CN115197968A

CN115197968A - Construction and screening method of chimeric antigen receptor modified cell library with automatically optimized antigen binding domain and application thereof

Info

Publication number: CN115197968A
Application number: CN202110383729.1A
Authority: CN
Inventors: 牛志远; 朱武凌; 郭长江; 支灵通
Original assignee: Xinxiang Medical University
Current assignee: Xinxiang Medical University
Priority date: 2021-04-09
Filing date: 2021-04-09
Publication date: 2022-10-18
Anticipated expiration: 2041-04-09
Also published as: CN115197968B

Abstract

The invention discloses a method for constructing and screening a chimeric antigen receptor modified cell library with an automatically optimized antigen binding domain and application thereof, wherein a chimeric antigen receptor sequence is inserted into a cell, and mutation is continuously induced in an extracellular antigen binding domain through a cis-acting element and a trans-acting factor to establish an antibody sequence dynamic library for subsequent screening; displaying the antibody sequence dynamic library on the cell surface by the receptor 2 to form a chimeric receptor library cell library; receptor 1 is able to recognize the tag on the target antigen and form a dimer with receptor 2, which is able to recognize the target antigen, thus initiating expression of the selection gene and simultaneously initiating the inhibition system to maintain stability of the antigen binding domain. Screening the chimeric antigen receptor library, and performing amplification sequencing on the antigen binding domain of the screened positive cell chimeric receptor to obtain the nucleic acid sequence of the antigen recognition region. The method can be used for preparing various antibodies, and is simple, convenient and efficient.

Description

Construction and screening method of chimeric antigen receptor modified cell library with automatically optimized antigen binding domain and application thereof

Technical Field

The invention relates to a method for constructing and screening a chimeric antigen receptor modified cell library with an automatically optimized antigen binding domain and application thereof, belonging to the technical field of genetic engineering.

Background

At present, the antibody plays an irreplaceable role in the fields of scientific research, detection, diagnosis, medical treatment and the like, and along with the development of chimeric antigen receptor T cell immunotherapy (CAR-T) and immune checkpoint therapy, the preparation of the antibody is in greater demand and higher requirement at present. For example, single chain antibodies (scFv) of the extracellular antigen-binding domain of a Chimeric Antigen Receptor (CAR) are prepared. The extracellular antigen-binding domain of CARs also mostly employs camelid and shark-derived single domain antibodies (sdabs) naturally devoid of light chain, known as heavy chain-only antibodies (hcabs), with antigen-binding fragments in each arm of the hcabs having a single heavy chain variable domain (VHH) that can have high affinity for antigen without the aid of a light chain, VHH is known as the smallest functional antigen-binding fragment, with a molecular weight of about 15Kd, and is widely used in CAR receptors. Furthermore, humanized antibodies are often required in the course of human therapy.

Currently, scFv and VHH are mostly obtained by conventional antibody preparation methods, which mainly comprise the following steps: 1. preparing an antigen and an adjuvant; 2. animal immunization; 3. collecting antiserum; 4.B lymphocyte isolation; 5. preparing hybridoma/amplifying antibody sequence and building a library; 6. screening antibodies; 7. obtaining an antibody sequence; further preparation of scFv and VHH sequences. In addition, the prepared antibody needs to be subjected to humanization treatment, the application of humanized mice is involved, or the splicing of the antibodies is involved, the whole process is complicated in steps, time-consuming and labor-consuming, and the cost is high.

Disclosure of Invention

The invention aims to provide a method for constructing a chimeric antigen receptor modified cell library with an automatically optimized antigen binding domain.

The second objective of the invention is to provide a library of chimeric antigen receptor-modified cells prepared by the above construction method.

The third purpose of the invention is to provide a method for screening the chimeric antigen receptor modified cell library with automatically optimized antigen binding domain.

The fourth purpose of the invention is to provide the application of the screening method in preparing the antibody.

The fifth object of the present invention is to provide an antibody screened by the above method.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a method for constructing a chimeric antigen receptor modified cell library with an automatically optimized antigen binding domain, wherein one or more vectors comprising a first gene element, a second gene element, a third gene element, a fourth gene element and a fifth gene element are transfected into cells to obtain the chimeric antigen receptor modified cell library;

the first gene element is a nucleotide sequence for coding a receptor 1, and the receptor 1 comprises an extracellular domain, a transmembrane domain and an intracellular domain; the extracellular domain may recognize a biotin or 6 × His tag labeled on a target antigen, and the intracellular domain includes tobacco etch virus protease TEVP;

the second genetic element is a nucleotide sequence encoding a receptor 2, the receptor 2 comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal domain; the antigen binding domain is an antibody sequence dynamic library, and the dynamic library is obtained by inducing a series of mutations under the action of a fourth gene element and introducing sequence diversity; the intracellular signaling domain comprises a tTA transcription factor domain and a linking sequence that connects the tTA transcription factor domain and the transmembrane domain, comprising a sequence that is recognizable by TEVP for cleavage;

the third gene element is a nucleotide sequence for coding a screening gene, and the tTA transcription factor can start the expression of the screening gene, so that a host generates fluorescence, obtains certain resistance or obtains a growth advantage under certain conditions;

the fourth gene element is a nucleotide sequence encoding one or more cis-regulatory elements and one or more trans-acting factors; the cis-form regulatory element can enable DNA to generate a DNA sequence with a special structure, the special structure refers to that the DNA structure contains a DNA single-chain structure without base complementary pairing, and the special structure comprises R-loop, G-triplet and G-quadruplet; the trans-acting factor is a Cas protein family and a variant of the Cas protein family, gRNA, uracil glycosylase inhibitor, adenine deaminase, cytidine deaminase, RNA-DNA hybrid chain binding protein, photosensitive protein, nucleolin, DNA helicase, error-prone DNA polymerase, recombinase, endonuclease, exonuclease and one or more combinations of alkyl adenine glycosylase;

the fifth gene element is a nucleotide sequence for coding a suppression system, and a tTA transcription factor can start the expression of the suppression system, so that a trans-acting factor is knocked down or knocked out, and an antibody sequence dynamic library is maintained to be stable; or the fifth genetic element is a nucleotide sequence encoding an photocontrol system capable of regulating expression of the fourth genetic element: starting the expression of the fourth gene element under the illumination condition, and starting to construct an antibody dynamic sequence library; after the light irradiation is stopped, the function of the fourth gene element is inhibited, and the antibody dynamic sequence library is maintained to be stable.

Preferably, the cell is a eukaryotic cell or a prokaryotic cell, further preferably, the cell is a cell of human or murine origin, and more preferably, the cell is 293T.

Preferably, the antibody sequence dynamic library comprises a traditional antibody library, a single-chain antibody library and a nano-antibody library.

Preferably, the nucleotide sequence of the receptor 1 is shown as SEQ ID NO. 1.

Preferably, the receptor 2 is a synNotch receptor modified from a Notch receptor, and the nucleotide sequence of the synNotch receptor is shown as SEQ ID NO.2 or SEQ ID NO. 3.

Preferably, the trans-acting factor comprises the following Cas protein family members and partial domains thereof: cas3-8s, cas10s, cas11s, cas9s, xCas9, cas12s, cas13s, and Cas14s.

Preferably, the trans-acting factors include the following adenine deaminases and partial domains thereof: ecTadA, mADA, hADAR2 and hADAT2.

Preferably, the trans-acting factors include the following cytidine deaminases and partial domains thereof: AID (activation-induced cytidine deaminase), APOBEC3G, APOBEC and CDA1.

Preferably, the nucleotide sequence encoding the light control system comprises the following photoproteins and partial domains thereof: bphS, cph1, PCB, phyB-PIFs, P Φ B, bphP-PpsR 2, bphP1-Q-PAS1, LOV2-J α, phyB, PIF6, epdz and Photometer visual (VVD).

Preferably, the Cas protein family is a family of proteins that recognize and direct specific sites on the genome through grnas.

Preferably, the light control system exerts a regulation effect on the fourth genetic element and requires coordination of illumination, wherein the illumination comprises visible light, invisible light, blue light, red light, far infrared light and ultraviolet light.

Preferably, the screening genes comprise GFP, mCherry, luciferase, puromycin resistance genes, bleomycin and neomycin resistance genes.

Preferably, the inhibition system enables the expression of negative regulatory protein, repressor protein, siRNA, CAS9/gRNA through tTA transcription factor or knockdown or knock-out of trans-acting factor through reverse transcription mode, so that the dynamic library of antibody sequences is kept stable.

In a second aspect, the present invention provides an antigen-binding domain auto-optimized chimeric antigen receptor-modified cell library obtained by the above-described construction method.

In a third aspect, the present invention provides a method for screening a library of chimeric antigen receptor-modified cells that are automatically optimized for the antigen binding domain described above, comprising the steps of:

1) Preparing a target antigen carrying a tag capable of being recognized by the chimeric receptor 2, the tag comprising biotin or 6 × His;

2) Co-incubating a target antigen and a chimeric antigen receptor modified cell library, and screening positive chimeric antigen receptor expression cells according to the expression condition of the screened genes;

3) Performing amplification sequencing on the chimeric antigen receptor extracellular antigen binding domain of the positive cell to obtain an antibody nucleotide sequence capable of being specifically bound with a target antigen;

preferably, after obtaining the nucleotide sequence of the antibody capable of specifically recognizing and binding to the target antigen, the antibody against the target antigen is obtained by genetic engineering.

Preferably, the target antigen is expressed on the surface of a cell, coated on the surface of a carrier, or is a soluble protein.

Preferably, the carrier comprises magnetic microspheres, non-magnetic microspheres, graphene, agarose microspheres, nanoparticles, silica-based magnetic beads, GMA magnetic beads and polystyrene magnetic microspheres.

In a fourth aspect, the present invention provides the use of the above screening method for the production of antibodies against a particular target antigen.

In a fifth aspect, the present invention provides an antibody screened by the above method.

The GFP nano antibody is obtained by using GFP-biotin as a target antigen and constructing a chimeric receptor modified cell library in different modes and screening, wherein an amino acid sequence of the anti-GFP nano antibody comprises any one of SEQ ID NO.4-13 or any combination of CDR1, CDR2 and CDR3 regions in SEQ ID NO. 4-13.

The invention has the beneficial effects that:

the library constructed by the construction method of the chimeric antigen receptor modified cell library is a dynamic library, can be screened and automatically optimized in an antigen binding domain, and overcomes the defect of limited sequence diversity of a static library in the prior art; and the whole process can be optimized by selecting a human antibody as a framework, so that the humanization of the antibody is directly realized. The dynamic library constructed by the method can be repeatedly utilized, and corresponding antigen binding domain sequences aiming at various antigens can be obtained according to the screening method of the chimeric antigen receptor modified cell library provided by the invention. Compared with the traditional antigen binding domain sequence/antibody sequence acquisition method, the efficiency is greatly improved, and the cost is greatly reduced.

Drawings

FIG. 1 is a schematic view of the present invention;

FIG. 2 is a schematic diagram of a gRNA-guided base editor;

FIG. 3 is a schematic diagram of a G-quadruplex guide base editor;

FIG. 4 is a schematic diagram of an optimized gRNA guide base editor;

FIG. 5 is a schematic view of the structure of the carrier 1;

FIG. 6 is a schematic structural view of the carrier 2;

FIG. 7 is a schematic structural view of the carrier 3;

FIG. 8 is a schematic view of the structure of the carrier 4;

FIG. 9 is a schematic view of the structure of the carrier 5;

FIG. 10 is a schematic diagram of the structure of dCas9 fused adenosine deaminase;

FIG. 11 is a schematic diagram of the structure of dCas9 fused cytidine deaminase;

FIG. 12 is a schematic structural view of dCas9-Vivid-Mcm 7;

FIG. 13 is a schematic representation of the structure of Vivid fused cytidine deaminase and adenosine deaminase;

FIG. 14 is a graph showing the results of eGFP recognition of VHH sequences obtained by ELISA;

in the figure, a, base editor positioning and library building are guided according to G-quadruplet of DNA, and screened nano-antibodies are identified; b, positioning and establishing a library according to a gRNA guide base editor and identifying the screened nano antibody; c, positioning and establishing a library according to a gRNA guide improved base editor and identifying the screened nano antibody.

FIG. 15 is a graph showing the results of PCR amplification of all antibody heavy chains;

FIG. 16 is a graph showing the results of a large number of PCR amplifications of all antibody heavy chains;

FIG. 17 is a graph showing the results of PCR amplification of nanobodies;

FIG. 18 is a flow cytometry validation of receptor 1 and receptor 2 expression, respectively;

FIG. 19 is a cell ELISA and immunofluorescence validation of receptor 1 and receptor 2 expression;

in the figure, a, the dimerization activated double receptor system expresses the structural pattern in the cell and the detection method is shown schematically; b, detecting the expression condition of streptavidin (receptor 1) by using cell ELISA; and c, incubating GFP and cells expressing the dimerization activation double-receptor system for 15 min under a fluorescence microscope to observe the expression condition of the receptor 2.

FIG. 20 shows the screening of nanobodies with biotin-labeled GFP protein;

FIG. 21 shows the results of the specific identification of nanobodies.

In the figure, a, the flow type of unlabelled GFP protein is used for detecting 5 positive cell clones screened; detecting the specificity of the 5 nano antibodies after prokaryotic expression and purification to GFP protein by ELISA, and taking the anti-CD19 nano antibody as a negative control; and c, incubating the positive cells carrying the VHH5 gene and unlabeled GFP for 30min, and observing the binding condition of the GFP and the cells under a fluorescence microscope.

Detailed Description

The invention will be further described with reference to specific embodiments, but the scope of the invention is not limited thereto; the equipment and reagents used in the examples are, unless otherwise specified, conventionally available commercially.

The invention constructs a vector for expressing a receptor 1 and a receptor 2, wherein the receptor 1 can recognize a biotin tag, an extracellular antigen binding domain of the receptor 2 can display a nano antibody library, when a target antigen with the biotin tag is incubated with a chimeric antigen receptor modified cell library, the target antigen can induce the receptor 1 and the receptor 2 to generate a dimer, and after dimerization, tobacco etch virus protease (TEV protease, TEVP) of an intracellular domain of the receptor 1 recognizes and cuts a tTA transcription factor structural domain which releases an intracellular signaling domain of the receptor 2, and the tTA transcription factor can start the expression of a screening gene and a suppression system (as shown in figure 1).

And then taking camel peripheral blood mononuclear cells to obtain a natural nano antibody sequence, replacing a nano antibody sequence region outside a receptor 2 cell with a natural nano antibody sequence library, packaging lentiviruses by using a vector and transfecting 293T cells, and screening and transfecting positive cells by using puromycin, so that the nano antibody library is displayed on the 293T cells through the receptor 2, and the chimeric antigen receptor modified cell library is obtained.

GFP protein is obtained through prokaryotic expression and purification, biotin-labeled GFP protein (GFP-biotin) is prepared to serve as a target antigen, the modified cell library is screened, and the fact that the chimeric receptor modified cell library activated through dimerization of the receptor 1 and the receptor 2 is successfully constructed and can be screened to obtain the GFP protein nano antibody is proved.

Furthermore, the invention introduces a fourth gene element into the chassis cells, namely, one or more cis-regulatory elements and one or more trans-acting factors are introduced, mutation is continuously induced in an antigen binding domain, and sequence diversity is introduced, so that a dynamic library of a nano antibody sequence is established, a dynamic chimeric antigen receptor modified cell library is constructed and screened, and a nano antibody of GFP protein is obtained, thereby proving the feasibility of the construction and screening methods of the chimeric antigen receptor modified cell library provided by the invention.

Example 1: vector construction

The vector inserted with a series of gene elements can express receptor 1 and receptor 2 in 293T cells, receptor 1 can recognize biotin tags, the extracellular antigen binding domain of receptor 2 can display a nanobody library, when the target antigen with the biotin tags is incubated with a library of chimeric antigen receptor modified cells, the target antigen can induce receptor 1 and receptor 2 to dimerize, and after dimerization, tobacco etch virus protease (TEV protease, TEVP) of the receptor 1 intracellular domain recognizes and cleaves a tTA transcription factor domain releasing the receptor 2 intracellular signaling domain, and the tTA transcription factor can start the expression of a screening gene and a suppression system (as shown in FIG. 1).

