CN112679617A - Mammal fusion protein display plasmid based on mesothelin anchoring, cell line and application - Google Patents

Abstract

The invention provides a mammal fusion protein display plasmid based on mesothelin anchoring, a cell line and application, belonging to the technical field of antibody screening. The mammal fusion protein display cell line based on mesothelin anchoring is obtained by transferring the mammal fusion protein display plasmid into a mammal antibody display cell. The display plasmid of the mammal fusion protein based on mesothelin anchoring provided by the invention realizes the display of the protein to be screened on mammal cells by utilizing the characteristic that the mesothelin fusion protein can be expressed on a membrane and can be expressed in a secretory way, and can greatly improve the screening efficiency of the protein to be screened; the fusion protein formed by the protein to be screened and the mesothelin has higher expression efficiency, and the mesothelin has a direct standard antibody and can assist in indicating the screening result.

Description

Mammal fusion protein display plasmid based on mesothelin anchoring, cell line and application

Technical Field

The invention belongs to the technical field of antibody screening, and particularly relates to a mammal fusion protein display plasmid based on mesothelin anchoring, a cell line and application.

Background

The monoclonal antibody medicament has higher targeting property, can directly reach diseased cells, can reduce damage of normal cells and reduce side effects, and is widely used in clinic. The research and development of new monoclonal antibody drugs mainly comprises the construction and screening of antibody libraries in vitro in a recombinant antibody mode. In the application of recombinant antibodies, common display technologies include a phage display technology, a ribosome/mRNA display technology, a yeast surface display technology and the like, wherein most of the display technologies are based on a prokaryotic expression system, because codons selected in the process of expressing proteins in the prokaryotic expression system are different from eukaryotic cells, the antibodies obtained by screening the prokaryotic expression system can not obtain high expression in vivo, and the post-translational modification processes such as glycosylation of the prokaryotic expression system are different from human expression, so that the recombinant antibodies cannot be well applied.

The adoption of the mammalian cell surface display technology can overcome the difference between the post-translational modification processes such as glycosylation and the like in the phage display process and the human expression. The direct antibody display by mammalian cells, especially human cells, not only can rapidly screen large-capacity antibody libraries, but also has no problem of post-translational modification, and is easy to rapidly enter the next expression and production.

However, the existing mammals display the structure of a fusion protein formed by a transmembrane region of a transmembrane protein such as PDGFR and an antibody molecule. When screening cells expressing the antibody by flow cytometry, only candidate cells expressing the antibody molecule across the membrane can be obtained. In order to further detect the activity and affinity of the antibody, sequencing and molecular cloning are required again to prepare secreted antibody.

Disclosure of Invention

In view of the above, the present invention provides a display plasmid, a cell line and applications of mesothelin-anchored-based mammalian fusion protein; the display plasmid can display protein structures in mammalian cells for screening for functional antibodies or proteins with affinity. The protein after the mesothelin fusion expression can also be secreted and expressed, and the membrane-borne expression protein can be screened by flow cytometry, and the secreted protein can be screened by liquid phase detection technologies such as ELISA and the like; the display plasmid provided by the invention has the dual functions of on-membrane expression and secretory expression, and greatly accelerates the screening speed of protein.

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

the invention provides an application of mesothelin in displaying of a mammal fusion protein, wherein the mesothelin and a protein to be screened are subjected to fusion expression in a mammal cell.

The invention provides a fusion protein for mammalian cell display, which comprises a signal peptide, a protein to be screened and mesothelin which are connected in sequence.

Preferably, the amino acid sequence of mesothelin is shown in SEQ ID No. 3.

Preferably, the amino acid sequence of the signal peptide is shown as SEQ ID No. 1.

The invention provides a fusion gene for coding the fusion protein, which comprises a gene for coding a signal peptide, a gene for coding a protein to be screened and a gene for coding mesothelin.

Preferably, the nucleotide sequence of the gene for coding the signal peptide is shown as SEQ ID No.4, and the nucleotide sequence of the gene for coding the mesothelin is shown as SEQ ID No. 6.

The invention provides a mammal fusion protein display plasmid based on mesothelin anchoring, which comprises a fusion gene and an initial plasmid.

Preferably, the initial plasmid is pcDNA-FRT plasmid, and the fusion gene is recombined between NheI enzyme cutting site and NotI enzyme cutting site of the pcDNA-FRT plasmid.

The invention provides a mammal fusion protein display cell line based on mesothelin anchoring, which is obtained by transferring a mammal fusion protein display plasmid into a mammal antibody display cell.

The invention provides the fusion protein, the fusion gene, the mammal fusion protein display plasmid and the application of the mammal fusion protein display cell line in drug screening.

The invention has the beneficial effects that: the display plasmid of the mammal fusion protein based on mesothelin anchoring provided by the invention realizes the display of the protein to be screened on mammal cells by utilizing the characteristic that the mesothelin fusion protein can be expressed on a membrane and can be expressed in a secretory way, and can greatly improve the screening efficiency of the protein to be screened; the fusion protein formed by the protein to be screened and the mesothelin has higher expression efficiency, and the mesothelin has a direct standard antibody and can assist in indicating the screening result. The fusion protein formed by the protein to be screened and the mesothelin provided by the invention not only can be used for library screening by a mammalian cell display technology, but also can be partially secreted and expressed, and can be used for further screening the activity and the affinity of an antibody by Western Blot or ELISA experiment by collecting cell culture supernatant.

Drawings

FIG. 1 shows the qPCR detection results of single-point integration efficiency (Lac gene) of monoclonal cells, and the corresponding table data is shown in Table 1;

FIG. 2 shows the result of detecting the expression of beta-galactosidase by monoclonal cells;

FIG. 3 shows the expression efficiency of mScalet and mTag BFP2 after 48h transfection of K562-PB-FRT monoclonal cells;

FIG. 4 shows the expression efficiency of mScalet and mTag BFP2 after transfection of K562-PB-FRT monoclonal cells for 15 d;

FIG. 5 is a structural map of the mesothelin-anchor-based mammalian fusion protein display plasmid of example 1;

FIG. 6 shows the results of flow analysis of the MSLN protein expressed by K562-PB-FRT-39-sifi-4D5-MSLN cells and the 4D5 antibody;

FIG. 7 shows the construction of YH antibody library in example 3;

FIG. 8 shows the results of construction of the S-RBD library in example 4, wherein A is the PCR results of Frist PCR 710-713 sample light chains; b is the PCR result of the heavy chain of the FristPCR 710-713 sample; c is the enzyme digestion result of the SOE-PCR product of the 710-713 samples; d is the enzyme digestion result of the MSLN in pcDNA5 vector;

FIG. 9 shows the results of 48h flow analysis of K562-PB-FRT-39-YH-MSLN-pcDNA5 antibody library cells by electroporation;

FIG. 10 shows the results of 72h flow sorting of K562-PB-FRT-39-YH-MSLN-pcDNA5 antibody library cells by electroporation;

FIG. 11 shows the result of PCR amplification of K562-PB-FRT-39-YH-MSLN-pcDNA5 antibody library cell cDNA;

FIG. 12 shows the result of detecting extracellular protein expressed by K562-PB-FRT-39-sifi-4D5-MSLN cells.

Detailed Description

The invention provides an application of mesothelin in displaying of a mammal fusion protein, wherein the mesothelin and a protein to be screened are subjected to fusion expression in a mammal cell.

In the present invention, Mesothelin (MSLN) is a cell surface glycoprotein, a biomarker of early stage pancreatic cancer, found in blood and urine. It was found that during the synthesis of mesothelin, a 69kD precursor was initially synthesized and then enzymatically hydrolyzed to yield a 30kD N-terminal secreted form and a 40kD GPI-linked membrane-bound form. The mesothelin and the protein to be screened are subjected to fusion expression in mammalian cells, so that the on-membrane expression and secretory expression of the protein to be screened can be realized simultaneously; greatly improves the screening efficiency of the protein to be screened and simplifies the screening steps.

The invention provides a fusion protein for mammalian cell display, which comprises a signal peptide, a protein to be screened and mesothelin which are connected in sequence. In the invention, the amino acid sequence of the mesothelin is preferably shown as SEQ ID No. 3; the amino acid sequence of the signal peptide is preferably shown as SEQ ID No. 1. The sequence of the protein to be screened is not particularly limited, any type of protein to be screened can be selected, and the protein to be screened can be selected from single-chain antibodies, such as human whole blood cell single-chain antibodies and mouse cell single-chain antibodies; in the implementation process of the invention, a 4D5ScFv is taken as an example, and the amino acid sequence of the 4D5ScFv is shown as SEQ ID No. 2.

The invention also provides a fusion gene for coding the fusion protein, which comprises a gene for coding a signal peptide, a gene for coding a protein to be screened and a gene for coding mesothelin. In the invention, the nucleotide sequence of the gene coding the signal peptide is preferably shown as SEQ ID No.4, and the nucleotide sequence of the gene coding the mesothelin is preferably shown as SEQ ID No. 6. In the present invention, when 4D5ScFv is used as the protein to be screened, the nucleotide sequence encoding 4D5ScFv is shown in SEQ ID No. 5. The method for preparing the fusion gene is not particularly limited, and the conventional artificial synthesis method in the field can be adopted.

The invention provides a mammal fusion protein display plasmid based on mesothelin anchoring, which comprises a fusion gene and an initial plasmid. In the present invention, the primary plasmid is preferably pcDNA-FRT plasmid, which is preferably purchased from ThermoFisher under the accession number V601020. In the present invention, the fusion gene is preferably recombined between the NheI cleavage site and the NotI cleavage site of the pcDNA-FRT plasmid. In the specific implementation process of the invention, the two ends of the fusion gene are preferably connected with NheI restriction enzyme sites and NotI restriction enzyme sites, so as to facilitate subsequent experimental operation. In the present invention, when 4D5ScFv is used as the protein to be screened, the nucleotide sequence of the mammalian fusion protein display plasmid is shown in SEQ ID No. 7. In the present invention, the method for preparing the mammalian fusion protein-displaying plasmid preferably comprises the steps of: artificially synthesizing the fusion gene, and performing double enzyme digestion on the fusion gene and the initial plasmid respectively and then connecting to obtain the recombinant plasmid. In the present invention, the double digestion is preferably performed with NheI enzyme and NotI enzyme, and the method and procedure for double digestion and ligation are not particularly limited, and may be performed by a conventional double digestion and ligation method in the art. In the present invention, the mammalian fusion protein display plasmid can also be synthesized directly by artificial synthesis.

The invention provides a mammal fusion protein display cell line based on mesothelin anchoring, which is obtained by transferring a mammal fusion protein display plasmid into a mammal antibody display cell. In the present invention, the mammalian antibody-displaying cells and the preparation method thereof are described in chinese patent CN202011525205.3, and the specific preparation method is described in the examples. In the invention, the transfer method is preferably electric transfer, and the specific conditions and parameters of the electric transfer are not particularly limited, and the conventional electric transfer method in the field can be adopted.

