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
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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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