Preferably, the vector 1 (shown in FIG. 5) has a key DNA sequence shown in SEQ ID NO.14 (the sequence of SEQ ID NO.14 is a sequence between the cPPT/CTS element and the EF-1. Alpha. Core promoter element shown in FIG. 5, and the sequence can be connected to any lentiviral vector with similar structure). The key DNA sequence of the vector 2 (see FIG. 6) is shown as SEQ ID NO.15 (SEQ ID NO.15 is a sequence between the cPPT/CTS element and the EF-1. Alpha. Core promoter element in FIG. 6, and this sequence can be linked to any lentiviral vector with similar structure). The vector was subjected to whole-gene synthesis by Gene Synthesis.

Example 2: construction of Nanobody libraries

Purchasing 200ml camel blood from Tianjin desert visitor, performing camel blood mononuclear cell separation experiment, and finally separating to obtain 2.03 × 10 ⁹ The mononuclear cells were resuspended in Trizol and then aliquoted into 10 centrifuge tubes (2 ml Trizol/centrifuge tube) and frozen in a-80 ℃ freezer.

RNA was extracted from cells In One of the tubes, and 95.8. Mu.g (A260/A280 = 2.0) In total was extracted, and All of them were reverse transcribed into cDNA using 5 × All In One RT MasterMix (Cat # G490, abm) and frozen In a freezer at-20 ℃.

1) First PCR amplification of all antibody heavy chains

Experimental materials:

Hieff Canace ^R gold High-Fidelity DNA Polymerase, product number 10148ES10;

an amplification primer:

CALL001:5′-GTCCTGGCTGCTCTTCTACAAGG-3′；

CALL002:5′-GGTACGTGCTGTTGAACTGTTCC-3′；

reaction system:

reaction procedure:

and (3) reaction results:

the PCR amplification results are shown in FIG. 15, which shows that the effect of 0.5. Mu.g of cDNA in a total amount of 5. Mu.l is the best.

re-PCR amplification: the cDNA was amplified once more with 5. Mu.l, and 8 tubes were amplified.

And (3) recovering PCR amplification products:

cutting 700bp band, using SanPrep column DNA gel recovery kit (REF B518131-0100, OLD # SK8132, LOT E710KA 8969) to recover target band DNA (as shown in FIG. 16), recovering 8-tube PCR product with total volume of 240 μ l, product concentration of 42ng/μ l, and purity (OD 260/280= 1.81).

2) Second PCR amplification of the Nanobody (8 tube PCR products from the recovery using nested primers amplification)

Experimental materials:

Hieff Canace ^R gold High-Fidelity DNA Polymerase, product number 10148ES10.

The primers are as follows: xba: i TCTAGA, ecoR: i GAATTC

Reaction system:

reaction procedure: in the same way as before

And (3) amplification results: as shown in fig. 17

Reaction system:

reaction procedure: in the same way as before

3) PCR product purification

Experimental materials: DNA product purification kit (D1300);

the experimental results are as follows: the concentration of the purified product is 150 ng/mu l, and the total volume is 240 mu l; purity a260/a280=1.84

4) And (3) carrying out enzyme digestion on the vector 1 or the vector 2, carrying out enzyme digestion on the PCR product and carrying out enzyme ligation. Experimental materials:

XmaI(NEB，R0180S)，

(NEB, R3136S), T4 DNA ligase (Takara, cat #2011A, lot # AIF 2071A), sanPrep column PCR product purification kit (REF # B518141-0100, LOT # B206KA 5188).

The experimental steps are as follows:

a) Enzyme digestion plasmid vector

b) Enzyme digestion of PCR product

c) Purification of the cleavage product after completion of the cleavage

The cleavage products were purified using a SanPrep column PCR product purification kit.

Carrier 1 gave 60 μ L of purified product 150ng/μ L A/A280 =1.86

VHH PCR product 40 μ L100 ng/μ L A/A280 =1.85

d) Enzymatic ligation

Ligation was carried out overnight at 16 ℃.

e) Ligation product purification

The ligation product was purified using a SanPrep column PCR product purification kit, using 100. Mu.L ddH ₂ And eluting with O buffer solution.

Example 3: preparation of competent cells of Escherichia coli

This study used the electrical transformation method to transfer the recombinant plasmid into the E.coli strain Stbl 3. The preparation method of competent cells of E.coli by electrotransformation (note: it needs to be operated before alcohol burner):

1) Streaking an escherichia coli Stbl3 bacterial liquid on an LB solid culture medium plate without antibiotics by using an inoculating loop (operation in an ultra-clean workbench), and inverting the plate and culturing at 37 ℃ for 24h to obtain a single colony with the diameter of about 1 mm;

2) Selecting a Stbl3 single colony to be placed in 3ml of LB liquid culture medium without antibiotics, placing the LB liquid culture medium in a constant temperature shaking table at 37 ℃, and shaking the LB liquid culture medium at 220rpm for overnight culture;

3) Transferring 3ml of overnight culture bacterial liquid into 250ml of LB liquid culture medium (without antibiotics), placing the mixture in a constant temperature shaking bed at 37 ℃, carrying out shaking culture at 220rpm for about 3-5h, and stopping culture when the OD value of the bacterial liquid reaches 0.4-0.5;

4) Respectively packaging 250ml of bacterial liquid into 2 85ml sterile centrifuge tubes (divided for 2 times), placing the tubes into a low-temperature centrifuge, centrifuging the tubes at 4 ℃ and 4000rpm for 10min, pouring out supernate, and placing the tubes into an ice box;

note that the following operations need to be performed at low temperature (ice bath operation).

5) Using 30ml sterile ddH ₂ O (precooling in refrigerator at 4 ℃) to fully suspend the bacterial sediment again, placing the bacterial sediment in a low-temperature centrifuge, centrifuging the bacterial sediment for 10min at the temperature of 4 ℃ and the rpm of 4000, pouring out the supernatant, and placing the centrifugal tube in a centrifugal tubeIn an ice box;

6) Repeating the previous step for 1 time;

7) Fully suspending the bacterial precipitate again by using 30ml of 10% glycerol (precooling in a refrigerator at 4 ℃), placing the bacterial precipitate in a low-temperature centrifuge, centrifuging the bacterial precipitate for 10min at 4 ℃ and 4000rpm, pouring out supernatant, and placing a centrifugal tube in an ice box;

8) Repeating the previous step for 1 time;

9) Resuspend the pellet with 0.5ml 10% glycerol (pre-chilled in a refrigerator at 4 ℃), gently blow it with a pipette to break up small clumps, dispense the pellet into 1.5ml sterile centrifuge tubes, 45 μ l per tube, dispense and place in an ice box.

10 The subpackaged centrifuge tubes are placed in a foam box, a proper amount of liquid nitrogen is poured into the foam box, and after freezing for 1min, the centrifuge tubes are stored at the temperature of minus 20 ℃ for standby.

Example 4: electrotransformation and plating of recombinant vectors

Experimental materials: U.S. Bio-Rad MicroPulser electroporator, bio-Rad electric rotor (cat # 165-2089).

The experimental method comprises the following steps: 100 μ L of purified ligation product +1mL of electrocompetent cells were electroporated 20 times (50 μ L each) by:

1) Pre-cooling the electric rotary cup (fresh and sterile) in a refrigerator at the temperature of-20 ℃;

2) Setting the spot punch instrument to be 'bacterio' and 'Time ms', and carrying out an experiment in an automatic mode;

3) Transferring the uniformly mixed competent cells into a precooled sterile electric transfer cup, covering a cover, and tapping for several times to ensure that the bacterial liquid fully enters an electric shock groove;

4) Placing the electric rotating cup on a point punching instrument, and clicking to normally display for 3-5 ms;

5) After the completion of the electroporation, 0.5mL of the preheated SOC recovery medium was added to each of the electroporation cuvettes, and then 20 cells were transferred to the same 50mL centrifuge tube and incubated at 37 ℃ and 170rpm for 1 hour.

6) 100 μ L of the cells after electroporation was diluted with LB medium at 10 × gradient (1,000,1, 1,000,1 ^-1 ampicillin，2％(wt/vol)glucose)，37℃，Overnight.

7) The remaining cells were divided into 4 portions and plated on 245mm square LB plates (100. Mu.g ml) ^-1 ampicillin and 2% (wt/vol) glucose), 37 ℃, overnight.

Example 5: computational electrotransfer efficiency collection library

1) The number of colonies on 9cm LB agar plates was counted, the library size was calculated by colony number × dilution factor, 20 single colonies were picked and colony PCR was performed using vF and vR as primers, and the PCR product size was analyzed to ensure that >75% of the colonies contained the correct size DNA fragment.

2) 100 single colonies are picked and used for vF and vR as primers for sequencing to obtain the library size of 100 single colonies, and then the size of the whole nano antibody library is calculated to be 2.15 multiplied by 10 ⁷ 。

3) Add 4mL LB media to each dish, collect cells with sterile cell scraper and transfer to 50mL centrifuge tube, rinse each dish once with 2mL LB media, collect cells, add 20% volume glycerol, measure OD ₆₀₀ And 20 aliquots of 150 μ L volume of cell suspension were separated and the remainder was kept in large centrifuge tubes and frozen at-80 ℃.

4) Taking out one part of the bacterial liquid to amplify and extract the plasmid, and the extraction step is carried out according to the kit instruction.

Example 6: preparation of lentivirus and selection of stable transformant

Mu.g of the ligated vector 1 or vector 2 plasmid and 17.5. Mu.g of the two lentiviral packaging plasmids PSPAX 2. Mu.g and PMD2. G5.8. Mu.g were added to 1.8mL of calcium chloride in the ratio of 4. Adding the mixed solution into 1.8mL BBS, standing for 20min, and adding 175cm BBS ² 293T cell culture flasks. The virus supernatants were harvested at 48h and 72h, respectively, and concentrated using 5 × PEG. The resulting virus was transfected with polybrene in 293T dishes. After 2 days, selection was performed with puromycin at 6. Mu.g/mL, and the puromycin concentration was maintained for 5 days by changing the solution every day.

Example 7: detection of stably transformed plants

Since the 293T stable transformant of the transfection vector 1 screened in example 6 expresses the receptor 1 and the receptor 2, and the extracellular domains of the two receptors carry Flag and HA tags, respectively, we verified the expression of the receptor 1 and the receptor 2 by flow cytometry by identifying the flow antibodies of the two tags, as shown in fig. 18, the two receptors are successfully expressed.

Since the extracellular domain of receptor 1 expressed by 293T stable transformant of transfection vector 2 screened in example 6 is single-domain streptavidin capable of recognizing biotin, we detected the expression of receptor 1 by cell ELISA using biotin-labeled antibody and HRP-labeled streptavidin (see FIG. 19 b). Receptor 2 extracellular domain has a nanobody domain, and we also detected the expression of receptor 2 with a Goat antibody (coat Anti-Llama IgG H & L, abcam, ab 112785) labeled with FITC that recognizes nanobodies (see FIG. 19 c).

Example 8: GFP protein purification and GFP-biotin ligand preparation

We pronucleus expressed and purified GFP protein by the following steps:

1) The eGFP protein expression vector is pET-28a (+), and the host bacterium is E.coli BL21 (DE 3);

2) Streaking an escherichia coli BL21 (DE 3) bacterial liquid on an LB solid culture medium plate containing kanamycin by using an inoculating loop (operation in an ultra-clean workbench), and inversely placing the plate at 37 ℃ for culturing for 24 hours to obtain a single bacterial colony with the diameter of about 1 mm;

3) Selecting a single colony to be placed in 3ml of LB liquid culture medium without antibiotics, placing the single colony in a constant temperature shaking table at 37 ℃, and shaking at 220rpm for overnight culture;

4) Transferring 3ml of overnight culture bacterial liquid into 200ml of LB liquid culture medium (without antibiotics), placing the mixture in a constant temperature shaking bed at 37 ℃, carrying out shaking culture at 220rpm for about 3-5h, and stopping culture when the OD value of the bacterial liquid reaches 0.8;

5) Adding 0.5mM IPTG to induce protein expression (IPTG mother liquor concentration is 1M,200ml LB liquid culture medium, adding 100. Mu.l), placing in a constant temperature shaking table at 16 ℃, and carrying out shake culture at 170rpm for 16h;

6) Centrifuging at 4000g for 5min, discarding the supernatant, and washing with 30ml Buffer A once;

7) Adding 25ml Buffer A, mixing, inserting on ice, performing ultrasonic crushing for 2s, 5s for gap time, and 40 times for working times.

8) Centrifuging at 10000rpm at 4 deg.C for 30min, and filtering the supernatant with 0.45 μm filter;

9) Washing the nickel column with 10 times volume of Buffer A, and adding a clear solution after washing;

10 ) after the supernatant had flowed through, 5 volumes of Buffer A washout protein containing 10mM imidazole were added once, 5 volumes of Buffer A washout protein containing 20mM imidazole were added once, 5 volumes of Buffer A washout protein containing 40mM imidazole were added once, 5 volumes of Buffer A washout protein containing 250mM imidazole were added once, and eluates were collected into different 1.5ml centrifuge tubes. Note that: when the protein is washed by imidazole at low concentration, quick Start is used ^TM The Bradford Protein Assay solution was used to observe whether any Protein was eluted.

11 Wash the nickel column with 10 volumes of Buffer a, then wash the nickel column with 10 volumes of ddH2O, and finally keep the nickel column in 20% ethanol for use.

12 SDS-PAGE gels to determine protein content and purity in each tube.

13 ) the protein solution in the target tube is combined, and the protein solution is concentrated by using a proper ultrafiltration tube, and 3 times of protein preservation solution is added to replace the eluent.

14 The elongated tip is used to transfer the protein solution.

15 Biotin-labeled GFP protein (GFP-Biotin) was prepared using a Biotin super Biotin rapid labeling kit (ARL 0020S-30K-0.5mL, available from forneddtechnology (wuhan) ltd.) and the Biotin labeling procedure was performed as described.

Example 9: nano antibody screening and specificity identification

Experimental materials: sequencing primer 5'-GAGGGCAGAGGAAGTCTGCT-3'; blastidin (Solibao);

and (2) incubating a GFP (GFP-biotin, 0.1 mu g/ml) ligand marked by biotin and the transfected 293T cells for 24h, screening positive cells (5 mu g/ml) by using blastcidin, carrying out monoclonal amplification on the positive cells, and sequencing a nano antibody region to obtain the GFP-specific nano antibody. On the left side of FIG. 20 are 293T cells not screened with blastcidin and one of the monoclonal cells after blast screening (on the right side of FIG. 20).

A total of 5 positive cell clones, VHH1, VHH2, VHH3, VHH4 and VHH5, were obtained by screening, and the results of flow assay with unlabeled GFP protein are shown in FIG. 21a, with staining positive for 2 cell clones, VHH3 and VHH 5.

The sequence of 5 nano-antibodies was obtained by sequencing, and the specificity of the 5 nano-antibodies to GFP protein was verified and screened by ELISA after prokaryotic expression and purification (step of prokaryotic expression and purification, expression vector, and induction expression conditions were as described in example 8), the results are shown in fig. 21b, which are consistent with the flow results, and the flow-positive VHH3 and VHH5 nano-antibody ELISA results corresponding to the 2 cell lines were also positive.

The ELISA assay procedure was as follows:

1) mu.L of PBS containing GFP protein (1. Mu.g/mL) was added to each well of the plate, mixed well by gentle shaking for 1min, the plate was sealed, and coated at room temperature for 2h.

2) The Solution was decanted, and 400. Mu.L of Wash Solution was added to each well for washing 4 times, 1min each time. After the last wash, the liquid was drained and the residue was blotted dry with clean absorbent paper.

3) mu.L of PBS containing VHH1-5 protein (1. Mu.g/mL) and PBS containing anti-CD19 VHH protein (1. Mu.g/mL) (control) were added to each well, the plates were sealed, and coated for 2h at room temperature.

4) mu.L of HRP-labeled anti-His tag antibody working solution was added to each well, the plate was sealed, and the plate was coated at room temperature for 2 hours.

5) The Solution was decanted and washed with 400 μ L of Wash Solution; repeating for three times; after the last wash, the liquid was drained and the residue was blotted dry with clean absorbent paper.

6) Add 200. Mu.L of Substrate Solution to each well and coat for 20-30min at room temperature in the dark.

