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
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[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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