ANTIBODY LIBRARY CONSTRUCTION METHOD AND APPLICATION THEREOF

Abstract

Disclosed by the present application are an antibody library construction method and an application thereof. The method comprises the following steps: inserting a first element and a second element into a same vector or different vectors, and transfecting the vectors into the cells to obtain an antibody expression cell library, i.e., the antibody library. The first element comprises CIS activators and selection marker genes; the second element comprises extracellular antibody library coding domain, Notch nuclear structure domain and intracellular transcription structure domain.

Claims

1. An antibody library construction method, comprising: inserting a first element and a second element into a same vector or different vectors, and transfecting the vector(s) into a cell to obtain an antibody expression cell library, i.e., the antibody library; wherein the first element comprises a CIS activator and a screening marker gene, and the second element comprises an extracellular antibody library coding domain, a Notch core domain and an intracellular transcription domain.

2. The method according to claim 1, wherein the screening marker gene comprises any one or a combination of at least two of a drug resistance gene, a suicide gene, a fluorescent protein gene, and a molecular tag.

3. The method according to claim 2, wherein the drug resistance gene comprises any one or a combination of at least two of a puromycin resistance gene, a neomycin resistance gene, a blasticidin resistance gene, and a hygromycin B resistance gene; the suicide gene comprises any one or a combination of at least two of a herpes simplex virus thymidine kinase gene, a cytosine deaminase gene, and an iCasp9 suicide system gene; the fluorescent protein gene comprises any one or a combination of at least two of EGFP, YFP, mCherry, DsRed, and BFP; and the molecular tag comprises any one or a combination of at least two of His-tag, Flag-tag, HA-tag, Myc-tag, and Strep-tag.

4. The method according to claim 1, wherein a screening method for the screening marker gene comprises any one or a combination of at least two of drug screening, flow cytometry detection and sorting, and magnetic-activated cell sorting.

5. The method according to claim 3, wherein a screening drug for the drug resistance gene comprises any one or a combination of at least two of puromycin, G418, blasticidin, and hygromycin B; and a screening drug for the suicide gene comprises any one or a combination of at least two of ganciclovir or FIAU, 5-fluorocytosine, AP1903, and AP20187.

6. The method according to claim 1, wherein the extracellular antibody library coding domain comprises any one or a combination of at least two of an antibody sequence, an antibody heavy chain sequence, an antibody light chain sequence, an antibody variable region sequence, a single chain antibody sequence, a single domain antibody sequence, and a Fab fragment sequence.

7. The method according to claim 1, wherein a source of the extracellular antibody library coding domain comprises any one or a combination of at least two of an immunized animal, a diseased human population, a healthy human population, a vaccinated human population, and artificial synthesis.

8. The method according to claim 1, wherein the Notch core domain comprises a human Notch, a mouse Notch, or a sequence having a similarity of not less than 85% to the human Notch or the mouse Notch; and wherein the human Notch has an amino acid sequence as shown in SEQ ID NO.1; and the mouse Notch has an amino acid sequence as shown in SEQ ID NO.2.

9. The method according to claim 1, wherein the CIS activator comprises pTet and/or UAS-pSV40.

10. The method according to claim 1, wherein the intracellular transcription domain comprises tTA and/or Ga14-VP64.

11. The method according to claim 1, wherein a method for transfecting comprises any one or a combination of at least two of viral transfection, transfection with a chemical transfection reagent, and electroporation transfection.

12. The method according to claim 1, wherein the coding domain, the domain or the gene contains any one or a combination of at least two of an amino acid sequence encoding a protein, a DNA sequence encoding a protein, and an RNA sequence encoding a protein.

13. An antibody library constructed by using the method according to claim 1.

14. A method for screening antibodies by using the antibody library according to claim 13, comprising the following steps: (1) contacting the antibody library with an antigen; and (2) screening a cell expressing a target antibody according to expression of the screening marker gene; wherein the antibody comprises a monoclonal antibody or a polyclonal antibody; and the antigen comprises any one or a combination of at least two of a wild cell, a cell transfected with a specific antigen gene, a cell bound to a specific antigen, an antigen dissolved in a culture medium, an antigen coated on a culture vessel, an antigen coated on a microsphere, and an antigen coated on a culture scaffold.

