Method for obtaining chlamydomonas reinhardtii source recombinant hirudin algae powder through fermentation culture method
The recombinant hirudin algae powder from Chlamydomonas reincarnation was obtained through fermentation and culture, which solved the problems of short half-life and lack of post-translational modification function of existing recombinant hirudin preparations, and achieved products with high efficiency expression, low cost and high biosafety.
Patent Information
- Application Number
- CN202411599756.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-11
- Publication Date
- 2025-02-11
AI Technical Summary
The existing recombinant hirudin preparations have short half-life in vivo and require frequent injections, which pose a risk of infection, and the commercially available products lack post-translational modification function, resulting in lower activity.
Recombinant hirudin algae powder from Chlamydomonas reincarnation was obtained through fermentation culture method. TAP culture medium and 50×TAP concentrate were used as fermentation broth, sodium acetate and acetic acid were added for pH adjustment, and the gradient step-by-step amplification was used for fermentation. Finally, through Algae powder was obtained by vacuum freeze drying at low temperature.
It achieves efficient expression of recombinant hirudin, has low production cost, high biosafety, and stable product quality, avoids the risk of infection caused by frequent injections, and improves the anticoagulation effect.
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Figure CN119410487A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of fermentation production, and in particular to a method for obtaining Chlamydomonas reinhardtii source recombinant hirudin algae powder through a fermentation culture method. Background Art
[0002] Chlamydomonas reinhardtii is a unicellular eukaryotic organism that usually uses light as an energy source for photosynthetic autotrophy, but in the dark, it can grow heterotrophically with acetate as the only carbon source. Its cultivation process is as simple as that of microorganisms such as yeast, which makes it known as "green yeast" and "photosynthetic yeast". Chlamydomonas reinhardtii is rich in nutrients, with a protein content of up to 46.9% in the dry weight state, which is comparable to Chlorella (45.3%) and Spirulina (50.4%), which are known as superfoods. It also has a crude polysaccharide content of 12.5% and a dietary fiber content of 11.9%. It contains eight essential amino acids, a variety of trace elements, unsaturated fatty acids and various biotins. Chlamydomonas reinhardtii is considered a safe food ingredient in many countries and regions. In May 2022, China's National Health Commission officially approved Chlamydomonas reinhardtii as a new food ingredient and "unlimited consumption". Due to its high nutritional value, it has broad development prospects in the fields of alternative protein raw materials, medicine, brewing, food additives, and feed.
[0003] Hirudin is a polypeptide extracted from the salivary glands of leeches. It can specifically bind to thrombin to prevent thrombosis and has been developed as an effective anticoagulant. The FDA has approved recombinant hirudin and its derivatives, such as bivalirudin, lepirudin, desirudin, etc., which are mainly used to prevent complications of thromboembolic diseases, prevent arterial thrombosis after surgery, and prevent thrombosis after thrombolysis or vascular reconstruction. However, as a polypeptide biological drug, ordinary recombinant hirudin and its derivatives have a short half-life in the body after gastrointestinal injection. If clinical patients need frequent injections to achieve the anticoagulant effect, there will be a risk of infection. The clinical use of anticoagulants generally has the side effect of bleeding, so the development of a long-acting preparation is extremely important. In addition, since similar recombinant hirudin products on the market are mostly derived from Escherichia coli or yeast engineering bacteria, they lack the function of post-translational modification, while Chlamydomonas reinhardtii, as a new chassis organism, has this function and undergoes sulfation modification at 87Tyr, making its activity higher than recombinant hirudin products from other yeast, Escherichia coli and other sources.
[0004] Recombinant hirudin from Chlamydomonas reinhardtii is cultivated by fermentation, which has lower production costs than other similar existing drugs or recombinant hirudin from other sources. It also has a short growth cycle and stable production batches (currently, plant-derived recombinant protein drugs on the market rely on rice cultivation, which occupies arable land, has a long growth cycle, and has unstable batch sources). Summary of the invention
[0005] In view of the above problems existing in the prior art, the problem to be solved by the present invention is to provide a fermentation culture method to obtain recombinant hirudin algae powder from Chlamydomonas reinhardtii. To solve the above technical problems, the technical solution adopted by the present invention is as follows:
[0006] The method for obtaining a recombinant hirudin algae strain derived from Chlamydomonas reinhardtii by fermentation culture is characterized in that the main body of the fermentation broth is TAP culture medium, the feed culture medium is a 50×TAP concentrate without Tirs, and sodium acetate and acetic acid are further added.
[0007] As a preferred embodiment, the concentration of the added sodium acetate is 0.5 mol / L; the concentration of the acetic acid is 20%.
[0008] As a preferred embodiment, 1 mol / L sodium hydroxide and 20% acetic acid are used to adjust the pH value.
[0009] As a preferred embodiment, the defoaming agent used in the culture process is corn oil.
[0010] As a preferred embodiment, the culture conditions are temperature: 23-28° C., pH: 6.0-8.0, dissolved oxygen: >15%, and stirring speed: >100 rpm.
[0011] As a preferred embodiment, the fermentation cultivation method is characterized in that the fermentation cultivation is carried out by gradient step-by-step amplification, such as Figure 1 / 2 as shown.
