Recombinant Abundant Protein in Late Stage of Embryo Development and Antifreeze Solution Containing It

An embryonic development, antifreeze technology, applied in fermentation, preservation of human or animal body, peptide source, etc., can solve the problems of cell loss, decreased cell viability, time-consuming and labor-intensive

Active Publication Date: 2020-10-27
THE FIRST AFFILIATED HOSPITAL OF ANHUI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The commonly used method to remove cryoprotectants is gradual dilution, but this method has disadvantages such as large osmotic pressure damage, time-consuming and labor-intensive, and easy to be contaminated by bacteria.
The addition / removal of cryoprotectant washing will not only cause cell loss, but also severely reduce cell viability

Method used

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  • Recombinant Abundant Protein in Late Stage of Embryo Development and Antifreeze Solution Containing It
  • Recombinant Abundant Protein in Late Stage of Embryo Development and Antifreeze Solution Containing It
  • Recombinant Abundant Protein in Late Stage of Embryo Development and Antifreeze Solution Containing It

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Experimental program
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preparation example Construction

[0026] The method for preparing the abundant protein in the late stage of recombinant embryonic development comprises the following steps:

[0027] First, the gene expression plasmid of the LEA protein is obtained.

[0028] LEA gene primers were designed by software, NdeI / BamHI restriction sites were introduced at both ends of the primers, and PCR amplification primers were synthesized by Invitrogen.

[0029] The double-stranded DNA fragment containing the LEA gene was obtained by PCR amplification. After the PCR amplified fragment was recovered, it was treated by the NdeI / BamHI double enzyme digestion system. The E. coli expression vector pET15b was also treated by the NdeI / BamHI double enzyme digestion system. DNA fragments are recovered. The LEA gene fragment was ligated to pET15b with T4 ligase, and the ligated product was transformed into Escherichia coli DH5α competent cells to obtain a recombinant plasmid. The recombinant plasmid was extracted for NdeI / BamHI double en...

Embodiment 1

[0034] Configure antifreeze:

[0035]

[0036] Freezing treatment:

[0037] Human mesenchymal stem cells (MSCs) in the logarithmic phase were taken, digested with trypsin, centrifuged to remove trypsin, and an appropriate amount of prepared cryopreservation solution was added. The final density of the cells was 2×10 6 / ml~1×10 7 / ml. Divide the cells into cryovials, 1.3ml per tube. Freeze according to the slow freezing procedure, and when the cooling rate is -1.5°C / min to -70°C, quickly immerse in liquid nitrogen and store for 15 days.

[0038] Rewarming:

[0039] Take out the cryopreservation tube from the liquid nitrogen container, immerse it directly in warm water at 37°C, rewarm for 5 minutes, take out the cell suspension in the cryopreservation tube, add the culture medium drop by drop, mix and centrifuge, and discard the supernatant. Add PBS containing resuspended cells.

[0040] After rewarming, flow cytometry was used to detect the cell survival rate and apoptos...

Embodiment 2

[0042] Configure antifreeze:

[0043]

[0044] Freezing treatment:

[0045] Human mesenchymal stem cells grown in logarithmic phase were taken, digested with trypsin, centrifuged to remove trypsin, and an appropriate amount of prepared cryopreservation solution was added. The final density of cells was 2×10 6 / ml~1×10 7 / ml. Divide the cells into cryovials, 1.3ml per tube. Freeze according to the slow freezing procedure, and when the cooling rate is -1.5°C / min to -70°C, quickly immerse in liquid nitrogen and store for 15 days.

[0046] Rewarming:

[0047] Take out the cryopreservation tube from the liquid nitrogen container, immerse it directly in warm water at 37°C, rewarm for 5 minutes, take out the cell suspension in the cryopreservation tube, add the culture medium drop by drop, mix and centrifuge, and discard the supernatant. Add PBS containing resuspended cells.

[0048] After rewarming, flow cytometry was used to detect the cell survival rate and apoptosis rate...

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Abstract

The invention provides a recombinant late embryogenesis abundant protein and anti-freezing liquid containing the recombinant late embryogenesis abundant protein. The recombinant late embryogenesis abundant protein has a sequence shown in SEQ ID NO.1 or a specific fragment of the sequence. The abundant protein is added into traditional cell anti-freezing liquid, so that the recovery survival rate of cryopreserved cells can be remarkably increased, and the survival rate of the cryopreserved cells can be remarkably increased; the abundant protein is non-toxic to cells and degradable in vivo, andthe low-temperature preservation process of the cells is simplified while low-temperature damage is reduced.

Description

technical field [0001] The invention relates to the technical field of recombinant protein and antifreeze, and relates to a recombinant protein abundant in the late stage of embryonic development and an antifreeze solution containing it. Background technique [0002] During the freezing process of the abundant protein in the late stage of recombinant embryonic development, the osmotic pressure of the extracellular solution will increase due to freezing. At this time, the water in the cell will flow out of the cell through the cell membrane, and the osmotic pressure of the intracellular solution will increase accordingly. Cells will shrink cell membranes or damage intracellular structures due to dehydration. If the water in the cells cannot seep out in time, a large number of ice crystals will be formed in the cells, which will damage the intracellular ice generated by the cells. At the same time, the intracellular ice crystals will continue to grow and cause damage when they...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29A01N1/02
CPCA01N1/0221C07K14/415
Inventor 曹云霞王建业章志国周平沈兵高大勇
Owner THE FIRST AFFILIATED HOSPITAL OF ANHUI MEDICAL UNIV
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