15. An antibody screened by using the method according to claim 14.

16. A system for screening antibodies, comprising: a first element, comprising a CIS activator and a screening marker gene; and a second element, comprising a gene for encoding an extracellular antibody library coding domain, a gene for encoding a Notch core domain and a gene for encoding an intracellular transcription domain.

17. A method for screening antibodies by using the system according to claim 16, comprising: transfecting a first element and a second element into a cell, wherein the first element comprises a CIS activator and a screening marker gene, and the second element comprises a gene for encoding an extracellular antibody library coding domain, a gene for encoding a Notch core domain and a gene for encoding an intracellular transcription domain; allowing the cell to express the extracellular antibody library coding domain; contacting the cell with an antigen; and screening a cell expressing a target antibody according to expression of the screening marker gene to screen the target antibody.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0073] FIG. 1 is a schematic diagram showing a principle that a synNotch system controls the gene expression in cells according to the present application;

[0074] FIG. 2 shows a synNotch-based antibody screening system according to the present application;

[0075] FIG. 3 is a schematic diagram showing a vector containing a CIS activator and a screening marker gene according to the present application;

[0076] FIG. 4 is a schematic diagram showing a vector containing a fluorescent protein fused with a puromycin resistance gene under the regulation of a CIS activator pTet according to the present application;

[0077] FIG. 5 is a schematic diagram showing a tTA expression vector according to the present application;

[0078] FIG. 6 is a diagram indicating that tTA activates a CIS activator pTet to express a green fluorescent protein, as shot by a fluorescence microscope according to the present application;

[0079] FIG. 7 is a diagram indicating that tTA activates a CIS activator pTet to express a red fluorescent protein, as shot by a fluorescence microscope according to the present application;

[0080] FIG. 8 is a schematic diagram showing a universal expression vector of an antibody library according to the present application;

[0081] FIG. 9 is a schematic diagram showing an expression vector of an anti-CD19 single chain antibody according to the present application;

[0082] FIG. 10 is a schematic diagram showing an expression vector of an anti-GPC3 single chain antibody according to the present application;

[0083] FIG. 11 is a schematic diagram showing an expression vector of a CD19 or GFPC3 antigen according to the present application;

[0084] FIG. 12 is a diagram showing expression of the CD19 and GFPC3 antigens in K562 cells, as detected through a flow cytometry according to the present application;

[0085] FIG. 13 is a graph showing results of screening CD19 antibody expression cells from a mixed antibody expression cell library by using the antibody screening system according to the present application;

[0086] FIG. 14 is a graph showing results of screening GPC3 antibody expression cells from a mixed antibody expression cell library by using the antibody screening system according to the present application;

[0087] FIG. 15 is a schematic diagram showing a vector containing a green fluorescent protein under the regulation of a CIS activator pTet according to the present application;

[0088] FIG. 16 is a schematic diagram showing an anti-Raji single chain antibody gene library expression vector according to the present application;

[0089] FIG. 17 is a schematic diagram showing an antigen expression vector used for negative screening according to the present application;

[0090] FIG. 18 is a graph showing results of expression of the CD19 antigen expression vector and the antigen expression vector used for negative screening in K562 cells, as detected by a flow cytometry according to the present application;

[0091] FIG. 19 is a graph showing induction and activation of an antibody expression cell library before screening, as detected through a flow cytometry according to the present application;

[0092] FIG. 20 is a graph showing induction and activation of an antibody expression cell library after screening, as detected through a flow cytometry according to the present application;

[0093] FIG. 21 is a schematic diagram illustrating a vector containing a fluorescent protein fused with a positive/negative screening gene under the regulation of a CIS activator pTet according to the present application;

[0094] FIG. 22 is a schematic diagram showing a single chain antibody gene library expression vector for primary drug screening according to the present application;

[0095] FIG. 23 is a schematic diagram showing a principle of drug positive screening by using an antibody screening system according to the present application;

[0096] FIG. 24 is a graph showing results of positively screening of antibody expression cells, as detected through a flow cytometry according to the present application;

[0097] FIG. 25 is a schematic diagram showing a principle of drug negative screening by using an antibody screening system according to the present application;

[0098] FIG. 26 is a graph showing results of negatively screening of antibody expression cells, as detected through a flow cytometry according to the present application;

[0099] FIG. 27 is a schematic diagram showing an expression vector of a single chain antibody fused with an antibody constant region according to the present application; and

[0100] FIG. 28 is a graph showing results of binding efficiency between antibodies screened by the antibody screening method of the present application and antigens, as detected through a flow cytometry according to the present application.