[0012] As a preferred embodiment, the fermentation and cultivation method is characterized in that the recombinant hirudin algae powder is obtained by low-temperature vacuum freeze-drying at a temperature of -20°C to -80°C.
[0013] Beneficial effects:
[0014] In order to achieve efficient expression of recombinant hirudin, the present invention designs a method for obtaining recombinant hirudin algae powder from Chlamydomonas reinhardtii by fermentation culture. Compared with the existing recombinant protein drug production system, this expression system has the following advantages:
[0015] 1. Low production cost; 2. Biosafety (in 2019 and 2022, it was certified and approved by the U.S. Food and Drug Administration and the National Health Commission of China for direct unlimited consumption), the metabolites do not contain toxic substances such as endotoxins, and the cells do not carry pathogenic microorganisms; 3. It adopts high-density fermentation culture, does not occupy arable land, and ensures the stability of product quality; 4. The cell culture operation is simple, the production cycle is short, and a lot of time is saved. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 Microalgae fermentation process
[0017] Figure 2 Algae powder obtained by fermentation and cultivation of recombinant hirudin-expressing algae strains Specific implementation methods
[0019] The terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified.
[0020] In the following examples, various processes and methods not described in detail are conventional methods well known in the art.
[0021] The reagents used in the following examples were purchased from common commercial sources, and the experimental operations and experimental conditions not specified were referred to the conventional operations and conventional conditions in the art.
[0022] 1. Preparation of fermentation medium: The main body of the fermentation medium is TAP medium; the pH is adjusted with 1 mol / L sodium hydroxide and 20% volume fraction of acetic acid; corn oil is used as a defoaming agent; the feed medium is 50×TAP concentrate without Tirs, and 0.5 mol / L sodium acetate and 20% volume fraction of acetic acid are added.
[0023] 2. Seed culture: The mutant strain activated by TAP plate was inoculated into liquid TAP medium of triangular shake flask (1 / 10 of seed tank) and placed on shaker for shaking culture at 200 rpm / min and 24℃ photoperiod (light:dark=14:10h) until the late logarithmic growth phase.
[0024] 3. Sterilize the fermentation tank body and empty pipeline tanks at 121℃ for 30 minutes.
[0025] 4. Electrode calibration: Place the dissolved oxygen electrode in saturated sodium sulfite for zero point calibration, and place the pH electrode in standard buffer for calibration.
[0026] 5. Inoculation: Inject anhydrous ethanol for inoculation, ignite it and then connect the seeds from the inoculation port.
[0027] 6. Fermentation tank parameter setting: transfer the Chlamydomonas reinhardtii liquid cultured to the late logarithmic growth stage in step 2 into a 5L fermentation tank for fermentation culture, set A. temperature: 23-28°C; B. pH: 6.0-8.0; C. dissolved oxygen: >15%; D. stirring speed: >100rpm, control the speed and ventilation volume, and start detecting the foam situation and defoaming 3 days after inoculation.
[0028] 7. Fermentation status: Detect biomass every 12 hours after 3 days of inoculation. When the biomass growth rate is less than 10% within 12 hours, the fermentation can be stopped.
[0029] 8. Fermentation cycle: The whole cycle is controlled within 15 days, and 2T tank is about 7 days;
[0030] 9. Collecting algae powder: Collect the algae mud after centrifugation and freeze-dry it at -20℃ to -80℃ to obtain Chlamydomonas reinhardtii recombinant hirudin algae powder.
[0031] Related recipes
[0032] 1. Prepare TAP medium in the following order:
[0033]
[0034] 2.Phosphate Buffer Ⅱ (for 100ml) is prepared in the following order:
[0035]
[0036] 3. Solution A (for 500 ml) is prepared in the following order:
[0037]
[0038] 4. Dissolve TAP trace elements (for 1L) in the following order and mix well:
[0039]
[0040] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.
Claims
1. A method for obtaining a recombinant hirudin algae strain derived from Chlamydomonas reinhardtii by fermentation culture, characterized in that: The main body of the fermentation broth is TAP medium, and the feed medium is 50×TAP concentrate without Tirs and sodium acetate and acetic acid are added.
2. The fermentation cultivation method according to claim 1, characterized in that: The concentration of the added sodium acetate was 0.5 mol / L, and the concentration of the acetic acid was 20%.
3. The fermentation cultivation method according to claim 1, characterized in that: The pH value was adjusted using 1 mol / L sodium hydroxide and 20% acetic acid.
4. The fermentation cultivation method according to claim 1, 2 or 3, characterized in that: Corn oil was used as the defoamer during the culture process.
5. The fermentation cultivation method according to claim 1, 2 or 3, characterized in that: The culture conditions are temperature: 23-28°C, pH: 6.0-8.0, dissolved oxygen: >15%, and stirring speed: >100 rpm.
6. The fermentation cultivation method according to claim 1, 2 or 3, characterized in that: The fermentation culture was carried out by gradient step-by-step amplification.
7. The fermentation cultivation method according to claim 1, 2 or 3, characterized in that: The recombinant hirudin algae powder is obtained by low-temperature vacuum freeze-drying at a temperature of -20°C to -80°C.