Add 50. Mu.L of Stop Solution to each well and shake well. And detecting the light absorption value within 30min, wherein the detection wavelength of the microplate reader is set to be 450nm, and the reference wavelength is set to be 540 or 570nm.

Finally, positive cells carrying the VHH5 gene were incubated with unlabeled GFP for 30min, and the results are shown in FIG. 21c, where the GFP protein was found to bind efficiently to the cells by observation under a fluorescence microscope.

Then, in order to increase the affinity of the VHH5 nanobody to GFP protein, we performed a series of artificial mutations to the three CDR regions of the VHH5 gene, and finally obtained a series of nanobodies with different affinities to GFP, including the VHH5 gene. Wherein the VHH5 nanobody comprises any one of SEQ ID No.16-25, or any combination of the CDR1, CDR2, CDR3 regions of SEQ ID No. 16-25.

Example 10: base editor positioning and library building are guided according to G-quadruplet of DNA, and nano antibody screening and identification are carried out

This example is to perform base editing and library building in vitro on the base editor guided by the G-quadruplet higher structure of DNA in the CDRs region of a nano antibody with a known sequence.

As shown in the principle of FIG. 3, since all base editors can only act on single-stranded DNA at present, one DNA single strand can be generated by the helicase action of dCas9 and gRNA, and in addition, a high-level structure, such as G-quadruplex, can be induced on one DNA single strand, so that another DNA single strand is generated, and the formation of G-quadruplex can be induced by a DNA sequence which is rich in G base and has a minimum length of 25 bp. Nucleolin-linked cytidine deaminase (promoting the mutation of G or C base to any base) and Nucleolin-linked adenosine deaminase (promoting the mutation of A base to any base) are designed at the same time, and because Nucleolin can recognize and combine with G-quadruplex, two base editors are positioned in CDRs. The two base editor genes connected by Nucleolin-Vivid and Vivid are transferred into 293T cells together through a sleeping beauty transposon system SB100X to construct a stable transformant. The method realizes the wide mutation induction of CDRs of VHH by optimizing the length of a Linker sequence between Vivid and deaminase and the blue light irradiation time.

Vector 2 (see example 1) was packaged into lentivirus and transferred into 293T cells and selected for stable transformants by puromycin screening.

Constructing a vector 3 (shown in FIG. 7), wherein the key DNA sequence of the vector 3 is shown as SEQ ID NO.26 (the sequence of SEQ ID NO.26 is the sequence between the cPPT/CTS element and the EF-1. Alpha. Core promoter element in FIG. 7, and the sequence can be connected to any lentiviral vector with similar structure);

constructing a vector 4 (as shown in FIG. 8), wherein the key DNA sequence of the vector 4 is shown as SEQ ID NO.27 (the sequence of SEQ ID NO.27 is a sequence between the cPPT/CTS element and the EF-1 alpha core promoter element in FIG. 8, and the sequence can be connected to any lentivirus vector with a similar structure); (vector delivery to Gene Synthesis Co., ltd for Total Gene Synthesis)

The vector 3 and the vector 4 are sequentially transferred into 293T cells by packaging lentiviruses and constructing 293T cell stable transformants.

After the vector transfection is finished, culturing for 5 days, then extracting cell genome DNA, and amplifying the nano antibody fragment after the library is built by using nested PCR, wherein the sequences of two pairs of nested PCR primers are as follows: f1:5'-CTAGAGCCACCATGGCCC-3'; r1: 5'-CCAGGATGTGGCACAGCA-3'; f2:5'-TCTAGATGGCCCTGCTGCTGCACG-3'; r2: 5'-GAATTCAGGTGCCCTGGTTGTAGC-3', after the fragment ends are amplified, the fragments are connected to pET-28a-c (+) vector (Novagen, cat. No. 69864-3) through enzyme digestion (Xba I/EcoR I) enzyme connection (T4 ligase), and the size of the cell display nano antibody library is calculated to be about 2.67 x 10 by the steps of electrotransformation, plate coating, monoclonal picking sequencing and the like ⁶ (see reference examples 2-5 for specific procedures).

And (3) incubating a GFP-biotin ligand and the transfected 293T cells for 24h, screening positive cells by using blastcidin, carrying out monoclonal amplification on the positive cells, and sequencing a nano antibody region to obtain the GFP specific nano antibody.

And selecting 3 nano antibody sequences in the obtained positive cells, and performing prokaryotic expression and purification (the protein expression and purification method is shown in example 8) to obtain the VHH antibody protein.

Detecting whether the obtained VHH sequence can identify eGFP by ELISA, wherein the specific steps comprise 1, coating a 96-hole plate with eGFP, and cleaning for 3 times after coating; 2. adding 3 purified VHH antibody proteins, simultaneously using the VHH antibody protein for resisting CD19 as negative control, and cleaning for 3 times after coating; 3. an HRP-labeled Anti-lama IgG (H + L) secondary antibody was added, coated and washed 3 times. 4. Adding TMB color development solution, and identifying whether the obtained VHH antibody protein can identify eGFP. As a result, 1 of the 3 antibodies showed better recognition of eGFP in FIG. 14a.

Then, in order to increase the affinity of the nanobody to GFP protein, we performed a series of artificial mutations on three CDR regions of the selected VHH3 gene, and finally obtained a series of nanobodies containing VHH3 gene with different affinity to GFP. Wherein the VHH5 nanobody comprises any one of SEQ ID No.4-13, or any combination of the CDR1, CDR2, CDR3 regions of SEQ ID No. 4-13.

Example 11: base editor positioning and library building are guided according to gRNA, and nano antibody screening and identification are carried out

In this example, base editing and library building are performed in vitro on a gRNA-guided base editor in a nano antibody CDRs region of a known sequence.

As shown in fig. 2, firstly, a VHH sequence is obtained, 3 grnas targeting CDR1, CDR2 and CDR3 regions of VHH are designed, a Vivid photosensitive protein (dCas 9-Vivid) linked to an inactivated Cas9 (dCas 9) is designed, and then two high-activity base editors, namely, a Vivid fused cytidine deaminase (promoting mutation of G or C base to any base) and a Vivid fused adenosine deaminase (promoting mutation of a base to any base), are designed, respectively, and under the condition of blue light irradiation, the Vivid photosensitive protein forms homodimers, so that the protein containing the Vivid domain randomly forms dimers, resulting in random binding of the two base editors to the dCas9-Vivid protein, thereby locating to the CDRs regions. gRNAs, dCas9-Vivid and two base editor genes were transferred into 293T cells together by the sleeping beauty transposon system SB100X to construct stable transformants. The method is characterized in that the method realizes the wide mutation induction of CDRs (complementary variable domain) regions of VHH (very high frequency) by optimizing the length of a Linker sequence between Vivid photosensitive protein and deaminase and the blue light irradiation time, so that a single VHH sequence is diversified, the establishment of a nano antibody is realized, and the length of the Linker sequence between Vivid and deaminase and the blue light irradiation time are determined by detecting the diversity of the nano antibody library.

Constructing a vector 5 (shown in figure 9), wherein the key DNA sequence of the vector 5 is shown as SEQ ID NO.28 (the sequence of SEQ ID NO.28 is the sequence between the U6 promoter element and the EF-1 alpha core promoter element in figure 9, and the sequence can be connected to any pLL7 series lentiviral vector with a similar structure);

constructing slow virus expressing carrier 6 (as shown in FIG. 10), the key DNA sequence of the carrier 6 is shown in SEQ ID NO.29 (the sequence can be connected to any proper position of the slow virus expressing carrier), in another preferred embodiment, the homology between the amino acid sequence expressed by the carrier 6 and the SEQ ID NO.29 sequence is more than or equal to 85%.

Constructing a lentivirus expression vector 7 (as shown in FIG. 11), wherein the key DNA sequence of the vector 7 is shown as SEQ ID NO.30 (the sequence can be connected to a proper position of any lentivirus expression vector), (the homology of the amino acid sequence expressed by the vector 7 and the sequence of the SEQ ID NO.30 is more than or equal to 85 percent in another preferred example);

the

vector

5,6,7 was submitted to gene synthesis.

Vector 5, vector 6 and vector 7 were sequentially transferred into 293T cells by packaging lentiviruses and constructing 293T cell stable transformants.

Specific procedure for detection of Nanobody library size referring to examples 2-5, the detected Nanobody library size is about 2.75X 10 ⁷ 。

And (3) incubating the GFP-biotin ligand and the transfected 293T cells for 24h, screening positive cells by using blastcidin, carrying out monoclonal amplification on the positive cells, and sequencing a nano antibody region to obtain the GFP specific nano antibody.

And selecting 5 nano antibody sequences in the obtained positive cells, and performing prokaryotic expression and purification (the protein expression and purification method is shown in example 8) to obtain the VHH antibody protein.

The resulting VHH sequences were tested for the ability to recognize eGFP by ELISA. The results are shown in FIG. 14b, with 2 of the 5 antibodies recognizing eGFP well.

Then, in order to increase the affinity of the nanobody to GFP protein, we performed a series of artificial mutations on the three CDR regions of the two VHH genes that were screened, and finally obtained a series of nanobodies with different affinities to GFP, including the two VHH genes. Wherein the VHH nanobody comprises any two of SEQ ID No.31-40, or any combination of CDR1, CDR2, CDR3 regions of SEQ ID No. 31-40.

Example 12: base editor positioning and library building are guided according to gRNA, and nano antibody screening and identification are carried out

In this example, base editing and library building can be performed in vitro on the basis of guide of gRNA in the CDRs region of a nanobody of unknown sequence.

As shown in fig. 4, since mutation library construction is performed on the CDRs region of the nanobody with a known sequence, and after several rounds of mutation, base editor can not recognize the CDRs region due to the sequence change of the gRNA recognition region or the G-quadruplet region, so that continuous library construction cannot be realized, and in addition, affinity maturation mutation cannot be performed after obtaining a specific nanobody, we improved on the basis of the scheme of fig. 1, 3 grnas respectively targeting the adjacent regions on the right side of CDR1, CDR2 and CDR3 of VHH are designed, and simultaneously, a complete hexamer containing an inactivated Cas9 (dCas 9) guide domain and a viid photoprotein and a helicase Mcm7 (with the N-terminal closer to the CDR region) are designed, wherein the Mcm7 and Mcm2-6 expressed in 293T cells form a cdrdinase activity, and the complete hexamer forms cdrdase activity, and the cddr-active protein hexamer is fused with the cdrd-active protein under the conditions of providing drive of ATP hydrolysis to open the double-stranded region (3 '-5's helicase activity) and guide the cdrd-6 expressed in 293T cells to promote the high base editing region, so that the cdrd-active protein is fused to the cdnas-active region, and the cdnas-active region, so that the cdnas-active protein is fused to the cdnas-active region through a-light-active region, and the random base deaminase, so that the cdnas-active region is fused to promote the cdnas-active region, so that two cdnas-active region, so that the high-active region is fused to form a high-active region under the high-active region, and the high-active deaminase is fused to promote the high-active site of the cdnas-active site. A stable transformant was constructed by transferring (dCas 9-Vivid-Mcm 7) and two base editor genes into 293T cells through the sleeping beauty transposon system SB 100X. The method realizes the wide induction mutation of CDRs of VHH by optimizing the length of Linker sequence between dCas9-Vivid-Mcm7, the length of Linker sequence between Vivid and deaminase and blue light irradiation time, and determines an optimization scheme by detecting the diversity of a nano antibody library.

Vector 2 was packaged into lentivirus and transferred into 293T cells and stable transformants were obtained by puromycin screening.

A vector 8 (shown in figure 12) is constructed, the vector 8 is a lentiviral vector same as the vector 1, the vector 8 can express a fusion protein containing three domains of Mcm7 (SEQ ID NO. 41), vivid (SEQ ID NO. 42) and dCAS9 (the sequence is consistent with or has homology of more than or equal to 85% with the inactivated CAS9, namely dCAS9, in Streptococcus pyogenes commonly used in the literature), wherein the fusion protein contains Mcm7 (the amino acid sequences of the two domains of Mcm7 and Vivid expressed by the vector 8 have homology of more than or equal to 85% with the sequences of SEQ ID NO.41 and SEQ ID NO. 42), and the three domains are sequentially connected by a5 xGGGGS Linker (SEQ ID NO. 43) (the length of the Linker can be optimized between 2 xGGGGS and 10 xGGS in another embodiment).

Constructing a lentivirus expression vector 9 (as shown in figure 13), wherein the vector 9 can express a fusion protein containing three structural domains of Vivid, rAPOBEC1 and ecTadA, the Vivid amino acid sequence is shown as SEQ ID NO.42, the ecTadA amino acid sequence is shown as SEQ ID NO.44 (in another preferred embodiment, the homology of the amino acid sequence expressed by the ecTadA and the SEQ ID NO.44 sequence is more than or equal to 85%), the rAPOBEC1 amino acid sequence is shown as SEQ ID NO.45 (in another preferred embodiment, the homology of the amino acid sequence expressed by the rAPOBEC1 and the SEQ ID NO.45 sequence is more than or equal to 85%, and in another preferred embodiment, the rAPOBEC1 can be replaced by cytidine deaminase AID and mutants thereof), and sequentially transferring the vector 8 and the vector 9 into 293T cells by packaging lentiviruses and constructing 293T cell stable strains.

Finally, vector 10 (vector structure is shown in FIG. 9) was transformed into 293T cells, wherein the vector 10 expresses 3 gRNAs to localize the protein comprising dCAS9 domain to 3 different positions, and the key DNA sequence of the vector 10 is shown in SEQ ID NO.46 (SEQ ID NO.46 is a sequence between the U6 promoter element and the EF-1. Alpha. Core promoter element in FIG. 9, and the sequence can be connected to any pLL7 series lentivirus vector with similar structure).

After 293T cells were irradiated with blue light for 2 days (10 s per 5s on, blue light intensity 0.84W/m ² ) Calculating the size of the nano antibody library, and the specific steps of the detection of the size of the nano antibody library are shown in the examples2-5, detected nanoconsubes size of about 6.75X 10 ⁷ . And (3) incubating a GFP-biotin ligand and the transfected 293T cells for 24h, screening positive cells by using blastcidin, selecting the positive cells, performing monoclonal amplification on the positive cells, and sequencing a nano antibody region to obtain the GFP specific nano antibody.

The resulting VHH sequences were tested for their ability to recognize eGFP by ELISA. The results are shown in fig. 14c, with 1 of 5 antibodies recognizing eGFP better.

Then, in order to increase the affinity of the nanobody to GFP protein, we performed a series of artificial mutations on the three CDR regions of the screened VHH gene, and finally obtained a series of nanobodies with different affinities to GFP, including the VHH gene. Wherein the VHH nanobody comprises any one of SEQ ID No.31-40, or any combination of the CDR1, CDR2, CDR3 regions of SEQ ID No. 31-40.