The invention also provides the fusion protein, the fusion gene, the mammal fusion protein display plasmid and the application of the mammal fusion protein display cell line in drug screening. In the invention, preferably, the fusion gene is used for displaying the mammalian fusion protein, constructing a mammalian antibody gene library and realizing the screening of antibody drugs. The specific screening method of the antibody drug is not particularly limited, and a conventional screening method of the antibody drug in the field can be adopted. In the practice of the present invention, the constructed antibody gene library is exemplified by a human ScFv antibody library and a murine ScFv antibody library.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Preparation of monoclonal cells of mammalian antibody-displaying cell K562-PB-FRT-39

Experimental Material

1. Cell: k562 cells

2. Culture medium: IMDM complete Medium, 1640 complete Medium

3. Plasmid: PB-FRT-LacZeo plasmid (nucleotide sequence shown in SEQ ID No. 8)

4. Reagent: Galacto-Star^TMSystem kit (from Applied Biosystems, Cat. No.: BM300S)

Experimental methods

Co-rotating the two plasmids:

take 1X 10⁷Individual K562 cells were centrifuged at 400g for 5min and the supernatant discarded, and K562 cells were resuspended in 120 μ l of serum-free 1640 medium, and 7 μ g PB-FRT-LacZeo plasmid and 2.8 μ g Super PB (PB200PA-1, purchased from SBI) were added and mixed well before transferring to 120 μ l shock tubes to avoid air bubbles. Setting the electric transfer parameters as follows: 560V, 30ms, and immediately after the electrotransfer is finished, 5ml of IMDM is used for completely culturing the cells in the electroporation tube, and then the cells are added into a T25 culture bottle for culturing to obtain K562-PB-FRT-LacZeo cells, wherein the culture conditions are as follows: 37 ℃ and 5% CO₂。

Drug screening:

after 48h of culture, centrifuging 400g of K562-PB-FRT-LacZeo cells for 5min to collect cell pellets, resuspending the K562-PB-FRT-LacZeo cell pellets with 5ml of fresh IMDM complete medium, counting 20. mu.l of cell suspension with a cell counter, and then adding fresh IMDM complete medium to adjust the cell density to 3X 10⁵One per ml, which was drug screened with puromycin at 5. mu.g/ml, and passaged once every 48 h. The drug is screened for more than one week for later use.

Single cloning:

10 round bottom 96 well plates were prepared, fresh IMDM complete medium was added to the round bottom 96 well plates at a volume of 150. mu.l/well, and 5X 10 wells were then removed⁶A10-piece 96-well plate monoclonal cell was prepared from each K562-PB-FRT-LacZeo cell by Sony flow sorter (SH800) according to the instructions of the apparatus.

Monoclonal cells in 5% CO₂The culture was performed in an incubator at 37 ℃ and on day 4, the single clones were supplemented with fresh IMDM complete medium at a volume of 100. mu.l/well. Day 7, monoclonal exchange, 100. mu.l of medium was discarded from each well and 100. mu.l of fresh IMDM complete medium was added. On the tenth day, monoclonal cells observed under a microscope were transferred to a 24-well plate for culture. Thereafter, the cells were expanded to prepare for screening of cell lines.

Molecular biological identification:

after the expansion culture of the monoclonal cells, 3X 10 cells were taken from each monoclonal⁶Genome extraction from individual cells (prepared using QIAamp DNA Mini Kit reference manual under the brand name of QIAGEN)Sending Ding Cheng becomes peptide source biotechnology limited company to carry out gene integration rate detection. The number of Lac genes in the genome was counted and 5 cells with the least Lac gene insertion were selected as RQ values.

Functional verification of inserted genes:

the PB-FRT-LacZeo carrier contains beta-galactosidase label. Thus, 1X 10 cells were taken from each of 5 monoclonal cells⁶The cells were examined for beta-galactosidase expression using the Galacto-StarTM System kit, according to the protocol, and cell lines with galactosidase activity were selected.

The experimental results are as follows:

the K562-PB-FRT monoclonal cells obtained by screening are products selected from 39 th, 58 th, 76 th, 86 th and 105 th times, and are named as K562-PB-FRT-LacZeo-86mono, K562-PB-FRT-LacZeo-76mono, K562-PB-FRT-LacZeo-105mono, K562-PB-FRT-LacZeo-58mono and K562-PB-FRT-LacZeo-39 mono.

As shown in FIG. 1, the qPCR detection result of K562-PB-FRT-LacZeo monoclonal cells shows that: the single-point integration rates of the 5 monoclonal cell PB-FRT-LacZeo plasmids are K562-PB-FRT-LacZeo-86mono, K562-PB-FRT-LacZeo-76mono, K562-PB-FRT-LacZeo-105mono, K562-PB-FRT-LacZeo-58mono and K562-PB-FRT-LacZeo-39mono in sequence from high to low.

TABLE 1 qPCR assay results data for K562-PB-FRT-LacZeo monoclonal cells

The results of detecting the expression of beta-galactosidase from 5 monoclonal cells of K562-PB-FRT-LacZeo (FIG. 2) show that: compared with a K562 control group, the above 5 monoclonals can express beta-galactosidase. Since the Lac gene relative copy number RQ of the K562-PB-FRT-LacZeo-86 mono-clone cells is higher than 0.5 and the beta-galactosidase expression level is highest compared with the reference gene of a pair of alleles, the K562-PB-FRT-LacZeo-86 mono-clone cells are considered to be the least likely to be single-point integrated cells and are eliminated.

Furthermore, the remaining 4K 562-PB-FRT-LacZeo: and 4, performing functional identification on a single FRT integration site by four monoclonal cell lines of K562-PB-FRT-LacZeo-39mono, K562-PB-FRT-LacZeo-58mono, K562-PB-FRT-LacZeo-76mono and K562-PB-FRT-LacZeo-105 mono.

Functional identification of a single FRT integration site was performed using a K562-PB-FRT host cell displaying a mixed pool of dual fluorescent protein plasmids.

Experimental Material

1. Cell:

K562-PB-FRT-39mono、K562-PB-FRT-58mono、K562-PB-FRT-76mono、K562-PB-FRT-105mono

2. plasmid:

by adopting a whole-gene synthesis technology, red fluorescent protein mSCarlet and blue fluorescent protein mtagBFP2 genes are synthesized and constructed to pcDNA5-FRT (purchased from Saimeri Fei Biotech Co., Ltd.) through upstream NheI and downstream NotI respectively, and endotoxin-free large-scale preparation is carried out (completed by the Kingzhi Biotech Co., Ltd.). Obtaining mScarlet inpcDNA5-FRT and mtagBFP2 in pcDNA5-FRT

mScalet (with red fluorescence, SEQ ID No.9)

atggtgagcaaaggcgaggccgtgatcaaggagttcatgaggttcaaggtgcacatggagggcagcatgaacggccacgagttcgagatcgagggcgagggcgaaggcagaccctatgagggcacccagaccgccaagctgaaggtgacaaagggcggccctctgcccttcagctgggatattctgtccccccagttcatgtacggcagcagagccttcaccaagcaccccgccgacatccccgactactataagcagagcttccccgagggctttaagtgggagagggtgatgaacttcgaggatggaggcgccgtgaccgtgacccaagacacatctctggaggacggcacactgatctacaaggtgaagctgaggggcaccaactttcctcccgacggccccgtgatgcagaagaagaccatgggctgggaggcttccaccgagagactgtaccccgaggacggcgtgctgaagggcgacattaagatggctctgagactgaaggacggcggaagatacctcgccgacttcaagaccacctacaaggccaagaagcccgtgcagatgcccggcgcctacaacgtggatagaaagctggacatcacatcccacaacgaggattacaccgtcgtggagcagtacgagagatccgagggcagacactccaccggcggcatggatgaactgtacaagtga

mtagBFP2 (with blue fluorescence, SEQ ID No.10)

atggtgagcaaaggcgaggagctgatcaaggagaacatgcacatgaagctgtacatggagggcaccgtcgacaaccaccacttcaagtgcacaagcgagggcgagggcaagccctacgaaggcacccagaccatgagaatcaaggtggtggagggaggccctctgcctttcgctttcgacattctggccacaagctttctgtacggcagcaagaccttcatcaaccacacacaaggcatccccgacttctttaagcagtccttccccgagggcttcacatgggagagggtgaccacctatgaggatggcggcgtgctgaccgccacacaagacacctctctgcaagacggctgtctgatctacaacgtgaagattagaggcgtgaacttcaccagcaacggacccgtgatgcagaagaagacactgggctgggaggccttcaccgagacactgtaccccgccgacggaggactggaaggaagaaatgacatggccctcaagctggtgggcggcagccatctgatcgccaacgccaagaccacctatagatccaagaagcccgccaagaatctgaagatgcccggcgtgtactacgtggactatagactggagagaatcaaggaggccaacaacgagacctacgtggagcagcatgaggtggccgtggccagatactgcgatctgccctccaagctgggccataagctgaattga

3. Culture medium:

IMDM Medium, 1640 Medium

The experimental method comprises the following steps:

1. amplifying and culturing the 4K 562-PB-FRT monoclonal cells obtained in the above step until the number of the cells is more than 1 × 10⁷When the cells were cultured, 1X 10 cells were taken for each monoclonal cell⁷The cell number is 400g, the cell sediment is collected by centrifugation for 5min, and then 5ml of 1640 culture medium (no FBS and no double antibody) is respectively added to wash the cells once, and residual serum is removed.

2. Separately resuspending each monoclonal cell in 120. mu.l 1640 medium (no FBS, no double antibody), 2.2. mu.g of mSacrlet in pcDNA5-FRT plasmid, 4.4. mu.g of mtagBFP2 in pcDNA5-FRT plasmid and 13.3. mu.g of pOG44 plasmid (Thermofisiher brand helper plasmid, cat No. V600520 expressing Flp recombinase, mSacrlet: mTag BFP 2: pOG 44) at a mass ratio of 1: 2: 6, setting the electrical transfer parameters 560V, 30ms, culturing the cells after complete basal weight suspension with 5ml of IMDM immediately after the electrical transfer in a T25 flask under the conditions of 37 ℃ and 5% CO₂。

3. After 48h of electrotransformation, 1X 10 cells were taken from each clone⁶The cells were centrifuged at 400g for 5min and the supernatant discarded, resuspended in 300. mu.l PBS and transferred to a correspondingly labeled flow tube, and the expression efficiency of mSacrlet and mTag BFP2 was measured by a BD LSRFortessa flow analyzer. The remaining cells were supplemented with a suitable volume of fresh IMDM complete medium to adjust the cell culture density to 3X 10⁵Per ml, 100. mu.gThey were subjected to drug screening with hygromycin/ml, and passaged once every 48 h. When the cells are passaged, the action concentration of hygromycin is slowly increased to 500. mu.g/ml according to the cell state.

4. After 15 days of drug screening, 1X 10 cells were collected from each clone⁶The cell number 400g was centrifuged for 5min and the supernatant discarded, the cells were resuspended in 300. mu.l PBS and transferred to the corresponding labeled flow tube, and the expression efficiency of mSacrlet and mTag BFP2 was again examined by BD LSRFortessa flow analyzer.

As shown in fig. 3: after 4 monoclonal cells such as K562-PB-FRT-39mono, K562-PB-FRT-58mono, K562-PB-FRT-76mono, K562-PB-FRT-105mono and the like are electrically transferred to mScarlet in pcDNA5-FRT and mTag BFP2 in pcDNA5-FRT plasmids for 48h, mScarlet and mTag BFP2 can be effectively expressed. The flow cytometry plot shows that two fluorescent proteins are expressed simultaneously, which is a sign that two plasmids enter cells simultaneously after transient transfection.

