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Biological cryoprotectant as well as preparation method and application thereof

A technology of antifreeze protein and sequence, applied in the field of biotechnology and medicine, can solve the problems of high cost and limited application of AFPIII

Pending Publication Date: 2021-04-16
XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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  • Abstract
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Problems solved by technology

[0006] Currently, there is only one company that can provide commercialized AFPIII, that is, A / F Protein Inc. The company is located in the United States and Canada. The company website is www.afprotein.com. AFPIII is derived from the separation and purification of deep-sea fish (Americanus) tissue. $10 / mg, the cost is high, and due to the import restrictions of biological products, the application of AFPIII in the medical field is limited

Method used

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  • Biological cryoprotectant as well as preparation method and application thereof
  • Biological cryoprotectant as well as preparation method and application thereof
  • Biological cryoprotectant as well as preparation method and application thereof

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Embodiment Construction

[0031] The artificial recombination of the natural antifreeze protein AFPⅢ of the deep-sea fish Zoarces americanus (ocean pout), the specific process is as follows:

[0032] 1. The amino acid sequence of natural antifreeze protein is derived from the pubmed database at: https: / / www.ncbi.nlm.nih.gov / protein / ABA41372.1? report=genpept, as follows: mksviltglflvllcvdhms sanqesvvat qlipintalt pimmkgkvvt pmgipfkemsqivgkqvnra vplgttimpemvkgyapn

[0033] 2. Cloning scheme: The amino acid sequence of the natural antifreeze protein AFPⅢ is codon-optimized and gene-synthesized according to the prokaryotic expression system. The nucleic acid sequence of the gene encoding the artificial recombinant antifreeze protein provided by the present invention is shown in sequence 4 in the sequence listing. NdeI / XhoI double restriction sites were cloned into the pET-28a cloning vector with a His tag at the N-terminal. For a schematic diagram of the cloning vector, see figure 2 .

[0034] 3. Con...

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Abstract

The invention provides a biological cryoprotectant and a preparation method thereof, and particularly relates to a pET28aAFPIII plasmid which is obtained by coding and amplifying a cDNA sequence by a pET28a cloning vector by taking a cDNA library derived from pubmed as a template by adopting an artificial recombination method for deep sea fish AFPIII. The plasmid is transformed into escherichia coli competent cells BL21, IPTG induction is carried out at different temperatures for different culture times, the temperature and the culture time with the optimal induction conditions are finally found to obtain prokaryotic expression of target protein, 2.8 mg of AFPIII can be finally separated from host cells with the culture volume of 200 mL through large-scale purification, the yield is 14 mg / L, and therefore, artificial recombination of antifreeze protein is achieved. The artificial recombinant antifreeze protein can be used as a biological cryoprotectant for cryopreservation of cells, tissues and organs.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine, and relates to antifreeze protein, in particular to an artificially recombined antifreeze protein, its preparation and its application as a biological cryoprotectant. Background technique [0002] Few biological systems can tolerate the cryocooling and rewarming process without the presence of cryoprotectants (CPA). The choice of CPA is a critical step in the cryopreservation process. The ideal CPA should penetrate into cells quickly and uniformly without causing excessive osmotic pressure difference, effectively bind intracellular water to inhibit the formation of ice crystals, and be safe, non-toxic or low-toxic. High permeability and low molecular weight are the basis for the protective effect of CPA during cryopreservation. Therefore, selecting and optimizing the composition of cryopreservation solution is crucial for the successful preservation of cells or tissues. [0003] For ...

Claims

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Application Information

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IPC IPC(8): C07K14/46C12N15/12C12N15/70A01N1/02
Inventor 潘峰刘迪
Owner XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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