<110> New countryside medical college

<120> construction and screening method of chimeric antigen receptor modified cell library with automatically optimized antigen binding domain and application thereof

<160> 46

<170> PatentIn version 3.5

<210> 1

<211> 1416

<212> DNA

<213> Artificial sequence

<221> receptor 1 sequence

<400> 1

atggccctgc tactggccct cagcctgctg gttctctgga cttccccagc cccaactctg 60

agtggcacca atgattaccc atacgatgtt ccagattacg ctgcggaagc gggtatcacc 120

ggcacgtggt acaaccagca tggttctacc ttcaccgtta ccgcgggtgc ggacggtaac 180

ctgaccggtc agtacgaaaa ccgtgcgcag ggcactggtt gccagaactc tccgtacacc 240

ctgaccggtc gttacaacgg taccaaactg gaatggcgtg ttgaatggaa caactctacc 300

gaaaactgcc actctcgtac cgaatggcgt ggtcagtacc agggtggtgc ggaagcgcgt 360

atcaacaccc agtggaacct gacctacgaa ggtggttctg gtccggcgac cgaacagggt 420

caggacacct tcaccaaagt taaaatgtac ttcagccact tcgtgccggt cttcctgcca 480

gcgaagccca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 540

cagcccctgt ccctgcgccc agaggcgagc cggccagcgg cggggggcgc agtgcacacg 600

agggggctgg acttcgccag cgatatctac atctgggcgc ccttggccgg gacttgtggg 660

gtccttctcc tgtcactggt tatcaccctt tactgcggag gcggggagag tttgtttaag 720

gggccaaggg actataaccc aatatcatcc actatttgcc acctcactaa cgagagcgat 780

ggacatacaa cctctctcta cgggataggc ttcggtcctt tcatcattac caataagcat 840

ctgtttcgcc gaaacaacgg tactcttctg gttcaatctc ttcatggtgt cttcaaggtg 900

aaaaacacca ctacgcttca gcaacacctg attgatggta gggatatgat aattatcaga 960

atgccgaaag atttcccacc ttttccacag aagctgaaat tcagggaacc gcagagagag 1020

gagaggattt gtttggtaac gaccaacttc cagacgaaga gtatgagttc tatggtgtcc 1080

gacactagct gcacgttccc ctcaagtgat gggatattct ggaaacactg gatacagact 1140

aaagacggac agtgtggaag cccattggtt tccacccgag atggttttat cgtgggtatc 1200

catagcgcct ctaatttcac aaacacgaac aactatttca cttcagtgcc caaaaacttt 1260

atggagctgc tcacaaacca agaagcgcag cagtgggtaa gcggttggag acttaacgct 1320

gactcagttc tctggggggg gcacaaagta ttcatggtaa agccagagga gccattccaa 1380

ccagtcaaag aagccacaca acttatgaac agctaa 1416

<210> 2

<211> 2394

<212> DNA

<213> Artificial sequence

<221> receptor 2 sequence 1

<400> 2

atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccagg 60

cccgactaca aggacgacga cgacaagccc gggatggccc aggtgcagct ggtggagagc 120

ggcggcggcc tggtgcaggc cggcggcagc ctgaggctga gctgcgccgc cccggggcgg 180

gccgggggcg gggtcccggc ggggtggttc aggcaggccc ccggcaagga gagggagttc 240

gtggccgcgg ggcgcttatg gggagggtgg ggagggtggg gaaggtgggg aggaggcagg 300

ttcaccatca gcagggacaa cgacaagaac accgtgtacg tgcagatgaa cagcctgatc 360

cccgaggaca ccgccatcta ctactgcgcc gcccgcggag gggcgggcgc gggaggaagg 420

gggcgggagc ggggctgtgg cgactactgg ggccagggca cccaggtgac cgtgggatcc 480

ccctgcgtgg gcagcaaccc ctgctacaac cagggcacct gcgagcccac cagcgagaac 540

cccttctaca ggtgcctgtg ccccgccaag ttcaacggcc tgctgtgcca catcctggac 600

tacagcttca ccggcggcgc cggcagggac atcccccccc cccagatcga ggaggcctgc 660

gagctgcccg agtgccaggt ggacgccggc aacaaggtgt gcaacctgca gtgcaacaac 720

cacgcctgcg gctgggacgg cggcgactgc agcctgaact tcaacgaccc ctggaagaac 780

tgcacccaga gcctgcagtg ctggaagtac ttcagcgacg gccactgcga cagccagtgc 840

aacagcgccg gctgcctgtt cgacggcttc gactgccagc tgaccgaggg ccagtgcaac 900

cccctgtacg accagtactg caaggaccac ttcagcgacg gccactgcga ccagggctgc 960

aacagcgccg agtgcgagtg ggacggcctg gactgcgccg agcacgtgcc cgagaggctg 1020

gccgccggca ccctggtgct ggtggtgctg ctgccccccg accagctgag gaacaacagc 1080

ttccacttcc tgagggagct gagccacgtg ctgcacacca acgtggtgtt caagagggac 1140

gcccagggcc agcagatgat cttcccctac tacggccacg aggaggagct gaggaagcac 1200

cccatcaaga ggagcaccgt gggctgggcc accagcagcc tgctgcccgg caccagcggc 1260

ggcaggcaga ggagggagct ggaccccatg gacatcaggg gcagcatcgt gtacctggag 1320

atcgacaaca ggcagtgcgt gcagagcagc agccagtgct tccagagcgc caccgacgtg 1380

gccgccttcc tgggcgccct ggccagcctg ggcagcctga acatccccta caagatcgag 1440

gcccataaga gcgagcccgt ggagcccccc ctgcccagcc agctgcacct gatgtacgtg 1500

gccgccgccg ccttcgtgct gctgttcttc gtgctccttt tctttctgct gagcaggaag 1560

aggaggaggc agctgtgcat ccagaagctg ctcgggatcg agggaagggg aggaggcgag 1620

ttcgctagcg agaacctgta tttccagggc atgtctagac tggacaagag caaagtcata 1680

aactctgctc tggaattact caatgaagtc ggtatcgaag gcctgacgac aaggaaactc 1740

gctcaaaagc tgggagttga gcagcctacc ctgtactggc acgtgaagaa caagcgggcc 1800

ctgctcgatg ccctggcaat cgagatgctg gacaggcatc atacccactt ctgccccctg 1860

gaaggcgagt catggcaaga ctttctgcgg aacaacgcca agtcattccg ctgtgctctc 1920

ctctcacatc gcgacggggc taaagtgcat ctcggcaccc gcccaacaga gaaacagtac 1980

gaaaccctgg aaaatcagct cgcgttcctg tgtcagcaag gcttctccct ggagaacgca 2040

ctgtacgctc tgtccgccgt gggccacttt acactgggct gcgtattgga ggatcaggag 2100

catcaagtag caaaagagga aagagagaca cctaccaccg attctatgcc cccacttctg 2160

agacaagcaa ttgagctgtt cgaccatcag ggagccgaac ctgccttcct tttcggcctg 2220

gaactaatca tatgtggcct ggagaaacag ctaaagtgcg aaagcggcgg gccggccgac 2280

gcccttgacg attttgactt agacatgctc ccagccgatg cccttgacga ctttgacctt 2340

gatatgctgc ctgctgacgc tcttgacgat tttgaccttg acatgctccc aggg 2394

<210> 3

<211> 1584

<212> DNA

<213> Artificial sequence

<221> receptor 2 sequence 2

<400> 3

atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccagg 60

ccctacccat acgatgttcc agattacgct cccgggatgg cccaggtgca gctggtggag 120

agcggcggcg gcctggtgca ggccggcggc agcctgaggc tgagctgcgc cgccagcggc 180

aggaccttca gcaactacgc catgggctgg ttcaggcagg cccccggcaa ggagagggag 240

ttcgtggccg ccatcagctg gaccggcgtg agcacctact acgccgacag cgtgaagggc 300

aggttcacca tcagcaggga caacgacaag aacaccgtgt acgtgcagat gaacagcctg 360

atccccgagg acaccgccat ctactactgc gccgccgtga gggccaggag cttcagcgac 420

acctacagca gggtgaacga gtacgactac tggggccagg gcacccaggt gaccgtggga 480

tccatgtact tcagccactt cgtgccggtc ttcctgccag cgaagcccac cacgacgcca 540

gcgccgcgac caccaacacc ggcgcccacc atcgcgtcgc agcccctgtc cctgcgccca 600

gaggcgagcc ggccagcggc ggggggcgca gtgcacacga gggggctgga cttcgccagc 660

gatatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 720

atcacccttt actgcaattc gagctcgaac aacaacaaca ataacaataa caacaacctc 780

gggatcgagg gaaggggagg aggcgagttc gctagcgaga acctgtattt ccagggcatg 840

tctagactgg acaagagcaa agtcataaac tctgctctgg aattactcaa tgaagtcggt 900

atcgaaggcc tgacgacaag gaaactcgct caaaagctgg gagttgagca gcctaccctg 960

tactggcacg tgaagaacaa gcgggccctg ctcgatgccc tggcaatcga gatgctggac 1020

aggcatcata cccacttctg ccccctggaa ggcgagtcat ggcaagactt tctgcggaac 1080

aacgccaagt cattccgctg tgctctcctc tcacatcgcg acggggctaa agtgcatctc 1140

ggcacccgcc caacagagaa acagtacgaa accctggaaa atcagctcgc gttcctgtgt 1200

cagcaaggct tctccctgga gaacgcactg tacgctctgt ccgccgtggg ccactttaca 1260

ctgggctgcg tattggagga tcaggagcat caagtagcaa aagaggaaag agagacacct 1320

accaccgatt ctatgccccc acttctgaga caagcaattg agctgttcga ccatcaggga 1380

gccgaacctg ccttcctttt cggcctggaa ctaatcatat gtggcctgga gaaacagcta 1440

aagtgcgaaa gcggcgggcc ggccgacgcc cttgacgatt ttgacttaga catgctccca 1500

gccgatgccc ttgacgactt tgaccttgat atgctgcctg ctgacgctct tgacgatttt 1560

gaccttgaca tgctcccagg gtaa 1584

<210> 4

<211> 127

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 1

<400> 4

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Arg Ala Gly Gly

20 25 30

Gly Val Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ala Ala Gly Arg Leu Trp Arg Gly Trp Gly Gly Trp Arg Arg

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Cys Ala Thr Arg Gly Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu

100 105 110

Arg Gly Cys Gly Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 5

<211> 121

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 2

<400> 5

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Arg Ala Gly Gly

20 25 30

Gly Val Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ala Ala Gly Arg Leu Trp Gly Arg Trp Gly Gly Gly Arg Phe

50 55 60

Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr Val Gln Met Asn

65 70 75 80

Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala Arg Gly

85 90 95

Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu Arg Gly Cys Gly Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val

115 120

<210> 6

<211> 124

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 3

<400> 6

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Arg Ala Gly Gly

20 25 30

Gly Val Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ala Ala Gly Arg Leu Trp Gly Gly Trp Gly Arg Trp Gly Gly

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr Val

65 70 75 80

Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Ala Arg Gly Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu Arg Gly Cys

100 105 110

Gly Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120

<210> 7

<211> 127

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 4

<400> 7

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Arg Ala Gly Gly

20 25 30

Gly Val Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ala Ala Ile Ser Trp Thr Gly Val Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

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

85 90 95

Tyr Cys Ala Ala Val Arg Ala Arg Ser Phe Ser Asp Thr Tyr Ser Arg

100 105 110

Val Asn Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 8

<211> 127

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 5

<400> 8

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Arg Ala Gly Gly

20 25 30

Gly Val Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ala Ala Ile Ser Trp Thr Gly Val Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

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

85 90 95

Tyr Cys Ala Ala Val Arg Ala Arg Ser Phe Ser Asp Thr Tyr Ser Arg

100 105 110

Val Asn Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 9

<211> 127

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 6

<400> 9

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser

20 25 30

Thr Ser Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Arg Glu Arg Glu

35 40 45

Phe Val Ala Ala Ile Thr Trp Thr Val Gly Asn Thr Ile Leu Gly Asp

50 55 60

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

65 70 75 80

Val Asp Leu Gln Met Asp Asn Leu Glu Pro Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ser Ala Arg Ser Arg Gly Tyr Val Leu Ser Val Leu Arg Ser

100 105 110

Val Asp Ser Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 10

<211> 124

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 7

<400> 10

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Arg Leu Val Gln Ala

1 5 10 15

Gly Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser

20 25 30

Thr Ser Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ala Ala Gly Arg Leu Trp Gly Gly Trp Gly Arg Trp Gly Gly

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr Val

65 70 75 80

Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Ala Arg Gly Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu Arg Gly Cys

100 105 110

Gly Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120

<210> 11

<211> 121

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 8

<400> 11

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser

20 25 30

Asn Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ala Ala Gly Arg Leu Trp Gly Arg Trp Gly Gly Gly Arg Phe

50 55 60

Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr Val Gln Met Asn

65 70 75 80

Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala Arg Gly

85 90 95

Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu Arg Gly Cys Gly Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val

115 120

<210> 12

<211> 127

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 9

<400> 12

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Arg Leu Val Gln Ala

1 5 10 15

Gly Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser

20 25 30

Thr Ser Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Arg Glu Arg Glu

35 40 45

Phe Val Ala Ala Ile Thr Trp Thr Val Gly Asn Thr Ile Leu Gly Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

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

85 90 95

Tyr Cys Ala Ala Arg Gly Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu

100 105 110

Arg Gly Cys Gly Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 13

<211> 121

<212> PRT

<213> Artificial sequence

<221> G4 Nanobody 10

<400> 13

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Tyr Ala Ala Pro Gly Arg Ala Gly Gly

20 25 30

Gly Val Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ala Ala Gly Arg Leu Trp Gly Arg Trp Gly Gly Gly Arg Phe

50 55 60

Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr Val Gln Met Asn

65 70 75 80

Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala Arg Gly

85 90 95

Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu Arg Gly Cys Gly Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val