DETAILED DESCRIPTION

[0101] To further elaborate on the technical means adopted and the effects achieved in the present application, the solutions of the present application are further described below through specific examples in conjunction with drawings, but the present application is not limited to the scope of the examples.

Example 1 Screening of Known CD19 and GPC3 Antibodies

[0102] FIG. 1 shows a principle that a synNotch system controls the gene expression in cells. When the extracellular single chain antibody of the synNotch system recognizes and binds to an antigen, the synNotch system undergoes the inducible transmembrane region shearing, thereby releasing the intracellular transcription domain into the cell nucleus, and the intracellular transcription domain binds to the upstream CIS activator to activate the expression of a regulated target gene.

[0103] The principle of a synNotch-based antibody screening system is shown in FIG. 2. Based on the synNotch system, the extracellular recognition domain is changed into an extracellular antibody library coding domain, and the regulated target gene is changed into a screening marker gene, so that an antigen-activated antibody screening system is obtained.

[0104] Antibodies specific for CD19 and GPC3 were screened from a known mixed antibody library of CD19 and GPC3, respectively.

[0105] (1) Construction of a Monoclonal Cell Line Stably Transfected with the CIS Activator and the Screening Marker Gene

[0106] In this example, pTet (SEQ ID NO.3) was used as the CIS activator. The pTet initiated the expression of the screening gene after receiving a tTA signal. A fluorescent protein fused with a puromycin resistance gene (SEQ ID NO.4) was used as the screening marker, and the fluorescent protein and the puromycin resistance gene was linked by a 2A (SEQ ID NO.5) sequence that could be automatically broken. The vector containing the CIS activator and the screening marker gene is schematically shown in FIG. 3.

[0107] In this example, two fluorescent proteins, i.e., EGFP (SEQ ID NO.6) and mCherry (SEQ ID NO.7), were used to fuse with the puromycin resistance gene to construct two screening markers, respectively. SV40 polyplex A (SEQ ID NO.8) was used as the terminator. The coding genes of entire sequences (FIG. 4) were directly artificially synthesized, and then constructed into a lentiviral vector, packaged into lentivirus, and then transfected into 293T cells, respectively. 3 days later, the transfected cells were monoclonalized.

[0108] To screen the monoclonal cell line to obtain correct monoclonal cells, a tTA expression vector (FIG. 5) needed to be constructed. The tTA expression vector consisted of an ef1-α promoter (SEQ ID NO.9), a tTA gene (SEQ ID NO.10) and a terminator. The tTA expression vector was constructed into a lentiviral vector and packaged into a lentivirus.

[0109] A portion of cells was got from monoclonal cells to be screened and then transfected with the tTA expression lentivirus. 2-3 days later, the cells were observed by a fluorescence microscope. Cell lines that expressed EGFP (FIG. 6, observed under the microscope for green fluorescent expression when monoclonal cells stably transfected with the green fluorescent protein fused with the puromycin resistance gene under the regulation of the CIS activator pTet) or mCherry (FIG. 7, observed under the microscope for red fluorescent expression when monoclonal cells stably transfected with the red fluorescent protein fused with the puromycin resistance gene under the regulation of the pTet CIS activator were screened), but had no fluorescent expression before transfection, were monoclonal cell lines that had been stably transfected with the pTet CIS activator and the screening marker gene.

