Method for constructing algal expression vector expressing recombinant hirudin
By constructing an expression vector containing an endogenous promoter and a 2A peptide self-cleavage sequence in Chlamydomonas reinhardtii, the problem of insufficient recombinant hirudin activity was solved, and efficient and low-cost recombinant hirudin production was achieved. Tyrosine sulfation modification was carried out using algal TPST enzyme.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- WUHAN YUANQI HE SHENG TECHNOLOGIAE SOCIETAS LTD
- Filing Date
- 2024-11-20
- Publication Date
- 2026-05-15
AI Technical Summary
In the existing technology, the recombinant hirudin has low activity, mainly due to the lack of tyrosine sulfation modification. Traditional plant and microbial systems cannot effectively achieve this modification, resulting in insufficient activity of recombinant hirudin. It also has problems such as long growth cycle, occupation of arable land, and endotoxin.
Using Chlamydomonas reinhardtii as the host, an expression vector containing the endogenous promoter beta2-tubulin and the RBCS2 intron sequence was constructed. Combined with the Hygromycin resistance gene, the 2A peptide self-cleavage sequence and the HA tag, the recombinant hirudin was efficiently expressed and isolated. Tyrosine sulfation modification was performed using the algal TPST enzyme.
This method achieves efficient expression of recombinant hirudin, improves activity, reduces production costs, avoids the limitations of traditional systems, and provides an efficient method for producing recombinant hirudin.
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Figure CN2024133226_15052026_PF_FP_ABST
Abstract
Description
A method for constructing an algal expression vector for recombinant hirudin. Technical Field
[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a method for constructing an algal expression vector for recombinant hirudin. Background Technology
[0002] Hirudin is the most potent known natural thrombin inhibitor and is widely used clinically for the treatment of cardiovascular diseases. However, its application in the medical field is severely limited by the low yield and high extraction cost of natural hirudin. Currently available recombinant hirudin has only one-tenth the efficacy of natural hirudin. The main reason for this difference is that natural hirudin is a short peptide composed of 63-65 amino acids, with a functional domain consisting of three disulfide bonds at its N-terminus. The tyrosine residue at position 63 of the C-terminus is modified by tyrosyl protein sulfotransferase (TPST) in the Golgi apparatus of the cell, forming a sulfonated tyrosine residue. This tyrosine sulfation modification is crucial for hirudin activity. Without this modification, most bioengineered hosts cannot complete the post-translational modification reaction of hirudin, resulting in a significant reduction in the activity of recombinant hirudin lacking sulfonation compared to natural hirudin.
[0003] Studies have shown that TPST exists only in higher plants, animals, and microalgae. Traditional plant and microbial production systems each have their limitations: while plant systems possess post-translational modification capabilities, they are greatly affected by the growing environment, have poor gene expression stability, and have long growth cycles and occupy arable land; microbial systems suffer from problems such as endotoxins and inclusion bodies, and most microorganisms lack post-translational modification capabilities, resulting in limited protein synthesis. In contrast, algae have advantages such as rapid growth, clear genetic background, ease of transformation and expression of exogenous genes, and contain TPST enzymes, making them suitable for the expression and production of recombinant hirudin. Currently, in the expression and production of recombinant hirudin, there is no known technical route that uses algae as a host, relies on genetic engineering and metabolic engineering, and induces the secretion of heterologous target proteins by introducing exogenous genes. Recombinant hirudin generated based on this technical route has the characteristic of natural hirudin with sulfation of the tyrosine residue at position 63 of the C-terminus, and has great application potential and economic value. Summary of the Invention
[0004] This invention enables efficient screening of positive transformants using the resistance gene Hyg+, the self-cleaving peptide P2A, the target gene insertion site EcoRV, and the protein tag 3×HA. Furthermore, the exogenous gene can self-cleave independently of proteases, resulting in stable expression of the target protein. By adding a 2A sequence after the resistance gene pHyg-3HA, followed by the target gene and a terminator, simultaneous expression of the resistance selection gene -Hyg and the target gene -CrHirudin on a single vector is achieved during target gene protein expression in *Chlamydomonas reinhardtii*. This couples the expression of the resistance selection gene -Hyg and the target gene -CrHirudin, increasing the positive transformant yield. Finally, the self-cleavage mechanism between the glycine and proline residues at the 2A peptide terminus ensures that the Hyg+ resistance target protein separates from the resistance protein after translation, eliminating interference from resistance proteins within the same reading frame and facilitating the study of the target protein's structure and function.
