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Method for preparing recombinant protein

A recombinant protein and production stage technology, applied in biochemical equipment and methods, chemical instruments and methods, recombinant DNA technology, etc., can solve the problems of less cells, more equipment requirements, complexity, etc., and achieve simple process and high output , the effect of improving output efficiency

Pending Publication Date: 2020-11-06
深圳市陆景生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, adherent cells need to be acclimatized for a long time when they are suspended, and a supporting suspension culture system is required
On the other hand, for the suspension culture conditions, the culture process and cell line also need to be optimized separately, which makes the suspension culture system a huge system, requiring a lot of equipment and complicated
However, when using the adherent culture method, despite the simple equipment requirements, the yield of recombinant protein in the same culture medium is lower due to the lower number of cells per unit volume

Method used

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  • Method for preparing recombinant protein
  • Method for preparing recombinant protein
  • Method for preparing recombinant protein

Examples

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Embodiment 1

[0027] This example provides a method for producing CD89 protein, which mainly expresses CD89 protein by plasmid transfection into HEK293 cells. Among them, HEK293 cells came from Shanghai Mingjin Biotechnology Co., Ltd., and the corresponding recombinant protein particles were donated by Professor Jin Jing from Northwestern University. In this method, Lipofectamine 2000 transfection reagent was used to transfect the corresponding recombinant protein plasmid in advance, and the HEK293 monoclonal cell line stably expressing the target protein was obtained through G418 screening, and then frozen and stored in liquid nitrogen for future use.

[0028] figure 1 and figure 2 Respectively, the time flow of the growth stage and the production stage of the method of the present embodiment, refer to figure 1 and figure 2 , the specific steps of the method are as follows:

[0029] 1. Growth stage

[0030] 1. Cell recovery and scale-up culture

[0031] 1) Resuscitation of frozen c...

Embodiment 2

[0062] This example provides a method for producing recombinant human ACE2-Fc protein, in which the ACE2-Fc protein is mainly expressed by plasmid transfection into HEK293 cells. Among them, HEK293 cells come from Shanghai Mingjin Biotechnology Co., Ltd., and the corresponding recombinant protein plasmid is synthesized by GenScript Biotechnology Co., Ltd. (The sequences of the target gene human ACE2 and Fc tag used in the plasmid can be obtained from NCBI or related literature. ). In this method, Lipofectamine 2000 transfection reagent was used to transfect the corresponding recombinant protein plasmid in advance, and the HEK293 monoclonal cell line stably expressing the target protein was obtained through G418 screening, and then frozen and stored in liquid nitrogen for future use.

[0063] The specific steps of the method are as follows:

[0064] 1. Growth stage

[0065] 1) Resuscitation of frozen cells: Carefully take out the frozen cells in liquid nitrogen, quickly put t...

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Abstract

The invention provides a method for preparing recombinant protein. The method comprises a growing stage and a production stage, wherein the production stage comprises the following steps: S1, culturing mammalian cells expressing recombinant protein in a serum-free culture medium; S2, collecting supernatant; S3, culturing the supernatant in a serum culture medium for 12-48 hours; and S4, replacingthe serum culture medium with a serum-free culture medium, and repeating S1-S2 for one or more times. The method at least has the following beneficial effects that, the serum-free culture medium is replaced by the serum culture medium after one round of supernatant collection from cells in a bioreactor is completed in the production stage, the state and vitality of cells can be quickly restored byutilizing ingredients beneficial to cell growth in serum, subsequent round of protein production can be constantly performed, and considerable production efficiency can be maintained, so that the cells at the terminal stage of the same round of growth stage can realize production of recombinant protein in multiple rounds and multiple batches in the same bioreactor, and one round of cell output efficiency can be effectively improved.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for producing recombinant protein. Background technique [0002] With the support of gene recombination technology, the artificially synthesized in vitro recombinant protein industry is booming, covering many fields. These recombinant proteins specifically include recombinant protein drugs and recombinant enzymes. Compared with Escherichia coli expression system, yeast expression system or insect baculovirus expression system, mammalian cell expression system can provide recombinant human protein with the post-translational modification closest to the natural state, and will form a natural state during protein expression. The protein folding and aggregation of the protein has the necessary spatial structure and modification of the active protein, so it is far superior to other eukaryotic expression systems in terms of activity and is closer to the natural protein. ...

Claims

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Application Information

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IPC IPC(8): C12P21/02C07K14/735C07K19/00C12N15/85
CPCC12P21/02C07K14/70535C12N9/485C12Y304/17023C12N15/85C07K2319/30C12N2800/107
Inventor 刘月鹏王朝阳蒋娟
Owner 深圳市陆景生物技术有限公司