[0110] (2) Construction of an antibody expression cell library: First, an intermediate vector including all components in the antibody gene library expression vector, other than the extracellular antibody library coding domain gene, was constructed to facilitate the subsequent insertion of the extracellular antibody library coding domain gene (FIG. 8, a schematic diagram showing a universal expression vector of an antibody library). Sequences of each component of the intermediate vector were synthesized. From the N terminus to C terminus, the components of the vector in series were an mPGK1 promoter (SEQ ID NO.11), a CD8α signal peptide (SEQ ID NO.12) coding sequence, an antibody gene library coding domain (a coding region sequence with a β-galactosidase (lacZ) N-terminus α fragment which was behind an Asc I restriction endonuclease site and was in front of a Not I restriction endonuclease site, for facilitating insertion of antibody library sequences and for displaying fragment insertion through blue-white spots), a NOTCH1 fragment (P1390-R1752 fragments containing a transmembrane region in SEQ ID NO.2) coding sequence, a tTA coding sequence, and a terminator. The synthesized sequences were constructed into a lentiviral vector for further insertion of the antibody gene sequence.

[0111] The coding sequences of the known anti-human CD19 single chain antibody (SEQ ID NO.13) and anti-human GPC3 single chain antibody (SEQ ID NO.14) with Asc I and Not I restriction endonuclease sites at both terminuses were synthesized and inserted between the Asc I and Not I restriction endonuclease sites of the intermediate vector, respectively. The vector inserted with the anti-human CD19 single chain antibody gene (FIG. 9) was packaged into a lentivirus and transfected into a monoclonal cell line with the EGFP screening marker to yield CD19 single chain antibody expression cells.

[0112] The vector inserted with the anti-human GPC3 single chain antibody gene (FIG. 10) was packaged into a lentivirus and transfected into a monoclonal cell line with the mCherry screening marker to yield GPC3 single chain antibody expression cells.

[0113] (3) Construction of CD19 and GFPC3 antigen expression cells: The coding sequences of CD19 (SEQ ID NO.15) and GPC3 (SEQ ID NO.16) antigens were synthesized and constructed into expression vectors respectively. In the expression vector, CD19 or GPC3, a blue fluorescent protein (SEQ ID NO.17), and a puromycin resistance gene were fused through two 2A sequences (FIG. 11).

[0114] The constructed expression vector was packaged into a lentivirus and transfected into K562 cells through virus. After 2 days of transfection, K562 cells expressing CD19 or GPC3 stably were screened by adding 1 μg/mL purinomycin into the culture medium. The expression of the blue fluorescent protein detected by the flow cytometry almost reached 100% (FIG. 12), indicating that the antigen had been stably expressed in K562 cells.

[0115] (4) Screening of CD19 antibody expression cells from the mixed antibody expression cell library: CD19 single chain antibody expression cells and GPC3 single chain antibody expression cells were mixed in a ratio of 1:1. The mixed cells were mixed with CD19 antigen expression cells in a ratio of 1:1. After the mixed cells were cultured for 2 days, 1 μg/mL puromycin was added to the culture medium for screening. After 8-10 days of screening, the antibody expression cells were almost cells with green fluorescence expressing the CD19 antibody (FIG. 13). When cells expressing both the CD19 single chain antibody and green fluorescence were mixed with cells expressing both the GPC3 single chain antibody and red fluorescence in a ratio of 1:1, and screened by mixing with CD19 antigen expression cells and adding puromycin, almost all of the cells obtained were cells expressing the CD19 antibody with green fluorescence.

[0116] (5) Screening of cells expressing the anti-GPC3 antibody from the mixed cells: CD19 antibody expression cells and GPC3 antibody expression cells were mixed in a ratio of 1:1. The mixed cells were mixed with GPC3 antigen expression cells in a ratio of 1:1. After the mixed cells were cultured for 2 days, 1 μg/mL of puromycin was added to the culture medium for screening. After 8-10 days of screening, the antibody expression cells were almost cells with red fluorescence expressing the GPC3 antibody (FIG. 14). When cells expressing both the CD19 single chain antibody and green fluorescence were mixed with cells expressing both the GPC3 single chain antibody and red fluorescence in a ratio of 1:1, and screened by mixing with GPC3 antigen expression cells and adding puromycin, almost all of the cells obtained were cells expressing the GPC3 antibody with red fluorescence.

[0117] These results of the above experiment indicate that a single chain antibody against a target antigen can be screened from a known antibody library according to this antibody screening method.

