Construction method and application of antigen-specific binding polypeptide gene display vector

1 . A method for screening an antigen-specific binding polypeptide or fragments thereof, comprising:

a) obtaining the antigen-specific binding VH sequences and the antigen-specific binding LC sequences from a phage display library;

b) constructing the antigen-specific binding polypeptide gene display vectors;

c) introducing the antigen-specific binding polypeptide gene display vectors into the first bacteria to obtain an antigen-specific binding polypeptide gene display bacterial library;

d) acquiring an antigen-specific binding polypeptide gene library from the antigen-specific binding polypeptide gene display bacterial library;

e) acquiring the antigen-specific binding polypeptide gene display vector DNA from the antigen-specific binding polypeptide gene library;

f) introducing the antigen-specific binding polypeptide display vector DNA into cells; optionally wherein the cell is a mammalian cell;

g) acquiring the gene of antigen-specific binding polypeptide from the cell;

wherein constructing the antigen-specific binding polypeptide gene display vectors comprises the following steps:

i. providing the first display vector polynucleotides comprising B2-display VH-B3 in the direction from 5′ to 3′, inserting the first display vector polynucleotides into the display component vector to form the display VH storage ligation products, and introducing the display VH storage ligation products into the first display bacteria to obtain a display VH component bacterial library, acquiring the display VH component plasmids containing the first display vector polynucleotides from the display VH component bacterial library, and digesting the display VH component plasmids with a restriction endonuclease that specifically recognizes the B2 and B3, thus obtaining the cleaved first display vector polynucleotides;

ii. providing the second display vector polynucleotides comprising S5-display LC-S6 in the direction from 5′ to 3′, inserting the second display vector polynucleotides into the display component vector to form the display LC storage ligation products, and introducing the display LC storage ligation products into the second display bacteria to obtain a display LC component bacterial library, acquiring the display LC component plasmids containing the second display vector polynucleotides from the display LC component bacterial library, and digesting the display LC component plasmids with a restriction endonuclease that specifically recognizes the S5 and S6, thus obtaining the cleaved second display vector polynucleotides;

iii. providing the third display vector polynucleotides comprising B3-display vector fragment I-S5 in the direction from 5′ to 3′, cleaving the third display vector polynucleotide with restriction endonucleases that specifically recognizes B3 or S5 to obtain a cleaved third display vector polynucleotide;

iv. providing the fourth display vector polynucleotides comprising S6-display vector fragment II-B2 in the direction from 5′ to 3′, cleaving the fourth display vector polynucleotide with restriction endonucleases that specifically recognizes S6 or B2 to obtain a cleaved fourth display vector polynucleotide;

v. mixing the cleaved first display vector polynucleotides, the cleaved second display vector polynucleotides, the cleaved third display vector polynucleotides and the cleaved fourth display vector polynucleotides so that they are able to ligate direction ally and cyclized to form the antigen-specific binding polypeptide gene display vectors;

wherein, the display VH encodes a heavy chain variable region of the antigen-specific binding polypeptide, and the display LC encodes the light chain of the antigen-specific binding polypeptide;

wherein the B2 and B3 are able to be specifically recognized and cleaved by an enzyme selected from the group consisting of: BsmBI and Esp3I; and wherein the S5 and S6 are able to be specifically recognized and cleaved by SfiI; and

wherein the B2 comprises a nucleic acid sequence as set forth in SEQ ID NO: 8, the B3 comprises a nucleic acid sequence as set forth in SEQ ID NO: 9, the S5 comprises a nucleic acid sequence as set forth in SEQ ID NO: 10, and/or the S6 comprises a nucleic acid sequence as set forth in SEQ ID NO: 11.

2 . The method according to claim 1 , wherein the display vector component vector is derived from a pUC vector.

3 . The method according to claim 1 , comprising:

a) providing the fifth polynucleotides each comprising a B-antigen-specific VH-B in the direction from 5′ to 3′;

b) providing a VH component vector, the VH component vector comprises a sixth polynucleotide comprising B3-VH component vector ligation fragment-B2 in the direction from 5′ to 3′;

c) cleaving the fifth polynucleotides and the VH component vector with the restriction endonuclease to obtain the cleaved fifth polynucleotides and a released sixth polynucleotide;

d) mixing the cleaved fifth polynucleotides and the released sixth polynucleotide so that they can be ligated directionally and cyclized to form an antigen-specific VH component library;

wherein the B is a recognition site for the restriction endonuclease that can specifically recognize B2 and/or B3, and the antigen-specific VH encodes the heavy chain variable region of the antigen-specific binding polypeptide.

4 . The method according to claim 1 , comprising:

a) providing the seventh polynucleotides each comprising a S-antigen-specific LC-S in the direction from 5′ to 3′;

b) providing an LC component vector, the LC component vector comprises an eighth polynucleotide comprising S6-LC component vector ligation fragment-S5 in the direction from 5′ to 3′;

c) cleaving the seventh polynucleotides and the LC component vector with the restriction endonuclease to obtain the cleaved seventh polynucleotides and a released eighth polynucleotide;

d) mixing the cleaved seventh polynucleotides and the released eighth polynucleotide so that they can be ligated directionally and cyclized to form an antigen-specific LC component library,

wherein the S is a recognition site for the restriction endonuclease that can specifically recognize S5 and/or S5, and the antigen-specific LC encodes the light chain of the antigen-specific binding polypeptide.

5 . The method according to claim 1 , comprising,

a) providing a ninth polynucleotide comprising B2-VH component vector tool fragment-B3 in the direction from 5′ to 3′;

b) inserting the ninth polynucleotide into an expression component vector to obtain the VH component vector.

6 . The method according to claim 1 , comprising,

a) providing a tenth polynucleotide comprising S5-LC component vector tool fragment-S6 in the direction from 5′ to 3′;

b) inserting the tenth polynucleotide into the expression component vector to obtain the LC component vector.

7 . The method according to claim 5 , wherein the expression component vector is derived from a pMD vector.

8 . The method according to claim 3 , comprising the steps of:

introducing the VH component vector into a ninth bacterium to obtain a VH component vector storage bacterial library; acquiring a VH component vector storage plasmid from the VH component vector storage bacterial library; acquiring the released sixth polynucleotide from the VH component vector storage plasmid; and/or

introducing the LC component vector into a tenth bacterium to obtain an LC component vector storage bacterial library; acquiring an LC component vector storage plasmid from the LC component vector storage bacterial library; acquiring the released eighth polynucleotide from the LC component vector storage plasmid.

9 . The method according to claim 8 , comprising digesting the VH component vector storage plasmid with the restriction endonuclease that specifically recognizes the B2 and B3, thus obtaining the released sixth polynucleotide; and/or digesting the LC component vector storage plasmid with the restriction endonuclease that specifically recognizes the S5 and S6, thus obtaining the released eighth polynucleotide.

10 . The method according to claim 1 , wherein the directional ligation involves using a ligase.