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A kind of endogenous l-asparaginase II gene knockout host bacteria, its preparation method and application

A technology of asparaginase and exogenous genes, applied in the field of preparation of Escherichia coli host strains, can solve the problems of ansB gene and host bacterial genome instability, low recombination efficiency, and treatment failure

Active Publication Date: 2021-05-14
JIANGSU HENGRUI MEDICINE CO LTD
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

But escherichia coli can background expression L-asparaginase II, the obtained L-asparaginase II product is mixed with the L-asparaginase II of Escherichia coli background expression, causes the inhomogeneity of product purity, has Potential immunogenic safety risk
In addition, impurities may also cause the body to form immunity to the drug, leading to treatment failure
[0006] Patent CN 101484181A In order to solve this problem, in BL21 (DE3) host bacteria, use the free recombinant expression vector to produce the L-asparaginase II of BL21 source, thereby obtain the L-asparaginase II of uniform purity, but, This strategy also has the risk of instability of the ansB gene and the genome of the host bacteria
In 2000, Kirill A. et al. (2000PANS.97(12):6640-6645.) found that the target gene was replaced or inserted into the large intestine by homologous recombination using a homologous sequence fragment or plasmid with the target gene of the host bacteria. Bacillus genome, thereby inactivating the E. coli target gene
However, not all genes in all bacteria can be successfully knocked out. The difficulty of gene knockout technology for bacteria lies in the fact that there are many factors that affect the recombination efficiency, such as bacterial species, subtype, gene to be knocked out, and competent cells. The recombination efficiency varies greatly depending on the state, operation method, etc. (Zhang Xue et al., 2008 China Biotechnology Journal. 28(12):89-93.)
In addition, improper selection of the targeting sequence of the targeting vector will also lead to low recombination efficiency and knockout failure. Appropriate targeting sequences and targeting vectors are crucial

Method used

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  • A kind of endogenous l-asparaginase II gene knockout host bacteria, its preparation method and application
  • A kind of endogenous l-asparaginase II gene knockout host bacteria, its preparation method and application
  • A kind of endogenous l-asparaginase II gene knockout host bacteria, its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Knockout of the ansB gene on the BL21 (DE3) genome

[0055] 1. Primer Design

[0056] According to the upstream and downstream sequences of the ansB gene on the BL21 (DE3) genome, primers for the upstream and downstream homologous recombination arms, connecting primers and identification primers for the upstream and downstream homologous recombination arms were designed. The primer sequences are shown in Table 3.

[0057] Table 3 Primer Sequence

[0058]

[0059]

[0060] 2. Amplification of targeting sequences and construction of targeting vectors

[0061] Using a genome extraction kit ( Genomic DNA Purification Kit, Promega) extracts the genome of BL21 (DE3) as a PCR template, primers are upstream and downstream homologous recombination arm primers (SEQ ID NO: 7 to SEQ ID NO: 10), using high-fidelity DNA polymerase (PlatinumTaq , Invitrogen) to amplify the upstream and downstream homologous recombination arms. Using the connecting primer (SEQ ID N...

Embodiment 2

[0068] Embodiment 2: Comparison of the growth characteristics of wild bacteria BL21 (DE3) and gene knockout bacteria BL21 (DE3) / ΔansB

[0069] Prepare LB medium, the formula is 1% Tryptone, 0.5% yeast extract, 1% NaCl. Inoculate in 5mL seed medium with 0.5% ratio, 50mL micro bioreactor (Mini Bioreactor, Corning), 37°C, 220rpm culture to logarithmic growth phase, transfer to 50mL LB medium with 2% ratio, do 3 parallel A 250mL shake flask (ErlenmeyerFlask, Corning), sampling every 30min or 1h, using a UV spectrophotometer to detect OD 600 value. Taking time as the abscissa, OD 600 Values ​​plot growth curves on the ordinate, see figure 2 .

[0070] Formula 1:

[0071]

[0072] In the formula, μ is the specific growth rate in h -1 , T2-T1 is the time spent by microorganisms from time point T1 to time point T2, in h, N1 is the cell mass of microorganisms at time point T1, and N2 is the cell mass of microorganisms at time point T2.

[0073] The obtained data was processe...

Embodiment 3

[0074] Example 3: Background expression of L-asparaginase II of wild-type bacteria BL21 (DE3) and gene knockout bacteria BL21 (DE3) / ΔansB

[0075] Prepare seed medium with the formula of 1% Tryptone, 0.5% yeast extract, 1% NaCl, 0.1% L-asparagine, prepare background expression medium with the formula of 1% Tryptone, 0.5% yeast extract, 1% NaCl, 0.6% L-asparagine. Get glycerol bacteria, inoculate 5mL seed culture medium with 0.1% ratio, 50mL miniature bioreactor (Mini Bioreactor, Corning), 25 ℃, 150rpm cultivate to OD 600 = around 4 (16-18 hours). Transfer to 30 mL background expression medium at a ratio of 4%, culture in a 125 mL flat-bottomed shaker flask (Erlenmeyer Flask, Corning) at 25° C., 150 rpm for 14 hours. Centrifuge at 8000rpm for 5min to collect the bacterial cells, extract periplasmic protein by osmotic pressure method (refer to pET manual), use 10kD concentration tube (Spin- UF 20, Corning) were properly concentrated and analyzed by SDS-PAGE electrophoresis. ...

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Abstract

The invention relates to a host bacterium for knocking out endogenous L-asparaginase II gene, its preparation method and its application. Specifically, the present invention relates to a method for knocking out the endogenous L-asparaginase II gene of Escherichia coli, and obtaining a host strain for knocking out the gene. By using the method of knocking out the targeting vector, the genome of the host bacterium does not contain the gene encoding L-asparaginase II and its variants. By adopting the host bacteria provided by the present invention, L-asparaginase II and variants thereof derived from different microorganisms such as Erwinia and Escherichia can be expressed recombinantly.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a method for preparing an Escherichia coli host strain for knocking out an endogenous L-asparaginase II gene and an application of the host strain. Background technique [0002] L-asparaginase II is a deaminase that catalyzes the hydrolysis of asparagine to aspartic acid and ammonia. The enzyme removes L-asparagine outside the cell, thereby preventing the growth of cancer cells that depend on L-asparagine for protein synthesis. Clinically, L-asparaginase II is mainly used in the treatment of acute lymphoblastic leukemia and lymphoma. [0003] The L-asparaginase II preparations currently on the market are derived from two microorganisms, namely Escherichia coli (Escherichia coli) and Erwinia (Erwinia chrysanthemi), and L-asparaginase II is a quadruple of the same subunit. Polymer with a molecular weight of 141kDa, the monomer has no enzymatic activity. These two sources of L-a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/11C12R1/19
CPCC12N9/82C12N15/70C12Y305/01001
Inventor 曾杰田静陈磊刘衍伟王宏伟
Owner JIANGSU HENGRUI MEDICINE CO LTD
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