Example 2 Preparation of the Antibody Library and Screening of CD19 Antibody Expression Cells Through the Flow Cytometry

[0118] (1) Construction of a Monoclonal Cell Line Stably Transfected with the CIS Activator and the Screening Marker Gene

[0119] In this example, the CIS activator pTet was used. The pTet initiated the expression of the screening marker green fluorescent protein gene after receiving a tTA signal. An entire sequence (FIG. 15) was artificially synthesized and constructed into a lentiviral vector. Lentivirus was packaged and then transfected into 293T cells. 3 days later, the transfected cells were monoclonalized. A portion of cells was got from monoclonal cells to be screened and then transfected with lentivirus expressing tTA. 2-3 days later, the cells were observed through a fluorescence microscope. A cell line with green fluorescent expression, which had no fluorescent expression before transfection, was selected as the monoclonal cell line stably transfected with the CIS activator and the screening marker gene.

[0120] (2) Construction of an Antibody Expression Cell Library

[0121] C57BL/6J mice were immunized with CD19-positive Raji cells. The booster immunization was performed once two weeks later and once at the fourth week. The mice were sacrificed three days later. Splenic lymphocytes of the mice were isolated. RNA was extracted from splenic lymphocytes by using an RNA extraction purification kit.

[0122] Reverse transcription was performed on the extracted RNA by using a reverse transcription primer of a light chain (SEQ ID NO.18) and a reverse transcription primer of a heavy chain (SEQ ID NO.19) with a reverse transcription kit, respectively. The heavy and light chains were amplified with degenerate primers, respectively. Then the light and heavy chains were linked by overlapping PCR to form a light chain-linker-heavy chain scFv library. DNA fragments of the scFv library were digested by both Asc I and Not I and then were inserted between the Asc I and Not I restriction endonuclease sites of the intermediate vector of the antibody gene library expression vector to yield the antibody gene library expression vector (FIG. 16). Single chain antibodies prepared from Raji cell-immunized mice were cloned into the antibody gene library expression vector.

[0123] The antibody gene library expression vector was packaged into a lentivirus and transfected into the monoclonal cell line stably transfected with the CIS activator and the screening marker gene obtained in the step (1), to obtain the antibody expression cell library.

[0124] (3) Construction of Antigen Expression Cells

[0125] The preparation of the antigen expression cells expressing CD19 was referred to Example 1. This antigen expression cell was used for positive screening of anti-CD19 antibodies. A coding gene of a transmembrane domain (SEQ ID NO.20) was synthesized and was used to replace the CD19 antigen gene in the positive screening vector to obtain an antigen expression vector for negative screening (FIG. 17).

[0126] The constructed antigen expression vectors were packaged into lentiviruses respectively and transfected into K562 cells through viruses. After 2 days of transfection, a positive screening antigen expression cell line and a negative screening antigen expression cell line were screened by adding 1 μg/mL purinomycin into the culture medium. The expression of the blue fluorescent protein detected through the flow cytometry almost reached 100% (FIG. 18), indicating that the antigen had been stably expressed in K562 cells.

[0127] (4) Screening of Cells Expressing the CD19 Antibody Through the Flow Cytometry

[0128] The antibody expression cell library was mixed with the positive-screening cells expressing the CD19 antigen in a ratio of 1:1. After the mixed cells were cultured for 2 days, a small number of cells expressing the green fluorescent protein appeared in the antibody expression cell library (FIG. 19). After the antibody expression cell library and the positive-screening cells expressing the CD19 antigen were mixed and then cultured for 2 days, a small number of cells expressing the green fluorescent protein appeared in the antibody expression cell library. The cells expressing the green fluorescent protein were sorted through a sorting flow cytometry and continued to be cultured until most of the green fluorescence disappeared.

[0129] The sorted antibody expression cell library was mixed with the negative-screening antigen expression cells in a ratio of 1:1. 3 days later, cells that were negative to both the green fluorescent protein and the blue fluorescent protein were sorted through the sorting flow cytometry, i.e., cells expressing the CD19 antibody. The antibody expression cells thus obtained, when co-cultured with the negative-screening antigen expression cells, expressed no green fluorescent protein, and, when co-cultured with the CD19 antigen expression cells, expressed the green fluorescent protein (FIG. 20). The antibody expression cells obtained after the positive-negative screening, when co-cultured with the negative-screening antigen expression cells, expressed no green fluorescent protein, and, when co-cultured with the CD19 antigen expression cells, expressed the green fluorescent protein.