[0005] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution:
[0006] A method for constructing an algal expression vector for recombinant hirudin, wherein the vector comprises a key promoter element consisting of an endogenous promoter beta2-tubulin and an RBCS2 intron sequence, which is linked to a Hygromycin resistance gene. A 2A peptide self-cleaving sequence is added between the recombinant hirudin gene and the Hygromycin resistance gene, and an HA tag is attached to the end.
[0007] Furthermore, the algal expression vector was constructed using Escherichia coli and examined using first-generation sequencing.
[0008] Furthermore, the algae mentioned is Chlamydomonas reinhardtii.
[0009] Furthermore, the gene encoding sequence of the recombinant hirudin is SEQ ID No. 1.
[0010] Furthermore, the nucleotide sequence of the Hygromycin resistance gene is SEQ ID No. 2.
[0011] Furthermore, the self-cleaving sequence of the 2A peptide is SEQ ID No. 3.
[0012] Furthermore, the endogenous promoter beta2-tubulin sequence is SEQ ID No. 8.
[0013] Furthermore, the RBCS2 intron sequence is SEQ ID No. 9.
[0014] Beneficial effects: To achieve efficient expression of recombinant hirudin, this invention presents a method for constructing an algal expression vector for recombinant hirudin. After multiple trials and comparisons, it was found that this expression vector can obtain the target protein, i.e., recombinant hirudin, more efficiently than other expression vectors. Attached Figure Description
[0015] 1. Figure 1. Design, modification, assembly and testing of Chlamydomonas reinhardtii expression system elements.
[0016] 2. Figure 2. Phyg3-FMDV2A-hirudin-HA plasmid map
[0017] 3. Figure 3 Screening of pHyg3-FMDV2A-Hirudin-HA positive transformed algal strains
[0018] Specific implementation methods
[0019] The terms used in this invention, unless otherwise stated, generally have the meanings commonly understood by those skilled in the art.
[0020] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art.
[0021] The reagents used in the following examples were obtained through common commercial channels. Experimental procedures and conditions not specified are in accordance with conventional procedures and conditions in the art.
[0022] To increase the yield of recombinant hirudin, reduce production costs, and produce recombinant protein products with background-derived activity, this invention provides a method for constructing a highly efficient microalgal recombinant protein drug expression vector using the single-celled eukaryotic organism Chlamydomonas reinhardtii as the chassis cell.
[0023] Amplifying the Hirudin target fragment
[0024] 1. Using the artificially synthesized CrHirudin fragment plasmid SEQ ID No. 1 as a template, PCR amplification was performed using high-fidelity enzyme (product number: P505) from Nanjing Novizan Biotechnology Co., Ltd. The amplification primer sequences Primer-S are SEQ ID No. 5 and Primer-AS are SEQ ID No. 6, with a fragment length of 267 bp.
[0025] 2. Add the following ingredients to the PCR amplification system in the following order:
[0026] 3. The PCR program should be set as follows:
[0027] 4. After 1% agarose gel electrophoresis, cut off the gel block of the target size and perform gel recovery using the gel recovery kit (B518131-0100) from Sangon Biotech (Shanghai) Co., Ltd.
[0028] Homologous recombination to obtain expression vectors
[0029] As shown in Figure 2, using the vector pHyg3 as a template, the fragment was inserted into the vector using homologous recombination. It was positioned after the sequence fragment of the self-cleaved polypeptide P2A and before the tag HA, thus constructing the final target gene expression plasmid SEQ ID No. 7, which was then confirmed by first-generation sequencing.
[0030] 1. Linearize the vector using restriction endonucleases from Takara, adding the following reagents in sequence:
[0031] 2. After 2% agarose gel electrophoresis, a 4.3kb gel block was cut off and the gel was recovered using the gel recovery kit (B518131-0100) from Sangon Biotech (Shanghai) Co., Ltd.