115 120

<210> 14

<211> 4946

<212> DNA

<213> Artificial sequence

<221> vector 1 Key sequence

<400> 14

tccctatcag tgatagagaa aagtgaaagt cgagtttacc actccctatc agtgatagag 60

aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt 120

taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg 180

atagagaaaa gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt 240

cgagtttacc actccctatc agtgatagag aactagttag gcgtgtacgg tgggaggcct 300

atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt 360

ttgacctcca tagaagacac cgggaccgat ccagcctctc gacattcgtt ggatcgccgc 420

tagcgccacc atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat 480

gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga 540

gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg 600

ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta 660

cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa 720

ttgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc 780

cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga 840

cggccccgta atgcagtgtc gtaccatggg ctgggaggcc tccactgagc ggatgtaccc 900

cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca 960

ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc 1020

ctacaacgtc gacatcaagt tggacatcct ttcccacaac gaggactaca ccatcgtgga 1080

acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagga 1140

gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc caatggccaa 1200

gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat 1260

ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt 1320

cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg 1380

cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa 1440

caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg 1500

gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt 1560

gctgccctct ggttatgtgt gggagggctg atacgtatta gtcatcgcta ttaccatggt 1620

gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc 1680

aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt 1740

tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg 1800

ggaggtttat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc 1860

acgctgtttt gacctccata gaagattcta gagccaccat ggccctgcta ctggccctca 1920

gcctgctggt tctctggact tccccagccc caactctgag tggcaccaat gatgactaca 1980

aagacgatga cgacaaggcg gaagcgggta tcaccggcac gtggtacaac cagcatggtt 2040

ctaccttcac cgttaccgcg ggtgcggacg gtaacctgac cggtcagtac gaaaaccgtg 2100

cgcagggcac tggttgccag aactctccgt acaccctgac cggtcgttac aacggtacca 2160

aactggaatg gcgtgttgaa tggaacaact ctaccgaaaa ctgccactct cgtaccgaat 2220

ggcgtggtca gtaccagggt ggtgcggaag cgcgtatcaa cacccagtgg aacctgacct 2280

acgaaggtgg ttctggtccg gcgaccgaac agggtcagga caccttcacc aaagttaaaa 2340

tgtacttcag ccacttcgtg ccggtcttcc tgccagcgaa gcccaccacg acgccagcgc 2400

cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg 2460

cgagccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc gccagcgata 2520

tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca ctggttatca 2580

ccctttactg cggaggcggg gagagtttgt ttaaggggcc aagggactat aacccaatat 2640

catccactat ttgccacctc actaacgaga gcgatggaca tacaacctct ctctacggga 2700

taggcttcgg tcctttcatc attaccaata agcatctgtt tcgccgaaac aacggtactc 2760

ttctggttca atctcttcat ggtgtcttca aggtgaaaaa caccactacg cttcagcaac 2820

acctgattga tggtagggat atgataatta tcagaatgcc gaaagatttc ccaccttttc 2880

cacagaagct gaaattcagg gaaccgcaga gagaggagag gatttgtttg gtaacgacca 2940

acttccagac gaagagtatg agttctatgg tgtccgacac tagctgcacg ttcccctcaa 3000

gtgatgggat attctggaaa cactggatac agactaaaga cggacagtgt ggaagcccat 3060

tggtttccac ccgagatggt tttatcgtgg gtatccatag cgcctctaat ttcacaaaca 3120

cgaacaacta tttcacttca gtgcccaaaa actttatgga gctgctcaca aaccaagaag 3180

cgcagcagtg ggtaagcggt tggagactta acgctgactc agttctctgg ggggggcaca 3240

aagtattcat ggtaaagcca gaggagccat tccaaccagt caaagaagcc acacaactta 3300

tgaacagcga gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc 3360

caatggccct gcccgtgacc gccctgctgc tgcccctggc cctgctgctg cacgccgcca 3420

ggccctaccc atacgatgtt ccagattacg ctcccgggat ggcccaggtg cagctggtgg 3480

agagcggcgg cggcctggtg caggccggcg gcagcctgag gctgagctgc gccgccagcg 3540

gcaggacctt cagcaactac gccatgggct ggttcaggca ggcccccggc aaggagaggg 3600

agttcgtggc cgccatcagc tggaccggcg tgagcaccta ctacgccgac agcgtgaagg 3660

gcaggttcac catcagcagg gacaacgaca agaacaccgt gtacgtgcag atgaacagcc 3720

tgatccccga ggacaccgcc atctactact gcgccgccgt gagggccagg agcttcagcg 3780

acacctacag cagggtgaac gagtacgact actggggcca gggcacccag gtgaccgtgg 3840

gatccatgta cttcagccac ttcgtgccgg tcttcctgcc agcgaagccc accacgacgc 3900

cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc 3960

cagaggcgag ccggccagcg gcggggggcg cagtgcacac gagggggctg gacttcgcca 4020

gcgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc ctgtcactgg 4080

ttatcaccct ttactgcaat tcgagctcga acaacaacaa caataacaat aacaacaacc 4140

tcgggatcga gggaagggga ggaggcgagt tcgctagcga gaacctgtat ttccagggca 4200

tgtctagact ggacaagagc aaagtcataa actctgctct ggaattactc aatgaagtcg 4260

gtatcgaagg cctgacgaca aggaaactcg ctcaaaagct gggagttgag cagcctaccc 4320

tgtactggca cgtgaagaac aagcgggccc tgctcgatgc cctggcaatc gagatgctgg 4380

acaggcatca tacccacttc tgccccctgg aaggcgagtc atggcaagac tttctgcgga 4440

acaacgccaa gtcattccgc tgtgctctcc tctcacatcg cgacggggct aaagtgcatc 4500

tcggcacccg cccaacagag aaacagtacg aaaccctgga aaatcagctc gcgttcctgt 4560

gtcagcaagg cttctccctg gagaacgcac tgtacgctct gtccgccgtg ggccacttta 4620

cactgggctg cgtattggag gatcaggagc atcaagtagc aaaagaggaa agagagacac 4680

ctaccaccga ttctatgccc ccacttctga gacaagcaat tgagctgttc gaccatcagg 4740

gagccgaacc tgccttcctt ttcggcctgg aactaatcat atgtggcctg gagaaacagc 4800

taaagtgcga aagcggcggg ccggccgacg cccttgacga ttttgactta gacatgctcc 4860

cagccgatgc ccttgacgac tttgaccttg atatgctgcc tgctgacgct cttgacgatt 4920

ttgaccttga catgctccca gggtaa 4946

<210> 15

<211> 5762

<212> DNA

<213> Artificial sequence

<221> vector 2 Key sequence

<400> 15

tccctatcag tgatagagaa aagtgaaagt cgagtttacc actccctatc agtgatagag 60

aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt 120

taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg 180

atagagaaaa gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt 240

cgagtttacc actccctatc agtgatagag aactagttag gcgtgtacgg tgggaggcct 300

atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt 360

ttgacctcca tagaagacac cgggaccgat ccagcctctc gacattcgtt ggatcgccgc 420

tagcgccacc atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat 480

gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga 540

gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg 600

ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta 660

cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa 720

ttgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc 780

cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga 840

cggccccgta atgcagtgtc gtaccatggg ctgggaggcc tccactgagc ggatgtaccc 900

cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca 960

ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc 1020

ctacaacgtc gacatcaagt tggacatcct ttcccacaac gaggactaca ccatcgtgga 1080

acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagga 1140

gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc caatggccaa 1200

gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat 1260

ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt 1320

cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg 1380

cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa 1440

caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg 1500

gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt 1560

gctgccctct ggttatgtgt gggagggctg atacgtatta gtcatcgcta ttaccatggt 1620

gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc 1680

aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt 1740

tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg 1800

ggaggtttat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc 1860

acgctgtttt gacctccata gaagattcta gagccaccat ggccctgccc gtgaccgccc 1920

tgctgctgcc cctggccctg ctgctgcacg ccgccaggcc cgactacaag gacgacgacg 1980

acaagcccgg gatggcccag gtgcagctgg tggagagcgg cggcggcctg gtgcaggccg 2040

gcggcagcct gaggctgagc tgcgccgccc cggggcgggc cgggggcggg gtcccggcgg 2100

ggtggttcag gcaggccccc ggcaaggaga gggagttcgt ggccgcgggg cgcttatggg 2160

gagggtgggg agggtgggga aggtggggag gaggcaggtt caccatcagc agggacaacg 2220

acaagaacac cgtgtacgtg cagatgaaca gcctgatccc cgaggacacc gccatctact 2280

actgcgccgc ccgcggaggg gcgggcgcgg gaggaagggg gcgggagcgg ggctgtggcg 2340

actactgggg ccagggcacc caggtgaccg tgggatcccc ctgcgtgggc agcaacccct 2400

gctacaacca gggcacctgc gagcccacca gcgagaaccc cttctacagg tgcctgtgcc 2460

ccgccaagtt caacggcctg ctgtgccaca tcctggacta cagcttcacc ggcggcgccg 2520

gcagggacat cccccccccc cagatcgagg aggcctgcga gctgcccgag tgccaggtgg 2580

acgccggcaa caaggtgtgc aacctgcagt gcaacaacca cgcctgcggc tgggacggcg 2640

gcgactgcag cctgaacttc aacgacccct ggaagaactg cacccagagc ctgcagtgct 2700

ggaagtactt cagcgacggc cactgcgaca gccagtgcaa cagcgccggc tgcctgttcg 2760

acggcttcga ctgccagctg accgagggcc agtgcaaccc cctgtacgac cagtactgca 2820

aggaccactt cagcgacggc cactgcgacc agggctgcaa cagcgccgag tgcgagtggg 2880

acggcctgga ctgcgccgag cacgtgcccg agaggctggc cgccggcacc ctggtgctgg 2940

tggtgctgct gccccccgac cagctgagga acaacagctt ccacttcctg agggagctga 3000

gccacgtgct gcacaccaac gtggtgttca agagggacgc ccagggccag cagatgatct 3060

tcccctacta cggccacgag gaggagctga ggaagcaccc catcaagagg agcaccgtgg 3120

gctgggccac cagcagcctg ctgcccggca ccagcggcgg caggcagagg agggagctgg 3180

accccatgga catcaggggc agcatcgtgt acctggagat cgacaacagg cagtgcgtgc 3240

agagcagcag ccagtgcttc cagagcgcca ccgacgtggc cgccttcctg ggcgccctgg 3300

ccagcctggg cagcctgaac atcccctaca agatcgaggc ccataagagc gagcccgtgg 3360

agccccccct gcccagccag ctgcacctga tgtacgtggc cgccgccgcc ttcgtgctgc 3420

tgttcttcgt gctccttttc tttctgctga gcaggaagag gaggaggcag ctgtgcatcc 3480

agaagctgct cgggatcgag ggaaggggag gaggcgagtt cgctagcgag aacctgtatt 3540

tccagggcat gtctagactg gacaagagca aagtcataaa ctctgctctg gaattactca 3600

atgaagtcgg tatcgaaggc ctgacgacaa ggaaactcgc tcaaaagctg ggagttgagc 3660

agcctaccct gtactggcac gtgaagaaca agcgggccct gctcgatgcc ctggcaatcg 3720

agatgctgga caggcatcat acccacttct gccccctgga aggcgagtca tggcaagact 3780

ttctgcggaa caacgccaag tcattccgct gtgctctcct ctcacatcgc gacggggcta 3840

aagtgcatct cggcacccgc ccaacagaga aacagtacga aaccctggaa aatcagctcg 3900

cgttcctgtg tcagcaaggc ttctccctgg agaacgcact gtacgctctg tccgccgtgg 3960

gccactttac actgggctgc gtattggagg atcaggagca tcaagtagca aaagaggaaa 4020

gagagacacc taccaccgat tctatgcccc cacttctgag acaagcaatt gagctgttcg 4080

accatcaggg agccgaacct gccttccttt tcggcctgga actaatcata tgtggcctgg 4140

agaaacagct aaagtgcgaa agcggcgggc cggccgacgc ccttgacgat tttgacttag 4200

acatgctccc agccgatgcc cttgacgact ttgaccttga tatgctgcct gctgacgctc 4260

ttgacgattt tgaccttgac atgctcccag gggagggcag aggaagtctg ctaacatgcg 4320

gtgacgtcga ggagaatcct ggcccaatgg ccctgctact ggccctcagc ctgctggttc 4380

tctggacttc cccagcccca actctgagtg gcaccaatga ttacccatac gatgttccag 4440

attacgctgc ggaagcgggt atcaccggca cgtggtacaa ccagcatggt tctaccttca 4500

ccgttaccgc gggtgcggac ggtaacctga ccggtcagta cgaaaaccgt gcgcagggca 4560

ctggttgcca gaactctccg tacaccctga ccggtcgtta caacggtacc aaactggaat 4620

ggcgtgttga atggaacaac tctaccgaaa actgccactc tcgtaccgaa tggcgtggtc 4680

agtaccaggg tggtgcggaa gcgcgtatca acacccagtg gaacctgacc tacgaaggtg 4740

gttctggtcc ggcgaccgaa cagggtcagg acaccttcac caaagttaaa atgtacttca 4800

gccacttcgt gccggtcttc ctgccagcga agcccaccac gacgccagcg ccgcgaccac 4860

caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag gcgagccggc 4920

cagcggcggg gggcgcagtg cacacgaggg ggctggactt cgccagcgat atctacatct 4980

gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc accctttact 5040

gcggaggcgg ggagagtttg tttaaggggc caagggacta taacccaata tcatccacta 5100

tttgccacct cactaacgag agcgatggac atacaacctc tctctacggg ataggcttcg 5160

gtcctttcat cattaccaat aagcatctgt ttcgccgaaa caacggtact cttctggttc 5220

aatctcttca tggtgtcttc aaggtgaaaa acaccactac gcttcagcaa cacctgattg 5280

atggtaggga tatgataatt atcagaatgc cgaaagattt cccacctttt ccacagaagc 5340

tgaaattcag ggaaccgcag agagaggaga ggatttgttt ggtaacgacc aacttccaga 5400

cgaagagtat gagttctatg gtgtccgaca ctagctgcac gttcccctca agtgatggga 5460

tattctggaa acactggata cagactaaag acggacagtg tggaagccca ttggtttcca 5520

cccgagatgg ttttatcgtg ggtatccata gcgcctctaa tttcacaaac acgaacaact 5580

atttcacttc agtgcccaaa aactttatgg agctgctcac aaaccaagaa gcgcagcagt 5640

gggtaagcgg ttggagactt aacgctgact cagttctctg gggggggcac aaagtattca 5700

tggtaaagcc agaggagcca ttccaaccag tcaaagaagc cacacaactt atgaacagct 5760

aa 5762

<210> 16

<211> 124

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 1

<400> 16

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ser Ala Ile Ser Ile Asp Gly Ser His Thr Thr Tyr Thr Ala Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Thr Leu Arg Leu Gln Tyr Gly Leu Ala Tyr Asp Leu Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 17

<211> 123

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 2

<400> 17

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

His Gly Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ser Asn Lys Asp Thr Gly Cys Tyr His Val Ala Arg Leu Pro Met Glu

50 55 60

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

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Thr Arg Glu Ile His Gly Lys Met Gln Trp Leu Phe Ser Val

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 18

<211> 123

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 3

<400> 18

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Pro Phe Ser Leu Gly Cys Gln Glu

20 25 30

Arg Ala Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

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

50 55 60

Met Cys Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Val Met Asn Arg Ala Ile Glu Asp Gly Phe Lys Cys Ser Trp Gln

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 19

<211> 123

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 4

<400> 19

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

Met Phe Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ser Thr Gln Ile Met Asp Arg Leu Phe Lys Val Pro Asn Ala Ser Glu

50 55 60

Gly Trp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Trp Cys Ala Arg Val His Gly Asp Pro Asn Phe Tyr Met Ile Glu

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 20

<211> 123

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 5

<400> 20

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Cys Tyr Gly Ile His Val Trp Asn

20 25 30

Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ser Asn Met Gln Pro Leu Ser Ala Ile Arg Glu Phe Trp Tyr Cys Val

50 55 60

His Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Val Cys Phe Pro Thr Asn Arg Tyr Glu His Met Asp Leu Gln Ile

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 21

<211> 123

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 6

<400> 21

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Tyr Phe Asn Glu Trp His Gln Pro

20 25 30

Thr Gly Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ser Cys Glu Ala Trp Pro Ile Ser Asp Thr Asn Met Phe Tyr Gln Leu

50 55 60

His Val Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Phe Ala Pro Gln Glu Ser Leu Asp Gly Ile Asn Arg Trp Tyr Lys

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 22

<211> 123

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 7

<400> 22

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala His Ile Phe Pro Thr Gly Trp Asn

20 25 30

Cys Lys Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ser Glu Met Thr Ile Tyr Ala Gly Val Trp His Asp Cys Pro Gln Lys

50 55 60

Asn Phe Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gln Met Cys Asn Arg Val Pro Phe Glu Ala Thr Gly Asp Leu Trp

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 23

<211> 123

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 8

<400> 23

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Lys Leu Phe Val Asn Gly Cys Tyr

20 25 30

Ser His Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

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

50 55 60

Tyr Asn Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Cys Arg Ser Phe Asn Ile Gln Gly Met Asp Thr Tyr Val Trp His

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 24

<211> 123

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 9

<400> 24

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Met Val Tyr Pro Ala Thr Glu Ile

20 25 30

His Trp Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ser Tyr Asp Ala Glu Met Trp Lys Gln Thr Asn Arg Val Gly His Cys

50 55 60

Ser Phe Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Tyr Val Ala Gly Gln Leu Asn Asp Cys Pro Glu Met Lys Phe His

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 25

<211> 123

<212> PRT

<213> Artificial sequence

<221> Natural Nanobody 10

<400> 25

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Phe Thr Trp Glu Arg Gly Ile Pro

20 25 30

Ser Ala Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ser Glu Lys Asn Pro Thr Ser Phe Cys Leu Tyr Ile Asp Ala Gly Met

50 55 60

Trp Arg Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Arg Lys Ala Cys Phe Ser Met Gln Val Pro Ile Asp His Glu

