ser Asp Trp
1 5
<210> 62
<211> 3
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 62
Arg Ala Val
1
<210> 63
<211> 9
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 63
Gln Gln Thr Asn Thr Phe Pro Ile Thr
1 5
<210> 64
<211> 120
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 64
Gly Ser Thr Gly Asp Ala Val Ile Trp Met Thr Gln Ser Pro Ile Gln
1 5 10 15
Met Thr Gln Ser Pro Ser Ser Val Ser Ala Tyr Val Gly Asp Arg Val
20 25 30
Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asp Trp Leu Ala Trp
35 40 45
Tyr Gln Gln Ala Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Arg Ala
50 55 60
Val Thr Leu Gln Asp Asp Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
65 70 75 80
Gly Thr Asp Phe Ser Leu Thr Ile Thr Gly Leu Gln Arg Glu Asp Phe
85 90 95
Ala Thr Tyr Tyr Cys Gln Gln Thr Asn Thr Phe Pro Ile Thr Phe Gly
100 105 110
His Gly Thr Arg Leu Glu Ile Lys
115 120
<210> 65
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 65
Glu Asp Thr Phe Thr Ser His Tyr
1 5
<210> 66
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 66
Ile Asn Pro Thr Gly Gly Ser Ile
1 5
<210> 67
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 67
Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val
1 5 10 15
<210> 68
<211> 127
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 68
Gly Ser Thr Gly Asp Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
1 5 10 15
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp
20 25 30
Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln
35 40 45
Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser
50 55 60
Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser
65 70 75 80
Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
85 90 95
Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala
100 105 110
Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 69
<211> 9
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 69
Ser Ser Asp Val Gly Ser Tyr Asn Leu
1 5
<210> 70
<211> 3
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 70
Glu Val Thr
1
<210> 71
<211> 10
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 71
Ile Ser Tyr Ala Gly Asn Asn Asn Leu Val
1 5 10
<210> 72
<211> 121
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 72
Gly Ser Thr Gly Asp Ala Gln Ser Ala Leu Thr Gln Pro Pro Ser Val
1 5 10 15
Ser Gly Ala Pro Gly Gln Thr Val Thr Ile Ser Cys Thr Gly Thr Ser
20 25 30
Ser Asp Val Gly Ser Tyr Asn Leu Val Ser Trp Tyr Gln Gln His Pro
35 40 45
Gly Lys Ala Pro Lys Leu Ile Ile Ile Glu Val Thr Lys Arg Pro Pro
50 55 60
Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser
65 70 75 80
Leu Thr Val Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr His Cys
85 90 95
Ile Ser Tyr Ala Gly Asn Asn Asn Leu Val Phe Gly Gly Gly Thr Gln
100 105 110
Leu Thr Val Leu Gly Gln Pro Lys Ala
115 120
<210> 73
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 73
Glu Asp Thr Phe Thr Ser His Tyr
1 5
<210> 74
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 74
Ile Asn Pro Thr Gly Gly Ser Ile
1 5
<210> 75
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 75
Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val
1 5 10 15
<210> 76
<211> 127
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 76
Gly Ser Thr Gly Asp Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
1 5 10 15
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp
20 25 30
Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln
35 40 45
Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser
50 55 60
Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser
65 70 75 80
Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
85 90 95
Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala
100 105 110
Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 77
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 77
Ser Ser Asp Ile Gly Arg Ser Ser
1 5
<210> 78
<211> 3
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 78
Arg Asn Asn
1
<210> 79
<211> 11
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 79
Ala Ala Trp Asp Asn Thr Leu Arg Gly Tyr Val
1 5 10
<210> 80
<211> 121
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 80
Gly Ser Thr Gly Asp Ala Ser Tyr Glu Leu Thr Gln Leu Pro Ser Ala
1 5 10 15
Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser
20 25 30
Ser Asp Ile Gly Arg Ser Ser Val Asn Trp Tyr Gln Gln Leu Pro Gly
35 40 45
Thr Ala Pro Lys Leu Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly
50 55 60
Val Pro Asp Arg Leu Ser Gly Ser Lys Ser Gly Thr Ser Gly Ser Leu
65 70 75 80
Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala
85 90 95
Ala Trp Asp Asn Thr Leu Arg Gly Tyr Val Phe Gly Thr Gly Thr Lys
100 105 110
Val Thr Val Leu Gly Gln Pro Lys Ala
115 120
<210> 81
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 81
Glu Asp Thr Phe Thr Ser His Tyr
1 5
<210> 82
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 82
Ile Asn Pro Thr Gly Gly Ser Ile
1 5
<210> 83
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 83
Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val
1 5 10 15
<210> 84
<211> 127
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 84
Gly Ser Thr Gly Asp Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
1 5 10 15
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp
20 25 30
Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln
35 40 45
Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser
50 55 60
Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser
65 70 75 80
Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
85 90 95
Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala
100 105 110
Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 85
<211> 12
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 85
Gln Ser Val Leu Phe Ser Pro Asn Asn Lys Asn Tyr
1 5 10
<210> 86
<211> 3
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 86
Trp Ala Ser
1
<210> 87
<211> 9
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 87
Gln Gln Tyr Asp Ser Ser Pro Trp Thr
1 5
<210> 88
<211> 119
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 88
Gly Ser Thr Gly Asp Ala Asp Ile Gln Leu Thr Gln Ser Pro Asp Ser
1 5 10 15
Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser
20 25 30
Gln Ser Val Leu Phe Ser Pro Asn Asn Lys Asn Tyr Leu Ala Trp Tyr
35 40 45
Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser
50 55 60
Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
65 70 75 80
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala
85 90 95
Val Tyr Tyr Cys Gln Gln Tyr Asp Ser Ser Pro Trp Thr Phe Gly Gln
100 105 110
Gly Thr Lys Val Glu Ile Lys
115
<210> 89
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 89
Glu Asp Thr Phe Thr Ser His Tyr
1 5
<210> 90
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 90
Ile Asn Pro Thr Gly Gly Ser Ile
1 5
<210> 91
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 91
Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val
1 5 10 15
<210> 92
<211> 127
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 92
Gly Ser Thr Gly Asp Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
1 5 10 15
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp
20 25 30
Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln
35 40 45
Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser
50 55 60
Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser
65 70 75 80
Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
85 90 95
Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala
100 105 110
Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 93
<211> 7
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 93
His Asn Val Asn Arg Asp Tyr
1 5
<210> 94
<211> 3
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 94
Gly Ala Ser
1
<210> 95
<211> 10
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 95
Gln His Phe Ser Gly Thr Pro Arg Leu Thr
1 5 10
<210> 96
<211> 115
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 96
Gly Ser Thr Gly Asp Ala Glu Thr Thr Leu Thr Gln Ser Pro Gly Thr
1 5 10 15
Leu Ser Leu Ser Pro Gly Asp Arg Val Thr Leu Ser Cys Arg Ala Ser
20 25 30
His Asn Val Asn Arg Asp Tyr Val Ala Trp Tyr Gln Gln Arg Arg Gly
35 40 45
Gln Ala Pro Arg Leu Leu Ile Ser Gly Ala Ser Lys Arg Ala Ala Gly
50 55 60
Val Pro Asp Arg Phe Thr Gly Ala Gly Ser Gly Ser Glu Phe Thr Leu
65 70 75 80
Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Gly Val Tyr Phe Cys Gln
85 90 95
His Phe Ser Gly Thr Pro Arg Leu Thr Phe Gly Gly Gly Thr Asn Val
100 105 110
Glu Met Gln
115
<210> 97
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 97
Glu Asp Thr Phe Thr Ser His Tyr
1 5
<210> 98
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 98
Ile Asn Pro Thr Gly Gly Ser Ile
1 5
<210> 99
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 99
Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val
1 5 10 15
<210> 100
<211> 127
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 100
Gly Ser Thr Gly Asp Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
1 5 10 15
Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp
20 25 30
Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln
35 40 45
Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser
50 55 60
Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser
65 70 75 80
Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
85 90 95
Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala
100 105 110
Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 101
<211> 6
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 101
His Asp Ile Ser Thr Phe
1 5
<210> 102
<211> 3
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 102
Asp Ala Ser
1
<210> 103
<211> 9
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 103
Gln Gln Arg Arg His Tyr Pro Val Thr
1 5
<210> 104
<211> 113
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 104
Gly Ser Thr Gly Asp Ala Ala Ile Gln Met Thr Gln Ser Pro Ser Phe
1 5 10 15
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
20 25 30
His Asp Ile Ser Thr Phe Leu Gly Trp Tyr Gln Lys Lys Pro Gly Lys
35 40 45
Pro Pro Lys Leu Leu Val Tyr Asp Ala Ser Thr Leu Gln Ser Gly Val
50 55 60
Pro Ser Arg Phe Arg Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Pro Glu Asp Phe Gly Ser Tyr Tyr Cys Gln Gln
85 90 95
Arg Arg His Tyr Pro Val Thr Phe Gly Gln Gly Thr Gly Leu Glu Ile
100 105 110
Lys
<210> 105
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 105
Glu Asp Thr Phe Thr Ser His Tyr
1 5
<210> 106
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 106
Ile Asn Pro Thr Gly Gly Ser Ile
1 5
<210> 107
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 107
Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val
1 5 10 15
<210> 108
<211> 127
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 108
Gly Ser Thr Gly Asp Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu
1 5 10 15
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp
20 25 30
Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln
35 40 45
Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser
50 55 60
Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser
65 70 75 80
Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
85 90 95
Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala
100 105 110
Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 109
<211> 6
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 109
Asn Ile Gly Ser Lys Asn
1 5
<210> 110
<211> 3
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 110
Arg Asp Ser
1
<210> 111
<211> 11
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 111
Gln Val Trp Asp Ser Ser Thr Ala Asp Val Val
1 5 10
<210> 112
<211> 119
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 112
Gly Ser Thr Gly Asp Ala Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val
1 5 10 15
Ser Val Ala Leu Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn
20 25 30
Ile Gly Ser Lys Asn Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala
35 40 45
Pro Val Leu Val Ile Tyr Arg Asp Ser Asn Arg Pro Ser Gly Ile Pro
50 55 60
Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile
65 70 75 80
Ser Arg Ala Gln Ala Gly Asp Glu Gly Asp Tyr Tyr Cys Gln Val Trp
85 90 95
Asp Ser Ser Thr Ala Asp Val Val Phe Gly Gly Gly Thr Gln Leu Thr
100 105 110
Val Leu Gly Gln Pro Lys Ala
115
<210> 113
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 113
Glu Asp Thr Phe Thr Ser His Tyr
1 5
<210> 114
<211> 8
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 114
Ile Asn Pro Thr Gly Gly Ser Ile
1 5
<210> 115
<211> 15
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 115
Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala Pro Met Asp Val
1 5 10 15
<210> 116
<211> 127
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 116
Gly Ser Thr Gly Asp Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu
1 5 10 15
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Arg Ala Ser Glu Asp
20 25 30
Thr Phe Thr Ser His Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln
35 40 45
Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Thr Gly Gly Ser Ile Ser
50 55 60
Tyr Ala Gln Lys Phe Gln Gly Arg Val Ala Met Thr Lys Asp Thr Ser
65 70 75 80
Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
85 90 95
Ala Val Tyr Tyr Cys Ala Arg Gly Gly Phe Thr Pro Asp Thr Ser Ala
100 105 110
Pro Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 117
<211> 6
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 117
Glu Ser Val Ser Ser Asn
1 5
<210> 118
<211> 3
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 118
Asp Ala Ser
1
<210> 119
<211> 9
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 119
His His Tyr Gly Ser Ser Pro Arg Pro
1 5
<210> 120
<211> 113
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 120