After 15 days of hygromycin drug selection, the plasmid was depleted from the cells and the host cells expressed only the gene introduced by pcDNA 5-FRT. If the host cell has only one FRT integration site, it can only flow cytometrically show a single positive population. That is, only blue or red fluorescence, and the double positive area (upper right quadrant) should have no apparent cell population.

The cell flow results after 15 days of hygromycin drug screening are shown in FIG. 4, and obvious grouping appears in 4 monoclonal cells, wherein the K562-PB-FRT-39mono cell grouping effect is the best. Three monoclonals of K562-PB-FRT-58mono, K562-PB-FRT-76mono and K562-PB-FRT-105mono have few cells In the double positive region, and can prove that all 4 monoclonals successfully have the Flp-In integration system and only have a single FRT integration site.

Example 2

MSLN-based anchored mammalian fusion protein-displaying cells were constructed using the mammalian display cell line K562-PB-FRT-39 (i.e., K562-PB-FRT-39mono) by antibody fragment 4D5 of known function, and MSLN-based antibody display function was examined.

Experimental Material

1. Cell: K562-PB-FRT-39 monoclonal cells

2. Culture medium: IMDM complete Medium, 1640 complete Medium

3. Display plasmids: 4D5 inpcDNA5-FRT-MSLN (nucleotide sequence as SEQ ID No.7, constructed by trusted biotechnology Co., Ltd.)

pOG44 plasmid (Thermo Fisher, cat # V600520)

4. Flow-through antibody:

rb mAb to Mesothelin (Abcam Co., PE fluorescein conjugated, cat # ab252136)

Biotinylated Human Her2(ACRO Corp., cat number HE2-H82E2)

PE-Streptavidin (Biolegend, cat # 405203).

Experimental methods

1. Take 1X 10⁷The displayed cells of clone K562-PB-FRT-39 were centrifuged at 400g for 5min to collect cell pellets, and then 5ml of 1640 medium (no FBS, no double antibody) was added to wash the cells once, and the residual serum was removed.

2. Mu.l of 1640 medium (no FBS, no double antibody) cells were added with 4. mu.g of 4D5 in pcDNA5-FRT-MSLN antibody library plasmid and 16. mu.g of pOG44 plasmid (4D5 in pcDNA 5-FRT-MSLN: pOG 44: 1:4), the cells were mixed well with the plasmid by pipette and then slowly added to a 120. mu.l electroporation cuvette to avoid air bubbles, and the electroporation parameters were set as follows: 560V, 30 ms. Note that ice bath is carried out for 5min before and after electric shock. Immediately after the ice bath is finished, the cells after electric shock are resuspended by 10ml of IMDM complete medium and then added into a T25 culture bottle for culture under the following culture conditions: 37 ℃ and 5% CO₂. The cell obtained by electrotransformation is the K562-PB-FRT-39-sifi-4D5-MSLN cell.

3. After 48h of electric conversion, respectively taking 2X 10⁶K562-PB-FRT-39-sifi-4D5-MSLN cells and K562-PB-FRT-39 cells which are not electrically transferred are centrifuged for 5min at 400g, supernatant is discarded, 1ml of PBS is added to wash the cells once respectively, each cell is averagely divided into two parts which are respectively marked as (i) K562-PB-FRT-39+ MSLN Control, (ii) K562-PB-FRT-39-sifi-4D5-MSLN + MSLN Antibody, (iii) K562-PB-FRT-39+ Streptavidin Control, and (iv) K562-PB-FRT-39-sifi-4D5-MSLN + Biotinylated 2. Mu.g of Biotinylated HER2 was added to tubeThe proteins were mixed well and incubated at room temperature for 40min, then washed once with 1ml PBS. After 40min, 1. mu.l of the flow antibody of Rb mAb to Mesothelin (abcam) was added to the tubes (first) and (second), and 2. mu.l of PE was added to the tubes (third) and (fourth), respectively^TMStreptavidin, gently mixed and dyed in the dark at room temperature for 30 min. After the staining, 1ml of PBS was added to each flow tube to wash the cells and remove antibody residues, then 300. mu.l of PBS was added to resuspend the cells, and the expression rates of MSLN fusion protein and 4D5 antibody in K562-PB-FRT-39-sifi-4D5-MSLN antibody pool cells were determined by BD LSRFortessa flow analyzer.

After electrotransfer for 48h, K562-PB-FRT-39-sifi-4D5-MSLN cells were supplemented with fresh IMDM complete medium to adjust the cell culture density to 3X 10⁵Hygromycin was added to 100. mu.g/ml for drug screening at 48h intervals. The concentration of hygromycin was slowly increased to 500. mu.g/ml depending on the cell state during cell passaging (the concentration of hygromycin used was adjusted to 200. mu.g/ml when the viability was greater than 60%, to 300. mu.g/ml when the viability was greater than 70%, and to 500. mu.g/ml when the viability was greater than 80%).

4. 10 days after drug screening, 1X 10 times of K562-PB-FRT-39-sifi-4D5-MSLN antibody bank cells and non-electrotransferred K563-FRT-B11 cells were again taken⁶The number of cells was stained by the same flow method as in the previous step, and the expression efficiency of MSLN fusion protein in K562-PB-FRT-39-sifi-4D5-MSLN antibody library cells was measured again by the BD LSRFortessa flow analyzer.

As shown in FIG. 6, the expression efficiency of MSLN protein was 3.63% and that of 4D5 antibody was 7.92% in K562-PB-FRT-39 monoclonal cells transfected with sifi-4D5-MSLN plasmid for 48 h. After 10 days of hygromycin drug screening, the expression level of MSLN protein in K562-PB-FRT-39-sifi-4D5-MSLN cells is obviously increased, the expression efficiency of MSLN protein is improved to 90.8%, and the expression efficiency of 4D5 antibody is improved to 75.9%. The results show that: K562-PB-FRT-39 cells can successfully express MSLN protein and 4D5 antibody, demonstrating that K562-PB-FRT-39 displays the success of 4D5 antibody.

Example 3

Construction of human ScFv antibody library

Experimental Material

1. Cell: K562-PB-FRT-39-cells, YH Whole blood cells (peripheral blood from healthy volunteers).

2. The kit comprises:

TRIzol(Thermo 15596026)

SuperScript^TM III Platinum^TM One-Step qRT-PCR Kit(Thermo 11732088)

Primer

Max DNA Polymerase(Takara R045A)

Kodaq 2XPCR Master Mix(abm G497)

Bestaq^TM DNA Polymerase Kits(abm G464-Dye)

Gel Extraction Kit(OMAGA D2500-03)

agarose (invitrogen 75510 and 019)

Animal tissue/cell total RNA extraction kit (Zhuang Union organism ZP404)

Bulk DNA product purification kit (Tiangen DP205)

SfiⅠ(NEB#R0123L)

T4 ligase (NEB # M0202S)

DH5 alpha competent cell (Shanghai Weidi Biotechnology Co., Ltd. DL1001)

DH10B competent cell (Shanghai Weidi Biotechnology Co., Ltd. DE1072)

Ultra-thin DNA product purification kit (Tiangen DP203)

EndoFree Plasmid Maxi Kit(QIAGEN 12362)

3. Instrument for measuring the position of a moving object

PCR instrument (BIO-RAD T100)

Electrophoresis apparatus (BIO-RAD POWERPAC HC)

Gel imager (BIO-RAD Universal Hood II)

High speed desk type centrifuge (Gene 1730R)

Centrifuge (eppendorf Minispin PLUS)

Water-bath (Shanghai Bo message HHS-11-2)

Enzyme mark instrument (Thermo 1510)

Metal bath

4. Primer sequences, see published literature (Generation of Human scFv Antibody primers: PCR Amplification and Assembly of Light-and Heavy-Chain Coding sequences. Cold Spring harbor protocol.2011 Sep 1; 2011(9))

Experimental methods

RNA extraction

(1) 10ml of peripheral blood was extracted from the human body, 10ml of TRIzol (dissociation of the ribosome, release of RNA) was added thereto, and the mixture was mixed and lysed on ice for 5 min.

(2) Adding 2ml of chloroform, covering the cover, placing the mixture on a vortex oscillator, oscillating the mixture for 15s, and standing the mixture on ice for 5-10 min (centrifuging the mixture when a layering phenomenon occurs).

(3) Precooling the centrifuge to 4 ℃ in advance and then centrifuging at 12000rpm for 15min, wherein after the centrifugation is finished, the solution in the centrifuge tube can be divided into 3 phases which are sequentially from top to bottom: RNA phase, DNA phase and organic phase.

(4) The RNA phase in the upper layer was transferred to a new 1.5ml EP tube, an equal volume of isopropanol (precooled at 4 ℃) was added, and after mixing by inversion, it was allowed to stand on ice for 5min, centrifuged at 12000rpm for 10min, and the supernatant was discarded.

(5) 1ml of ddH with RNase Free was added₂75% ethanol (precooled at 4 ℃) prepared in O, and after being inverted and mixed evenly, the mixture is centrifuged at 12000rpm and 4 ℃ for 5min, and the supernatant is discarded. And (3) inverting the 1.5ml EP tube on dust-free paper for 10-15 min, and removing residual ethanol.

(6) Adding 15-30 mu L of ddH of RNase Free₂The pellet was dissolved O, 2. mu.l was taken out and tested for concentration and purity, and the remaining RNA was reverse transcribed according to the method provided in the reverse transcription kit (Thermo).

2. Reverse transcription of cDNA

According to SuperScript^TM III Platinum^TMThe One-Step qRT-PCR Kit specification carries out reverse transcription on the RNA, and the specific steps are as follows:

(1) an RNA Primer was prepared and preannealed as follows

(2) Incubating at 65 deg.C for 5min, and freezing for 1 min;

(3) cDNA Synthesis Mix, system as follows:

(4) adding cDNA Synthesis Mix into each RNA Primer system, mixing uniformly, centrifuging and collecting;

(5) the PCR procedure was as follows:

(6) the reaction product was placed on ice for a period of time and briefly centrifuged to obtain a cDNA solution of the YH sample.

3. First PCR

The primer Sequences involved are from published literature (Generation of Human scFv Antibody Libraries: PCR Amplification and Assembly of Light-and Heavy-Chain Coding Sequences). The specific primer names used are shown in Table 1.

TABLE 1 primer sequence names

The primers in the above tables were prepared to have a concentration of 10 μm using nuclease-free water, and then the primers were paired in the order shown in the following tables, and then the corresponding reagents were sequentially added to prepare PCR reaction systems according to the instructions of Kodaq 2 XPCR Master Mix.

TABLE 2 human ScFv antibody library primer pairing scheme (heavy chain)

TABLE 3 human ScFv antibody library primer pairing scheme (light chain)

(1) And (3) PCR system:

(2) PCR amplification conditions:

(3) PCR product-precipitation

The PCR products of the heavy and light chains were combined, respectively, and 1/10 volumes of 3M sodium acetate and 2.5 times the volume of 95% ethanol were added thereto, mixed well, left to stand on ice for 15min, 14,000 Xg, centrifuged at 4 ℃ for 10min, and the supernatant was discarded. Washed once with 75% ethanol and 50. mu.l ddH₂Dissolving O to obtain a DNA solution.

