Clarifying reagent for thallus lysate and application of clarifying reagent in plasmid extraction process

Abstract

The invention discloses a clarifying reagent for a thallus lysate, which comprises a high-concentration salt solution, salts in the salt solution comprise one or more of zinc chloride, magnesium chloride, calcium chloride, copper chloride, magnesium sulfate, calcium acetate, magnesium acetate and ammonium acetate, the salts are weighed according to the proportion, water is added, the materials are fully stirred and dissolved, and the pH is adjusted to 4.0-7.2. The clarifying reagent for the thallus lysatedisclosed by the invention is added into a lysate after thalli are lysed, the lysate is left to stand for several hours, genome residues, RNA residues and host protein residues can be removed through centrifugation or clarification filtration, and a plasmid extraction process is simplified; the problem that the service life of a chromatographic column is shortened due to blockage of the chromatographic column when the chromatographic column is used for chromatography can be avoided; in a plasmid extraction process, when the plasmid is used after thallus cracking, the chromatography frequency can be reduced, the process steps can be reduced, the production efficiency can be improved, the production cost can be reduced, plasmid loss caused in the chromatography process can be avoided, and the plasmid recovery rate can be improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a clarification reagent for bacterial cell lysate. Background technique [0002] When extracting plasmid DNA on a large scale, the following steps are usually followed: The first step is to collect bacteria. That is, the culture supernatant is removed by filtration or high-speed centrifugation, and E. coli is collected. In the second step, the bacteria are lysed. That is, under certain conditions, the cells are partially or completely broken, and some or all of the intracellular substances such as plasmid DNA, chromosomal DNA, protein, and endotoxin are released outside the cell. At present, the industrial large-scale production of plasmids basically adopts the method of alkaline lysis, adding a certain concentration of sodium hydroxide to the system, and mixing it evenly in time to avoid local excessive alkalinity. In the third step, the samples are clarified after lysis. After ...

AI Technical Summary

Problems solved by technology

The production process is complicated, and most of the chromatography materials used in the production process are imported materials, and the material cost is quite expensive

During the chromatography process, the high content of impurities (host protein, host DNA, RNA) in the raw feed solution will cause the chromatography column to be easily blocked during the chromatography process, resulting in the inability to continue the chromatography

It is difficult to regenerate the used chromatographic column, the service life is short, and the number of repeatable times is small

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