Gly Ser Thr Gly Asp Ala Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
1 5 10 15
Leu Ser Leu Ser Pro Gly Glu Thr Ala Thr Leu Ser Cys Arg Ala Ser
20 25 30
Glu Ser Val Ser Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile
50 55 60
Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys His His
85 90 95
Tyr Gly Ser Ser Pro Arg Pro Phe Gly Gln Gly Thr Lys Val Glu Ile
100 105 110
Lys
<210> 121
<211> 381
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 121
ggttccactg gtgaccaggt ccagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtca aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc a 381
<210> 122
<211> 339
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 122
ggttccactg gtgacgccga aattgtaatg acacagtctc catcctccct gtctgcttct 60
gaaggagaca gagtcatcat tacttgccgg gcgagtcagg gcattaggaa ttctttagcc 120
tggtatcagc agaaaccagg gaaagcccct aagctcctac tctatgatgc atccaaattg 180
gaaagtggag tcccatccag gttcagtggc agtggatctg ggacgcactt cactctcacc 240
atcgacagcc tgcagcctga agattttgca acttattact gtcaacacta ttttggtact 300
cctctcactt tcggcggagg gaccaaggtg gagatcaag 339
<210> 123
<211> 381
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 123
ggttccactg gtgacgaggt gcagctggtg gagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc a 381
<210> 124
<211> 339
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 124
ggttccactg gtgacgccgc catccggttg acccagtctc catcctccct gtctgcctct 60
gtaggagaca cagtcaccat cacttgccgg gcaagtcaga ccattagtaa atatttacat 120
tggtatcagc agaaaccggg ggaagcccct aaactcctga tttctgaagc ctccactttc 180
caaggtgggg tctcatcacg gttcagcggc agtagatctg ggacagattt cactctcacc 240
atctacagtc tgcaacctga agattctgca acttattact gtcaacagag ttacagttcc 300
cgattcactt tcggccctgg gaccaaagtg gagatcaaa 339
<210> 125
<211> 381
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 125
ggttccactg gtgacgaggt gcagctggtg gagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc a 381
<210> 126
<211> 339
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 126
ggttccactg gtgacgccgc catccagatg acccagtctc catcttctgt gtctgcatct 60
gtaggagaca gagtcaccat cacttgtcgg gcgagtcagg gtattagcag ctggttagcc 120
tggtatcagc agaaaccagg gaaagcccct aagctcctga tctatgctgc atccagtttg 180
caaagtgggg tcccatcaag gttcagcggc agtggatctg ggacagattt cactctcact 240
atcagcagcc tgcagcctga agattttgca acttactatt gtcaacaggc taacagtttc 300
cctctcactt ttggccaggg gaccaagctg gagatcaaa 339
<210> 127
<211> 378
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 127
ggttccactg gtgaccaggt gcagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
tcctcggtga aggtctcctg caaggcttct ggaggcacct tcagcagcat tgcaatcaac 120
tgggtgcgac aggcccctgg acaagggctt gcgtggatgg gaaaaatcat ccctatcttt 180
ggtacagcaa actacgcaca gaagttccag ggcagagtca cgatgaccgc ggacgaatcc 240
acgaacacag cctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattat 300
tgtgcgagag atgttataga ggctacgata tatggtatgg acgtctgggg ccaagggacc 360
acggtcaccg tctcctca 378
<210> 128
<211> 339
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 128
ggttccactg gtgacgccga catcgtgatg acccagtctc catcctccct gcctgcatct 60
gtgggagaca gagtcacaat cacttgccgg acaagtcaga gcattagcac ctatgtaaat 120
tggtatcagc agaagtcagg caatgcccct gagctcctga tgtatggtgc ttccatttta 180
caaagtgggg tcccatcaag gttcagtggc agtgggtctg ggacagactt cactctaacc 240
atcagcagtc tgcaacctga agattttgca acttactact gtcaacagag ttacagtgcc 300
ccttacactt ttgcccaggg gaccaagttg gagatcaga 339
<210> 129
<211> 381
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 129
ggttccactg gtgaccaggt tcagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc a 381
<210> 130
<211> 339
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 130
ggttccactg gtgacgccgt catctggatg acccagtctc catcctccct gtctgcatct 60
atgggagaca gagtcaccat cacttgccgg gcgagtcagg gcgtcagcaa ttatttagcc 120
tggtatcagc acaaaccagg caaagcacct gaattactga tctatgctgc ttccactttg 180
caatcagggg tcccatctcg gttcagcgcc agtagatctg ggacagattt cactctcacc 240
atcagcagcc tgcagcctga agatattgca acttattact gtcaacacta tgacagtccc 300
ccgtacactt ttggccaggg gaccaagctg gaggtcaaa 339
<210> 131
<211> 381
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 131
ggttccactg gtgacgaggt gcagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc a 381
<210> 132
<211> 351
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 132
ggttccactg gtgacgcctc ctatgagctg acacagccac cctcggtgtc agtgtcccca 60
ggacagacgg ccaggatcac ctgctctgga gatgcattgg caaagcactt tggtcattgg 120
taccagcaga ggccaggcca ggcccctgtt ctagtgatat ataaagacac tgagaggccc 180
ttagggatcc ctgagcgatt ctctggctcc agctcagggg caacagtcac gctgaccatc 240
agtgcagtcg aggcagagga cgaggctgac tattactgtc aatcaccaga caccactggt 300
aggattttcg gcggagggac caaggtcacc gtcctaggtc agcccaaggc t 351
<210> 133
<211> 381
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 133
ggttccactg gtgaccaggt ccagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc a 381
<210> 134
<211> 339
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 134
ggttccactg gtgacgccgc catccggttg acccagtctc catcttccgt gtctgcatct 60
gtaggagaca gagtcaccat cacttgtcgg gcgagtcagg gtattagcag ctggttagcc 120
tggtatcagc agaaaccagg gaaagcccct aagctcctga tctatgctgc atccagtttg 180
caaagtgggg tcccatcaag gttcagcggc agtggatctg ggacagattt cactctcacc 240
atcagcagcc tgcagcctga agattttgca acttactatt gtcaacagag ttacagtatc 300
cctcgcactt ttggccaggg gaccaagctg gagatcaaa 339
<210> 135
<211> 366
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 135
caggttcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60
tcctgcaggg catctgaaga caccttcacc agccactata tacactgggt gcgacaggcc 120
cctggacaag ggcttgagtg gatgggaata atcaatccta ctggtggcag tataagctac 180
gcacagaagt tccagggcag agtcgccatg acgaaggaca cgtccacgag cacagtctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagaggaggc 300
ttcactcctg atactagtgc ccctatggac gtctggggcc aagggacaat ggtcaccgtc 360
tcttca 366
<210> 136
<211> 360
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 136
ggttccactg gtgacgccgt catctggatg acccagtctc ccatccagat gacccagtct 60
ccatcttctg tgtctgcata tgtaggagac agagtcacca ttacttgtcg ggcgagtcag 120
gatattagcg actggttagc ctggtatcag caggctccag ggaaagcccc taaactcctg 180
atctatcgtg cagtcacttt acaagatgat gtcccatcaa ggttcagcgg cagcggatct 240
gggacagatt tcagtctcac tatcaccggc ctgcagcgtg aagatttcgc aacttattat 300
tgtcaacaga ctaacacttt cccgatcacc ttcggccacg ggacacgact ggagattaaa 360
<210> 137
<211> 381
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 137
ggttccactg gtgacgaggt ccagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc a 381
<210> 138
<211> 363
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 138
ggttccactg gtgacgccca gtctgccctg actcagccgc cctcagtgtc tggggcccca 60
gggcagacgg tcaccatctc ctgcactgga accagcagtg atgttgggag ttataacctt 120
gtctcctggt accaacagca cccaggcaaa gcccccaaac tcatcatcat tgaggtcact 180
