Method for obtaining stable cell populations for expressing target protein in CHO cells through piggyBac transposons

A target protein and transposon technology, applied in the field of biopharmaceuticals, can solve the problems of difficult to adapt to the construction time, slow recovery of cell groups, low production efficiency, etc., so as to save the cost of liquid nitrogen storage and management and cell expression Stable, easy-to-operate results

Pending Publication Date: 2017-05-31
SHANGHAI WUXI BIOLOGIC TECH CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This integration probability is usually less than 1 in 10,000, resulting in slow recovery of the post-transfection cell population during the antibiotic selection phase
Although the traditional stable cell population has a high expression level, its construction time of several months is difficult to meet the needs of today's industry development
[0004] In the prior art, there are also reports of using other transposons to construct a method for stably expressing proteins in cells, but from the experimental results, it generally takes more than one month for the method using the Tol2 transposon in the prior art to construct a stable protein. Cell populations, the method used to produce the target protein still suffers from low production efficiency

Method used

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  • Method for obtaining stable cell populations for expressing target protein in CHO cells through piggyBac transposons
  • Method for obtaining stable cell populations for expressing target protein in CHO cells through piggyBac transposons
  • Method for obtaining stable cell populations for expressing target protein in CHO cells through piggyBac transposons

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] In this example, piggyBac transposons were used to construct different stable cell populations. After 2 days, 5 days or 7 days of antibiotic selection, batch feeding experiments were performed to express proteins, so as to obtain a suitable antibiotic selection time. Specifically, the following steps are included:

[0031] Step 1, the gene of the target protein and the antibiotic gene are cloned between a terminal inverted repeat sequence recognized by a pair of piggyBac transposase. For target proteins containing multiple expression units, such as antibodies or Fc fusion proteins, different expression units can be cloned into vectors containing different antibiotic genes.

[0032] Step 2, use the endotoxin-free plasmid extraction kit to extract the plasmids containing transposase and target protein respectively. All plasmids do not need to be digested and linearized.

[0033] In step 3, the host cells are CHO cells that have been acclimated to suspension. The expect...

Embodiment 2

[0042] In this example, piggyBac transposons were used to construct four different stable cell populations of B, C, D, and E encoding human IgG1 antibodies. After being screened with antibiotics for one week, a batch fed-feed experiment was performed to express the protein, and the number of feeding days was recorded as 10 days and 14-day protein expression data. Specifically, the following steps are included:

[0043] Step 1, the gene of the target protein and the antibiotic gene are cloned between a terminal inverted repeat sequence recognized by a pair of piggyBac transposase. For target proteins containing multiple expression units, such as antibodies or Fc fusion proteins, different expression units can be cloned into vectors containing different antibiotic genes.

[0044] Step 2, use the endotoxin-free plasmid extraction kit to extract the plasmids containing transposase and target protein respectively. All plasmids do not need to be digested and linearized.

[0045] ...

Embodiment 3

[0054] This example estimates the applicable production volumes of this method by evaluating the stability of expression in stable cell populations constructed with piggyBac transposons. Specifically, the following steps are included:

[0055] Step 1, the gene of the target protein and the antibiotic gene are cloned between a terminal inverted repeat sequence recognized by a pair of piggyBac transposase. For target proteins containing multiple expression units, such as antibodies or Fc fusion proteins, different expression units can be cloned into vectors containing different antibiotic genes.

[0056] Step 2, use the endotoxin-free plasmid extraction kit to extract the plasmids containing transposase and target protein respectively. All plasmids do not need to be digested and linearized.

[0057] In step 3, the host cells are CHO cells that have been acclimated to suspension. On the day of transfection, the host cell density is expected to be between 1 and 2E6 / ml.

[0058...

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Abstract

The invention discloses a method for obtaining stable cell populations for expressing target protein in CHO cells through piggyBac transposons. The method includes the steps that 1) plasmid of gene segments containing to-be-expressed coding target protein genes and antibiotic screening genes and plasmid containing piggyBac transposons sequences are established; 2) the plasmid established in the step 1) is transfected and led into the CHO cells at the same time; 3) the cells after being transfected in the step 2) are directly subjected to antibiotic screening in a shake flask. By means of the method, the stable cell populations can be established in one week to two weeks, and the target protein can be efficiently expressed. The invention also discloses a method for obtaining the stable cell populations for expressing the target protein in the CHO cells through the piggyBac transposons and directly producing the target protein in an enlarged mode, and production of 50 liters or above can be met.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a method for rapidly and efficiently expressing proteins using piggyBac transposons in CHO (Chinese Hamster Ovary, Chinese hamster ovary cells) cells. Background technique [0002] DNA transposons are repetitive sequences naturally present in the genome of animal cells. PiggyBac transposons were originally discovered in insect cells, and the core elements include transposase and terminal inverted repeat sequence. The gene sequence and terminal inverted repeat sequence of PiggyBac transposon have been published, and its numbers in GENBANK are GU937109.1 and ABS12111.1. The PiggyBac transposition system can transfer DNA sequences between genomes and plasmids by "cut and paste". Compared with other transposition systems, piggyBac transposase can accurately insert two terminal inverted repeat sequences and the sequence between them into the position where the genomic nucleic acid ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/65
Inventor 张峥李晓璐蔡洁行周伟昌陈智胜
Owner SHANGHAI WUXI BIOLOGIC TECH CO LTD
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