Recombinant engineered Escherichia coli for expressing anti-TNF antibody Fab fragment

A technology for recombinant Escherichia coli, Escherichia coli, applied in bacteria, microorganism-based methods, enzymes, etc., can solve problems such as mispairing of disulfide bonds, misfolding, etc.

Active Publication Date: 2016-12-07
BEIJING TRI PRIME GENE PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, even if the Fab antibody is expressed in the periplasmic space of E. coli, if it is overexpressed, there will still be problems of misfolding and mispairing of disulfide bonds. Therefore, the amount of Fab antibody expressed and the correct structure represented by disulfide bond formation Formation and biological activity have great influence

Method used

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  • Recombinant engineered Escherichia coli for expressing anti-TNF antibody Fab fragment
  • Recombinant engineered Escherichia coli for expressing anti-TNF antibody Fab fragment
  • Recombinant engineered Escherichia coli for expressing anti-TNF antibody Fab fragment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Construction of recombinant Escherichia coli engineering bacteria that co-express hPDI and the heavy chain amino acid sequence of SEQ ID No:7, the Fab fragment of the light chain amino acid sequence of SEQ ID No:8

[0038] The secretion and expression of Fab antibody in the periplasmic space of Escherichia coli is achieved by introducing the target gene into the empty plasmid expression vector, constructing the recombinant plasmid expression vector, and then transferring it into the Escherichia coli engineering bacteria to construct the recombinant Escherichia coli engineering bacteria.

[0039] 1) Construction and amplification of the target gene

[0040] Before the heavy chain gene sequence of the Fab antibody (the corresponding amino acid sequence is shown in SEQ ID No: 7), a section of OmpA signal peptide gene sequence (the corresponding amino acid sequence is shown in SEQ ID No: 9) is introduced, and then a section containing ribose is inserted The conne...

Embodiment 2

[0073] Example 2: Construction of recombinant Escherichia coli engineering bacteria that co-express hPDI and the heavy chain amino acid sequence of SEQ ID No:1, the Fab fragment of the light chain amino acid sequence of SEQ ID No:2

[0074] Method basically with reference to embodiment 1, but difference is as follows:

[0075]1) When constructing and expressing the target gene of the Fab antibody, the heavy chain gene sequence of the Fab antibody is the nucleotide sequence corresponding to the amino acid sequence of SEQ ID No: 1, and the light chain gene sequence of the Fab antibody is corresponding to the amino acid sequence of SEQ ID No: 2 the nucleotide sequence.

[0076] 2) The empty plasmid vector is pET23b, and the recombinant plasmid expression vector thus prepared is named HL-pET23b.

[0077] 3) HL-pET23b was transformed into hPDI-pCDFduet / BL21(DE3) competent cells to obtain recombinant Escherichia coli engineering strain HL-pET23b / hPDI-pCDFduet / BL21(DE3).

Embodiment 3

[0078] Embodiment 3: the construction of the recombinant Escherichia coli engineering bacterium of the Fab fragment of the heavy chain amino acid sequence of coexpressing hPDI and SEQ ID No:3, the light chain amino acid sequence of SEQ ID No:4

[0079] Method basically with reference to embodiment 1, but difference is as follows:

[0080] 1) When constructing and expressing the target gene of the Fab antibody, the heavy chain gene sequence of the Fab antibody is the nucleotide sequence corresponding to the amino acid sequence of SEQ ID No: 3, and the light chain gene sequence of the Fab antibody is corresponding to the amino acid sequence of SEQ ID No: 4 the nucleotide sequence.

[0081] 2) The empty plasmid vector is pET24a, and the recombinant plasmid expression vector thus prepared is named HL-pET24a.

[0082] 3) HL-pET24a was transformed into hPDI-pCDFduet / BL21(DE3) competent cells to obtain recombinant Escherichia coli engineering strain HL-pET24a / hPDI-pCDFduet / BL21(DE3)...

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Abstract

The invention belongs to the field of biomedicine and relates to recombinant engineered Escherichia coli that converts a recombinant expression vector, and the recombinant expression vector comprises genes for expressing anti-TNF antibody Fab fragment and genes for expressing protein disulfide isomerase. By expressing the ant-TNF Fab fragment with the recombinant engineered Escherichia coli, it is possible to significantly improve correct formation of disulfide bonds at the premise of significantly improving periplasmic space expression quantity.

Description

technical field [0001] The present invention generally relates to recombinant Escherichia coli engineering bacteria, and particularly relates to recombinant Escherichia coli engineering bacteria expressing antibody Fab fragments. Background technique [0002] Antibody (Ab) is an immune system induced by antigens (such as bacteria, viruses and their components, heterologous proteins), synthesized and secreted by lymphocytes (plasma cells) in the body, and used to identify and neutralize antigens Globulin substances. Antibodies are divided into polyclonal antibody (polyclonal antibody, pAb) and monoclonal antibody (monoclonal antibody, mAb), in which the former is produced by the body stimulated by heterologous antigens or different epitopes, and the latter is produced by homologous antigens or even the same The antigenic epitopes stimulate the body to produce. Therefore, more monoclonal antibodies have stronger specificity and higher sensitivity, but the difficulty and cost...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12R1/19
CPCC07K16/241C12N9/90C12Y503/04001
Inventor 童梅刘美君莫婷张宇萌徐晨周敏毅
Owner BEIJING TRI PRIME GENE PHARMA CO LTD
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