[0032] 3. Homologous recombination was performed using the Nanjing Novizan Biotechnology Co., Ltd. reagent kit (C112-02). Reagents were added in the following order:
[0033] 4. Maintain a constant temperature of 37℃ for 30 minutes.
[0034] 5. Add 10 μL of the recombinant product to E. coli competent cells DH5α, gently tap the tube wall, and let stand on ice for 30 min.
[0035] 6. Incubate in a 42.0℃ water bath for 45 seconds, then incubate on ice for 2 minutes. After that, add SOC, mix well, and incubate at 37℃ and 200 rpm for 1 hour.
[0036] 7. After 1 hour, spread the liquid from the centrifuge tube onto a solid culture plate containing 100 ng / uL Amp+, dry it, and then incubate it upside down in a 37°C incubator for 8 hours.
[0037] 8. Select positive single-clone colonies for liquid culture, shake to mix, and then perform first-generation sequencing for confirmation.
[0038] Preparation of expression vector DNA
[0039] 1. Inoculate an appropriate amount of bacterial culture into a medium containing 100 ng / mL Amp+ and incubate at 37°C and 200 rpm for 12 h.
[0040] 2. Centrifuge at 8000xg for 2 minutes to collect bacteria.
[0041] 3. Using the plasmid extraction kit (B515109-0100) from Sangon Biotech (Shanghai) Co., Ltd., add 250 μL of Buffer P1 to suspend the bacterial culture, then add 250 μL of Buffer P2, let stand at room temperature for 4 min, and finally add 300 μL of Buffer P3 while inverting the container 5-10 times. Centrifuge at 12000 x g for 5-10 min, and pour the supernatant into the adsorption column.
[0042] 4. Add 500uL of wash buffer, centrifuge at 9000xg for 30s, and repeat once.
[0043] 5. Centrifuge the empty adsorption column at 9000xg for 1 min.
[0044] 6. Place the adsorption column into a 1.5 mL centrifuge tube, add 50-100 μL of ddH2O, let stand for 1 min, and centrifuge for 1 min.
[0045] 7. Linearization was performed using NEB KPnI-HF endonuclease (R3142L), with reagents added in the following order:
[0046] 8. After product purification using the Nanjing Novizan Biotechnology Co., Ltd. kit (DC301-01), the product was electroporated into Chlamydomonas reinhardtii cells.
[0047] Screening for positive transformed algal strains
[0048] 1. Spread the electrolyzed cells evenly onto TAP plates containing Hygromycin resistance and culture them under low light for transformant selection.
[0049] 2. Select successfully grown cell transformants and transfer them to TAP plates containing Hygromycin resistance for further culture. Then, select a portion of the cell transformants and transfer them to 24-well plates (containing TAP liquid medium with Hygromycin resistance). Culture at 24°C with shaking until the plateau phase.
[0050] 3. Collect the algal solution, centrifuge and extract the protein. Use Western blot to screen the cultured Chlamydomonas reinhardtii cells. The expected size of the recombinant hirudin protein is 20.2 kDa, as shown in Figure 3.
[0051] 4. Screen out recombinant hirudin-containing Chlamydomonas reinhardtii strains that overexpress hirudin.
[0052] Related formulas
[0053] 1. TAP medium should be prepared in the following order:
[0054] 2. Prepare Phosphate Buffer II (for 100ml) in the following order:
[0055] 3. Solution A (for 500ml) should be prepared in the following order:
[0056] 4. Dissolve and mix the TAP trace elements (for 1L) separately in the following order:
[0057] 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 invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.
[0058] Sequence list information:
[0059] DTD Version: V1_3
[0060] Filename: A method for constructing an algal expression vector for recombinant hirudin.xml
[0061] Software Name: WIPO Sequence
[0062] Software version: 2.3.0
[0063] Generation Date: 2024-11-07
[0064] Basic Information:
[0065] Current application / Intellectual Property Office: CN
[0066] Current application / applicant file name: Wuhan Yuanqi He Sheng Technologiae Societas Limitata
[0067] Applicant's Name or Company Name: Wuhan Yuanqi Hesheng Technology Co., Ltd.
