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Method for increasing expression quantity of Fab antibody

An expression level and antibody technology, applied in the field of genetic engineering, can solve the problems of losing industrial application prospects, achieve good practical value and scientific research significance, and improve the effect of Fab expression

Active Publication Date: 2017-07-21
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these defective strains often have some non-specific effects on the growth of host cells (Escherichia coli), such as: bacterial cell growth rate, stability, recombinant protein expression yield and toxicity, etc., resulting in the loss of industry application prospect
[0011] In short, it is precisely because of the absence of specific strains and the diversification of transformation possibilities that for the expression of Fab antibodies, there is still a lack of a more suitable genetic engineering transformation method to increase its expression in the prior art.

Method used

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  • Method for increasing expression quantity of Fab antibody
  • Method for increasing expression quantity of Fab antibody
  • Method for increasing expression quantity of Fab antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0046] The method for increasing the expression of Fab antibody provided in this application mainly depends on the transformation of Escherichia coli. In this example, the original host cell strain Escherichia coli W3110 (W3110(F-l-rph-1 INV(rrnD,rrnE)ilvG) was taken as an example. The preparation process is briefly described as follows.

[0047] (1) Construct recombinant plasmids pKO3-pepN, pKO3-DegQ, pKO3-dcp and pKO3-pepP respectively

[0048] The artificially synthesized pepN, DegQ, dcp, and pepP integration cassettes (recombined gene sequences) were subjected to SalI and NotI double enzyme digestion, and the pKO3 plasmid was subjected to SalI and NotI double enzyme digestion, and then the digested products were ligated, respectively. Construction of recombinant plasmids pKO3-pepN, pKO3-DegQ, pKO3-dcp and pKO3-pepP;

[0049] It needs to be explained that there are three main reasons for choosing the pKO3 plasmid: first, the pKO3 plasmid uses the replication origin (RepA)...

Embodiment 2

[0090] According to the current routine transgenic operation method, the recombinant plasmid pVEGF-Fab was constructed for the expression of Fab antibody.

[0091] Since it is necessary to use pETDuet-1 with two independent promoters as the expression vector for double-gene co-expression, it is necessary to properly optimize the coding gene of Fab. The principles of gene optimization are as follows:

[0092] An independent leader peptide sequence from the periplasm of Gram-negative bacteria is added to the N-terminus of the light chain and the heavy chain Fd of the Fab coding gene, specifically:

[0093]The amino acid sequence of OmpA was added before the light chain: KKTAIAIAVALAGFATVAQA, after codon optimization according to the E. coli prokaryotic expression system, the full-length light chain DNA was synthesized by the solid-phase phosphoramidite method, and inserted into after the first promoter;

[0094] The OmpA amino acid sequence was added before the Fd segment of th...

Embodiment 3

[0097] Pick the transformed positive single colony in Example 2 and add it to 5 mL of 2×PY (1% plant peptone, 0.5% yeast extract, 0.5% NaCl) culture solution containing 10 μg / mL tetracycline, at 37°C, 250rpm Incubate overnight with shaking;

[0098] Inoculate 100 μL of the overnight culture into 200 mL of SM6E medium supplemented with tetracycline (10 μg / mL), culture overnight at 30°C with shaking at 250 rpm; repeat this step once until the culture reaches OD 600 About 2, and then directly used or long-term storage for future use (the long-term storage procedure is: centrifuge, collect the cells, then resuspend the cells in 100mL of SM6E, add glycerol at a final concentration of 12.5%, store at -80°C)

[0099] Take 2 mL of the culture, add it to 200 mL of SM6E medium containing 10 μg / mL tetracycline, and culture it at 30°C with shaking at 250 rpm until OD 600 about 2.

[0100] Add IPTG to a final concentration of 200 μM to induce recombinant protein production. At 1, 2, 4, ...

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Abstract

The invention specifically relates to a method for increasing the expression quantity of a Fab antibody in gene engineering, belonging to the technical field of gene engineering. The method comprises the following steps: construction of a recombinant plasmid pVEGF-FAB; preparation of specific protease defective cell mutants (delta pepN, delta DegQ, delta dcp and delta pepP); transformation; inducible expression of the Fab antibody; etc. The method provided by the invention is special and innovative in reconstruction of host cell strains; prepared host cell strains can eliminate inhibition or degradation effect on expressed Fab due to deletion of specific protease, so the expression quantity of Fab antibody can be increased; thus, the method has good application value in expression and preparation of specific Fab antibodies, provides good reference for expression and preparation of other antibody proteins, and thus has good practical value and scientific research significance.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for increasing the expression of Fab antibodies in genetic engineering. Background technique [0002] Bacterial cells (e.g. Escherichia coli, E. coli ) are commonly used to produce recombinant proteins. Compared with the eukaryotic cell expression system, the bacterial expression system has the advantages of low cost, easy large-scale fermentation, and easy automatic control of conditions. Expressing recombinant proteins through E. coli is an efficient and economical way. There are many other advantages to using bacterial cells such as E. coli to produce recombinant proteins, especially due to the versatile nature of bacterial cells as host cells allowing gene insertion via plasmids. E. coli is used to produce many recombinant proteins, including human insulin and Fab antibody proteins, among others. [0003] Fab is an antigen-binding fragment ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/66C12R1/19
CPCC12N9/54C12N15/66C12N15/70
Inventor 张守涛郭亚楠田庆南
Owner ZHENGZHOU UNIV
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