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 26

<211> 3378

<212> DNA

<213> Artificial sequence

<221> vector 3

<400> 26

gactacaaag acgatgacga caagtccgaa gtcgagtttt cccatgagta ctggatgaga 60

cacgcattga ctctcgcaaa gagggcttgg gatgaacgcg aggtgcccgt gggggcagta 120

ctcgtgcata acaatcgcgt aatcggcgaa ggttggaata ggccgatcgg acgccacgac 180

cccactgcac atgcggaaat catggccctt cgacagggag ggcttgtgat gcagaattat 240

cgacttatcg atgcgacgct gtacgtcacg cttgaacctt gcgtaatgtg cgcgggagct 300

atgattcact cccgcattgg acgagttgta ttcggtgccc gcgacgccaa gacgggtgcc 360

gcaggttcac tgatggacgt gctgcatcac ccaggcatga accaccgggt agaaatcaca 420

gaaggcatat tggcggacga atgtgcggcg ctgttgtccg acttttttcg catgcggagg 480

caggagatca aggcccagaa aaaagcacaa tcctctactg actctggtgg ttcttctggt 540

ggttctagcg gcagcgagac tcccgggacc tcagagtccg ccacacccga aagttctggt 600

ggttcttctg gtggttcttc cgaagtcgag ttttcccatg agtactggat gagacacgca 660

ttgactctcg caaagagggc tcgagatgaa cgcgaggtgc ccgtgggggc agtactcgtg 720

ctcaacaatc gcgtaatcgg cgaaggttgg aatagggcaa tcggactcca cgaccccact 780

gcacatgcgg aaatcatggc ccttcgacag ggagggcttg tgatgcagaa ttatcgactt 840

atcgatgcga cgctgtacgt cacgtttgaa ccttgcgtaa tgtgcgcggg agctatgatt 900

cactcccgca ttggacgagt tgtattcggt gttcgcaacg ccaagacggg tgccgcaggt 960

tcactgatgg acgtgctgca ttacccaggc atgaaccacc gggtagaaat cacagaaggc 1020

atattggcgg acgaatgtgc ggcgctgttg tgttactttt ttcgcatgcc caggcaggtc 1080

tttaacgccc agaaaaaagc acaatcctct actgactctg gtggttcttc tggtggttct 1140

agcggcagcg agactcccgg gacctcagag tccgccacac ccgaaagttc tggtggttct 1200

tctggtggtt ctatggtgaa gctcgcgaag gcaggtaaaa atcaaggtga ccccaagaaa 1260

atggctcctc ctccaaagga ggtagaagaa gatagtgaag atgaggaaat gtcagaagat 1320

gaagaagatg atagcagtgg agaagaggtc gtcatacctc agaagaaagg caagaaggct 1380

gctgcaacct cagcaaagaa ggtggtcgtt tccccaacaa aaaaggttgc agttgccaca 1440

ccagccaaga aagcagctgt cactccaggc aaaaaggcag cagcaacacc tgccaagaag 1500

acagttacac cagccaaagc agttaccaca cctggcaaga agggagccac accaggcaaa 1560

gcattggtag caactcctgg taagaagggt gctgccatcc cagccaaggg ggcaaagaat 1620

ggcaagaatg ccaagaagga agacagtgat gaagaggagg atgatgacag tgaggaggat 1680

gaggaggatg acgaggacga ggatgaggat gaagatgaaa ttgaaccagc agcgatgaaa 1740

gcagcagctg ctgcccctgc ctcagaggat gaggacgatg aggatgacga agatgatgag 1800

gatgacgatg acgatgagga agatgactct gaagaagaag ctatggagac tacaccagcc 1860

aaaggaaaga aagctgcaaa agttgttcct gtgaaagcca agaacgtggc tgaggatgaa 1920

gatgaagaag aggatgatga ggacgaggat gacgacgacg acgaagatga tgaagatgat 1980

gatgatgaag atgatgagga ggaggaagaa gaggaggagg aagagcctgt caaagaagca 2040

cctggaaaac gaaagaagga aatggccaaa cagaaagcag ctcctgaagc caagaaacag 2100

aaagtggaag gcacagaacc gactacggct ttcaatctct ttgttggaaa cctaaacttt 2160

aacaaatctg ctcctgaatt aaaaactggt atcagcgatg tttttgctaa aaatgatctt 2220

gctgttgtgg atgtcagaat tggtatgact aggaaatttg gttatgtgga ttttgaatct 2280

gctgaagacc tggagaaagc gttggaactc actggtttga aagtctttgg caatgaaatt 2340

aaactagaga aaccaaaagg aaaagacagt aagaaagagc gagatgcgag aacacttttg 2400

gctaaaaatc tcccttacaa agtcactcag gatgaattga aagaagtgtt tgaagatgct 2460

gcggagatca gattagtcag caaggatggg aaaagtaaag ggattgctta tattgaattt 2520

aagacagaag ctgatgcaga gaaaaccttt gaagaaaagc agggaacaga gatcgatggg 2580

cgatctattt ccctgtacta tactggagag aaaggtcaaa atcaagacta tagaggtgga 2640

aagaatagca cttggagtgg tgaatcaaaa actctggttt taagcaacct ctcctacagt 2700

gcaacagaag aaactcttca ggaagtattt gagaaagcaa cttttatcaa agtaccccag 2760

aaccaaaatg gcaaatctaa agggtatgca tttatagagt ttgcttcatt cgaagacgct 2820

aaagaagctt taaattcctg taataaaagg gaaattgagg gcagagcaat caggctggag 2880

ttgcaaggac ccaggggatc acctaatgcc agaagccagc catccaaaac tctgtttgtc 2940

aaaggcctgt ctgaggatac cactgaagag acattaaagg agtcatttga cggctccgtt 3000

cgggcaagga tagttactga ccgggaaact gggtcctcca aagggtttgg ttttgtagac 3060

ttcaacagtg aggaggatgc caaagctgcc aaggaggcca tggaagacgg tgaaattgat 3120

ggaaataaag ttaccttgga ctgggccaaa cctaagggtg aaggtggctt cgggggtcgt 3180

ggtggaggca gaggcggctt tggaggacga ggtggtggta gaggaggccg aggaggattt 3240

ggtggcagag gccggggagg ctttggaggg cgaggaggct tccgaggagg cagaggagga 3300

ggaggtgacc acaagccaca aggaaagaag acgaagtttg aatctggtgg ttctcccaag 3360

aagaagagga aagtctag 3378

<210> 27

<211> 3183

<212> DNA

<213> Artificial sequence

<221> vector 4

<400> 27

gactacaaag acgatgacga caagagctca gagactggcc cagtggctgt ggaccccaca 60

ttgagacggc ggatcgagcc ccatgagttt gaggtattct tcgatccgag agagctccgc 120

aaggagacct gcctgcttta cgaaattaat tgggggggcc ggcactccat ttggcgacat 180

acatcacaga acactaacaa gcacgtcgaa gtcaacttca tcgagaagtt cacgacagaa 240

agatatttct gtccgaacac aaggtgcagc attacctggt ttctcagctg gagcccatgc 300

ggcgaatgta gtagggccat cactgaattc ctgtcaaggt atccccacgt cactctgttt 360

atttacatcg caaggctgta ccaccacgct gacccccgca atcgacaagg cctgcgggat 420

ttgatctctt caggtgtgac tatccaaatt atgactgagc aggagtcagg atactgctgg 480

agaaactttg tgaattatag cccgagtaat gaagcccact ggcctaggta tccccatctg 540

tgggtacgac tgtacgttct tgaactgtac tgcatcatac tgggcctgcc tccttgtctc 600

aacattctga gaaggaagca gccacagctg acattcttta ccatcgctct tcagtcttgt 660

cattaccagc gactgccccc acacattctc tgggccaccg ggttgaaaag cggcagcgag 720

actcccggga cctcagagtc cgccacaccc gaaagtatgg tgaagctcgc gaaggcaggt 780

aaaaatcaag gtgaccccaa gaaaatggct cctcctccaa aggaggtaga agaagatagt 840

gaagatgagg aaatgtcaga agatgaagaa gatgatagca gtggagaaga ggtcgtcata 900

cctcagaaga aaggcaagaa ggctgctgca acctcagcaa agaaggtggt cgtttcccca 960

acaaaaaagg ttgcagttgc cacaccagcc aagaaagcag ctgtcactcc aggcaaaaag 1020

gcagcagcaa cacctgccaa gaagacagtt acaccagcca aagcagttac cacacctggc 1080

aagaagggag ccacaccagg caaagcattg gtagcaactc ctggtaagaa gggtgctgcc 1140

atcccagcca agggggcaaa gaatggcaag aatgccaaga aggaagacag tgatgaagag 1200

gaggatgatg acagtgagga ggatgaggag gatgacgagg acgaggatga ggatgaagat 1260

gaaattgaac cagcagcgat gaaagcagca gctgctgccc ctgcctcaga ggatgaggac 1320

gatgaggatg acgaagatga tgaggatgac gatgacgatg aggaagatga ctctgaagaa 1380

gaagctatgg agactacacc agccaaagga aagaaagctg caaaagttgt tcctgtgaaa 1440

gccaagaacg tggctgagga tgaagatgaa gaagaggatg atgaggacga ggatgacgac 1500

gacgacgaag atgatgaaga tgatgatgat gaagatgatg aggaggagga agaagaggag 1560

gaggaagagc ctgtcaaaga agcacctgga aaacgaaaga aggaaatggc caaacagaaa 1620

gcagctcctg aagccaagaa acagaaagtg gaaggcacag aaccgactac ggctttcaat 1680

ctctttgttg gaaacctaaa ctttaacaaa tctgctcctg aattaaaaac tggtatcagc 1740

gatgtttttg ctaaaaatga tcttgctgtt gtggatgtca gaattggtat gactaggaaa 1800

tttggttatg tggattttga atctgctgaa gacctggaga aagcgttgga actcactggt 1860

ttgaaagtct ttggcaatga aattaaacta gagaaaccaa aaggaaaaga cagtaagaaa 1920

gagcgagatg cgagaacact tttggctaaa aatctccctt acaaagtcac tcaggatgaa 1980

ttgaaagaag tgtttgaaga tgctgcggag atcagattag tcagcaagga tgggaaaagt 2040

aaagggattg cttatattga atttaagaca gaagctgatg cagagaaaac ctttgaagaa 2100

aagcagggaa cagagatcga tgggcgatct atttccctgt actatactgg agagaaaggt 2160

caaaatcaag actatagagg tggaaagaat agcacttgga gtggtgaatc aaaaactctg 2220

gttttaagca acctctccta cagtgcaaca gaagaaactc ttcaggaagt atttgagaaa 2280

gcaactttta tcaaagtacc ccagaaccaa aatggcaaat ctaaagggta tgcatttata 2340

gagtttgctt cattcgaaga cgctaaagaa gctttaaatt cctgtaataa aagggaaatt 2400

gagggcagag caatcaggct ggagttgcaa ggacccaggg gatcacctaa tgccagaagc 2460

cagccatcca aaactctgtt tgtcaaaggc ctgtctgagg ataccactga agagacatta 2520

aaggagtcat ttgacggctc cgttcgggca aggatagtta ctgaccggga aactgggtcc 2580

tccaaagggt ttggttttgt agacttcaac agtgaggagg atgccaaagc tgccaaggag 2640

gccatggaag acggtgaaat tgatggaaat aaagttacct tggactgggc caaacctaag 2700

ggtgaaggtg gcttcggggg tcgtggtgga ggcagaggcg gctttggagg acgaggtggt 2760

ggtagaggag gccgaggagg atttggtggc agaggccggg gaggctttgg agggcgagga 2820

ggcttccgag gaggcagagg aggaggaggt gaccacaagc cacaaggaaa gaagacgaag 2880

tttgaatctg gtggttctac taatctgtca gatattattg aaaaggagac cggtaagcaa 2940

ctggttatcc aggaatccat cctcatgctc ccagaggagg tggaagaagt cattgggaac 3000

aagccggaaa gcgatatact cgtgcacacc gcctacgacg agagcaccga cgagaatgtc 3060

atgcttctga ctagcgacgc ccctgaatac aagccttggg ctctggtcat acaggatagc 3120

aacggtgaga acaagattaa gatgctctct ggtggttctc ccaagaagaa gaggaaagtc 3180

taa 3183

<210> 28

<211> 589

<212> DNA

<213> Artificial sequence

<221> vector 5

<400> 28

gaaacaccga acaaagcacc agtggtctag tggtagaata gtaccctgcc acggtacaga 60

cccgggttcg attcccggct ggtgcacccc gccgggaccc cgccccgttt tagagctaga 120

aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt 180

gcaacaaagc accagtggtc tagtggtaga atagtaccct gccacggtac agacccgggt 240

tcgattcccg gctggtgcag agggtgggga gggtggggag ttttagagct agaaatagca 300

agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcaacaa 360

agcaccagtg gtctagtggt agaatagtac cctgccacgg tacagacccg ggttcgattc 420

ccggctggtg cactcccgcg cccgcccctc cggttttaga gctagaaata gcaagttaaa 480

ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttggatcc 540

agcgcttagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagt 589

<210> 29

<211> 5328

<212> PRT

<213> Artificial sequence

<221> vector 6

<400> 29

atgtccgaag tcgagttttc ccatgagtac tggatgagac acgcattgac tctcgcaaag 60

agggcttggg atgaacgcga ggtgcccgtg ggggcagtac tcgtgcataa caatcgcgta 120

atcggcgaag gttggaatag gccgatcgga cgccacgacc ccactgcaca tgcggaaatc 180

atggcccttc gacagggagg gcttgtgatg cagaattatc gacttatcga tgcgacgctg 240

tacgtcacgc ttgaaccttg cgtaatgtgc gcgggagcta tgattcactc ccgcattgga 300

cgagttgtat tcggtgcccg cgacgccaag acgggtgccg caggttcact gatggacgtg 360

ctgcatcacc caggcatgaa ccaccgggta gaaatcacag aaggcatatt ggcggacgaa 420

tgtgcggcgc tgttgtccga cttttttcgc atgcggaggc aggagatcaa ggcccagaaa 480

aaagcacaat cctctactga ctctggtggt tcttctggtg gttctagcgg cagcgagact 540

cccgggacct cagagtccgc cacacccgaa agttctggtg gttcttctgg tggttcttcc 600

gaagtcgagt tttcccatga gtactggatg agacacgcat tgactctcgc aaagagggct 660

cgagatgaac gcgaggtgcc cgtgggggca gtactcgtgc tcaacaatcg cgtaatcggc 720

gaaggttgga atagggcaat cggactccac gaccccactg cacatgcgga aatcatggcc 780

cttcgacagg gagggcttgt gatgcagaat tatcgactta tcgatgcgac gctgtacgtc 840

acgtttgaac cttgcgtaat gtgcgcggga gctatgattc actcccgcat tggacgagtt 900

gtattcggtg ttcgcaacgc caagacgggt gccgcaggtt cactgatgga cgtgctgcat 960

tacccaggca tgaaccaccg ggtagaaatc acagaaggca tattggcgga cgaatgtgcg 1020

gcgctgttgt gttacttttt tcgcatgccc aggcaggtct ttaacgccca gaaaaaagca 1080

caatcctcta ctgactctgg tggttcttct ggtggttcta gcggcagcga gactcccggg 1140

acctcagagt ccgccacacc cgaaagttct ggtggttctt ctggtggttc tgataaaaag 1200

tattctattg gtttagccat cggcactaat tccgttggat gggctgtcat aaccgatgaa 1260

tacaaagtac cttcaaagaa atttaaggtg ttggggaaca cagaccgtca ttcgattaaa 1320

aagaatctta tcggtgccct cctattcgat agtggcgaaa cggcagaggc gactcgcctg 1380

aaacgaaccg ctcggagaag gtatacacgt cgcaagaacc gaatatgtta cttacaagaa 1440

atttttagca atgagatggc caaagttgac gattctttct ttcaccgttt ggaagagtcc 1500

ttccttgtcg aagaggacaa gaaacatgaa cggcacccca tctttggaaa catagtagat 1560

gaggtggcat atcatgaaaa gtacccaacg atttatcacc tcagaaaaaa gctagttgac 1620

tcaactgata aagcggacct gaggttaatc tacttggctc ttgcccatat gataaagttc 1680

cgtgggcact ttctcattga gggtgatcta aatccggaca actcggatgt cgacaaactg 1740

ttcatccagt tagtacaaac ctataatcag ttgtttgaag agaaccctat aaatgcaagt 1800

ggcgtggatg cgaaggctat tcttagcgcc cgcctctcta aatcccgacg gctagaaaac 1860

ctgatcgcac aattacccgg agagaagaaa aatgggttgt tcggtaacct tatagcgctc 1920

tcactaggcc tgacaccaaa ttttaagtcg aacttcgact tagctgaaga tgccaaattg 1980

cagcttagta aggacacgta cgatgacgat ctcgacaatc tactggcaca aattggagat 2040

cagtatgcgg acttattttt ggctgccaaa aaccttagcg atgcaatcct cctatctgac 2100

atactgagag ttaatactga gattaccaag gcgccgttat ccgcttcaat gatcaaaagg 2160

tacgatgaac atcaccaaga cttgacactt ctcaaggccc tagtccgtca gcaactgcct 2220

gagaaatata aggaaatatt ctttgatcag tcgaaaaacg ggtacgcagg ttatattgac 2280

ggcggagcga gtcaagagga attctacaag tttatcaaac ccatattaga gaagatggat 2340

gggacggaag agttgcttgt aaaactcaat cgcgaagatc tactgcgaaa gcagcggact 2400

ttcgacaacg gtagcattcc acatcaaatc cacttaggcg aattgcatgc tatacttaga 2460

aggcaggagg atttttatcc gttcctcaaa gacaatcgtg aaaagattga gaaaatccta 2520

acctttcgca taccttacta tgtgggaccc ctggcccgag ggaactctcg gttcgcatgg 2580

atgacaagaa agtccgaaga aacgattact ccatggaatt ttgaggaagt tgtcgataaa 2640

ggtgcgtcag ctcaatcgtt catcgagagg atgaccaact ttgacaagaa tttaccgaac 2700

