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

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.