[0068] Applicant's name or title / language:zh
[0069] Applicant's Name or Title / Latin Name: Wuhan Yuanqi He Sheng Technologiae Societas Limitata
[0070] Inventor's Name: Tian Aijie
[0071] Inventor's name / language: zh
[0072] Inventor's Name / Latin Name: Tian Aijie
[0073] Invention Title: A Method for Constructing an Algal Expression Vector for Recombinant Hirudin (zh)
[0074] Total number of sequences: 9
[0075] sequence:
[0076] Serial Number (ID): 1
[0077] Length: 267
[0078] Molecular type: DNA
[0079] Feature location / qualifier:
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[0081] >mol_type, other DNA
[0082] >organism, synthetic construct
[0083] residues:
[0084] Serial Number (ID): 2
[0085] Length: 909
[0086] Molecular type: DNA
[0087] Feature location / qualifier:
[0088] -source,1..909
[0089] >mol_type, other DNA
[0090] >organism, synthetic construct
[0091] residues:
[0092] Serial Number (ID): 3
[0093] Length: 72
[0094] Molecular type: DNA
[0095] Feature location / qualifier:
[0096] -source,1..72
[0097] >mol_type, other DNA
[0098] >organism, synthetic construct
[0099] residues:
[0100] Serial Number (ID): 4
[0101] residues:
[0102] 000
[0103] Serial Number (ID): 5
[0104] Length: 22
[0105] Molecular type: DNA
[0106] Feature location / qualifier:
[0107] -source,1..22
[0108] >mol_type, other DNA
[0109] >organism, synthetic construct
[0110] residues:
[0111] Serial Number (ID): 6
[0112] Length: 23
[0113] Molecular type: DNA
[0114] Feature location / qualifier:
[0115] -source,1..23
[0116] >mol_type, other DNA
[0117] >organism, synthetic construct
[0118] residues:
[0119] Serial Number (ID): 7
[0120] Length: 1948
[0121] Molecular type: DNA
[0122] Feature location / qualifier:
[0123] -source,1..1948
[0124] >mol_type, other DNA
[0125] >organism, synthetic construct
[0126] residues:
[0127] Serial Number (ID): 8
[0128] Length: 330
[0129] Molecular type: DNA
[0130] Feature location / qualifier:
[0131] -source,1..330
[0132] >mol_type, other DNA
[0133] >organism, synthetic construct
[0134] residues:
[0135] Serial Number (ID): 9
[0136] Length: 145
[0137] Molecular type: DNA
[0138] Feature location / qualifier:
[0139] -source,1..145
[0140] >mol_type, other DNA
[0141] >organism, synthetic construct
[0142] residues:
[0143] END
Claims
1. A method for constructing an algal expression vector for recombinant hirudin, characterized in that, The vector contains the endogenous promoter beta2-tubulin and the RBCS2 intron sequence as key promoter elements, which are linked to the Hygromycin resistance gene. A 2A peptide self-cleaving sequence is added between the recombinant hirudin gene and the Hygromycin resistance gene, and an HA tag is attached to the end.
2. The method for constructing an algal expression vector for recombinant hirudin according to claim 1, characterized in that, The algal expression vector was constructed using Escherichia coli and examined using first-generation sequencing.
3. The method for constructing an algal expression vector for recombinant hirudin according to claim 1, characterized in that, The algae mentioned is Chlamydomonas reinhardtii.
4. The method for constructing an algal expression vector for recombinant hirudin according to claim 1, characterized in that, The recombinant hirudin gene coding sequence is SEQ ID No.
1.
5. The method for constructing an algal expression vector for recombinant hirudin according to claim 1, characterized in that, The nucleotide sequence of the Hygromycin resistance gene is SEQ ID No.
2.
6. The method for constructing an algal expression vector for recombinant hirudin according to claim 1, characterized in that, The self-cleavage sequence of the 2A peptide is SEQ ID No.
3.
7. The method for constructing an algal expression vector for recombinant hirudin according to claim 1, characterized in that, The endogenous promoter beta2-tubulin sequence is SEQ ID No.
8.
8. The method for constructing an algal expression vector for recombinant hirudin according to claim 1, characterized in that, The RBCS2 intron sequence is SEQ ID No. 9.