(4) The above DNA solution was Gel recovered according to the method provided by Gel Extraction Kit (OMAGA) Kit.

4. Second PCR

(1)PCR-like：

And (3) taking VH and VL obtained by recovering the first PCR glue as templates, configuring the following system, and carrying out PCR-like:

VH PCR Product 5μl(10ng/μl)

VL PCR Product 5μl(10ng/μl)

Kodaq 2×Master Mix 10μl

the PCR-like reaction procedure was as follows:

(2) the PCR-like product was diluted 100 times (20/2000) as a template, RSC-F (sense) and RSC-B (reverse) as primers, and the following PCR reaction systems were prepared by sequentially adding the corresponding reagents according to the instruction of Kodaq 2 XPCR Master Mix:

(3) PCR amplification conditions:

(4) PCR products were purified according to the method provided by the bulk DNA product purification kit (Tiangen).

5. Enzyme digestion

(1) Digestion of the SOE-PCR product:

an enzyme digestion system is configured according to the instruction of Sfi1(NEB), and the SOE-PCR product is subjected to enzyme digestion.

The enzyme digestion system is as follows:

50 ℃ overnight enzyme digestion

(2) Plasmid vector digestion

An enzyme digestion system is configured according to the instruction of Sfi1(NEB), and the 4D5 in pcDNA5-FRT-MSLN vector is subjected to enzyme digestion.

The enzyme digestion system is as follows:

50 ℃ overnight enzyme digestion

(3) After agarose Gel electrophoresis of the digestion product, the target band was excised, and then Gel recovered according to the method provided by Gel Extraction Kit (OMAGA).

(4) Enzyme linking: the enzyme digestion product of YH sample and the enzyme digestion product of 4D5 in pcDNA5-FRT-MSLN vector are referred to T4 ligase (ABClonal) instruction, the following ligation system is configured, and YH-pcDNA5-FRT-MSLN ligation product is obtained by overnight ligation at 16 ℃.

6. Ligation product conversion

(1) Ligation product transformation-transformation

Transformation of the T4 ligation products was performed according to the instructions of DH5 alpha competent cells (purchased from Shanghai Diego Biotechnology Ltd.).

(2) Ligation product conversion-purification of ligation product

To ensure ligation efficiency, the YH-pcDNA5-FRT-MSLN ligation product was purified first, and DNA was purified according to the protocol provided in the ultra-thin DNA product purification kit (Tiangen).

(3) Ligation product conversion-electrotransformation

Transformation of the T4 ligation purified product was performed according to the instructions of DH10B-Plus competent cells (purchased from Shanghai Tokyo Biotechnology Ltd.).

(4) Shaking a large amount of bacteria liquid

The remaining bacterial liquid of the electroporation was transferred to 200ml of LB liquid medium resistant to AMP at 37 ℃ and 220rpm, and shaken overnight.

(5) Plasmid major grape

The Plasmid was extracted according to the EndoFree Plasmid Maxi Kit (QIAGEN) Kit instructions.

Results of the experiment

As shown in FIG. 7, the heavy chain and light chain (about 400bp) of YH sample are amplified by ordinary PCR, then the heavy chain and light chain (about 800bp) of YH sample are connected by SOE-PCR, then the combined SOE-PCR product of the heavy chain and light chain of YH sample and 4D5 in pcDNA5-FRT-MSLN vector are cut by enzyme, finally the heavy chain and light chain of YH sample are connected with MSLN in pcDNA5 vector by T4 ligase, and the successful construction of YH-pcDNA5-FRT-MSLN antibody library can be confirmed according to the bands in the figure.

Example 4

The light chain and heavy chain coding sequences in the murine splenocytes were amplified and assembled by PCR technique to construct murine ScFv antibody library.

Experimental Material

1. Animals: 710 mice, 711 mice, 712 mice, 713 mice; all mice were immunized with S-RBD antigen, resulting in the mice producing natural antibodies against the S-RBD antigen in vivo, from the laboratory animals technologies, Inc., of Wei Tony Hua, Beijing, the strain BALA/c.

2. The kit comprises:

TRIzol(Thermo 15596026)

SuperScript^TM III Platinum^TM One-Step qRT-PCR Kit(Thermo 11732088)

Primer

Max DNA Polymerase(Takara R045A)

Kodaq 2X PCR Master Mix(abm G497)

Bestaq^TM DNA Polymerase Kits(abm G464-Dye)

Gel Extraction Kit(OMAGA D2500-03)

agarose (invitrogen 75510 and 019)

Animal tissue/cell total RNA extraction kit (Zhuang Union organism ZP404)

Bulk DNA product purification kit (Tiangen DP205)

SfiⅠ(NEB#R0123L)

T4 ligase (NEB # M0202S)

DH5 alpha competent cell (Shanghai Weidi Biotechnology Co., Ltd. DL1001)

DH10B competent cell (Shanghai Weidi Biotechnology Co., Ltd. DE1072)

Ultra-thin DNA product purification kit (Tiangen DP203)

EndoFree Plasmid Maxi Kit(QIAGEN 12362)

3. The instrument comprises the following steps:

PCR instrument (BIO-RAD T100)

Electrophoresis apparatus (BIO-RAD POWERPAC HC)

Gel imager (BIO-RAD Universal Hood II)

High speed desk type centrifuge (Gene 1730R)

Centrifuge (eppendorf Minispin PLUS)

Water-bath (Shanghai Bo message HHS-11-2)

Enzyme mark instrument (Thermo 1510)

Metal bath

4. The primer sequence is as follows:

VL-for-k1 catggcggactacaaagacawtgttctcacccagtc(SEQ ID No.11)

VL-for-k2 catggcggactacaaagacatccagatgacacagwc(SEQ ID No.12)

VL-for-k3 catggcggactacaaagatrttgtgatgacccagwc(SEQ ID No.13)

VL-for-k4 catggcggactacaaagacattstgmtgacccagtc(SEQ ID No.14)

VL-for-k5 catggcggactacaaagatgttgtgvtgacccaaac(SEQ ID No.15)

VL-for-k6 catggcggactacaaagacacaactgtgacccagtc(SEQ ID No.16)

VL-for-k7 catggcggactacaaagayattktgctcactcagtc(SEQ ID No.17)

VL-for-k8 catggcggactacaaagatattgtgatracccaggm(SEQ ID No.18)

VL-for-k9 catggcggactacaaagacattgtaatgacccaatc(SEQ ID No.19)

VL-for-k10 catggcggactacaaagacattgtgatgwcacagtc(SEQ ID No.20)

VL-for-k11 catggcggactacaaagatrtccagatgamccagtc(SEQ ID No.21)

VL-for-k12 catggcggactacaaagatggagaaacaacacaggc(SEQ ID No.22)

VL-revK1 ggagccgccgccgccagaaccaccaccaccagaaccaccaccaccgcgtttbatttccagcttgg(SEQ ID No.23)

VL-revK2 ggagccgccgccgccagaaccaccaccacagaaccaccaccaccgcgttttatttccaattttg(SEQ ID No.24)

VH-for-1 ggcggcggcggctccggtggtggtggatccgaggttcdsctgcaacagty(SEQ ID No.25)

VH-for-2 ggcggcggcggctccggtggtggtggatcccaggtgcaamtgmagsagtc(SEQ ID No.26)

VH-for-3 ggcggcggcggctccggtggtggtggatccgavgtgmwgctggtggagtc(SEQ ID No.27)

VH-for-4 ggcggcggcggctccggtggtggtggatcccaggttaytctgaaagagtc(SEQ ID No.28)

VH-for-5 ggcggcggcggctccggtggtggtggatccgakgtgcagcttcagsagtc(SEQ ID No.29)

VH-for-6 ggcggcggcggctccggtggtggtggatcccagatccagttsgygcagtc(SEQ ID No.30)

VH-for-7 ggcggcggcggctccggtggtggtggatcccagrtccaactgcagcagyc(SEQ ID No.31)

VH-for-8 ggcggcggcggctccggtggtggtggatccgaggtgmagctasttgagwc(SEQ ID No.32)

VH-for-9 ggcggcggcggctccggtggtggtggatccgaagtgaagmttgaggagtc(SEQ ID No.33)

VH-for-10 ggcggcggcggctccggtggtggtggatccgatgtgaacctggaagtgtc(SEQ ID No.34)

VH-for-11 ggcggcggcggctccggtggtggtggatcccagatkcagcttmaggagtc(SEQ ID No.35)

VH-for-12 ggcggcggcggctccggtggtggtggatcccaggcttatctgcagcagtc(SEQ ID No.36)

VH-for-13 ggcggcggcggctccggtggtggtggatcccaggttcacctacaacagtc(SEQ ID No.37)

VH-for-14 ggcggcggcggctccggtggtggtggatcccaggtgcagcttgtagagac(SEQ ID No.38)

VH-for-15 ggcggcggcggctccggtggtggtggatccgargtgmagctgktggagac(SEQ ID No.39)

VH-rev1 cggagtcaggcccccgaggccgaggagacggtgacmgtgg(SEQ ID No.40)

VH-rev2 cggagtcaggcccccgaggccgcagagacagtgaccagag(SEQ ID No.41)

VH-rev3 cggagtcaggcccccgaggccgaggagactgtgagastgg(SEQ ID No.42)

Outer-for ctacagcaggcccaggcggccatggcggactacaaa(SEQ ID No.43)

Outer-rev cggagtcaggcccccgag(SEQ ID No.44)。

experimental methods

RNA extraction

(1) Mice were sacrificed, spleens removed from their bodies, placed in petri dishes, ground with 2ml TRIzol (to dissociate the nucleoprotein bodies and release the RNA), lysed for tissues and cells, and the lysates transferred to a clean 1.5ml EP tube;

(2) adding 0.4ml of chloroform, covering the cover, placing the cover on a vortex oscillator, oscillating for 15s violently, and standing the mixture on ice for 5-10 min (layering phenomenon occurs);

(3) precooling the centrifuge to 4 ℃ in advance and then centrifuging at 12000rpm for 15min, wherein after the centrifugation is finished, the solution in the centrifuge tube can be divided into 3 phases which are sequentially from top to bottom: RNA phase, DNA phase and organic phase.

(4) The RNA phase in the upper layer was transferred to a new 1.5ml EP tube, an equal volume of isopropanol (precooled at 4 ℃) was added, and after mixing by inversion, it was allowed to stand on ice for 5min, centrifuged at 12000rpm for 10min, and the supernatant was discarded.

(5) 1ml of 75% ethanol prepared with RNase Free ddH2O (precooled at 4 ℃) was added thereto, and the mixture was mixed by inversion and centrifuged at 12000rpm at 4 ℃ for 5min to discard the supernatant. And (3) inverting the 1.5ml EP tube on dust-free paper for 10-15 min, and removing residual ethanol.

(6) Adding 15-30 mu L of ddH of RNase Free₂The pellet was dissolved O, 2. mu.l was taken out and tested for concentration and purity, and the remaining RNA was reverse transcribed according to the method provided in the reverse transcription kit (Thermo 11732088).

2. First PCR

The primer sequences involved in this experiment are from published literature (Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly. antibody Engineering, (2010) pp.21-44).

