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Recombinant vector for enhancing ability of displaying Fab fragment antigen binding on yeast cell surface by using endoplasmic reticulum retrieval signal sequence

A yeast cell and surface display technology, which is applied in the direction of introducing foreign genetic material using vectors, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems of weak yeast display efficiency, insufficient effective assembly and folding, etc., to promote folding and Assembly efficiency, improvement of antigen-binding ability, effect of improving antigen-binding ability

Active Publication Date: 2019-09-10
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has been reported that the functional Fab yeast display efficiency of Infliximab is relatively weak [Sivelle C., Sierocki R., Ferreira-Pinto K., et al., "Fab is the most efficient format to express functional antibodies by yeast surface display", mAbs,2018,10(5):720-9], which may be related to the insufficient efficient assembly and folding of the Fab's VH-CH1 domain and VL-CL domain in the endoplasmic reticulum

Method used

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  • Recombinant vector for enhancing ability of displaying Fab fragment antigen binding on yeast cell surface by using endoplasmic reticulum retrieval signal sequence
  • Recombinant vector for enhancing ability of displaying Fab fragment antigen binding on yeast cell surface by using endoplasmic reticulum retrieval signal sequence

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1: the construction of the genetically engineered bacteria containing plasmid pFab-1

[0034] The gene fragments of VH-CH1-FLAG-Aga2 and VL-CL-HA-ERS complexes of D2E7 and Infliximab were obtained by PCR cloning method;

[0035] PCR reaction system: 10×KOD buffer, 5 μl; dNTP (2.5mM), 4 μl; primer F (10 μM), 3 μl; primer R (10 μM), 3 μl; Pfu polymerase, 2 μl; template, 1 μl; add ddH 2 0 to 50 μl. PCR amplification system: 95°C, 5min; 95°C, 30s, 55°C, 30s, 72°C, 30s, 25 cycles; 72°C, 5min; 12°C, 10min;

[0036] After the PCR product of the VH-CH1-FLAG-Aga2 complex obtained above was recovered with 0.8% agarose gel, it was double-digested with restriction endonucleases PstI and EcoRI. The enzyme digestion system was: PstI, 1 μl; EcoRI , 1μl; 10×Cutsmartbuffer, 5μl; PCR recovered product, 30μl, add ddH 2 0 to 50 μl. After digestion at 37°C for 5 hours, recover with 0.8% agarose gel;

[0037] Then it was ligated with the pESD vector that had been digested wit...

Embodiment 2

[0040] Embodiment 2: Fab-induced expression of genetically engineered bacteria containing plasmid pFab-1

[0041] The yeast transformant transformed with pFab-1 plasmid in Example 1 was inoculated in YNB-CAA-Glucose liquid medium, and cultured overnight at 30°C until OD 600 3.0-4.0, change YNB-CAA-Galactose medium to induce, initial OD600 0.8, induced at 18°C ​​for 48h.

Embodiment 3

[0042] Example 3: Fab yeast cell surface display efficiency and activity detection of genetically engineered bacteria containing plasmid pFab-1

[0043] For the yeast cells induced in Example 2, two samples were taken from each sample, one was used for the detection of Fab display efficiency, and the other was used for the detection of activity. Take 10 respectively 6 For each yeast cell, wash once with solution A, then wash once with solution B, centrifuge at 4°C, 3000rpm for 2min. One of the cells used for Fab activity detection was incubated with different concentrations of 6×His-TNF-α (expressed and purified in Escherichia coli) at 25°C for 30 min, then washed twice with solution B, and finally washed with 20 μl solution B and 0.15 Resuspend in μl fluorescent antibody Anti-6×His-iFluor 647. Another portion of cells used for Fab display efficiency detection was directly resuspended with 20 μl solution B and 0.15 μl fluorescent antibody Anti-HA-FITC. The resuspended cells...

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Abstract

The invention discloses a recombinant vector for enhancing the ability of displaying Fab fragment antigen binding on the yeast cell surface by using endoplasmic reticulum retrieval signal sequences, and belongs to the technical field of antibody engineering. A GAL1-GAL10 bidirectional promoter vector is used for expressing two structures domains at the same time, namely VH-CH1 and VL-CL, of Adalimumab or Infliximab, and the amino terminal endoplasmic reticulum retrieval signal sequences are located in an endoplasmic reticulum and assembled into natural Fab form fragments to be displayed on thecell surface through an Aga1-Aga2 yeast cell display system. Meanwhile, by fusing the saccharomyces cerevisiae retrieval signal sequences into the carboxyl terminal of the Fab fragment VL-CL structure domain, the ability of the yeast cell surface to display Fab fragment bonded antigens is greatly improved, displaying of functional Fab fragments is promoted, the vector is more suitable for displaying the Fab fragments on the yeast cell surface, and an optimization strategy is provided for Fab modification in antibody engineering.

Description

technical field [0001] The invention relates to a recombinant carrier which utilizes endoplasmic reticulum retention signal peptide to improve the ability of yeast cell surface to display Fab fragment antigen binding, and belongs to the technical field of antibody engineering. Background technique [0002] Antibodies are immunoglobulins secreted by immune cells under antigen stimulation that can specifically bind to corresponding antigens. Monoclonal antibodies are currently one of the most economically valuable biotherapeutic drugs, and are widely used in the treatment of viral infections, immune diseases, and cancer. At present, there are two main ways to produce antibodies, traditional animal immunization and antibody engineering. Most of the methods for immunizing animals to produce antibodies are time-consuming and expensive. Therefore, antibody engineering technology has gradually developed, including phage display, ribosome display and cell display technology, etc.,...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/66C12N15/62C12R1/865
CPCC12N15/81C12N15/66C07K16/241C07K2319/04C07K2317/55
Inventor 易犁梅萌李俊红汪声晨张桂敏
Owner HUBEI UNIV
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