aagcggcccc caggggtccc tgatcgtttc tctggctcca agtctggcaa cacggcctcc 240
ctgaccgtca ctggactcca ggctgaggat gaggctgatt atcactgcat ctcatatgca 300
ggcaacaaca atttggtatt cggcggaggc acccagctga ccgtcctcgg tcagcccaag 360
gct 363
<210> 139
<211> 381
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 139
ggttccactg gtgaccaggt ccagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc a 381
<210> 140
<211> 363
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 140
ggttccactg gtgacgcctc ctatgagctg acacagctac cctcagcgtc tgggaccccc 60
gggcagaggg tcaccatctc ttgttctggg agcagttccg acatcggaag gagtagtgta 120
aactggtacc aacagctccc aggaacggcc cccaaactcc tcatctatcg taataatcag 180
cggccttcag gggtccctga ccgactctct ggctccaagt ctggcacctc aggctccctg 240
gccatcagtg ggctccagtc tgaggatgag gctgattatt actgtgcagc atgggataac 300
accctgcgtg gttatgtctt cggaactggg accaaggtca ccgtcctagg tcagcccaag 360
gct 363
<210> 141
<211> 381
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 141
ggttccactg gtgaccaggt ccagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc a 381
<210> 142
<211> 357
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 142
ggttccactg gtgacgccga catccagttg acccagtctc cagactccct ggctgtgtct 60
ctgggcgaga gagccaccat caactgcaag tccagccaga gtgttttatt cagtcccaac 120
aataagaact acttagcttg gtaccagcag aaaccaggac agcctcctaa gctgctcatt 180
tactgggcat ctacccggga atccggggtc cctgaccgat tcagtggcag cgggtctggg 240
acagatttca ctctcaccat cagcagcctg caggctgaag atgtggcagt ttattactgt 300
cagcaatatg atagtagtcc gtggacgttc ggccaaggga ccaaggtgga aatcaaa 357
<210> 143
<211> 381
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 143
ggttccactg gtgaccaggt ccagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc a 381
<210> 144
<211> 345
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 144
ggttccactg gtgacgccga aacgacactc acgcagtctc caggcaccct gtctttgtct 60
ccaggggaca gagtcaccct ttcctgcagg gccagtcaca atgttaatag agactacgtg 120
gcctggtacc agcagagacg tggccaggct cccaggctcc tcatctccgg tgcatctaag 180
agggccgctg gcgtcccgga caggttcacc ggcgctgggt ctgggtcaga gtttacgctc 240
accatcagca gattggagcc tgaagatttt ggagtctatt tctgtcaaca ttttagtgga 300
acacctcggc tcactttcgg cggagggacc aacgtggaga tgcag 345
<210> 145
<211> 381
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 145
ggttccactg gtgaccaggt gcagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
tcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc a 381
<210> 146
<211> 339
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 146
ggttccactg gtgacgccgc catccagatg acccagtctc catccttcct gtctgcatct 60
gtaggagaca gagtcaccat cacttgccgg gccagtcacg acatcagcac ttttttaggc 120
tggtatcaaa aaaaaccggg gaaaccccct aaactcctgg tctatgatgc atccactttg 180
cagagtgggg tcccatcaag gttccgcggc agtggatctg ggacagactt cactctcaca 240
atcagcagcc tgcagcctga agattttggt tcttattact gtcaacaacg aagacattac 300
ccggtcacct ttggccaagg gacaggactg gagattaaa 339
<210> 147
<211> 381
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 147
ggttccactg gtgaccaggt gcagctggtg caatctgggt ctgagttgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggtcaaggg 360
acaatggtca ccgtctcttc a 381
<210> 148
<211> 357
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 148
ggttccactg gtgacgcctc ctatgagctg actcagccac tctcagtgtc agtggccctg 60
ggacagacgg ccaggattac ctgtggggga aacaacattg gaagtaaaaa tgtgcactgg 120
taccagcaga agccaggcca ggcccctgtg ctggtcatct atagggatag caaccggccc 180
tctgggatcc ctgagcgatt ctctggctcc aactcgggga acacggccac cctgaccatc 240
agcagagccc aagccgggga tgagggtgac tattactgtc aggtgtggga cagcagcact 300
gcagatgtgg tattcggcgg aggcacccag ctgaccgtcc taggtcagcc caaggct 357
<210> 149
<211> 381
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 149
ggttccactg gtgaccaggt acagctggtg caatctgggt ctgagttgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggtcaaggg 360
acaatggtca ccgtctcttc a 381
<210> 150
<211> 339
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 150
ggttccactg gtgacgccga aattgtgttg acgcagtctc cagccaccct gtctttgtct 60
ccaggggaaa cagccaccct ctcctgcagg gccagtgaga gcgttagcag caacttagcc 120
tggtaccagc agaaacctgg ccaggctccc aggctcctca tctatgatgc atccaacagg 180
gccactggca tcccagccag gttcagtggc agtgggtctg ggacagactt cactctcacc 240
atcagcagac tggagcctga agattttgca gtttattact gtcaccacta tggtagctca 300
cctcgaccgt tcggccaagg gaccaaggtg gaaatcaaa 339
<210> 151
<211> 1374
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 151
ggttccactg gtgaccaggt ccagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtca aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc agcctccacc aagggcccat cggtcttccc cttggccccg 420
tcggccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa ggactacttc 480
cccgaaccgg tgacggtgtc gtggaactca ggcgccctga ccagcggcgt gcacaccttc 540
ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 600
agcagcttgg gcacccagac ctacatctgc aacgtgaatc acaagcccag caacaccaag 660
gtggacaaga aagttgagcc caaatcttgt gacaaaactc acacatgccc accgtgccca 720
gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 780
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 840
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 900
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 960
caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 1020
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 1080
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 1140
ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 1200
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 1260
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1320
gctctgcaca accactacac acagaagagc ctctccctgt ctccgggtaa atga 1374
<210> 152
<211> 663
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 152
ggttccactg gtgacgccga aattgtaatg acacagtctc catcctccct gtctgcttct 60
gaaggagaca gagtcatcat tacttgccgg gcgagtcagg gcattaggaa ttctttagcc 120
tggtatcagc agaaaccagg gaaagcccct aagctcctac tctatgatgc atccaaattg 180
gaaagtggag tcccatccag gttcagtggc agtggatctg ggacgcactt cactctcacc 240
atcgacagcc tgcagcctga agattttgca acttattact gtcaacacta ttttggtact 300
cctctcactt tcggcggagg gaccaaggtg gagatcaagc gaactgtggc tgcaccatct 360
gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 420
ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 480
caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540
ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600
gaagtcaccc atcagggcct gagcttgccc gtcacaaaga gcttcaacag gggagagtgt 660
tag 663
<210> 153
<211> 1374
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 153
ggttccactg gtgacgaggt gcagctggtg gagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc agcctccacc aagggcccat cggtcttccc cttggccccg 420
tcggccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa ggactacttc 480
cccgaaccgg tgacggtgtc gtggaactca ggcgccctga ccagcggcgt gcacaccttc 540
ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 600