Example 3 Preparation of the Antibody Library and Screening of CD19 Antibody Expression Cells Using Drugs

[0130] (1) Construction of a Monoclonal Cell Line Stably Transfected with the CIS Activator and the Screening Marker Gene

[0131] In this example, pTet was used as the CIS activator. An iCasp9 negative screening system (SEQ ID NO.21), a green fluorescent protein and a puromycin resistance gene were used as a screening marker, in which the iCasp9 negative screening system, the green fluorescent protein and the puromycin resistance gene were linked by 2A sequences that could be automatically broken.

[0132] An entire sequence (FIG. 21) was artificially synthesized, constructed into a lentiviral vector, packaged into lentivirus and then transfected into 293T cells. 3 days later, the transfected cells were monoclonalized. A portion of cells was got from monoclonal cells to be screened and then transfected with the lentivirus expressing tTA in Example 1. 2-3 days later, the cells were observed through a fluorescence microscope. A cell line with green fluorescent expression, which had no fluorescent expression before transfection, was selected as the monoclonal cell line stably transfected with the CIS activator and the screening marker gene.

[0133] (2) Construction of an Antibody Expression Cell Library

[0134] First, an intermediate vector including all components in the antibody gene library expression vector, other than the extracellular antibody library coding domain gene, was constructed to facilitate the subsequent insertion of the extracellular antibody library coding domain gene (FIG. 22). Sequences of each component of the intermediate vector were synthesized. From the N terminus to C terminus, the components of the vector in series were an mPGK1 promoter, a CD8α signal peptide coding sequence, an antibody gene library coding domain (a coding region sequence with a β-galactosidase (lacZ) N-terminus α fragment which was behind an Asc I restriction endonuclease site and was in front of a Not I restriction endonuclease site, for facilitating insertion of antibody library sequences and for displaying fragment insertion through blue-white spots), a NOTCH1 fragment coding sequence, a tTA coding sequence, a terminator, an EFS promoter (SEQ ID NO.22), a blasticidin resistance (SEQ ID NO.23) gene, and a terminator.

[0135] The synthesized sequences were constructed into a lentiviral vector for further insertion of the antibody gene library sequence. DNA fragments of the scFv library of the Raji-immunized mice were digested by both Asc I and Not I and then were inserted between the Asc I and Not I restriction endonuclease sites of the intermediate vector of the antibody gene library expression vector to yield the antibody gene library expression vector. The antibody gene library expression vector was packaged into a lentivirus and then transfected to the monoclonal cell line stably transfected with the CIS activator and the screening marker gene. 2 days later, 10 m/mL blasticidin was added to the culture medium for primary screening to obtain the antibody expression cell library.

[0136] (3) Screening of Cells Expressing the CD19 Antibody Using Drugs

[0137] The antibody expression cell library and positive-screening cells expressing the CD19 antigen were mixed in a ratio of 1:1. After the mixed cells were cultured for 2 days, 1 μg/mL puromycin was added to the culture medium for screening. After 6-8 days of positive screening (the principle of positive screening was shown in FIG. 23), almost all of the antibody library expression cells were cells expressing the green fluorescence protein (FIG. 24). When the mixed antibody library expression cells and positive-screening cells expressing the CD19 antigen were subjected to positive screening through puromycin, almost all of the antibody expression cells were cells expressing the green fluorescence protein.

[0138] 10 μg/mL blasticidin was added to the culture medium of the mixed cells until blue fluorescent positive-screening antigen cells completely disappeared. The antibody library expression cells subjected to positive screening were continued to be cultured until the green fluorescent protein was almost not expressed. Then the antibody library expression cells and negative-screening antigen expression cells were mixed in a ratio of 1:1. 10 nM of AP1903 was added to the culture medium for negative screening (the principle of negative screening was shown in FIG. 25) until the green fluorescent protein was not expressed in the cultured antibody expression cells (FIG. 26). When the mixed antibody library expression cells and negative-screening cells were subjected to AP1903 negative selection, almost all of the antibody expression cells were cells that did not express green fluorescent protein.

[0139] Finally, 10 μg/mL blasticidin was added to the culture medium of the mixed cells until blue fluorescent negative-screening antigen cells completely disappeared. In this case, the antibody library expression cells were cells expressing the CD19 antibody.