gaaaaagtat tgcctaagca cagtttactt tacgagtatt tcacagtgta caatgaactc 2760

acgaaagtta agtatgtcac tgagggcatg cgtaaacccg cctttctaag cggagaacag 2820

aagaaagcaa tagtagatct gttattcaag accaaccgca aagtgacagt taagcaattg 2880

aaagaggact actttaagaa aattgaatgc ttcgattctg tcgagatctc cggggtagaa 2940

gatcgattta atgcgtcact tggtacgtat catgacctcc taaagataat taaagataag 3000

gacttcctgg ataacgaaga gaatgaagat atcttagaag atatagtgtt gactcttacc 3060

ctctttgaag atcgggaaat gattgaggaa agactaaaaa catacgctca cctgttcgac 3120

gataaggtta tgaaacagtt aaagaggcgt cgctatacgg gctggggacg attgtcgcgg 3180

aaacttatca acgggataag agacaagcaa agtggtaaaa ctattctcga ttttctaaag 3240

agcgacggct tcgccaatag gaactttatg cagctgatcc atgatgactc tttaaccttc 3300

aaagaggata tacaaaaggc acaggtttcc ggacaagggg actcattgca cgaacatatt 3360

gcgaatcttg ctggttcgcc agccatcaaa aagggcatac tccagacagt caaagtagtg 3420

gatgagctag ttaaggtcat gggacgtcac aaaccggaaa acattgtaat cgagatggca 3480

cgcgaaaatc aaacgactca gaaggggcaa aaaaacagtc gagagcggat gaagagaata 3540

gaagagggta ttaaagaact gggcagccag atcttaaagg agcatcctgt ggaaaatacc 3600

caattgcaga acgagaaact ttacctctat tacctacaaa atggaaggga catgtatgtt 3660

gatcaggaac tggacataaa ccgtttatct gattacgacg tcgatcacat tgtaccccaa 3720

tcctttttga aggacgattc aatcgacaat aaagtgctta cacgctcgga taagaaccga 3780

gggaaaagtg acaatgttcc aagcgaggaa gtcgtaaaga aaatgaagaa ctattggcgg 3840

cagctcctaa atgcgaaact gataacgcaa agaaagttcg ataacttaac taaagctgag 3900

aggggtggct tgtctgaact tgacaaggcc ggatttatta aacgtcagct cgtggaaacc 3960

cgccaaatca caaagcatgt tgcacagata ctagattccc gaatgaatac gaaatacgac 4020

gagaacgata agctgattcg ggaagtcaaa gtaatcactt taaagtcaaa attggtgtcg 4080

gacttcagaa aggattttca attctataaa gttagggaga taaataacta ccaccatgcg 4140

cacgacgctt atcttaatgc cgtcgtaggg accgcactca ttaagaaata cccgaagcta 4200

gaaagtgagt ttgtgtatgg tgattacaaa gtttatgacg tccgtaagat gatcgcgaaa 4260

agcgaacagg agataggcaa ggctacagcc aaatacttct tttattctaa cattatgaat 4320

ttctttaaga cggaaatcac tctggcaaac ggagagatac gcaaacgacc tttaattgaa 4380

accaatgggg agacaggtga aatcgtatgg gataagggcc gggacttcgc gacggtgaga 4440

aaagttttgt ccatgcccca agtcaacata gtaaagaaaa ctgaggtgca gaccggaggg 4500

ttttcaaagg aatcgattct tccaaaaagg aatagtgata agctcatcgc tcgtaaaaag 4560

gactgggacc cgaaaaagta cggtggcttc gatagcccta cagttgccta ttctgtccta 4620

gtagtggcaa aagttgagaa gggaaaatcc aagaaactga agtcagtcaa agaattattg 4680

gggataacga ttatggagcg ctcgtctttt gaaaagaacc ccatcgactt ccttgaggcg 4740

aaaggttaca aggaagtaaa aaaggatctc ataattaaac taccaaagta tagtctgttt 4800

gagttagaaa atggccgaaa acggatgttg gctagcgccg gagagcttca aaaggggaac 4860

gaactcgcac taccgtctaa atacgtgaat ttcctgtatt tagcgtccca ttacgagaag 4920

ttgaaaggtt cacctgaaga taacgaacag aagcaacttt ttgttgagca gcacaaacat 4980

tatctcgacg aaatcataga gcaaatttcg gaattcagta agagagtcat cctagctgat 5040

gccaatctgg acaaagtatt aagcgcatac aacaagcaca gggataaacc catacgtgag 5100

caggcggaaa atattatcca tttgtttact cttaccaacc tcggcgctcc agccgcattc 5160

aagtattttg acacaacgat agatcgcaaa cgatacactt ctaccaagga ggtgctagac 5220

gcgacactga ttcaccaatc catcacggga ttatatgaaa ctcggataga tttgtcacag 5280

cttgggggtg actctggtgg ttctcccaag aagaagagga aagtctaa 5328

<210> 30

<211> 5130

<212> DNA

<213> Artificial sequence

<221> vector 7

<400> 30

atgagctcag agactggccc agtggctgtg gaccccacat tgagacggcg gatcgagccc 60

catgagtttg aggtattctt cgatccgaga gagctccgca aggagacctg cctgctttac 120

gaaattaatt gggggggccg gcactccatt tggcgacata catcacagaa cactaacaag 180

cacgtcgaag tcaacttcat cgagaagttc acgacagaaa gatatttctg tccgaacaca 240

aggtgcagca ttacctggtt tctcagctgg agcccatgcg gcgaatgtag tagggccatc 300

actgaattcc tgtcaaggta tccccacgtc actctgttta tttacatcgc aaggctgtac 360

caccacgctg acccccgcaa tcgacaaggc ctgcgggatt tgatctcttc aggtgtgact 420

atccaaatta tgactgagca ggagtcagga tactgctgga gaaactttgt gaattatagc 480

ccgagtaatg aagcccactg gcctaggtat ccccatctgt gggtacgact gtacgttctt 540

gaactgtact gcatcatact gggcctgcct ccttgtctca acattctgag aaggaagcag 600

ccacagctga cattctttac catcgctctt cagtcttgtc attaccagcg actgccccca 660

cacattctct gggccaccgg gttgaaaagc ggcagcgaga ctcccgggac ctcagagtcc 720

gccacacccg aaagtgataa aaagtattct attggtttag ccatcggcac taattccgtt 780

ggatgggctg tcataaccga tgaatacaaa gtaccttcaa agaaatttaa ggtgttgggg 840

aacacagacc gtcattcgat taaaaagaat cttatcggtg ccctcctatt cgatagtggc 900

gaaacggcag aggcgactcg cctgaaacga accgctcgga gaaggtatac acgtcgcaag 960

aaccgaatat gttacttaca agaaattttt agcaatgaga tggccaaagt tgacgattct 1020

ttctttcacc gtttggaaga gtccttcctt gtcgaagagg acaagaaaca tgaacggcac 1080

cccatctttg gaaacatagt agatgaggtg gcatatcatg aaaagtaccc aacgatttat 1140

cacctcagaa aaaagctagt tgactcaact gataaagcgg acctgaggtt aatctacttg 1200

gctcttgccc atatgataaa gttccgtggg cactttctca ttgagggtga tctaaatccg 1260

gacaactcgg atgtcgacaa actgttcatc cagttagtac aaacctataa tcagttgttt 1320

gaagagaacc ctataaatgc aagtggcgtg gatgcgaagg ctattcttag cgcccgcctc 1380

tctaaatccc gacggctaga aaacctgatc gcacaattac ccggagagaa gaaaaatggg 1440

ttgttcggta accttatagc gctctcacta ggcctgacac caaattttaa gtcgaacttc 1500

gacttagctg aagatgccaa attgcagctt agtaaggaca cgtacgatga cgatctcgac 1560

aatctactgg cacaaattgg agatcagtat gcggacttat ttttggctgc caaaaacctt 1620

agcgatgcaa tcctcctatc tgacatactg agagttaata ctgagattac caaggcgccg 1680

ttatccgctt caatgatcaa aaggtacgat gaacatcacc aagacttgac acttctcaag 1740

gccctagtcc gtcagcaact gcctgagaaa tataaggaaa tattctttga tcagtcgaaa 1800

aacgggtacg caggttatat tgacggcgga gcgagtcaag aggaattcta caagtttatc 1860

aaacccatat tagagaagat ggatgggacg gaagagttgc ttgtaaaact caatcgcgaa 1920

gatctactgc gaaagcagcg gactttcgac aacggtagca ttccacatca aatccactta 1980

ggcgaattgc atgctatact tagaaggcag gaggattttt atccgttcct caaagacaat 2040

cgtgaaaaga ttgagaaaat cctaaccttt cgcatacctt actatgtggg acccctggcc 2100

cgagggaact ctcggttcgc atggatgaca agaaagtccg aagaaacgat tactccatgg 2160

aattttgagg aagttgtcga taaaggtgcg tcagctcaat cgttcatcga gaggatgacc 2220

aactttgaca agaatttacc gaacgaaaaa gtattgccta agcacagttt actttacgag 2280

tatttcacag tgtacaatga actcacgaaa gttaagtatg tcactgaggg catgcgtaaa 2340

cccgcctttc taagcggaga acagaagaaa gcaatagtag atctgttatt caagaccaac 2400

cgcaaagtga cagttaagca attgaaagag gactacttta agaaaattga atgcttcgat 2460

tctgtcgaga tctccggggt agaagatcga tttaatgcgt cacttggtac gtatcatgac 2520

ctcctaaaga taattaaaga taaggacttc ctggataacg aagagaatga agatatctta 2580

gaagatatag tgttgactct taccctcttt gaagatcggg aaatgattga ggaaagacta 2640

aaaacatacg ctcacctgtt cgacgataag gttatgaaac agttaaagag gcgtcgctat 2700

acgggctggg gacgattgtc gcggaaactt atcaacggga taagagacaa gcaaagtggt 2760

aaaactattc tcgattttct aaagagcgac ggcttcgcca ataggaactt tatgcagctg 2820

atccatgatg actctttaac cttcaaagag gatatacaaa aggcacaggt ttccggacaa 2880

ggggactcat tgcacgaaca tattgcgaat cttgctggtt cgccagccat caaaaagggc 2940

atactccaga cagtcaaagt agtggatgag ctagttaagg tcatgggacg tcacaaaccg 3000

gaaaacattg taatcgagat ggcacgcgaa aatcaaacga ctcagaaggg gcaaaaaaac 3060

agtcgagagc ggatgaagag aatagaagag ggtattaaag aactgggcag ccagatctta 3120

aaggagcatc ctgtggaaaa tacccaattg cagaacgaga aactttacct ctattaccta 3180

caaaatggaa gggacatgta tgttgatcag gaactggaca taaaccgttt atctgattac 3240

gacgtcgatc acattgtacc ccaatccttt ttgaaggacg attcaatcga caataaagtg 3300

cttacacgct cggataagaa ccgagggaaa agtgacaatg ttccaagcga ggaagtcgta 3360

aagaaaatga agaactattg gcggcagctc ctaaatgcga aactgataac gcaaagaaag 3420

ttcgataact taactaaagc tgagaggggt ggcttgtctg aacttgacaa ggccggattt 3480

attaaacgtc agctcgtgga aacccgccaa atcacaaagc atgttgcaca gatactagat 3540

tcccgaatga atacgaaata cgacgagaac gataagctga ttcgggaagt caaagtaatc 3600

actttaaagt caaaattggt gtcggacttc agaaaggatt ttcaattcta taaagttagg 3660

gagataaata actaccacca tgcgcacgac gcttatctta atgccgtcgt agggaccgca 3720

ctcattaaga aatacccgaa gctagaaagt gagtttgtgt atggtgatta caaagtttat 3780

gacgtccgta agatgatcgc gaaaagcgaa caggagatag gcaaggctac agccaaatac 3840

ttcttttatt ctaacattat gaatttcttt aagacggaaa tcactctggc aaacggagag 3900

atacgcaaac gacctttaat tgaaaccaat ggggagacag gtgaaatcgt atgggataag 3960

ggccgggact tcgcgacggt gagaaaagtt ttgtccatgc cccaagtcaa catagtaaag 4020

aaaactgagg tgcagaccgg agggttttca aaggaatcga ttcttccaaa aaggaatagt 4080

gataagctca tcgctcgtaa aaaggactgg gacccgaaaa agtacggtgg cttcgatagc 4140

cctacagttg cctattctgt cctagtagtg gcaaaagttg agaagggaaa atccaagaaa 4200

ctgaagtcag tcaaagaatt attggggata acgattatgg agcgctcgtc ttttgaaaag 4260

aaccccatcg acttccttga ggcgaaaggt tacaaggaag taaaaaagga tctcataatt 4320

aaactaccaa agtatagtct gtttgagtta gaaaatggcc gaaaacggat gttggctagc 4380

gccggagagc ttcaaaaggg gaacgaactc gcactaccgt ctaaatacgt gaatttcctg 4440

tatttagcgt cccattacga gaagttgaaa ggttcacctg aagataacga acagaagcaa 4500

ctttttgttg agcagcacaa acattatctc gacgaaatca tagagcaaat ttcggaattc 4560

agtaagagag tcatcctagc tgatgccaat ctggacaaag tattaagcgc atacaacaag 4620

cacagggata aacccatacg tgagcaggcg gaaaatatta tccatttgtt tactcttacc 4680

aacctcggcg ctccagccgc attcaagtat tttgacacaa cgatagatcg caaacgatac 4740

acttctacca aggaggtgct agacgcgaca ctgattcacc aatccatcac gggattatat 4800

gaaactcgga tagatttgtc acagcttggg ggtgactctg gtggttctac taatctgtca 4860

gatattattg aaaaggagac cggtaagcaa ctggttatcc aggaatccat cctcatgctc 4920

ccagaggagg tggaagaagt cattgggaac aagccggaaa gcgatatact cgtgcacacc 4980

gcctacgacg agagcaccga cgagaatgtc atgcttctga ctagcgacgc ccctgaatac 5040

aagccttggg ctctggtcat acaggatagc aacggtgaga acaagattaa gatgctctct 5100

ggtggttctc ccaagaagaa gaggaaagtc 5130

<210> 31

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA Nanobody 1

<400> 31

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Leu Thr Ala Trp Phe

20 25 30

Arg Gln Ile His Cys Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ser Trp Val Pro Ala Lys Met Phe Cys Gln Ile Leu Thr Asp

50 55 60

Glu Arg Asn Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Ala Asn Gln Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 32

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA Nanobody 2

<400> 32

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Val Asn Leu Gln Gly

20 25 30

Ala Tyr Met Ile Cys Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ile Ala Ser Gly Lys Val Glu Asp Tyr Met His Cys Trp Leu

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Cys Ala Ile Gln Tyr His Trp Glu Asn Val Phe Thr Cys Pro Ser

100 105 110

Gly Arg Met Ala Leu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 33

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA Nanobody 3

<400> 33

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Asp Ser Val Ile Pro

20 25 30

Glu Ala Asn Cys Met Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Met Ser Asn Gln Tyr Lys Phe Cys Asp His Ile Arg Gly Pro

50 55 60

Val Glu Ala Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

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

85 90 95

Tyr Cys Ala Met Ala Glu Ile His Arg Thr Pro Val Leu Ser Tyr Lys

100 105 110

Phe Asn Cys Gln Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 34

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA nanobody 4

<400> 34

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Pro Thr Cys Ser Gly

20 25 30

Ile Asn Tyr Val Glu Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Arg Gln Tyr Ala Pro Glu Val Asn Ser Gly Thr Phe Met Leu

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Cys Ala Gln Trp Ser Arg Ile Asn His Gly Phe Asp Leu Tyr Met

100 105 110

Ala Thr Lys Val Cys Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 35

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA Nanobody 5

<400> 35

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Asp Pro Ile Met Trp

20 25 30

Asn Gln Ala Tyr Arg Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Val Ser Phe Gln Pro Cys Tyr Leu His Glu Lys Ile Asn Trp

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Cys Ala Gln Met Ser Asp Arg Ala Tyr Pro His Ile Asn Phe Val

100 105 110

Leu Thr Trp Cys Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 36

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA Nanobody 6

<400> 36

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Trp Arg Gln Pro Glu

20 25 30

Met Cys Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Gln Arg Phe Ile Gly Pro Asn Leu His Lys Ala Met Trp Glu

