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Method for removing endotoxin in phage preparation and application of method

A bacteriophage and endotoxin technology, applied in the direction of bacteriophage, virus/bacteriophage, microorganism-based method, etc., can solve the problems of limitation, different molecular size, density, sedimentation coefficient, etc., to achieve simple equipment, good removal effect, and wide market. Effect

Active Publication Date: 2021-01-05
SHANGHAI PUBLIC HEALTH CLINICAL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] The disadvantage of this method is: due to the different forms of LPS, the molecular size, density and sedimentation coefficient are different, the application of this method is limited
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Method used

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  • Method for removing endotoxin in phage preparation and application of method
  • Method for removing endotoxin in phage preparation and application of method
  • Method for removing endotoxin in phage preparation and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 Removal of free and small molecule LPS in the solution

[0081] 1. Phage amplification

[0082] Streak the host bacteria on the LB solid medium, place it in a constant temperature incubator at 37°C and cultivate it for about 12 hours, pick a single colony that conforms to the shape of the bacteria, place it in 5ml LB liquid medium, and culture it with shaking at 37°C and 220rpm About 8 hours. Transplantation was carried out at a ratio of 1%, and the host bacteria were cultivated to the logarithmic growth phase.

[0083] The host bacteria in the logarithmic growth phase were transferred according to the ratio of 1%, and an equal proportion of the target phage was added at the same time, and then placed at 37° C. and 220 rpm for shaking culture for about 2 hours. In addition, two groups of control experiments were set up, one group was only inoculated with bacteria, and the other group was neither inoculated with host bacteria nor added with phage. Comparing ...

Embodiment 2

[0099] Embodiment 2 removes macromolecule LPS and improves the titer of bacteriophage

[0100] 5. PEG concentration

[0101] Put the dialyzed solution in Step 4 of Example 1 into a suitable centrifuge tube (15ml or 50ml), add solid NaCl at 5.84g / 100ml, and fully dissolve. Then add PEG8000 according to the ratio of 10% (W / V) to dissolve it, so that the solution becomes a homogeneous solution. Then place it at 4°C overnight or for more than 8 hours, then centrifuge at 12000 rpm / min for 20 minutes, discard the supernatant after centrifugation, and invert the centrifuge tube for 2-3 minutes to remove the supernatant solution as much as possible. According to 10% of the original solution, add washing solution (0.9% physiological saline or PBS solution or SM buffer, etc.) for resuspension, and fully wash the tube wall. The resulting liquid is the phage concentrate.

[0102] Purpose of this step:

[0103] (1) Removal of macromolecular LPS in phage preparations.

[0104] (2) Remo...

Embodiment 3

[0106] Embodiment 3 removes the residue of organic reagent

[0107] 6. Chloroform extraction

[0108] Add the solution resuspended in Step 5 of Example 2 to the chloroform solution at a ratio of 1:1, vortex for 1 min, mix thoroughly, and then centrifuge at 5000 rpm / min for 5 min, the solution has obvious layers, take the supernatant and repeat the extraction once.

[0109] Purpose of this step:

[0110] (1) Further remove residual TritonX-100 reagent.

[0111] (2) Remove PEG8000 and other impurities in the solution.

[0112] 7. Dialysis

[0113] Dilute the solution obtained in step 6 according to clinical needs in an appropriate ratio, transfer it into the dialysis membrane of BiotechCE Tubing MWCO:1000kD and place it in 100 times the volume of normal saline for dialysis, and dialyze twice to remove the chloroform in the solution solution.

[0114] Purpose of this step:

[0115] (1) The main function is to remove various reagents introduced in the solution.

[0116] (2...

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Abstract

The invention relates to a method for removing endotoxin (LPS) in a phage preparation and application of the method. The method comprises the following steps: killing bacteria with chloroform, removing impurities such as bacterial fragments and the like in the phage preparation in a centrifugal mode, dissociating LPS bound to phage and degrading large molecule LPS into small molecule LPS with an active agent TritonX-100, removing free small molecule LPS by dialysis,increasing phage titer and removing large molecule LPS by PEG8000 concentration, removing introduced impurities by chloroform, andremoving an organic solvent by dialysis. The method has advantages that (1) the method has low cost and simple equipment, and can be carried out in routine laboratories; (2) LPS content in the phagepreparation is removed to be less than 5 EU / ml without affecting activity of the phage; (3) titer of active phage after recovery can reach up to 120% of that before recovery; and (4) the method has universal applicability for removal of endotoxins from phage or protein preparations.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for removing endotoxin in a phage preparation and its application. Background technique [0002] (1) Phage module: [0003] Bacterial drug resistance has become a huge challenge in the field of anti-infection, and it is also one of the issues of widespread concern in the medical field all over the world. According to the data released by China Information Drug Resistance Network in 2019, about 70% of the pathogenic bacteria that caused the disease were Gram-negative bacteria. Among the main clinical isolates in 2019, the top five were Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Acinetobacter baumannii and Pseudomonas aeruginosa. Many of these pathogenic bacteria have produced varying degrees of resistance to different types of antibiotics. As a result, the clinical treatment effect is getting worse and worse, the side effects of drugs are getting bigge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/02A61K35/76A61P31/04C12R1/92
CPCC12N7/00A61K35/76A61P31/04C12N2795/10251C12N2795/10232C12N2795/10151C12N2795/10132C12N2795/10351C12N2795/10332Y02A50/30
Inventor 朱同玉赵运泽陈立光顾敬敏程梦珺吴楠楠郭晓奎郭明权乐率秦金红
Owner SHANGHAI PUBLIC HEALTH CLINICAL CENT
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