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Method for preparing penicillin binding protein combined with beta-lactam antibiotic and special recombinant bacterium

A technology of binding protein and recombinant bacteria, which is applied in the field of penicillin binding protein, can solve the problem of low efficiency of penicillin binding protein, and achieve the effect of high yield and low solution yield

Inactive Publication Date: 2011-09-14
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the efficiency of producing penicillin-binding proteins is not high at present

Method used

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  • Method for preparing penicillin binding protein combined with beta-lactam antibiotic and special recombinant bacterium
  • Method for preparing penicillin binding protein combined with beta-lactam antibiotic and special recombinant bacterium
  • Method for preparing penicillin binding protein combined with beta-lactam antibiotic and special recombinant bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, the preparation of recombinant bacteria

[0031] The Streptococcus pneumoniae R6 genome was used as a template, and the following primers were used for PCR amplification to obtain PCR products; the PCR products were digested with restriction endonucleases NdeI and XhoI, and the PCR products were subjected to 1% agarose gel electrophoresis to recover about 2250bp Target gene fragment (i.e. PBP2x target gene); electrophoresis results are as follows figure 1 As shown, lanes 1-6 are the target genes, and lane 7 is the marker.

[0032] Upstream primer: 5'-TT CATATG ATGAAGTGGACAAAAAGA-3' (5' end contains NdeI restriction site and protective base)

[0033] Downstream primer: 5'-TT CTCGAG TTAGTCTCCTAAAGTTAAT-3' (3' end contains XhoI restriction site and protective base)

[0034] Recover the target gene fragment, connect it to the PMD19-T vector, transform the ligated product into Escherichia coli DH5α, screen the positive single clone through resistance, cul...

Embodiment 2

[0038] Embodiment 2, preparation and purification of protein

[0039] 1. Protein preparation

[0040] Inoculate the recombinant strain BL21-pET28b-PBP2x into LB liquid medium containing 30ug / mL kanamycin, shake at 250rpm at 37°C, and culture overnight (12h) to obtain seed liquid; take 1mL seed liquid and inoculate 100mL kanamycin-containing Namycin 30ug / mL LB liquid medium, shake at 250rpm at 37°C, to the A of the culture system 600 When it is 0.6, take out 1mL of the bacterial liquid as the uninduced control, add IPTG to the rest of the bacterial liquid to a final concentration of 1mM, shake the bacteria at 200pm 30°C, and start from the 3rd hour of induction (recorded as the 0th hour when adding IPTG), every Take out 1mL of bacterial liquid every hour, and the induction time is 3h, 4h, 5h, 6h, 7h, 8h and overnight (12h); centrifuge the collected bacterial liquid at 10000rpm at 4°C for 5min to collect the bacterial cells; use Tris-HCL (10mM Tris pH7.0) to resuspend the bact...

Embodiment 3

[0064] Embodiment 3, protein activity detection

[0065] 1. ELISA plate coated with penicillin-binding protein: the penicillin-binding protein prepared in Example 2 is configured with coating buffer to 1 ug / mL; 100 uL per well.

[0066] 2. Enzyme labeling substance: HRP-labeled ampicillin, its working concentration is 1ug / mL, and the working solution of the enzyme labeling substance is obtained by diluting the enzyme labeling substance with the sample diluent.

[0067] 3. Standard product of β-lactam antibiotics: the standard product is penicillin G freeze-dried powder, which was purchased from Sigma-Aldrich Company in the United States; the product catalog number is 46616; the penicillin G was dissolved in the sample diluent to obtain the following different concentrations Standard solution: 8.1μg / L, 2.7μg / L, 0.9μg / L, 0.3μg / L, 0.1μg / L.

[0068] 4. Substrate chromogenic solution: composed of liquid A and liquid B, liquid A is an aqueous solution of 2% carbamide peroxide, liqu...

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Abstract

The invention discloses a method for preparing a penicillin binding protein combined with beta-lactam antibiotic and a special recombinant bacterium. The recombinant bacterium is prepared by the following steps of: importing the coding gene of the penicillin binding protein into initial escherichia coli to obtain recombinant escherichia coli. Experiments prove that the method for preparing the penicillin binding protein has high yield, and the problems of low yield and difficulty in preparation in the prior art are solved. Therefore, the method has significance.

Description

technical field [0001] The invention relates to a method for preparing penicillin-binding protein combined with β-lactam antibiotics and special recombinant bacteria. Background technique [0002] Residual antibiotics in food will destroy the balance of gastrointestinal flora, and can cause long-term diarrhea, vitamin deficiency and affect the efficacy of other drugs, thereby affecting human health. At present, the problem of antibiotic residues has attracted widespread attention. As a necessity in people's life, the problem of antibiotic residues in milk has attracted more and more attention. How to ensure the quality and safety of milk, in addition to strictly controlling the use of antibiotics in the industrial chain, it is particularly important to establish a rapid detection method for antibiotics in milk. [0003] Antibiotic residues in milk mainly come from the feed of dairy cows and the treatment of diseases of dairy cows. The main disease of dairy cows is mastitis,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P21/02C12R1/19
Inventor 沈建忠吴聪明汪洋曾昆江海洋张静丁双阳史为民
Owner CHINA AGRI UNIV
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