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Cys Ala Trp Gly Met Pro Arg Phe Tyr Cys Asp Thr Ser Lys His

100 105 110

Val Gln Asn Leu Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 37

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA Nanobody 7

<400> 37

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Val Ser Thr Cys Tyr

20 25 30

Lys Pro Trp Leu Phe Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Asp Phe His Thr Gln Gly Ala Leu Ser Glu Met Asn Pro Tyr

50 55 60

Cys Lys Ile Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

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

85 90 95

Tyr Cys Ala Gln Tyr Met Glu Arg Val His Lys Cys Gly Ala Leu Thr

100 105 110

Ser Phe Trp Asn Ile Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 38

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA Nanobody 8

<400> 38

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Thr His Gly Leu Ser

20 25 30

Ile Asn Phe Met Cys Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Pro Met Tyr Ser Ala Lys Glu Ile Thr Asn Trp Cys Val Leu

50 55 60

Asp Arg Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

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

85 90 95

Tyr Cys Ala Gln Glu Phe Thr Asp Arg Trp Ala Leu Cys Ser His Met

100 105 110

Pro Lys Ile Asn Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 39

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA Nanobody 9

<400> 39

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Arg Gln Glu His

20 25 30

Cys Asn Lys Ala Tyr Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

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

50 55 60

His Trp Ile Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr

65 70 75 80

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

85 90 95

Tyr Cys Ala Gln Phe Pro Thr Ala Lys Leu Met Gly His Glu Trp Tyr

100 105 110

Cys Arg Asn Ser Ile Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 40

<211> 127

<212> PRT

<213> Artificial sequence

<221> gRNA nanobody 10

<400> 40

Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Arg Lys Thr Cys Tyr

20 25 30

Asn Pro Asp Phe His Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Pro Val Gln Arg Met Ala Asp Asn Lys Cys Phe Leu Ile Thr

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Cys Ala Ile Arg Gln Pro Cys His Leu Tyr Thr Lys Val Phe Asn

100 105 110

Met Ser Glu Ala Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

<210> 41

<211> 719

<212> PRT

<213> person (Homo sapiens)

<221> Mcm7

<400> 41

Met Ala Leu Lys Asp Tyr Ala Leu Glu Lys Glu Lys Val Lys Lys Phe

1 5 10 15

Leu Gln Glu Phe Tyr Gln Asp Asp Glu Leu Gly Lys Lys Gln Phe Lys

20 25 30

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

35 40 45

Tyr Val Asp Leu Asp Asp Val Ala Glu Asp Asp Pro Glu Leu Val Asp

50 55 60

Ser Ile Cys Glu Asn Ala Arg Arg Tyr Ala Lys Leu Phe Ala Asp Ala

65 70 75 80

Val Gln Glu Leu Leu Pro Gln Tyr Lys Glu Arg Glu Val Val Asn Lys

85 90 95

Asp Val Leu Asp Val Tyr Ile Glu His Arg Leu Met Met Glu Gln Arg

100 105 110

Ser Arg Asp Pro Gly Met Val Arg Ser Pro Gln Asn Gln Tyr Pro Ala

115 120 125

Glu Leu Met Arg Arg Phe Glu Leu Tyr Phe Gln Gly Pro Ser Ser Asn

130 135 140

Lys Pro Arg Val Ile Arg Glu Val Arg Ala Asp Ser Val Gly Lys Leu

145 150 155 160

Val Thr Val Arg Gly Ile Val Thr Arg Val Ser Glu Val Lys Pro Lys

165 170 175

Met Val Val Ala Thr Tyr Thr Cys Asp Gln Cys Gly Ala Glu Thr Tyr

180 185 190

Gln Pro Ile Gln Ser Pro Thr Phe Met Pro Leu Ile Met Cys Pro Ser

195 200 205

Gln Glu Cys Gln Thr Asn Arg Ser Gly Gly Arg Leu Tyr Leu Gln Thr

210 215 220

Arg Gly Ser Arg Phe Ile Lys Phe Gln Glu Met Lys Met Gln Glu His

225 230 235 240

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

245 250 255

Val Glu Gly Glu Asn Thr Arg Ile Ala Gln Pro Gly Asp His Val Ser

260 265 270

Val Thr Gly Ile Phe Leu Pro Ile Leu Arg Thr Gly Phe Arg Gln Val

275 280 285

Val Gln Gly Leu Leu Ser Glu Thr Tyr Leu Glu Ala His Arg Ile Val

290 295 300

Lys Met Asn Lys Ser Glu Asp Asp Glu Ser Gly Ala Gly Glu Leu Thr

305 310 315 320

Arg Glu Glu Leu Arg Gln Ile Ala Glu Glu Asp Phe Tyr Glu Lys Leu

325 330 335

Ala Ala Ser Ile Ala Pro Glu Ile Tyr Gly His Glu Asp Val Lys Lys

340 345 350

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

355 360 365

Met Lys Ile Arg Gly Asn Ile Asn Ile Cys Leu Met Gly Asp Pro Gly

370 375 380

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

385 390 395 400

Ser Gln Tyr Thr Thr Gly Arg Gly Ser Ser Gly Val Gly Leu Thr Ala

405 410 415

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

420 425 430

Ala Leu Val Leu Ala Asp Gln Gly Val Cys Cys Ile Asp Glu Phe Asp

435 440 445

Lys Met Ala Glu Ala Asp Arg Thr Ala Ile His Glu Val Met Glu Gln

450 455 460

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

465 470 475 480

Arg Cys Ser Ile Leu Ala Ala Ala Asn Pro Ala Tyr Gly Arg Tyr Asn

485 490 495

Pro Arg Arg Ser Leu Glu Gln Asn Ile Gln Leu Pro Ala Ala Leu Leu

500 505 510

Ser Arg Phe Asp Leu Leu Trp Leu Ile Gln Asp Arg Pro Asp Arg Asp

515 520 525

Asn Asp Leu Arg Leu Ala Gln His Ile Thr Tyr Val His Gln His Ser

530 535 540

Arg Gln Pro Pro Ser Gln Phe Glu Pro Leu Asp Met Lys Leu Met Arg

545 550 555 560

Arg Tyr Ile Ala Met Cys Arg Glu Lys Gln Pro Met Val Pro Glu Ser

565 570 575

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

580 585 590

Trp Ala Ser Lys Asp Ala Thr Tyr Thr Ser Ala Arg Thr Leu Leu Ala

595 600 605

Ile Leu Arg Leu Ser Thr Ala Leu Ala Arg Leu Arg Met Val Asp Val

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

Arg Ser Val Arg Phe Ser Glu Ala Glu Gln Arg Cys Val Ser Arg Gly

675 680 685

Phe Thr Pro Ala Gln Phe Gln Ala Ala Leu Asp Glu Tyr Glu Glu Leu

690 695 700

Asn Val Trp Gln Val Asn Ala Ser Arg Thr Arg Ile Thr Phe Val

705 710 715

SEQ ID NO. 76

<210> 42

<211> 150

<212> PRT

<213> Neurospora crassa species (Neurospora crassa)

<221> Vivid

<400> 42

His Thr Leu Tyr Ala Pro Gly Gly Tyr Asp Ile Met Gly Tyr Leu Ile

1 5 10 15

Gln Ile Met Asn Arg Pro Asn Pro Gln Val Glu Leu Gly Pro Val Asp

20 25 30

Thr Ser Cys Ala Leu Ile Leu Cys Asp Leu Lys Gln Lys Asp Thr Pro

35 40 45

Ile Val Tyr Ala Ser Glu Ala Phe Leu Tyr Met Thr Gly Tyr Ser Asn

50 55 60

Ala Glu Val Leu Gly Arg Asn Cys Arg Phe Leu Gln Ser Pro Asp Gly

65 70 75 80

Met Val Lys Pro Lys Ser Thr Arg Lys Tyr Val Asp Ser Asn Thr Ile

85 90 95

Asn Thr Met Arg Lys Ala Ile Asp Arg Asn Ala Glu Val Gln Val Glu

100 105 110

Val Val Asn Phe Lys Lys Asn Gly Gln Arg Phe Val Asn Phe Leu Thr

115 120 125

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

130 135 140

Phe Gln Cys Glu Thr Glu

145 150

<210> 43

<211> 25

<212> PRT

<213> Artificial sequence

<221> Linker

<400> 43

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25

SEQ ID NO. 79

<210> 44

<211> 166

<212> PRT

<213> Escherichia coli (Escherichia coli)

<221> ecTadA*

<400> 44

Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr

1 5 10 15

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

20 25 30

Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile

35 40 45

Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln

50 55 60

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

65 70 75 80

Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser

85 90 95

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

100 105 110

Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg

115 120 125

Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu

130 135 140

Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys

145 150 155 160

Ala Gln Ser Ser Thr Asp

165

<210> 45

<211> 228

<212> PRT

<213> rat (ratus norvegicus)

<221> rAPOBEC1

<400> 45

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

1 5 10 15

Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu Arg

20 25 30

Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His Ser

35 40 45

Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val Asn

50 55 60

Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr Arg

65 70 75 80

Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys Ser

85 90 95

Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu Phe

100 105 110

Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg Gln

115 120 125

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

130 135 140

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

145 150 155 160

Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg Leu

165 170 175

Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys Leu

180 185 190

Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile Ala

195 200 205

Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp Ala

210 215 220

Thr Gly Leu Lys

225

<210> 46

<211> 589

<212> DNA

<213> Artificial sequence

<221> vector 10 Key sequence

<400> 46

gaaacaccga acaaagcacc agtggtctag tggtagaata gtaccctgcc acggtacaga 60

cccgggttcg attcccggct ggtgcagttc aggcaggccc ccggcagttt tagagctaga 120

aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt 180

gcaacaaagc accagtggtc tagtggtaga atagtaccct gccacggtac agacccgggt 240

tcgattcccg gctggtgcaa ggcaggttca ccatcagcag ttttagagct agaaatagca 300

agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcaacaa 360

agcaccagtg gtctagtggt agaatagtac cctgccacgg tacagacccg ggttcgattc 420

ccggctggtg cactactggg gccagggcac ccgttttaga gctagaaata gcaagttaaa 480

ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttggatcc 540

agcgcttagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagt 589

Claims

1. A construction method of a chimeric antigen receptor modified cell library with an automatically optimized antigen binding domain is characterized in that a vector containing a first gene element, a second gene element, a third gene element, a fourth gene element and a fifth gene element is transfected into a cell, and the number of the vectors is one or more, so that the chimeric antigen receptor modified cell library is obtained;

the first gene element is a nucleotide sequence for coding a receptor 1, and the receptor 1 comprises an extracellular domain, a transmembrane domain and an intracellular domain; the extracellular domain may recognize a biotin or 6 × His tag tagged on a target antigen, the intracellular domain including tobacco etch virus protease TEVP;

the third gene element is a nucleotide sequence for coding a screening gene, and a tTA transcription factor can start the expression of the screening gene, so that a host generates fluorescence, obtains certain resistance or obtains a growth advantage under certain conditions;

the fourth gene element is a nucleotide sequence encoding one or more cis-regulatory elements and one or more trans-acting factors; the cis-form regulatory element can enable DNA to generate a DNA sequence with a special structure, the special structure refers to that the DNA structure contains a DNA single-chain structure without base complementary pairing, and the special structure comprises R-loop, G-triplet and G-quadruplet; the trans-acting factor is a Cas protein family and a variant of the Cas protein family, gRNA, a uracil glycosylase inhibitor, adenine deaminase, cytidine deaminase, RNA-DNA hybrid chain binding protein, photosensitive protein, nucleolin, DNA helicase, error-prone DNA polymerase, recombinase, endonuclease, exonuclease and one or more combinations of alkyl adenine glycosylase;

the fifth gene element is a nucleotide sequence for coding a suppression system, and a tTA transcription factor can start the expression of the suppression system, so that a trans-acting factor is knocked down or knocked out, and an antibody sequence dynamic library is maintained to be stable; or the fifth genetic element is a nucleotide sequence encoding an photocontrol system capable of regulating expression of the fourth genetic element: starting a fourth gene element to play a role under the illumination condition, and starting to construct an antibody dynamic sequence library; after the light is stopped, the function of the fourth gene element is inhibited, and the dynamic sequence library of the antibody is maintained to be stable.

2. The method of claim 1, wherein the cells are human or murine cells.

3. The method of claim 1, wherein the dynamic library of antibody sequences comprises a classical antibody library, a single chain antibody library, and a nanobody library.

4. The method for constructing the library of chimeric antigen receptor-modified cells according to claim 1, wherein the nucleotide sequence of the receptor 1 is represented by SEQ ID No. 1.

5. The method for constructing the library of chimeric antigen receptor-modified cells according to claim 1, wherein the receptor 2 is a synNotch receptor modified from a Notch receptor, and the nucleotide sequence of the synNotch receptor is shown in SEQ ID No.2 or SEQ ID No. 3.

6. The method of constructing a library of chimeric antigen receptor-modified cells according to claim 1, wherein the trans-acting factor comprises the following Cas protein family members and their partial domains: cas3-8s, cas10s, cas11s, cas9s, xCas9, cas12s, cas13s, and Cas14s.

7. The method for constructing a library of chimeric antigen receptor-modified cells according to claim 1, wherein the trans-acting factor comprises the following adenine deaminase and partial domain thereof: ecTadA, mADA, hADAR2 and hADAT2.

8. The method for constructing a library of chimeric antigen receptor-modified cells according to claim 1, wherein the trans-acting factor comprises the following cytidine deaminase and partial domain thereof: AID (activation-induced cytidine deaminase), APOBEC3G, APOBEC and CDA1.

9. The method for constructing the library of chimeric antigen receptor-modified cells according to claim 1, wherein the nucleotide sequence encoding the light control system comprises the following photoproteins and partial domains thereof: bphS, cph1, PCB, phyB-PIFs, P Φ B, bphP-PpsR 2, bphP1-Q-PAS1, LOV2-J α, phyB, PIF6, epdz and Photometer visual (VVD).

10. The method of claim 1, wherein the light control system is adapted to control the fourth genetic element in response to light selected from the group consisting of visible light, invisible light, blue light, red light, far infrared light, and ultraviolet light.

11. The method for constructing a library of chimeric antigen receptor-modified cells according to claim 1, wherein the selection gene comprises GFP, mCherry, luciferase, puromycin resistance gene, bleomycin, and neomycin resistance gene.

12. The method for constructing the library of chimeric antigen receptor-modified cells according to claim 1, wherein the suppression system is used to stabilize the dynamic library of antibody sequences by activating a negative regulatory protein, repressor, siRNA, CAS9/gRNA expression via tTA transcription factor or by knocking down or knocking out a trans-acting factor via reverse transcription.

13. A library of chimeric antigen receptor-modified cells whose antigen binding domain is automatically optimized, wherein the library of chimeric antigen receptor-modified cells is constructed by the method of any one of claims 1 to 12.

14. A method for screening a library of chimeric antigen receptor-modified cells whose antigen binding domain is automatically optimized, wherein the screening is performed using the library of chimeric antigen receptor-modified cells according to claim 13, comprising the steps of:

1) Preparing a target antigen, wherein the target antigen is marked with a label capable of being recognized by a receptor 1, and the label comprises biotin or 6 XHis;

2) Co-incubating a target antigen and a chimeric antigen receptor modified cell library, and screening positive chimeric antigen receptor expression cells according to the expression condition of a screened gene;

3) And performing amplification sequencing on the chimeric antigen receptor extracellular antigen binding domain of the positive cell to obtain an antibody nucleotide sequence capable of being specifically bound with a target antigen.

15. The method for screening a library of chimeric antigen receptor-modified cells according to claim 14, further comprising the following steps after obtaining the nucleotide sequence of the antibody capable of specifically binding to the target antigen: the antibody aiming at the target antigen is obtained by adopting a genetic engineering method.

16. The method of claim 14, wherein the target antigen is expressed on the surface of a cell, coated on the surface of a vector, or is a soluble protein.

17. The method of claim 16, wherein the carrier comprises magnetic microspheres, non-magnetic microspheres, graphene, agarose microspheres, nanoparticles, silica-based magnetic beads, GMA magnetic beads, and polystyrene magnetic microspheres.

18. Use of a method of screening a library of chimeric antigen receptor-modified cells according to any one of claims 14 to 17 for the production of antibodies to a particular target antigen.

19. An antibody selected by the method of any one of claims 14 to 17.