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Production technology for preparing freeze-drying genetic engineering bacterium competence cell and protective agent formula

A technology of competent cells and genetically engineered bacteria, used in gene therapy, freeze-dried delivery, pharmaceutical formulations, etc., can solve problems affecting product quality stability and market promotion and application, product unstable biological activity, dry ice fixed-point preparation and sales personnel inconvenience And other issues

Inactive Publication Date: 2008-09-17
袁红杰 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most domestic agents and distributors do not provide -80°C refrigeration facilities, and the fixed-point preparation and delivery of dry ice also brings great inconvenience to sales staff
In addition, some scientific research institutes may cause product instability or even complete loss of biological activity during storage due to limited conditions, which have seriously affected the product's quality stability and market promotion and application.

Method used

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  • Production technology for preparing freeze-drying genetic engineering bacterium competence cell and protective agent formula
  • Production technology for preparing freeze-drying genetic engineering bacterium competence cell and protective agent formula

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Specifications 100

[0062] 1 Seed preparation

[0063] The dry powder strain Ecoli.DH5α was activated under aseptic conditions and inoculated with LB solid medium (10g trptone, 10g NaCl, 5gyeast extract, 15g agar pH7.01L) and cultivated at 37°C for 24hr. Pick several single clones separately and inoculate 25ml SOB medium (2% trptone, 10mM NaCl, 0.5% yeast extract, 2.5mM KCl, 10mM MgCl 2 , 10mM MgSO 4 ) Cultivate in a shaker at 275 rpm at 37°C, monitor the bacterial concentration when the OD550 is about 0.5, mix the cultured bacterial solution with glycerin at the ratio of 6ml bacterial solution to 4ml glycerin, mix and dispense into 1ml / tube, store at -80°C .

[0064] 2 Activation and cultivation of competent cells

[0065] The strains stored at -80°C were thawed in an ice-water bath, and 0.45ml seed solution was transferred to a large 2L large shake flask for amplification and culture. The medium was 300ml with 0.001% PEG 4000 SOB. Incubate on a shaker at 23°C ...

Embodiment 2

[0077] Example 2: 1000 specifications

[0078] 1 Seed preparation

[0079] The dry powder strain Ecoli.DH5α was activated under aseptic conditions and inoculated with LB solid medium (10g trptone, 10g NaCl, 5gyeast extract, 15g agar pH7.01L) and cultivated at 37°C for 24hr. Pick several single clones separately, and inoculate 25ml SOB medium (2% trptone, 10mM NaCl, 0.5% yeast extract, 2.5mM KCl, 10mM MgCl2, 10mM MgSO4) at 37℃, 275rpm shaker culture, and monitor the bacterial concentration OD550 at about 0.5 At the same time, mix the cultured bacterial liquid with glycerol at the ratio of 6ml bacterial liquid to 4ml glycerin, mix well and distribute into 1ml / tube, and store at -80℃.

[0080] 2 Activation and cultivation of competent cells

[0081] The strains stored at -80°C were thawed in an ice-water bath, and 0.45ml×10 kinds of liquid were transferred to a large 2L×10 large shake flask for amplification and culture. The medium was 300ml each bottle with 0.001% PEG 4000 SOB. Incu...

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Abstract

The invention discloses a manufacturing technique for preparing frozen-out gene engineering bacteria competent cells. The invention also relates to a prescription of protecting agent, which is wide in application by taking as the host bacteria for transferring outer DNA in researching, developing, producing and checking of biotechnology. The protecting agent is one of the most fundamental matching consumption preparations. The invention utilizes vacuum freeze drying technology to deal with the poor gene engineering bacteria competent cells, and can save the cells steadily at a wide temperature range of 20 DEG C below zero to 4 DEG C for long time while keeping high transformation efficiency. The invention enables the cells to store and long-distance transport conveniently. The invention relates to a manufacturing technique for preparing frozen-out competent cells, quality inspection regulation and the frozen-out protecting agent, comprising culture conditions of gene engineering bacteria, technological processes of freezing and drying and the composition and matching of the protecting agent. The protecting agent is formed by water and one or arbitrary combination of the following materials: gelatin, degreasing milk, dextran, trehalose, sucrose, sorbitol or mannitol.

Description

Technical field [0001] The invention relates to a genetically engineered host bacteria live bacteria freeze-dried powder, which is widely used in biotechnology research, development, production and testing, and belongs to one of the most basic supporting consumable preparations in biotechnology. Background technique [0002] Genetic engineering is a new biotechnology science born in the 1970s based on the comprehensive development of molecular biology and molecular genetics. An important feature of it is that foreign genetic material-nucleic acid molecules can reproduce in different host organisms, can cross the natural species barrier, and place genes from any organism into a new organism, and this organism can It has no relationship with the original organism, and this ability also determines that it will be used in a wide range of scientific fields. [0003] Generally speaking, the specific experimental process of genetic engineering is to use artificial methods to extract the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/19A61K47/42A61K48/00
Inventor 袁红杰王翰斌王翰腾
Owner 袁红杰
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