Example 4 Characterization of Antibody Binding

[0140] (1) Cloning of Antibody Coding Sequences of Screened CD19 Antibody Expression Cells

[0141] Genomes of the screened CD19 antibody expression cells were extracted by using the DNeasy Blood & Tissue Kit (manufactured by Qiagen) according to the instructions of the kit. With the extracted genomes as a template, the coding gene of scFv was amplified by using a primer pair (SEQ ID NO.24, SEQ ID NO.25). The amplified product was digested by both Asc I and Not I, then inserted into an expression vector digested by both Asc I and Not I, and fused with an IgG1 constant region (SEQ ID NO.26) of the human (FIG. 27).

[0142] The linked vector was transfected into competent cells. The competent cells were coated on a petri dish containing an LB solid medium with 100 mg/L ampicillin. In the next day, about 20 clones were picked out from the petri dish, then inoculated in an LB liquid medium containing 100 mg/L ampicillin respectively and cultured in shaking flasks. The remaining colonies on the petri dish were eluted with the LB culture medium. Polyclonal scFv expression plasmids were extracted by using a plasmid extraction kit according to the instructions of the kit. Monoclonal scFv expression plasmids were also extracted after the monoclonal bacteria in shake flasks were cultured overnight, respectively.

[0143] (2) Expression and Purification of the Antibody

[0144] The polyclonal scFv expression plasmids and the monoclonal scFv expression plasmids were transfected into 293T cells by using PEI respectively. The culture supernatant was collected after 72 hours of transfection. The culture supernatant was mixed with binding buffer in a ratio of 1:1, and then filtered for later use. The Pteoein A column was equilibrated with 5-10 volumes of binding buffer, and the prepared culture supernatant samples were loaded. The column was rinsed with the binding buffer until the binding buffer did not contain the protein. Finally, the eluate was passed through the column, and the eluate was collected until the eluate did not contain the protein. The collected antibody was dialyzed and concentrated.

[0145] (3) Detection of Binding of Antibodies by Using the Flow Cytometry

[0146] 100 μL of suspension of K562 cells expressing CD19 and not expressing CD19 were plated in V cell plates, centrifuged at 500 g for 3 min to precipitate cells. Then the supernatant was discarded. 100 μL of purified antibody solution (IgG concentration was controlled to be 0.5 μg/mL) was added, then incubated at 4° C. for 30 min, centrifuged at 500 g for 3 min, and washed with 200 μL of PBS for three times.

[0147] The well was designed according to the experimental. 80 μl of FITC-labeled goat anti-human second antibody (1:150 dilution) was added to each well, then incubated at 4° C. for 30 min, centrifuged at 500 g for 3 min, and after the supernatant was discarded, washed with 200 μL PBS for three times. 200 μL PBS was added to suspend the cells after the last wash. The suspended cells were filtered with a strainer and then detected by the flow cytometry. Results showed that both polyclonal scFv and monoclonal scFv were able to specifically bind to K562 cells expressing the CD19 antigen to varying degrees (FIG. 28). The screened polyclonal scFv and monoclonal scFv were both able to specifically bind to K562 cells expressing the CD19 antigen to varying degrees but not to K562 cells without CD19 antigen expression.

[0148] In summary, the present application provides an antibody library construction method and an application thereof. The method is designed based on the principle that a synNotch system controls the gene expression in cells. Through a large number of experiments, the whole scheme flow process is optimized; and after repeated design and verification, the extracellular recognition domain of the synNotch system is changed into an antibody library, and the regulated target gene is changed into a screening marker gene, so that a technology for screening polyclonal antibodies against complex antigens is obtained. In such a way, the problems of complexity, diversity and mutability of tumor antigen and the limited available targets are solved, and this method has a wide application prospect and huge market value.

[0149] The applicant has stated that although the detailed method of the present application is described through the examples described above, the present application is not limited to the detailed method described above, which means that implementation of the present application does not necessarily depend on the detailed method described above. It should be apparent to those skilled in the art that any improvements made to the present application, equivalent replacements of raw materials of the product of the present application, additions of adjuvant ingredients to the product of the present application, and selections of specific manners, etc., all fall within the protection scope and the disclosed scope of the present application.