agcagcttgg gcacccagac ctacatctgc aacgtgaatc acaagcccag caacaccaag 660
gtggacaaga aagttgagcc caaatcttgt gacaaaactc acacatgccc accgtgccca 720
gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 780
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 840
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 900
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 960
caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 1020
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 1080
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 1140
ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 1200
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 1260
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1320
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa atga 1374
<210> 154
<211> 663
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 154
ggttccactg gtgacgccgc catccggttg acccagtctc catcctccct gtctgcctct 60
gtaggagaca cagtcaccat cacttgccgg gcaagtcaga ccattagtaa atatttacat 120
tggtatcagc agaaaccggg ggaagcccct aaactcctga tttctgaagc ctccactttc 180
caaggtgggg tctcatcacg gttcagcggc agtagatctg ggacagattt cactctcacc 240
atctacagtc tgcaacctga agattctgca acttattact gtcaacagag ttacagttcc 300
cgattcactt tcggccctgg gaccaaagtg gagatcaaac gaactgtggc tgcaccatct 360
gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 420
ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 480
caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540
ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600
gaagtcaccc atcagggcct gagcttgccc gtcacaaaga gcttcaacag gggagagtgt 660
tag 663
<210> 155
<211> 1362
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 155
ggttccactg gtgacgaggt gcagctggtg gagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc agcctccacc aagggcccat cggtcttccc cttggccccg 420
tcggccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa ggactacttc 480
cccgaaccgg tgacggtgtc gtggaactca ggcgccctga ccagcggcgt gcacaccttc 540
ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 600
agcagcttgg gcacccagac ctacatctgc aacgtgaatc acaagcccag caacaccaag 660
gtggacaaga aagttgagcc caaatcttgt gtcgagtgcc caccgtgccc agcaccacct 720
gtggcaggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780
cggacccctg aggtcacgtg cgtggtggtg gacgtgagcc acgaagaccc cgaggtccag 840
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc acgggaggag 900
cagttcaaca gcacgttccg tgtggtcagc gtcctcaccg ttgtgcacca ggactggctg 960
aacggcaagg agtacaagtg caaggtctcc aacaaaggcc tcccagcccc catcgagaaa 1020
accatctcca aaaccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080
cgggaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320
cactacacgc agaagagcct ctccctgtct ccgggtaaat ga 1362
<210> 156
<211> 663
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 156
ggttccactg gtgacgccgc catccagatg acccagtctc catcttctgt gtctgcatct 60
gtaggagaca gagtcaccat cacttgtcgg gcgagtcagg gtattagcag ctggttagcc 120
tggtatcagc agaaaccagg gaaagcccct aagctcctga tctatgctgc atccagtttg 180
caaagtgggg tcccatcaag gttcagcggc agtggatctg ggacagattt cactctcact 240
atcagcagcc tgcagcctga agattttgca acttactatt gtcaacaggc taacagtttc 300
cctctcactt ttggccaggg gaccaagctg gagatcaaac gaactgtggc tgcaccatct 360
gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 420
ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 480
caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540
ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600
gaagtcaccc atcagggcct gagcttgccc gtcacaaaga gcttcaacag gggagagtgt 660
tag 663
<210> 157
<211> 1371
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 157
ggttccactg gtgaccaggt gcagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
tcctcggtga aggtctcctg caaggcttct ggaggcacct tcagcagcat tgcaatcaac 120
tgggtgcgac aggcccctgg acaagggctt gcgtggatgg gaaaaatcat ccctatcttt 180
ggtacagcaa actacgcaca gaagttccag ggcagagtca cgatgaccgc ggacgaatcc 240
acgaacacag cctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattat 300
tgtgcgagag atgttataga ggctacgata tatggtatgg acgtctgggg ccaagggacc 360
acggtcaccg tctcctcagc ctccaccaag ggcccatcgg tcttcccctt ggccccgtcg 420
gccaagagca cctctggggg cacagcggcc ctgggctgcc tggtcaagga ctacttcccc 480
gaaccggtga cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca caccttcccg 540
gctgtcctac agtcctcagg actctactcc ctcagcagcg tggtgaccgt gccctccagc 600
agcttgggca cccagaccta catctgcaac gtgaatcaca agcccagcaa caccaaggtg 660
gacaagaaag ttgagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccagca 720
cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 780
atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 840
gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 900
cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 960
gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc 1020
atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg 1080
cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 1140
ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac 1200
aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc 1260
gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct 1320
ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaatg a 1371
<210> 158
<211> 663
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 158
ggttccactg gtgacgccga catcgtgatg acccagtctc catcctccct gcctgcatct 60
gtgggagaca gagtcacaat cacttgccgg acaagtcaga gcattagcac ctatgtaaat 120
tggtatcagc agaagtcagg caatgcccct gagctcctga tgtatggtgc ttccatttta 180
caaagtgggg tcccatcaag gttcagtggc agtgggtctg ggacagactt cactctaacc 240
atcagcagtc tgcaacctga agattttgca acttactact gtcaacagag ttacagtgcc 300
ccttacactt ttgcccaggg gaccaagttg gagatcagac gaactgtggc tgcaccatct 360
gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 420
ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 480
caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540
ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600
gaagtcaccc atcagggcct gagcttgccc gtcacaaaga gcttcaacag gggagagtgt 660
tag 663
<210> 159
<211> 1374
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 159
ggttccactg gtgaccaggt tcagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc agcctccacc aagggcccat cggtcttccc cttggccccg 420
tcggccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa ggactacttc 480
cccgaaccgg tgacggtgtc gtggaactca ggcgccctga ccagcggcgt gcacaccttc 540
ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 600
agcagcttgg gcacccagac ctacatctgc aacgtgaatc acaagcccag caacaccaag 660
gtggacaaga aagttgagcc caaatcttgt gacaaaactc acacatgccc accgtgccca 720
gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 780
ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 840
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 900
ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 960
caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 1020
cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 1080
ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 1140
ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 1200
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 1260
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1320
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa atga 1374
<210> 160
<211> 663
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 160
ggttccactg gtgacgccgt catctggatg acccagtctc catcctccct gtctgcatct 60
atgggagaca gagtcaccat cacttgccgg gcgagtcagg gcgtcagcaa ttatttagcc 120
tggtatcagc acaaaccagg caaagcacct gaattactga tctatgctgc ttccactttg 180
caatcagggg tcccatctcg gttcagcgcc agtagatctg ggacagattt cactctcacc 240
atcagcagcc tgcagcctga agatattgca acttattact gtcaacacta tgacagtccc 300
ccgtacactt ttggccaggg gaccaagctg gaggtcaaac gaactgtggc tgcaccatct 360
gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 420
ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 480
caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540
ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600
gaagtcaccc atcagggcct gagcttgccc gtcacaaaga gcttcaacag gggagagtgt 660
tag 663
<210> 161
<211> 1374
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 161
ggttccactg gtgacgaggt gcagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc agcctccacc aagggcccat cggtcttccc cttggccccg 420
tcggccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa ggactacttc 480
cccgaaccgg tgacggtgtc gtggaactca ggcgccctga ccagcggcgt gcacaccttc 540
ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 600
agcagcttgg gcacccagac ctacatctgc aacgtgaatc acaagcccag caacaccaag 660
gtggacaaga aagttgagcc caaatcttgt gacacacctc ccccatgccc acggtgccca 720
gcacctgaac tcctgggagg accgtcagtc ttcctcttcc ccccaaaacc caaggatacc 780
cttatgatct cccggacccc tgaggtcacg tgcgtggtgg tggacgtgag ccacgaagac 840
cccgaggtcc agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 900
ccacgggagg agcagttcaa cagcacgttc cgtgtggtca gcgtcctcac cgtcgtgcac 960
caggactggc tgaacggcaa ggagtacaag tgcaagatct ccaacaaagg cctcccagcc 1020
cccatcgaga aaaccatctc caaaaccaaa gggcagcccc gagaaccaca ggtgtacacc 1080
ctgcccccat cccgggagga gatgaccaag aaccaggtca gcctgacctg cctggtcaaa 1140
ggcttctacc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 1200
tacaagacca cgcctcccat gctggactcc gacggctcct tcttcctcta cagcaagctc 1260
accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1320
gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa atga 1374
<210> 162
<211> 657
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 162
ggttccactg gtgacgcctc ctatgagctg acacagccac cctcggtgtc agtgtcccca 60
ggacagacgg ccaggatcac ctgctctgga gatgcattgg caaagcactt tggtcattgg 120
taccagcaga ggccaggcca ggcccctgtt ctagtgatat ataaagacac tgagaggccc 180
ttagggatcc ctgagcgatt ctctggctcc agctcagggg caacagtcac gctgaccatc 240
agtgcagtcg aggcagagga cgaggctgac tattactgtc aatcaccaga caccactggt 300
aggattttcg gcggagggac caaggtcacc gtcctaggtc agcccaaggc tgccccctcg 360
gtcactctgt tcccgccctc ctctgaggag cttcaagcca acaaggccac actggtgtgt 420
ctcataagtg acttctaccc gggagccgtg acagtggcct ggaaggcaga tagcagcccc 480
gtcaaggcgg gagtggagac caccacaccc tccaaacaaa gcaacaacaa gtacgcggcc 540
agcagctacc tgagcctgac gcctgagcag tggaagtccc acagaagcta cagctgccag 600
gtcacgcatg aagggagcac cgtggagaag acagtggccc ctacagaatg ttcatag 657
<210> 163
<211> 1362
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 163
ggttccactg gtgaccaggt ccagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc agcctccacc aagggcccat cggtcttccc cttggccccg 420
tcggccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa ggactacttc 480
cccgaaccgg tgacggtgtc gtggaactca ggcgccctga ccagcggcgt gcacaccttc 540
ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 600
agcagcttgg gcacccagac ctacatctgc aacgtgaatc acaagcccag caacaccaag 660
gtggacaaga aagttgagcc caaatcttgt gtcgagtgcc caccgtgccc agcaccacct 720
gtggcaggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960
aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320
cactacacgc agaagagcct ctccctgtct ccgggtaaat ga 1362
<210> 164
<211> 663
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 164
ggttccactg gtgacgccgc catccggttg acccagtctc catcttccgt gtctgcatct 60
gtaggagaca gagtcaccat cacttgtcgg gcgagtcagg gtattagcag ctggttagcc 120
tggtatcagc agaaaccagg gaaagcccct aagctcctga tctatgctgc atccagtttg 180
caaagtgggg tcccatcaag gttcagcggc agtggatctg ggacagattt cactctcacc 240
atcagcagcc tgcagcctga agattttgca acttactatt gtcaacagag ttacagtatc 300
cctcgcactt ttggccaggg gaccaagctg gagatcaaac gaactgtggc tgcaccatct 360
gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 420
ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 480
caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540
ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600
gaagtcaccc atcagggcct gagcttgccc gtcacaaaga gcttcaacag gggagagtgt 660
tag 663
<210> 165
<211> 1347
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 165
caggttcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60
tcctgcaggg catctgaaga caccttcacc agccactata tacactgggt gcgacaggcc 120
cctggacaag ggcttgagtg gatgggaata atcaatccta ctggtggcag tataagctac 180
gcacagaagt tccagggcag agtcgccatg acgaaggaca cgtccacgag cacagtctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagaggaggc 300
ttcactcctg atactagtgc ccctatggac gtctggggcc aagggacaat ggtcaccgtc 360
tcttcagcct ccaccaaggg cccatcggtc ttccccttgg ccccgtcggc caagagcacc 420
tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 480
gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 540
tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 600
cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt 660
gagcccaaat cttgtgtcga gtgcccaccg tgcccagcac cacctgtggc aggaccgtca 720
gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 780
acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840
gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900
taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960
aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020
aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 1080
aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140
gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200
tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 1260
gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320
agcctctccc tgtctccggg taaatga 1347
<210> 166
<211> 684
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 166
ggttccactg gtgacgccgt catctggatg acccagtctc ccatccagat gacccagtct 60
ccatcttctg tgtctgcata tgtaggagac agagtcacca ttacttgtcg ggcgagtcag 120
gatattagcg actggttagc ctggtatcag caggctccag ggaaagcccc taaactcctg 180
atctatcgtg cagtcacttt acaagatgat gtcccatcaa ggttcagcgg cagcggatct 240
gggacagatt tcagtctcac tatcaccggc ctgcagcgtg aagatttcgc aacttattat 300
tgtcaacaga ctaacacttt cccgatcacc ttcggccacg ggacacgact ggagattaaa 360
cgaactgtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 420
ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 480
tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 540
agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 600
aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagcttgcc cgtcacaaag 660
agcttcaaca ggggagagtg ttag 684
<210> 167
<211> 1362
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 167
ggttccactg gtgacgaggt ccagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc agcctccacc aagggcccat cggtcttccc cttggccccg 420
tcggccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa ggactacttc 480
cccgaaccgg tgacggtgtc gtggaactca ggcgccctga ccagcggcgt gcacaccttc 540
ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 600
agcagcttgg gcacccagac ctacatctgc aacgtgaatc acaagcccag caacaccaag 660
gtggacaaga aagttgagcc caaatcttgt gtcgagtgcc caccgtgccc agcaccacct 720
gtggcaggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960
aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320
cactacacgc agaagagcct ctccctgtct ccgggtaaat ga 1362
<210> 168
<211> 669
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 168
ggttccactg gtgacgccca gtctgccctg actcagccgc cctcagtgtc tggggcccca 60
gggcagacgg tcaccatctc ctgcactgga accagcagtg atgttgggag ttataacctt 120
gtctcctggt accaacagca cccaggcaaa gcccccaaac tcatcatcat tgaggtcact 180
aagcggcccc caggggtccc tgatcgtttc tctggctcca agtctggcaa cacggcctcc 240
ctgaccgtca ctggactcca ggctgaggat gaggctgatt atcactgcat ctcatatgca 300
ggcaacaaca atttggtatt cggcggaggc acccagctga ccgtcctcgg tcagcccaag 360
gctgccccct cggtcactct gttcccgccc tcctctgagg agcttcaagc caacaaggcc 420
acactggtgt gtctcataag tgacttctac ccgggagccg tgacagtggc ctggaaggca 480
gatagcagcc ccgtcaaggc gggagtggag accaccacac cctccaaaca aagcaacaac 540
aagtacgcgg ccagcagcta cctgagcctg acgcctgagc agtggaagtc ccacagaagc 600
tacagctgcc aggtcacgca tgaagggagc accgtggaga agacagtggc ccctacagaa 660
tgttcatag 669
<210> 169
<211> 1362
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 169
ggttccactg gtgaccaggt ccagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc agcctccacc aagggcccat cggtcttccc cttggccccg 420
tcggccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa ggactacttc 480
cccgaaccgg tgacggtgtc gtggaactca ggcgccctga ccagcggcgt gcacaccttc 540
ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 600
agcagcttgg gcacccagac ctacatctgc aacgtgaatc acaagcccag caacaccaag 660
gtggacaaga aagttgagcc caaatcttgt gtcgagtgcc caccgtgccc agcaccacct 720
gtggcaggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960
aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320
cactacacgc agaagagcct ctccctgtct ccgggtaaat ga 1362
<210> 170
<211> 669
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 170
ggttccactg gtgacgcctc ctatgagctg acacagctac cctcagcgtc tgggaccccc 60
gggcagaggg tcaccatctc ttgttctggg agcagttccg acatcggaag gagtagtgta 120
aactggtacc aacagctccc aggaacggcc cccaaactcc tcatctatcg taataatcag 180
cggccttcag gggtccctga ccgactctct ggctccaagt ctggcacctc aggctccctg 240
gccatcagtg ggctccagtc tgaggatgag gctgattatt actgtgcagc atgggataac 300
accctgcgtg gttatgtctt cggaactggg accaaggtca ccgtcctagg tcagcccaag 360
gctgccccct cggtcactct gttcccgccc tcctctgagg agcttcaagc caacaaggcc 420
acactggtgt gtctcataag tgacttctac ccgggagccg tgacagtggc ctggaaggca 480
gatagcagcc ccgtcaaggc gggagtggag accaccacac cctccaaaca aagcaacaac 540
aagtacgcgg ccagcagcta cctgagcctg acgcctgagc agtggaagtc ccacagaagc 600
tacagctgcc aggtcacgca tgaagggagc accgtggaga agacagtggc ccctacagaa 660
tgttcatag 669
<210> 171
<211> 1362
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 171
ggttccactg gtgaccaggt ccagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc agcctccacc aagggcccat cggtcttccc cttggccccg 420
tcggccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa ggactacttc 480
cccgaaccgg tgacggtgtc gtggaactca ggcgccctga ccagcggcgt gcacaccttc 540
ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 600
agcagcttgg gcacccagac ctacatctgc aacgtgaatc acaagcccag caacaccaag 660
gtggacaaga aagttgagcc caaatcttgt gtcgagtgcc caccgtgccc agcaccacct 720
gtggcaggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960
aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320
cactacacgc agaagagcct ctccctgtct ccgggtaaat ga 1362
<210> 172
<211> 681
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 172
ggttccactg gtgacgccga catccagttg acccagtctc cagactccct ggctgtgtct 60
ctgggcgaga gagccaccat caactgcaag tccagccaga gtgttttatt cagtcccaac 120
aataagaact acttagcttg gtaccagcag aaaccaggac agcctcctaa gctgctcatt 180
tactgggcat ctacccggga atccggggtc cctgaccgat tcagtggcag cgggtctggg 240
acagatttca ctctcaccat cagcagcctg caggctgaag atgtggcagt ttattactgt 300
cagcaatatg atagtagtcc gtggacgttc ggccaaggga ccaaggtgga aatcaaacga 360
actgtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga 420
actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg 480
gaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc 540
aaggacagca cctacagcct cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa 600
cacaaagtct acgcctgcga agtcacccat cagggcctga gcttgcccgt cacaaagagc 660
ttcaacaggg gagagtgtta g 681
<210> 173
<211> 1362
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 173
ggttccactg gtgaccaggt ccagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc agcctccacc aagggcccat cggtcttccc cttggccccg 420
tcggccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa ggactacttc 480
cccgaaccgg tgacggtgtc gtggaactca ggcgccctga ccagcggcgt gcacaccttc 540
ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 600
agcagcttgg gcacccagac ctacatctgc aacgtgaatc acaagcccag caacaccaag 660
gtggacaaga aagttgagcc caaatcttgt gtcgagtgcc caccgtgccc agcaccacct 720
gtggcaggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960
aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320
cactacacgc agaagagcct ctccctgtct ccgggtaaat ga 1362
<210> 174
<211> 669
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 174
ggttccactg gtgacgccga aacgacactc acgcagtctc caggcaccct gtctttgtct 60
ccaggggaca gagtcaccct ttcctgcagg gccagtcaca atgttaatag agactacgtg 120
gcctggtacc agcagagacg tggccaggct cccaggctcc tcatctccgg tgcatctaag 180
agggccgctg gcgtcccgga caggttcacc ggcgctgggt ctgggtcaga gtttacgctc 240
accatcagca gattggagcc tgaagatttt ggagtctatt tctgtcaaca ttttagtgga 300
acacctcggc tcactttcgg cggagggacc aacgtggaga tgcagcgaac tgtggctgca 360
ccatctgtct tcatcttccc gccatctgat gagcagttga aatctggaac tgcctctgtt 420
gtgtgcctgc tgaataactt ctatcccaga gaggccaaag tacagtggaa ggtggataac 480
gccctccaat cgggtaactc ccaggagagt gtcacagagc aggacagcaa ggacagcacc 540
tacagcctca gcagcaccct gacgctgagc aaagcagact acgagaaaca caaagtctac 600
gcctgcgaag tcacccatca gggcctgagc ttgcccgtca caaagagctt caacagggga 660
gagtgttag 669
<210> 175
<211> 1362
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 175
ggttccactg gtgaccaggt gcagctggtg cagtctgggg ctgaggtgaa gaagcctggg 60
tcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggccaaggg 360
acaatggtca ccgtctcttc agcctccacc aagggcccat cggtcttccc cttggccccg 420
tcggccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa ggactacttc 480
cccgaaccgg tgacggtgtc gtggaactca ggcgccctga ccagcggcgt gcacaccttc 540
ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 600
agcagcttgg gcacccagac ctacatctgc aacgtgaatc acaagcccag caacaccaag 660
gtggacaaga aagttgagcc caaatcttgt gtcgagtgcc caccgtgccc agcaccacct 720
gtggcaggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960
aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320
cactacacgc agaagagcct ctccctgtct ccgggtaaat ga 1362
<210> 176
<211> 663
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 176
ggttccactg gtgacgccgc catccagatg acccagtctc catccttcct gtctgcatct 60
gtaggagaca gagtcaccat cacttgccgg gccagtcacg acatcagcac ttttttaggc 120
tggtatcaaa aaaaaccggg gaaaccccct aaactcctgg tctatgatgc atccactttg 180
cagagtgggg tcccatcaag gttccgcggc agtggatctg ggacagactt cactctcaca 240
atcagcagcc tgcagcctga agattttggt tcttattact gtcaacaacg aagacattac 300
ccggtcacct ttggccaagg gacaggactg gagattaaac gaactgtggc tgcaccatct 360
gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 420
ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 480
caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540
ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600
gaagtcaccc atcagggcct gagcttgccc gtcacaaaga gcttcaacag gggagagtgt 660
tag 663
<210> 177
<211> 1362
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 177
ggttccactg gtgaccaggt gcagctggtg caatctgggt ctgagttgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggtcaaggg 360
acaatggtca ccgtctcttc agcctccacc aagggcccat cggtcttccc cttggccccg 420
tcggccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa ggactacttc 480
cccgaaccgg tgacggtgtc gtggaactca ggcgccctga ccagcggcgt gcacaccttc 540
ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 600
agcagcttgg gcacccagac ctacatctgc aacgtgaatc acaagcccag caacaccaag 660
gtggacaaga aagttgagcc caaatcttgt gtcgagtgcc caccgtgccc agcaccacct 720
gtggcaggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960
aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320
cactacacgc agaagagcct ctccctgtct ccgggtaaat ga 1362
<210> 178
<211> 663
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 178
ggttccactg gtgacgcctc ctatgagctg actcagccac tctcagtgtc agtggccctg 60
ggacagacgg ccaggattac ctgtggggga aacaacattg gaagtaaaaa tgtgcactgg 120
taccagcaga agccaggcca ggcccctgtg ctggtcatct atagggatag caaccggccc 180
tctgggatcc ctgagcgatt ctctggctcc aactcgggga acacggccac cctgaccatc 240
agcagagccc aagccgggga tgagggtgac tattactgtc aggtgtggga cagcagcact 300
gcagatgtgg tattcggcgg aggcacccag ctgaccgtcc taggtcagcc caaggctgcc 360
ccctcggtca ctctgttccc gccctcctct gaggagcttc aagccaacaa ggccacactg 420
gtgtgtctca taagtgactt ctacccggga gccgtgacag tggcctggaa ggcagatagc 480
agccccgtca aggcgggagt ggagaccacc acaccctcca aacaaagcaa caacaagtac 540
gcggccagca gctacctgag cctgacgcct gagcagtgga agtcccacag aagctacagc 600
tgccaggtca cgcatgaagg gagcaccgtg gagaagacag tggcccctac agaatgttca 660
tag 663
<210> 179
<211> 1362
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 179
ggttccactg gtgaccaggt acagctggtg caatctgggt ctgagttgaa gaagcctggg 60
gcctcagtga aggtttcctg cagggcatct gaagacacct tcaccagcca ctatatacac 120
tgggtgcgac aggcccctgg acaagggctt gagtggatgg gaataatcaa tcctactggt 180
ggcagtataa gctacgcaca gaagttccag ggcagagtcg ccatgacgaa ggacacgtcc 240
acgagcacag tctacatgga gctgagcagc ctgagatctg aggacacggc cgtgtattac 300
tgtgcgagag gaggcttcac tcctgatact agtgccccta tggacgtctg gggtcaaggg 360
acaatggtca ccgtctcttc agcctccacc aagggcccat cggtcttccc cttggccccg 420
tcggccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa ggactacttc 480
cccgaaccgg tgacggtgtc gtggaactca ggcgccctga ccagcggcgt gcacaccttc 540
ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 600
agcagcttgg gcacccagac ctacatctgc aacgtgaatc acaagcccag caacaccaag 660
gtggacaaga aagttgagcc caaatcttgt gtcgagtgcc caccgtgccc agcaccacct 720
gtggcaggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960
aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320
cactacacgc agaagagcct ctccctgtct ccgggtaaat ga 1362
<210> 180
<211> 663
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 180
ggttccactg gtgacgccga aattgtgttg acgcagtctc cagccaccct gtctttgtct 60
ccaggggaaa cagccaccct ctcctgcagg gccagtgaga gcgttagcag caacttagcc 120
tggtaccagc agaaacctgg ccaggctccc aggctcctca tctatgatgc atccaacagg 180
gccactggca tcccagccag gttcagtggc agtgggtctg ggacagactt cactctcacc 240
atcagcagac tggagcctga agattttgca gtttattact gtcaccacta tggtagctca 300
cctcgaccgt tcggccaagg gaccaaggtg gaaatcaaac gaactgtggc tgcaccatct 360
gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 420
ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 480
caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540
ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600
gaagtcaccc atcagggcct gagcttgccc gtcacaaaga gcttcaacag gggagagtgt 660
tag 663

Claims (7)

1. A novel coronavirus antibody characterized by being XY4;
the variable region amino acid of the XY4 antibody heavy chain comprises:
CDRH1 amino acid sequence: GGTFSSIA
CDRH2 amino acid sequence: IIPIFGTA
CDRH3 amino acid sequence: ardvietiygmdv;
the variable region amino acid of the XY4 antibody light chain comprises:
CDRL1 amino acid sequence: QSISTY
CDRL2 amino acid sequence: GAS
CDRL3 amino acid sequence: QQSYSAPYT.
2. The novel coronavirus antibody of claim 1,
amino acid sequence of heavy chain variable region of XY4 antibody:
GSTGDQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSIAINWVRQAPGQGLAWMGKIIPIFGTANYAQKFQGRVTMTADESTNTAYMELSSLRSEDTAVYYCARDVIEATIYGMDVWGQGTTVTVSS；
amino acid sequence of light chain variable region of XY4 antibody:
GSTGDADIVMTQSPSSLPASVGDRVTITCRTSQSISTYVNWYQQKSGNAPELLMYGASILQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTFAQGTKLEIR。
3. the novel coronavirus antibody of claim 1,
XY4 is an antibody for use in the treatment of novel coronaviruses.
4. The antibody of any one of claims 1-3, of the type Fab, fab '-SH, fv, scFv, (Fab') 2 fragments.
5. Any nucleic acid capable of expressing the novel coronavirus antibody of any one of claims 1-3.
6. An agent for detecting, preventing or treating a novel coronavirus, comprising the novel coronavirus antibody according to any one of claims 1 to 3.
7. Use of the novel coronavirus antibody of any one of claims 1-3, comprising any one or more of the following;
(1) Used for preparing a novel coronavirus detection preparation;
(2) For the preparation of a formulation for the prevention of a novel coronavirus infection;
(3) Can be used for preparing preparations for treating novel coronavirus infection.
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