Design and construction of fully humanized antibody yeast display technology

A yeast and construct technology, applied in the field of yeast display library constructs, can solve problems such as low efficiency, low antibody library capacity, and the impact of antibody diversity

Pending Publication Date: 2021-04-13
ADLAI NORTYE BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Second, the diversity of the phage antibody library is easily lost in the continuous expansion of bacteria
For example, the capacity of the current antibody library is relatively low, which has a great impact on the diversity of antibodies
Relatively inefficient recombination between yeast expression plasmids and antibody fragments also limits antibody diversity

Method used

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  • Design and construction of fully humanized antibody yeast display technology
  • Design and construction of fully humanized antibody yeast display technology
  • Design and construction of fully humanized antibody yeast display technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0109] CLAIMS 1. A construct comprising a promoter, a multiple cloning site region, a CH1 fragment of a human IgG heavy chain constant region, a linker sequence, a yeast surface localization sequence and optionally a tag sequence.

[0110] 2. The construct according to embodiment 1, which comprises the structure described in expression (I):

[0111] Expression (I):

[0112] 5'-A-B-C-D-E-F-3' (I);

[0113] in,

[0114] A stands for promoter;

[0115] B represents the multiple cloning site region;

[0116] C represents the CH1 fragment of the human IgG heavy chain constant region;

[0117] D represents the linker sequence;

[0118] E represents the yeast surface localization sequence; and

[0119] F stands for no or tag sequence.

[0120] 3. The construct according to embodiment 1 or 2, wherein the promoter is selected from the group consisting of yeast GAL1 promoter, yeast GAL10 promoter and yeast GAL1 / GAL10 bidirectional promoter.

[0121] 4. The construct according to...

Embodiment 1

[0169] Example 1 Construction of recombinant expression vector pAN-HC

[0170] A recombinant expression vector with a human IgG heavy chain constant region was constructed using commercial plasmid pYD1 (purchased from Invitrogen). A brief operation is as follows:

[0171] (1) The commercialized plasmid pYD1 was digested with endonucleases AgeI and PmeI at 37°C for one hour; then larger fragments were recovered with agarose gel.

[0172] (2) Artificially synthesize a nucleic acid sequence as shown in SEQ ID NO:2 (purchased from ThermoFisher). This sequence contains elements such as figure 1 shown.

[0173] (3) Using an In-fusion Cloning kit (purchased from Takara Biotechnology Co., Ltd.), the vector fragment recovered and purified in step (1) was ligated with the fragment synthesized in step (2).

[0174] (4) Transform the ligation product in step (3) into bacteria, and carry out screening and sequencing identification of positive clones.

[0175] The recombinant expressio...

Embodiment 2

[0176] Example 2 Expression vector constructed using recombinant vector pAN-HC to express antibody

[0177] The known TIGIT antibody sequence was used to test the pAN-HC recombinant vector obtained in Example 1 to evaluate the antibody expression ability of the recombinant vector.

[0178] (1) Provide a nucleic acid sequence of a human TIGIT antibody (purchased from ThermoFisher).

[0179] (2) Digest the recombinant vector pAN-HC with DNA restriction enzymes NheI and Eco47III, and recover the large fragment.

[0180] (3) Transfer the antibodies and carrier sequences in steps (1) and (2) to yeast strain EBY100 by yeast electroporation.

[0181] (4) Use 100 ml of tryptophan-deficient medium SD-CAA (tyrosine 5g / L, glucose 20g / L, yeast nitrogen source base 1.7g / L without amino acid and ammonium sulfate, ammonium sulfate 5.3g / L , Na 2 HPO 4 -7H 2 O 10.2g / L, NaH 2 PO 4 -H 2 (08.6g / L) was cultivated overnight, and the yeast containing the recombinant plasmid was screened out....

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Abstract

The invention relates to a construct for a yeast display library and a preparation method of the construct. The construct comprises a promoter, a multiple cloning site region, a CH1 fragment of a human IgG heavy chain constant region, a linker sequence, a yeast surface positioning sequence and an optional tag sequence. The invention specifically relates to the construct for the yeast display library, an expression vector, a host cell as well as a construction method and application of the construct. The yeast display library provided by the invention has high conversion efficiency and rich diversity.

Description

technical field [0001] This application relates to the technical field of genetic engineering, in particular to a construct for yeast display library, expression vector, host cell and its construction method and application. Background technique [0002] Currently, the commonly used antibody screening techniques include in vivo screening and in vitro screening, and in vitro screening is favored due to its simplicity and rapidity. The in vitro screening technology is based on the construction of an antibody library, and the current relatively mature technologies mainly include phage display technology and yeast display technology. As the earliest antibody screening technology in vitro, phage display technology has been widely used in the research and development of antibody drugs, and a large number of marketed drugs have been approved for use. The principle is to amplify the antibody heavy chain variable region (VH) and light chain variable region (VL) of B lymphocytes by P...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/10C12R1/84C12R1/865
CPCC12N15/81C12N15/815C12N15/1037C40B40/02C12N2800/102C40B40/08
Inventor 何南海陈丹丹路杨杨东晖
Owner ADLAI NORTYE BIOPHARMA CO LTD
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