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Method for obtaining high-vitality superspectrum beta-lactam enzyme by directional anagenesis in vitro

A technology of lactamase and high activity, applied in the field of medical bioengineering

Inactive Publication Date: 2008-01-16
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The TEM-1 enzyme gene exists widely in natural bacteria, and has been able to be efficiently expressed in Escherichia coli (Sosa-PeinadoA, Mustafi D, Makinen MW. Overexpression and biosynthetic deuterium enrichment of TEM-1 beta-lactamase for structural characterization by magnetic resonance methods , Protein Expr Purif.2000, 19 (2): 235-245.), but there are few TEM-type enzyme genes that can degrade CAZ and other third-generation cephalosporins and can be expressed efficiently in Escherichia coli

Method used

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  • Method for obtaining high-vitality superspectrum beta-lactam enzyme by directional anagenesis in vitro
  • Method for obtaining high-vitality superspectrum beta-lactam enzyme by directional anagenesis in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: Cloning of TEM-1 gene

[0030] (1) PCR amplification of TEM-1 fragment

[0031] Using the TEM-1 gene as a template, design primers and add enzyme cutting sites. The above-mentioned enzyme-producing E.coli plasmid was extracted and used as a template for PCR reaction. The reaction conditions were: pre-denaturation at 94°C for 4 min, followed by 20 cycles with the following parameters: denaturation at 94°C for 1 min, annealing at 58°C for 50 s, extension at 72°C for 1 min, and the last cycle of extension at 72°C for 10 min.

[0032] (2) Purification of PCR products

[0033] The low melting point agarose (Low melting point agarose, LMPA) method was used for recovery. After the PCR product was subjected to 1% low-melting point agarose gel electrophoresis, the gel piece containing the target fragment was cut out, put into a 1.5ml Eppendorf tube, and about 200 μl of TE was added, and an equal volume of Tris saturated phenol was added to melt at 70°C for 10 C...

Embodiment 2

[0046] Embodiment 2: TEM-1 gene error-prone PCR

[0047] Error prone PCR:

[0048] 10× error-prone PCR buffer: containing 4.4mmoL / L MgCl2, 500mmoL / L KCl, 100mmoL / LTris-HCl (pH7.4), 0.1% (w / v) gelatin.

[0049] 10×dNTP mixture: contains 2mmoL / L dGTP, 2mmoL / L dATP, 10mmoL / L dCTP, 10mmoL / L dTTP.

[0050] 5mmoL / L MnCl2 solution.

[0051] Reaction system: volume

[0052] 10×Taq DNA polymerase 1μl

[0053] 10× error-prone PCR buffer 10μl

[0054] 10×dNTP mix 10μl

[0055] dCTP, dTTP 100mM 1μl each

[0056] Template DNA 4μl

[0057] Upstream primer 1μl

[0058] Downstream primer 1μl

[0059] Ultrapure water 51μl

[0060] 2.5mmol / L MgCl 2 10μl

[0061] 5mmoL / L MnCl 2 10μl

[0062] Total 100μl

[0063] PCR reaction conditions: time

[0064] Melting temperature 95°C 1min

[0065] Extension temperature 50℃ 1min

[0066] Annealing temperature 72℃ 3min

[0067] React 40 cycles,

[0068] Annealing temperature 72℃ 10min

[0...

Embodiment 3

[0070] Embodiment 3: Construction and screening of error-prone PCR mutation library

[0071] (1) Purification of mismatched PCR products is the same as the second step in Example 1

[0072] (2) Restriction enzyme digestion of mismatched PCR products

[0073] Digested with restriction enzymes Nde I and Xho I

[0074] Purify mismatched PCR product 8μl

[0075] XhoI 1μl

[0076] Nde I 1μl

[0077] 10×Universal Buffer 2μl

[0078] Deionized water 8μl

[0079] Total 20μl

[0080] Mix and incubate at 37° C. for 1 hour, and perform 2% agarose gel electrophoresis.

[0081] (3) Restriction digestion of pET24a vector DNA

[0082] Digest the plasmid vector with restriction enzymes Nde I and Xho I:

[0083] Plasmid DNA 4μl (about 2μg)

[0084] Nde I 1μl

[0085] XhoI 1μl

[0086] 10×Universal Buffer 2μl

[0087] Deionized water 12μl

[0088] Mix and incubate at 37°C for 1 hour, total 20μl

[0089] Perform 1% agarose gel electrophoresis to detect ...

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Abstract

The method to prepare high active extended spectrum beta-lactamase through directed evolution in vitro of the invention belongs to medical bioengineering technical field. In particular, the invention adopts two directed evolution strategies of error-prone PCR and DNA shuffling to transform a lab strain plant which contains TEM-1 gene. The substrate spectrum enzymes of the mutated strain plant and the wild strain plant, and the activity of the substrate spectrum enzymes are compared, showing that the original substrate spectrum is enlarged because of mutation and the degradability of the third representative generation of beta-lactam antibiotics ceftazidime (CAZ) and the cefotaxime (CTX) is improved from zero to a specific activity of 43IU / mg to 78IU / mg.

Description

technical field [0001] The method for obtaining high-activity extended-spectrum β-lactamases (extended-pectrum β-lactamases, ESBL) by in vitro directed evolution of TEM-1β-lactamases of the present invention belongs to the technical field of medical bioengineering, and more specifically mainly adopts Two strategies of directed evolution, error-prone PCR and DNA shuffling, transformed a strain containing TEM-1 gene constructed in our laboratory. Comparing the substrate spectrum enzyme and its total activity of the mutant strain and its wild strain, the substrate spectrum of the mutation is expanded, comparing the substrate spectrum enzyme and its total activity of the mutant strain and its wild strain, and the mutation The original substrate spectrum is expanded, and for the representative third-generation β-lactam antibiotic CAZ, CTX is improved from non-degradable to specific activities of 43IU / mg and 78IU / mg. Background technique [0002] Directed evolution in vitro is a ...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N1/21C12N15/10C12N9/78
Inventor 吕建新
Owner WENZHOU MEDICAL UNIV
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