The primers were prepared to 10 μm using nuclease-free water, and then the primers were paired in the order shown in the following table, and the corresponding reagents were added in order according to the instructions of Bestag 2 XPCRMaster Mix to prepare a PCR reaction system.

TABLE 4 murine ScFv antibody library primer pairing protocol (heavy chain)

TABLE 5 murine ScFv antibody library primer pairing scheme (light chain)

(1) And (3) PCR system:

(2) PCR amplification conditions:

(3) PCR products of the heavy chain and the light chain were combined, subjected to agarose Gel electrophoresis, and a target band of about 400bp was excised, followed by Gel recovery according to the method provided by Gel Extraction Kit (OMAGA) Kit.

3. Second PCR

(1)PCR-like：

mVH and mVL obtained by recovering First PCR glue are used as templates, the following system is configured, and PCR-like is carried out

mVH PCR product 5. mu.l (10 ng/. mu.l);

mVLPCR product 5. mu.l (10 ng/. mu.l);

Kodaq 2×MasterMix 10μl。

PCR-like reaction procedure:

(2) the PCR-like product was diluted 100 times (20/2000) as a template, and Outer-for and Outer-rev were primers, and the following PCR reaction system was prepared by sequentially adding the corresponding reagents according to the instruction of Kodaq 2 × PCR Master Mix:

(3) the PCR amplification conditions were as follows:

(4) the SOE-PCR product was subjected to agarose Gel electrophoresis, and the 800bp band of interest was excised and recovered by Gel Extraction Kit (OMAGA) according to the instructions provided in the Kit.

4. Enzyme digestion

(1) Digestion of the SOE-PCR product:

an enzyme digestion system is configured according to the instruction of Sfi1(NEB), and the SOE-PCR product is subjected to enzyme digestion.

Enzyme digestion system:

50 ℃ overnight enzyme digestion

(2) Plasmid vector digestion

Configuring an enzyme digestion system according to the specification of Sfi1(NEB), and carrying out enzyme digestion on the 4D5 in pcDNA5-FRT-MSLN vector, wherein the enzyme digestion system is as follows:

the digestion was carried out overnight at 50 ℃.

(5) After agarose Gel electrophoresis of the digestion product, the target band was excised, and then Gel recovered according to the method provided by Gel Extraction Kit (OMAGA).

(6) Enzyme linking: the SOE-PCR product of the combined heavy chain and light chain of the S-RBD immune mouse sample and the enzyme digestion product of the 4D5 in pcDNA5-FRT-MSLN vector are referred to T4 ligase (NEB) specification, a ligation system is configured, and the ligation product S-RBD-pcDNA5-FRT-MSLN is obtained by overnight ligation at 16 ℃.

The linking system is as follows:

6. ligation product conversion

(1) Ligation product transformation-transformation

Transformation of the T4 ligation products was performed according to the instructions of DH5 alpha competent cells (purchased from Shanghai Diego Biotechnology Ltd.).

(2) Ligation product conversion-purification of ligation product

To ensure ligation efficiency, S-RBD-pcDNA5-FRT-MSLN ligation product was purified according to the ultra-thin DNA product purification kit:

(3) ligation product conversion-electrotransformation

Transformation of the T4 ligation purified product was performed according to the instructions of DH10B-Plus competent cells (purchased from Shanghai Tokyo Biotechnology Ltd.).

(4) A large amount of bacteria liquid is shaken: the remaining cell suspension was transferred to 200ml of AMP-resistant LB liquid medium, and shaken overnight at 37 ℃ and 220 rpm.

(5) Plasmid major grape

The Plasmid was extracted according to the EndoFree Plasmid Maxi Kit (QIAGEN) Kit instructions.

(6) The transformed and electrotransferred plate was sequenced by picking 100 single clones.

Results of the experiment

As shown in FIG. 8, the heavy chain and the light chain (400bp) of the S-RBD sample are amplified by common PCR, then the heavy chain and the light chain (800bp) of the S-RBD sample are connected by SOE-PCR, then the SOE-PCR product formed by combining the heavy chain and the light chain of the S-RBD sample and the 4D5 in pcDNA5-FRT-MSLN vector are both subjected to enzyme digestion, finally the enzyme digestion product of the S-RBD sample is connected with the enzyme digestion product of the 4D5 in pcDNA5-FRT-MSLN vector by T4 ligase, and the S-RBD-pcDNA5-FRT-MSLN antibody library can be successfully constructed according to the bands in the figure.

Example 5

A human ScFv antibody-displaying cell library was constructed based on K562-PB-FRT-39 cells.

Experimental Material

1. Cell: K562-PB-FRT-39 cells

2. Plasmid: YH-pcDNA5-FRT-MSLN antibody library plasmid

3. Culture medium:

IMDM Medium, 1640 Medium

4. Flow-through antibody:

biotinylated 2019 n-Cov Spike S-His (KACTUS, cat # COV-VM4SSB), hereinafter referred to as Biotinylated-Spike.

APC-Streptavidin (Biolegend, cat # 405207)

Experimental methods

1. Take 2X 10⁸The K562-PB-FRT-39 cells are centrifuged at 400g for 5min to collect cell precipitates, and then 10ml of 1640 medium (no FBS and no double antibody) is added to wash the cells once, so as to remove residual serum.

2. Resuspending the monoclonal cells in 2ml 1640 medium (no FBS, no double antibody), adding 40 μ g of YH-pcDNA5-FRT-MSLN antibody library plasmid and 360 μ g of POG44 plasmid (YH-pcDNA 5-FRT-MSLN: POG 44: 1:9), after the cells and plasmid were mixed well, transferring them to 2 1000 μ l shock tubes, and setting the electrotransformation parameters: 950V, 30 ms. Note that ice bath is carried out for 5min before and after electric shock. Immediately after the ice bath is finished, suspending the shocked cells by using 100ml of IMDM complete culture medium, and adding the cells into a T175 culture flask for culture under the following culture conditions: 37 ℃ and 5% CO₂。

3. After 48h of electric conversion, respectively taking 2X 10⁶K562-PB-FRT-39 cells and K562-PB-FRT-39-YH-pcDNA5-FRT-MSLN antibody library cells, centrifuged at 400g for 5min, the supernatant was discarded, 1ml of PBS was added to each of the cells to wash the cells, and each cell was washed withAveragely divided into two parts, marked as (i) K562-PB-FRT-39+ Streptavidin Control, (ii) K562-PB-FRT-39-YH-pcDNA5-FRT-MSLN + Biotinylated-Spike, ((iii) K562-PB-FRT-39+ MSLN antibiotic Control, ((iii) K562-PB-FRT-39-YH-pcDNA5-FRT-MSLN + MSLN antibiotic. Add 1. mu.g of Biotinylated-Spike protein into the No. two tube, mix well and incubate for 40min at room temperature, then add 1ml of PBS to wash once. After 40min, 3 μ l of APC-Streptavidin flow Antibody was added to tube III and tube II, 1 μ l of PE-MSLN Antibody flow Antibody was added to tube III and tube IV, and after flicking and mixing, the mixture was stained in dark at room temperature for 30 min. After the staining, 1ml PBS was added to each flow tube to wash the cells and remove antibody residues, then 300. mu.l PBS was added to resuspend the cells, and the expression efficiency of S antibody and MSLN protein in K562-PB-FRT-39-YH-pcDNA5-FRT-MSLN antibody pool cells was tested by BD LSRFortessa flow analyzer. The remaining cells were supplemented with fresh IMDM complete medium to adjust the cell culture density to 3X 10⁵/ml。

4. According to the flow analysis result of K562-PB-FRT-39-YH-pcDNA5-FRT-MSLN antibody library cells after 48h of electrotransformation, the remaining K562-PB-FRT-39-YH-pcDNA5-FRT-MSLN antibody library cells were stained with the former flow staining method at 72h and sorted by a Sony flow sorter.

5. 1X 10 of the sorted K562-PB-FRT-39-YH-pcDNA5-FRT-MSLN antibody bank cells⁵The number of cells was determined by extracting nucleic acid and reverse transcribing to obtain cDNA according to the method provided by the 5 × All-In-One RT Master Mix (with ExCellenCT lysine Kit).

6. The newly synthesized first strand cDNA was used as a template for downstream PCR amplification directly, and RSC-F (sense) and RSC-B (reverse) in the YH library amplification process were used as primers, and the corresponding reagents were added in sequence to prepare a PCR reaction system according to the instruction of Kodaq 2 × PCR Master Mix.

PCR amplification conditions:

7. and (3) carrying out agarose gel electrophoresis on the PCR product, recovering a target fragment of about 800bp, sending the target fragment to a sequencing company for TA cloning, selecting 20 effective clones, and analyzing the sequences of the effective clones.

Results of the experiment

As shown in FIG. 9, the flow analysis results after K562-PB-FRT-39 cells were transfected with YH-pcDNA5-FRT-MSLN antibody pool plasmid for 48h showed: the expression level of MSLN protein in K562-PB-FRT-39-YH-pcDNA5-FRT-MSLN antibody pool cells was 99.7%, and the expression level of S antibody was 10.2%, indicating that the transfection efficiency of K562-PB-FRT-39 transfection of YH-pcDNA5-FRT-MSLN antibody pool plasmid was 99.7%, but the successful expression of S antibody in YH-pcDNA5-FRT-MSLN antibody pool plasmid was 10.2%. As shown in FIG. 10, the K562-PB-FRT-39-YH-pcDNA5-FRT-MSLN antibody pool cells were selected for 1% of double positive area (MSLN protein positive and S antibody positive) selection cells when they were flow sorted by electroporation for 72 h.

As shown in FIG. 11, there was a band around 800bp, and the antibody sequence of ScFv was successfully obtained from K562-PB-FRT-39-YH-pcDNA5-FRT-MSLN antibody library cells, which has the following sequence:

DNVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSFSLTISSMEAEDAATYYCQQWSRTPPTFGSGTKLEIKRGG。

comparative example

The transmembrane region of the PDGFR protein is a design commonly used in the art to express the protein as a fusion on the membrane. Now, the transmembrane expression amounts of 4D5 antibodies in K562-PB-FRT-39-sifi-4D5-MSLN cells and K562-PB-FRT-39-4D5-PDGFR cells were compared.

Experimental Material

1. Cell: k562, K562-PB-FRT-39-sifi-4D5-MSLN, K562-PB-FRT-39-4D5-PDGFR

2. Culture medium: IMDM complete medium

Western Blot antibody:

ErbB2 monoclonal antibody 19G9(Anti-4D5-FC)1:10000 (see published patent: CN201910784230.4 for details)

Experimental methods

1. Take 1X 10⁷The K562 cells were centrifuged at 400g for 5min to collect cell pellets, and then 10ml of 1640 medium (no FBS, no double antibody) was added to wash the cells once, and the residual serum was removed.

2. Resuspend monoclonal cells in 120 μ l 1640 medium (no FBS, no diabodies), add 2 μ g of 4D5-PDGFR antibody library plasmid and 18 μ g of POG44 plasmid (4D 5-PDGFR: POG44 ═ 1:9), transfer the cells and plasmid to 120 μ l shock tubes after mixing well, set the electrotransfer parameters: 560V, 30 ms. Note that ice bath is carried out for 5min before and after electric shock. Immediately after the ice bath is finished, the cells after electric shock are suspended by 10ml of IMDM complete culture medium and then are added into a T25 culture bottle for culture under the following culture conditions: 37 ℃ and 5% CO₂。

3. After electrotransfer for 48h, K562-PB-FRT-39-4D5-PDGFR cells are passaged, and the density of the passaged cells is 3 x 10⁵And then adding 500 mu g/ml hygromycin B drug for screening for 2 weeks to obtain K562-PB-FRT-39-4D5-PDGFR cells.

