Plasmid for displaying antibody Fab segment on yeast surface

A yeast surface display and antibody technology, applied in the introduction of foreign genetic material, application, fermentation and other directions using a carrier, can solve the problems of easy inactivation, easy residual yeast cells, yeast display technology relying on flow sorting, etc., to achieve convenient operation. Effect

Inactive Publication Date: 2017-06-13
SHANGHAI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Yeast cell surface display antibody library technology is widely used, but there are still two problems. First, due to the low efficiency of yeast cell plasmid transformation, the capacity of antibody library construction in yeast is smaller than the capacity of phage display antibody library. At the same time, yeast display technology Relying on flow sorting, there is also a certain limit on the size of the library that can be manipulated; second, the existing yeast system generally displays scFv, but the scFv is not stable enough and easy to inactivate; third, Fab antibodies are relatively stable, but the screening is difficult, and the process It is complex and needs to express the heavy chain region and light chain region of Fab separately. At present, the complete Fab antibody is displayed on the surface of yeast by mating yeast cells of different types carrying the heavy chain region and light chain region of Fab respectively.
In addition to the time-consuming and labor-intensive disadvantages of this method, it is easy to leave yeast cells that only display the heavy chain region of the Fab antibody, which interferes with the screening or transformation of the Fab antibody
When scFv is converted into full-length IgG, the affinity for antigen will be weakened or even inactivated.

Method used

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  • Plasmid for displaying antibody Fab segment on yeast surface
  • Plasmid for displaying antibody Fab segment on yeast surface
  • Plasmid for displaying antibody Fab segment on yeast surface

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The map of the plasmid pYD-AH vector used to display antibody Fab fragments on the surface of yeast in this example is as follows figure 1 As shown in A, the map of the plasmid pYD-HA vector used to display antibody Fab fragments on the surface of yeast in this example is as follows figure 1 Shown in B. The sequences of the plasmids pYD-AH vector and pYD-HA vector include the yeast GAL1 / GAL10 bidirectional promoter, the light chain region of the Fab antibody expressed with the GAL10 promoter, and the heavy chain region of the Fab antibody expressed with the GAL1 promoter, wherein, The heavy chain region of the Fab antibody expressed with GAL1 as the promoter is fused with Aga2.

[0025] The sequences of the plasmids pYD-AH vector and pYD-HA.vector specifically include:

[0026] 1. Yeast TRP1 selection marker ORF (TRP1): Yeast auxotrophic selection marker TRP1.

[0027] 2. f1origin of ss-DNA replication (f1ori): a promoter that guides single-stranded DNA replication. ...

Embodiment 2

[0043] In order to detect whether the plasmids pYD-AH vector and pYD-HA vector in Example 1 can correctly display Fab antibodies in the yeast display screening system, thrombopoietin receptor (Thrombopoietin Receptor, TpoR) antibody 3D9 (this antibody is described in WO In 2014 / 035693A2, the sequence of the light chain region is SEQ ID NO: 3, the sequence of the heavy chain region is SEQ ID NO: 4) and human epidermal growth factor receptor 2 (Human Epidermal Growth Factor Receptor 2, Her2, also known as c-erb2 ) antibody 4D5 as an example for verification. The specific steps are:

[0044] (1) Subclone the 3D9 light chain region into the plasmid pYD-AH vector using restriction endonucleases PacI and SacI (NEB) according to the instructions of the plasmid map:

[0045] Add restriction endonuclease sites PacI (sequence ttaattaa) and SacI (sequence gagctc) to the 5' end and 3' end of the 3D9 light chain region respectively, and send to Gene Synthesis Company Shanghai Jierui Bioen...

Embodiment 3

[0055] Construct and detect a Fab antibody phage display plasmid consistent with the multiple cloning sites of the pYD-AH / pYD-HA plasmid system:

[0056] (1) The restriction site of the plasmid pcome3xss, which is commonly used in phage antibody display libraries, was transformed into a restriction site consistent with the pYD-AH and pYD-HA plasmid systems, and Fab 3D9 and Fab 4D5 were respectively constructed into the pcomb3xss plasmid. The constructed plasmid was transformed into Xl-1blue cells to produce antibodies displaying Fab 3D9, ScFv 3D9 ( image 3 .A), Fab4D5, ScFv 4D5 ( image 3 .B) the phage;

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Abstract

The invention provides a plasmid for displaying an antibody Fab segment on a yeast surface. The plasmid is characterized in that a sequence contains yeast GAL1/GAL10 bidirectional promoter, a light chain area and a heavy chain area, wherein the light chain area takes GAL10 as the promoter for expressing Fab antibody; the heavy chain area takes GAL1 as the promoter for expressing Fab antibody; the heavy chain area which takes GAL1 as the promoter for expressing Fab antibody is fused with Aga2. The Fab segment screened by using the plasmid can be directly converted into whole-length antibody and the problem of inactivation is avoided.

Description

technical field [0001] The invention relates to a plasmid for displaying antibody Fab fragments on the surface of yeast. Background technique [0002] Yeast cell surface display antibody library technology is widely used, but there are still two problems. First, due to the low efficiency of yeast cell plasmid transformation, the capacity of antibody library construction in yeast is smaller than the capacity of phage display antibody library. At the same time, yeast display technology Relying on flow sorting, there is also a certain limit on the size of the library that can be manipulated; second, the existing yeast system generally displays scFv, but the scFv is not stable enough and easy to inactivate; third, Fab antibodies are relatively stable, but the screening is difficult, and the process It is complex and needs to express the heavy chain region and light chain region of Fab separately. At present, the complete Fab antibody is displayed on the surface of yeast mainly b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/13C12N1/19
CPCC07K16/00C07K2317/55C12N15/81C12N2800/102C12N2830/205
Inventor 理查德·乐纳张宏恺杜明娟
Owner SHANGHAI TECH UNIV
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