4. Preparing a protein sample: each is 1 × 10⁷The K562-PB-FRT-39-sifi-4D5-MSLN cells and the K562-PB-FRT-39-4D5-PDGFR cells were resuspended in 15ml of serum-free and double-antibody-free IMDN medium, and then added to a T25 cell culture flask, and then placed at 37 ℃ with 5% CO₂An incubator is used for collecting 10000g of cell suspension after 72 hours and centrifuging for 5 minutes, the supernatant is transferred to a 30KDa ultrafiltration tube and centrifuged for 10 minutes at 5000rpm, 10ml of PBS is added to replace protein in the supernatant into PBSA, finally the volume of the protein solution is maintained at 200 mul, 50 mul of 5 Xloading Buffer is added and mixed evenly, and then the mixture is heated for 10 minutes in a 100 ℃ metal bath.

5. Protein loading: marker, 20 mu g K562-PB-FRT-39-4D5-PDGFR supernatant, 20 mu g K562-PB-FRT-39-sifi-4D5-MSLN supernatant, 25ng4D5-FC protein.

SDS-PAGE gel electrophoresis: constant voltage electrophoresis is adopted, and electrophoresis is performed for 10min at 100V and then is adjusted to electrophoresis at 180V for 50 min.

7. Semi-dry transfer printing: firstly soaking a PVDF membrane by methanol for 30s, then putting the PVDF membrane and 6 pieces of transfer printing filter paper into a transfer printing solution for soaking for 20min, and then sequentially placing the PVDF membrane and the 6 pieces of transfer printing filter paper according to the sequence from bottom to top: three layers of filter paper, PVDF membrane, SDS-PAGE gel, three layers of filter paper (note that no air bubble can exist between PVDF membrane and gel), and finally inserting electrodes and adopting 56mA constant current transfer printing for 90 min.

8. And (3) sealing: after the transfer, the PVDF film was removed, the front and back sides of the next film were marked, and then washed in TBST wash to remove the residual transfer solution, and finally blocked with 5% skim milk for 1 hour.

9. Antibody incubation: the 19G9-HRP antibody was diluted to 5ml with an antibody diluent at a ratio of 1:10000, and the PVDF membrane was soaked in the 19G9-HRP antibody solution and incubated overnight on a shaker at 4 ℃.

10. Washing the membrane: the 19G9-HRP is recycled into a labeled 15ml centrifuge tube, the PVDF membrane is put into a membrane washing box containing TBST washing liquor, then the TBST washing liquor is placed on a shaking table to shake for 15min, and the membrane washing is repeated for 3 times at intervals of 15 min.

11. And (3) developing: and (3) uniformly mixing the solution A and the solution B of the developing solution according to the ratio of 1:1, and developing by using a chemiluminescence imaging analyzer, wherein the developing solution needs to be prepared for use.

Results of the experiment

As shown in FIG. 12, after the PVDF membrane after the transfer was incubated with the 19G9-HRP antibody against 4D5 protein, it was found that both K562-PB-FRT-39-4D5-PDGFR supernatant and K562-PB-FRT-39-sifi-4D5-MSLN supernatant had the expression of 4D5 protein, and that the content of 4D5 protein in K562-PB-FRT-39-sifi-4D5-MSLN supernatant was significantly higher than that of K562-PB-FRT-39-4D5-PDGFR supernatant when the protein loading amounts were the same. The results show that: the efficiency of expressing the 4D5 protein by the K562-PB-FRT-39-sifi-4D5-MSLN cell is obviously superior to that of the K562-PB-FRT-39-4D5-PDGFR cell.

According to the embodiment, the display plasmid of the mammal fusion protein based on mesothelin anchoring provided by the invention realizes the display of the protein to be screened on the mammal cell by utilizing the characteristic that the mesothelin fusion protein can be expressed on a membrane and can be expressed in a secretory way, and can greatly improve the screening efficiency of the protein to be screened; the expression efficiency of the fusion protein formed by the protein to be screened and the mesothelin is higher.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Beijing ancient cooking peptide source Biotechnology Ltd

Jiao Shunchang

<120> mammal fusion protein display plasmid based on mesothelin anchoring, cell line and application

<160> 44

<170> SIPOSequenceListing 1.0

<210> 1

<211> 36

<212> PRT

<213> Artificial Sequence

<400> 1

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

1 5 10 15

Ala Leu Gly Ser Leu Leu Phe Leu Leu Phe Ser Leu Gly Trp Val Gln

20 25 30

Pro Ser Arg Thr

35

<210> 2

<211> 247

<212> PRT

<213> Artificial Sequence

<400> 2

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Gly Ser Thr

100 105 110

Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser

210 215 220

Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Val Trp Gly Gln Gly

225 230 235 240

Thr Leu Val Thr Val Ser Ser

245

<210> 3

<211> 289

<212> PRT

<213> Artificial Sequence

<400> 3

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

1 5 10 15

Asp Val Leu Lys His Lys Leu Asp Glu Leu Tyr Pro Gln Gly Tyr Pro

20 25 30

Glu Ser Val Ile Gln His Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro

35 40 45

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

50 55 60

Leu Leu Glu Val Asn Lys Gly His Glu Met Ser Pro Gln Val Ala Thr

65 70 75 80

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

85 90 95

Leu Asp Thr Leu Thr Ala Phe Tyr Pro Gly Tyr Leu Cys Ser Leu Ser

100 105 110

Pro Glu Glu Leu Ser Ser Val Pro Pro Ser Ser Ile Trp Ala Val Arg

115 120 125

Pro Gln Asp Leu Asp Thr Cys Asp Pro Arg Gln Leu Asp Val Leu Tyr

130 135 140

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

145 150 155 160

Val Lys Ile Gln Ser Phe Leu Gly Gly Ala Pro Thr Glu Asp Leu Lys

165 170 175

Ala Leu Ser Gln Gln Asn Val Ser Met Asp Leu Ala Thr Phe Met Lys

180 185 190

Leu Arg Thr Asp Ala Val Leu Pro Leu Thr Val Ala Glu Val Gln Lys

195 200 205

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

210 215 220

Pro Val Arg Asp Trp Ile Leu Arg Gln Arg Gln Asp Asp Leu Asp Thr

225 230 235 240

Leu Gly Leu Gly Leu Gln Gly Gly Ile Pro Asn Gly Tyr Leu Val Leu

245 250 255

Asp Leu Ser Met Gln Glu Ala Leu Ser Gly Thr Pro Cys Leu Leu Gly

260 265 270

Pro Gly Pro Val Leu Thr Val Leu Ala Leu Leu Leu Ala Ser Thr Leu

275 280 285

Ala

<210> 4

<211> 110

<212> DNA

<213> Artificial Sequence

<400> 4

atggccttgc caacggctcg acccctgttg gggtcctgtg ggacccccgc cctcggcagc 60

ctcctgttcc tgctcttcag cctcggatgg gtgcagccct cgaggaccgg 110

<210> 5

<211> 741

<212> DNA

<213> Artificial Sequence

<400> 5

gatatccaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga tagggtcacc 60

atcacctgcc gtgccagtca ggatgtgaat actgctgtag cctggtatca acagaaacca 120

ggaaaagctc cgaaactact gatttactcg gcatccttcc tttattctgg agtcccttct 180

cgcttctctg gatctagatc tgggacggat ttcactctga ccatcagcag tctgcagccg 240

gaagacttcg caacttatta ctgtcagcaa cattatacta ctcctcccac gttcggacag 300

ggtaccaagg tggagatcaa acgcactggc tccactagcg gttccggcaa acctggcagc 360

ggagaaggca gcaccaaagg ggaggttcag ctggtggagt ctggcggtgg cctggtgcag 420

ccagggggct cactccgttt gtcctgtgca gcttctggct tcaacattaa agacacctat 480

atacactggg tgcgtcaggc cccgggtaag ggcctggaat gggttgcaag gatttatcct 540

acgaatggtt atactagata tgccgatagc gtcaagggcc gtttcactat aagcgcagac 600

acatccaaaa acacagccta cctgcagatg aacagcctgc gtgctgagga cactgccgtc 660

tattattgtt ctagatgggg aggggacggc ttctatgcta tggacgtgtg gggtcaagga 720

accctggtca ccgtctcctc g 741

<210> 6

<211> 870

<212> DNA

<213> Artificial Sequence

<400> 6

acccagatgg accgcgtgaa cgccatcccc ttcacctacg agcagctgga cgtcctaaag 60

cataaactgg atgagctcta cccacaaggt taccccgagt ctgtgatcca gcacctgggc 120

tacctcttcc tcaagatgag ccctgaggac attcgcaagt ggaatgtgac gtccctggag 180

accctgaagg ctttgcttga agtcaacaaa gggcacgaaa tgagtcctca ggtggccacc 240

ctgatcgacc gctttgtgaa gggaaggggc cagctagaca aagacaccct agacaccctg 300

accgccttct accctgggta cctgtgctcc ctcagccccg aggagctgag ctccgtgccc 360

cccagcagca tctgggcggt caggccccag gacctggaca cgtgtgaccc aaggcagctg 420

gacgtcctct atcccaaggc ccgccttgct ttccagaaca tgaacgggtc cgaatacttc 480

gtgaagatcc agtccttcct gggtggggcc cccacggagg atttgaaggc gctcagtcag 540

cagaatgtga gcatggactt ggccacgttc atgaagctgc ggacggatgc ggtgctgccg 600

ttgactgtgg ctgaggtgca gaaacttctg ggaccccacg tggagggcct gaaggcggag 660

gagcggcacc gcccggtgcg ggactggatc ctacggcagc ggcaggacga cctggacacg 720

ctggggctgg ggctacaggg cggcatcccc aacggctacc tggtcctaga cctcagcatg 780

caagaggccc tctcggggac gccctgcctc ctaggacctg gacctgttct caccgtcctg 840

gcactgctcc tagcctccac cctggcctaa 870

<210> 7

<211> 6754

<212> DNA

<213> Artificial Sequence

<400> 7

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gccgccacca tggccttgcc aacggctcga cccctgttgg ggtcctgtgg gacccccgcc 960

ctcggcagcc tcctgttcct gctcttcagc ctcggatggg tgcagccctc gaggaccggg 1020

gcccaggcgg ccgatatcca gatgacccag tccccgagct ccctgtccgc ctctgtgggc 1080

gatagggtca ccatcacctg ccgtgccagt caggatgtga atactgctgt agcctggtat 1140

caacagaaac caggaaaagc tccgaaacta ctgatttact cggcatcctt cctttattct 1200

ggagtccctt ctcgcttctc tggatctaga tctgggacgg atttcactct gaccatcagc 1260

agtctgcagc cggaagactt cgcaacttat tactgtcagc aacattatac tactcctccc 1320

acgttcggac agggtaccaa ggtggagatc aaacgcactg gctccactag cggttccggc 1380

aaacctggca gcggagaagg cagcaccaaa ggggaggttc agctggtgga gtctggcggt 1440

ggcctggtgc agccaggggg ctcactccgt ttgtcctgtg cagcttctgg cttcaacatt 1500

aaagacacct atatacactg ggtgcgtcag gccccgggta agggcctgga atgggttgca 1560

aggatttatc ctacgaatgg ttatactaga tatgccgata gcgtcaaggg ccgtttcact 1620

ataagcgcag acacatccaa aaacacagcc tacctgcaga tgaacagcct gcgtgctgag 1680

gacactgccg tctattattg ttctagatgg ggaggggacg gcttctatgc tatggacgtg 1740

tggggtcaag gaaccctggt caccgtctcc tcgggccagg ccggccagac ccagatggac 1800

cgcgtgaacg ccatcccctt cacctacgag cagctggacg tcctaaagca taaactggat 1860

gagctctacc cacaaggtta ccccgagtct gtgatccagc acctgggcta cctcttcctc 1920

aagatgagcc ctgaggacat tcgcaagtgg aatgtgacgt ccctggagac cctgaaggct 1980

ttgcttgaag tcaacaaagg gcacgaaatg agtcctcagg tggccaccct gatcgaccgc 2040

tttgtgaagg gaaggggcca gctagacaaa gacaccctag acaccctgac cgccttctac 2100

cctgggtacc tgtgctccct cagccccgag gagctgagct ccgtgccccc cagcagcatc 2160

tgggcggtca ggccccagga cctggacacg tgtgacccaa ggcagctgga cgtcctctat 2220

cccaaggccc gccttgcttt ccagaacatg aacgggtccg aatacttcgt gaagatccag 2280

tccttcctgg gtggggcccc cacggaggat ttgaaggcgc tcagtcagca gaatgtgagc 2340

atggacttgg ccacgttcat gaagctgcgg acggatgcgg tgctgccgtt gactgtggct 2400

gaggtgcaga aacttctggg accccacgtg gagggcctga aggcggagga gcggcaccgc 2460

ccggtgcggg actggatcct acggcagcgg caggacgacc tggacacgct ggggctgggg 2520

ctacagggcg gcatccccaa cggctacctg gtcctagacc tcagcatgca agaggccctc 2580

tcggggacgc cctgcctcct aggacctgga cctgttctca ccgtcctggc actgctccta 2640

gcctccaccc tggcctaata agcggccgct cgagtctaga gggcccgttt aaacccgctg 2700

atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc 2760

ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc 2820

atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa 2880

gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc 2940

tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc 3000

attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 3060

agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 3120

tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 3180

ccccaaaaaa cttgattagg gtgatggttc acgtacctag aagttcctat tccgaagttc 3240

ctattctcta gaaagtatag gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc 3300

tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga 3360

gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt 3420

aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc 3480

cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg 3540

catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc 3600

tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac 3660

gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt 3720

catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt 3780

cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga 3840

agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg 3900

cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc 3960

caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga 4020

gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg 4080

tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca 4140

gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc 4200

ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa 4260

ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac gtactacgag atttcgattc 4320

caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat 4380

gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc 4440

agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt 4500

ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat 4560

accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa 4620

ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg 4680

gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca 4740

gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 4800

tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4860

gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4920

ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4980

ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 5040

acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 5100

tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 5160

ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 5220

ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 5280

ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 5340

actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 5400

gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 5460

tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 5520

caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 5580

atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 5640

acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 5700

ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 5760

ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 5820

tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 5880

tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 5940

gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 6000

tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 6060

tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 6120

ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 6180

tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 6240

ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 6300

gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 6360

ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 6420

cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 6480

ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 6540

ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 6600

gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 6660

ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 6720

gcgcacattt ccccgaaaag tgccacctga cgtc 6754

<210> 8

<211> 9491

<212> DNA

<213> Artificial Sequence

<400> 8

actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 60

catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 120

agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa 180

atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa 240

tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac 300

gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa 360

ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct 420

aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa 480

gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc 540

gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc 600

aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 660

gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 720

cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg 780

gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg 840

ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg 900

tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag 960

gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata 1020

ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa 1080

aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag 1140

ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc 1200

cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg 1260

ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga 1320

cacctggggg gatacgggga aaaggcctcc acggccacta gtggtgtgga aagtccccag 1380

gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg 1440

gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag 1500

caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc 1560

attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg 1620

cctctgagct attccagaag tagtgaggag gcttttttgg aggctaccat ggagaagtta 1680

ctattccgaa gttcctattc tctagaaagt ataggaactt caagcttggc actggccgtc 1740

gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca 1800

catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa 1860

cagttgcgca gcctgaatgg cgaatggcgc tttgcctggt ttccggcacc agaagcggtg 1920

ccggaaagct ggctggagtg cgatcttcct gaggccgata ctgtcgtcgt cccctcaaac 1980

tggcagatgc acggttacga tgcgcccatc tacaccaacg tgacctatcc cattacggtc 2040

aatccgccgt ttgttcccac ggagaatccg acgggttgtt actcgctcac atttaatgtt 2100

gatgaaagct ggctacagga aggccagacg cgaattattt ttgatggcgt taactcggcg 2160

tttcatctgt ggtgcaacgg gcgctgggtc ggttacggcc aggacagtcg tttgccgtct 2220

gaatttgacc tgagcgcatt tttacgcgcc ggagaaaacc gcctcgcggt gatggtgctg 2280

cgctggagtg acggcagtta tctggaagat caggatatgt ggcggatgag cggcattttc 2340

cgtgacgtct cgttgctgca taaaccgact acacaaatca gcgatttcca tgttgccact 2400

cgctttaatg atgatttcag ccgcgctgta ctggaggctg aagttcagat gtgcggcgag 2460

ttgcgtgact acctacgggt aacagtttct ttatggcagg gtgaaacgca ggtcgccagc 2520

ggcaccgcgc ctttcggcgg tgaaattatc gatgagcgtg gtggttatgc cgatcgcgtc 2580

acactacgtc tgaacgtcga aaacccgaaa ctgtggagcg ccgaaatccc gaatctctat 2640

cgtgcggtgg ttgaactgca caccgccgac ggcacgctga ttgaagcaga agcctgcgat 2700

gtcggtttcc gcgaggtgcg gattgaaaat ggtctgctgc tgctgaacgg caagccgttg 2760

ctgattcgag gcgttaaccg tcacgagcat catcctctgc atggtcaggt catggatgag 2820

cagacgatgg tgcaggatat cctgctgatg aagcagaaca actttaacgc cgtgcgctgt 2880

tcgcattatc cgaaccatcc gctgtggtac acgctgtgcg accgctacgg cctgtatgtg 2940

gtggatgaag ccaatattga aacccacggc atggtgccaa tgaatcgtct gaccgatgat 3000

ccgcgctggc taccggcgat gagcgaacgc gtaacgcgaa tggtgcagcg cgatcgtaat 3060

cacccgagtg tgatcatctg gtcgctgggg aatgaatcag gccacggcgc taatcacgac 3120

gcgctgtatc gctggatcaa atctgtcgat ccttcccgcc cggtgcagta tgaaggcggc 3180

ggagccgaca ccacggccac cgatattatt tgcccgatgt acgcgcgcgt ggatgaagac 3240

cagcccttcc cggctgtgcc gaaatggtcc atcaaaaaat ggctttcgct acctggagag 3300

acgcgcccgc tgatcctttg cgaatacgcc cacgcgatgg gtaacagtct tggcggtttc 3360

gctaaatact ggcaggcgtt tcgtcagtat ccccgtttac agggcggctt cgtctgggac 3420

tgggtggatc agtcgctgat taaatatgat gaaaacggca acccgtggtc ggcttacggc 3480

ggtgattttg gcgatacgcc gaacgatcgc cagttctgta tgaacggtct ggtctttgcc 3540

gaccgcacgc cgcatccagc gctgacggaa gcaaaacacc agcagcagtt tttccagttc 3600

cgtttatccg ggcaaaccat cgaagtgacc agcgaatacc tgttccgtca tagcgataac 3660

gagctcctgc actggatggt ggcgctggat ggtaagccgc tggcaagcgg tgaagtgcct 3720

ctggatgtcg ctccacaagg taaacagttg attgaactgc ctgaactacc gcagccggag 3780

agcgccgggc aactctggct cacagtacgc gtagtgcaac cgaacgcgac cgcatggtca 3840

gaagccggcc acatcagcgc ctggcagcag tggcgtctgg cggaaaacct cagtgtgacg 3900

ctccccgccg cgtcccacgc catcccgcat ctgaccacca gcgaaatgga tttttgcatc 3960

gagctgggta ataagcgttg gcaatttaac cgccagtcag gctttctttc acagatgtgg 4020

attggcgata aaaaacaact gctgacgccg ctgcgcgatc agttcacccg tgcaccgctg 4080

gataacgaca ttggcgtaag tgaagcgacc cgcattgacc ctaacgcctg ggtcgaacgc 4140

tggaaggcgg cgggccatta ccaggccgaa gcagcgttgt tgcagtgcac ggcagataca 4200

cttgctgacg cggtgctgat tacgaccgct cacgcgtggc agcatcaggg gaaaacctta 4260

tttatcagcc ggaaaaccta ccggattgat ggtagtggtc aaatggcgat taccgttgat 4320

gttgaagtgg cgagcgatac accgcatccg gcgcggattg gcctgaactg ccagctggcg 4380

caggtagcag agcgggtaaa ctggctcgga ttagggccgc aagaaaacta tcccgaccgc 4440

cttactgccg cctgttttga ccgctgggat ctgccattgt cagacatgta taccccgtac 4500

gtcttcccga gcgaaaacgg tctgcgctgc gggacgcgcg aattgaatta tggcccacac 4560

cagtggcgcg gcgacttcca gttcaacatc agccgctaca gtcaacagca actgatggaa 4620

accagccatc gccatctgct gcacgcggaa gaaggcacat ggctgaatat cgacggtttc 4680

catatgggga ttggtggaga cgactcctgg agcccgtcag tatcggcgga attacagctg 4740

agcgccggtc gctaccatta ccagttggtc tggtgtcaaa aagcgctttc gaaagatccc 4800

aacgaaaagc gtgaccacat ggtccttctt gagtttgtaa ctgctgctgg gattacacat 4860

ggcatggatg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga 4920

gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc 4980

gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg 5040

ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg 5100

tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc 5160

gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc 5220

gtggccgagg agcaggactg acacccgagc gaaaacggtc tgcgctgcgg gacgcgcgaa 5280

ttgaattatg gcccacacca gtggcgcggc gacttccagt tcaacatcag ccgctacagt 5340

caacagcaac tgatggaaac cagccatcgc catctgctgc acgcggaaga aggcacatgg 5400

ctgaatatcg acggtttcca tatggggatt ggtggcgacg actcctggag cccgtcagta 5460

tcggcggaat tccagctgag cgccggtcgc taccattacc agttggtctg gtgtcaaaaa 5520

taataataac cgggcagggg ggatctgcat ggatctttgt gaaggaacct tacttctgtg 5580

gtgtgacata attggacaaa ctacctacag agatttaaag ctctaaggta aatataaaat 5640

ttttaagtgt ataatgtgtt aaactactga ttctaattgt ttgtgtattt tagattccaa 5700

cctatggaac tgatgaatgg gagcagtggt ggaatgcctt taatgaggaa aacctgtttt 5760

gctcagaaga aatgccatct agtgatgatg aggctactgc tgactctcaa cattctactc 5820

ctccaaaaaa gaagagaaag gtagaagacc ccaaggactt tccttcagaa ttgctaagtt 5880

ttttgagtca tgctgtgttt agtaatagaa ctcttgcttg ctttgctatt tacaccacaa 5940

aggaaaaagc tgcactgcta tacaagaaaa ttatggaaaa atattctgta acctttataa 6000

gtaggcataa cagttataat cataacatac tgttttttct tactccacac aggcatagag 6060

tgtctgctat taataactat gctcaaaaat tgtgtacctt tagcttttta atttgtaaag 6120

gggttaataa ggaatatttg atgtatagtg ccttgactag agatcataat cagccatacc 6180

acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa 6240

cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa 6300

taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt 6360

ggtttgtcca aactcatcaa tgtatcttat catgtctgga tcgctagcga attcgaattt 6420

aaatcggatc cgcggccgct tttccccgta tccccccagg tgtctgcagg ctcaaagagc 6480

agcgagaagc gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc 6540

cgcacgctgc cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg 6600

ctcgctgctg ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg 6660

ggctgtccct gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga 6720

acatgaaata acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa 6780

tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat 6840

tgacaagcac gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga 6900

cggattcgcg ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac 6960

gcagactatc tttctagggt taatctagct gcatcaggat catatcgtcg ggtctttttt 7020

ccggctcagt catcgcccaa gctggcgcta tctgggcatc ggggaggaag aagcccgtgc 7080

cttttcccgc gaggttgaag cggcatggaa agagtttgcc gaggatgact gctgctgcat 7140

tgacgttgag cgaaaacgca cgtttaccat gatgattcgg gaaggtgtgg ccatgcacgc 7200

ctttaacggt gaactgttcg ttcaggccac ctgggatacc agttcgtcgc ggcttttccg 7260

gacacagttc cggatggtca gcccgaagcg catcagcaac ccgaacaata ccggcgacag 7320

ccggaactgc cgtgccggtg tgcagattaa tgacagcggt gcggcgctgg gatattacgt 7380

cagcgaggac gggtatcctg gctggatgcc gcagaaatgg acatggatac cccgtgagtt 7440

acccggcggg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 7500

ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 7560

taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 7620

aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 7680

attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 7740

cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 7800

gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 7860

ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 7920

agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 7980

tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 8040

ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 8100

gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 8160

ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 8220

gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 8280

aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 8340

aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 8400

ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 8460

gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 8520

gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 8580

tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 8640

ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 8700

ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 8760

atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 8820

ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 8880

tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 8940

attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 9000

tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 9060

ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 9120

gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 9180

gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 9240

gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 9300

aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 9360

taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 9420

tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 9480

tgaatactca t 9491

<210> 9

<211> 699

<212> DNA

<213> Artificial Sequence

<400> 9

atggtgagca aaggcgaggc cgtgatcaag gagttcatga ggttcaaggt gcacatggag 60

ggcagcatga acggccacga gttcgagatc gagggcgagg gcgaaggcag accctatgag 120

ggcacccaga ccgccaagct gaaggtgaca aagggcggcc ctctgccctt cagctgggat 180

attctgtccc cccagttcat gtacggcagc agagccttca ccaagcaccc cgccgacatc 240

cccgactact ataagcagag cttccccgag ggctttaagt gggagagggt gatgaacttc 300

gaggatggag gcgccgtgac cgtgacccaa gacacatctc tggaggacgg cacactgatc 360

tacaaggtga agctgagggg caccaacttt cctcccgacg gccccgtgat gcagaagaag 420

accatgggct gggaggcttc caccgagaga ctgtaccccg aggacggcgt gctgaagggc 480

gacattaaga tggctctgag actgaaggac ggcggaagat acctcgccga cttcaagacc 540

acctacaagg ccaagaagcc cgtgcagatg cccggcgcct acaacgtgga tagaaagctg 600

gacatcacat cccacaacga ggattacacc gtcgtggagc agtacgagag atccgagggc 660

agacactcca ccggcggcat ggatgaactg tacaagtga 699

<210> 10

<211> 714

<212> DNA

<213> Artificial Sequence

<400> 10

atggtgagca aaggcgagga gctgatcaag gagaacatgc acatgaagct gtacatggag 60

ggcaccgtcg acaaccacca cttcaagtgc acaagcgagg gcgagggcaa gccctacgaa 120

ggcacccaga ccatgagaat caaggtggtg gagggaggcc ctctgccttt cgctttcgac 180

attctggcca caagctttct gtacggcagc aagaccttca tcaaccacac acaaggcatc 240

cccgacttct ttaagcagtc cttccccgag ggcttcacat gggagagggt gaccacctat 300

gaggatggcg gcgtgctgac cgccacacaa gacacctctc tgcaagacgg ctgtctgatc 360

tacaacgtga agattagagg cgtgaacttc accagcaacg gacccgtgat gcagaagaag 420

acactgggct gggaggcctt caccgagaca ctgtaccccg ccgacggagg actggaagga 480

agaaatgaca tggccctcaa gctggtgggc ggcagccatc tgatcgccaa cgccaagacc 540

acctatagat ccaagaagcc cgccaagaat ctgaagatgc ccggcgtgta ctacgtggac 600

tatagactgg agagaatcaa ggaggccaac aacgagacct acgtggagca gcatgaggtg 660

gccgtggcca gatactgcga tctgccctcc aagctgggcc ataagctgaa ttga 714

<210> 11

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 11

catggcggac tacaaagaca wtgttctcac ccagtc 36

<210> 12

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 12

catggcggac tacaaagaca tccagatgac acagwc 36

<210> 13

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 13

catggcggac tacaaagatr ttgtgatgac ccagwc 36

<210> 14

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 14

catggcggac tacaaagaca ttstgmtgac ccagtc 36

<210> 15

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 15

catggcggac tacaaagatg ttgtgvtgac ccaaac 36

<210> 16

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 16

catggcggac tacaaagaca caactgtgac ccagtc 36

<210> 17

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 17

catggcggac tacaaagaya ttktgctcac tcagtc 36

<210> 18

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 18

catggcggac tacaaagata ttgtgatrac ccaggm 36

<210> 19

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 19

catggcggac tacaaagaca ttgtaatgac ccaatc 36

<210> 20

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 20

catggcggac tacaaagaca ttgtgatgwc acagtc 36

<210> 21

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 21

catggcggac tacaaagatr tccagatgam ccagtc 36

<210> 22

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 22

catggcggac tacaaagatg gagaaacaac acaggc 36

<210> 23

<211> 65

<212> DNA

<213> Artificial Sequence

<400> 23

ggagccgccg ccgccagaac caccaccacc agaaccacca ccaccgcgtt tbatttccag 60

cttgg 65

<210> 24

<211> 64

<212> DNA

<213> Artificial Sequence

<400> 24

ggagccgccg ccgccagaac caccaccaca gaaccaccac caccgcgttt tatttccaat 60

tttg 64

<210> 25

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 25

ggcggcggcg gctccggtgg tggtggatcc gaggttcdsc tgcaacagty 50

<210> 26

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 26

ggcggcggcg gctccggtgg tggtggatcc caggtgcaam tgmagsagtc 50

<210> 27

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 27

ggcggcggcg gctccggtgg tggtggatcc gavgtgmwgc tggtggagtc 50

<210> 28

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 28

ggcggcggcg gctccggtgg tggtggatcc gavgtgmwgc tggtggagtc 50

<210> 29

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 29

ggcggcggcg gctccggtgg tggtggatcc gakgtgcagc ttcagsagtc 50

<210> 30

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 30

ggcggcggcg gctccggtgg tggtggatcc cagatccagt tsgygcagtc 50

<210> 31

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 31

ggcggcggcg gctccggtgg tggtggatcc cagrtccaac tgcagcagyc 50

<210> 32

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 32

ggcggcggcg gctccggtgg tggtggatcc gaggtgmagc tasttgagwc 50

<210> 33

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 33

ggcggcggcg gctccggtgg tggtggatcc gaagtgaagm ttgaggagtc 50

<210> 34

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 34

ggcggcggcg gctccggtgg tggtggatcc gatgtgaacc tggaagtgtc 50

<210> 35

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 35

ggcggcggcg gctccggtgg tggtggatcc cagatkcagc ttmaggagtc 50

<210> 36

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 36

ggcggcggcg gctccggtgg tggtggatcc caggcttatc tgcagcagtc 50

<210> 37

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 37

ggcggcggcg gctccggtgg tggtggatcc caggttcacc tacaacagtc 50

<210> 38

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 38

ggcggcggcg gctccggtgg tggtggatcc caggtgcagc ttgtagagac 50

<210> 39

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 39

ggcggcggcg gctccggtgg tggtggatcc gargtgmagc tgktggagac 50

<210> 40

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 40

cggagtcagg cccccgaggc cgaggagacg gtgacmgtgg 40

<210> 41

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 41

cggagtcagg cccccgaggc cgcagagaca gtgaccagag 40

<210> 42

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 42

cggagtcagg cccccgaggc cgaggagact gtgagastgg 40

<210> 43

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 43

ctacagcagg cccaggcggc catggcggac tacaaa 36

<210> 44

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 44

cggagtcagg cccccgag 18

Claims (10)

1. The application of mesothelin in displaying of mammal fusion protein is characterized in that the mesothelin and protein to be screened are subjected to fusion expression in mammal cells.

2. A fusion protein for mammalian cell display, comprising a signal peptide, the protein to be screened of claim 1, and mesothelin, all linked in sequence.

3. The fusion protein of claim 2, wherein the mesothelin has an amino acid sequence as set forth in SEQ ID No. 3.

4. The fusion protein of claim 2, wherein the signal peptide has the amino acid sequence shown in SEQ ID No. 1.

5. A fusion gene encoding the fusion protein of any one of claims 2 to 4, comprising a gene encoding a signal peptide, a gene encoding a protein to be screened, and a gene encoding mesothelin, which are linked in this order.

6. The fusion gene of claim 5, wherein the nucleotide sequence of the gene encoding the signal peptide is shown in SEQ ID No.4, and the nucleotide sequence of the gene encoding mesothelin is shown in SEQ ID No. 6.

7. A mesothelin-anchored-based mammalian fusion protein display plasmid comprising the fusion gene of claim 6 and a starting plasmid.

8. The mammalian fusion protein display plasmid of claim 7, wherein the initial plasmid is a pcDNA-FRT plasmid and the fusion gene is recombined between the NheI and NotI cleavage sites of the pcDNA-FRT plasmid.

9. A mesothelin-anchorage-based mammalian fusion protein-displaying cell line obtained by transferring the mammalian fusion protein-displaying plasmid according to claim 7 or 8 into mammalian antibody-displaying cells.

10. Use of the fusion protein of any one of claims 2 to 4, the fusion gene of claim 5 or 6, the mammalian fusion protein display plasmid of claim 7 or 8, or the mammalian fusion protein display cell line of claim 9 for drug screening.

Images