Construction method of algae expression vector for expressing recombinant hirudin

Abstract

The invention relates to the technical field of genetic engineering, in particular to a construction method and culture of a recombinant hirudin expression vector with algae as chassis cells. An endogenous promoter beta2-tubulin and an RBCS2 intron sequence form a key starting element, the key starting element is linked with a Hygromycin resistance gene, a 2A peptide self-cleavage sequence is added between a recombinant hirudin gene and the Hygromycin resistance gene, and the tail end is connected with an HA tag to construct the algae expression vector. In order to improve the safety and synthesis yield of the recombinant hirudin and reduce the production cost, the invention successfully constructs an algae expression vector for expressing the recombinant hirudin; the expression vector can significantly improve the activity and yield of recombinant hirudin.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for constructing an algae expression vector for expressing recombinant hirudin. Background Art

[0002] Hirudin is the most effective natural thrombin inhibitor known and is widely used in 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. The efficacy of recombinant hirudin currently available on the market is only one-tenth of that of natural hirudin. The main reason for this difference is that natural hirudin (Sulfo hirudin) is a short peptide composed of 63 to 65 amino acids, with three disulfide-bonded functional domains at its N-terminus. The tyrosine at position 63 at the C-terminus is modified by tyrosylprotein sulfotransferase (TPST) in the Golgi apparatus of the cell to form sulfonated tyrosine. The occurrence of tyrosine sulfation modification is very important for the activity of hirudin. Without this modification, most bioengineering hosts cannot complete the post-translational modification reaction of hirudin, which makes the activity of recombinant hirudin lacking sulfonation significantly reduced compared with natural hirudin.

[0003] Studies have shown that TPST only exists in higher plants and animals and microalgae. Traditional plant and microbial production systems each have their own limitations: although the plant system has the ability of post-translational modification, it is greatly affected by the planting environment, has poor gene expression stability, and has a long growth cycle and occupies arable land; the microbial system has problems such as endotoxins and inclusion bodies, and most microorganisms lack the ability of post-translational modification, resulting in limited synthetic protein categories. In contrast, algae have the advantages of rapid growth, clear genetic background, easy transformation and expression of foreign genes, and contain TPST enzymes, which are suitable for the expression and production of recombinant hirudin. At present, in the expression and production of recombinant hirudin, there is no technical route that uses algae as a host, genetic engineering and metabolic engineering as a technical basis, and introduces foreign genes to induce secretion and expression of heterologous target proteins. The recombinant hirudin generated based on this technical route has the characteristics of tyrosine sulfation at the 63rd position of the C-terminus of natural hirudin, and has great application potential and economic value. Summary of the invention

[0004] The present invention can efficiently screen positive transformants through the resistance gene Hyg+, the self-cleaving polypeptide P2A, the target gene insertion site EcoRV and the protein tag 3×HA, and the exogenous gene can be self-cleaved independently of the protease, so as to obtain stable target protein expression. A 2A sequence is added after the resistance gene of pHyg-3HA, and then the target gene and the terminator are added, so that when the target gene protein is expressed in Chlamydomonas reinhardtii, the resistance screening gene-Hyg and the target gene-CrHirudin are simultaneously expressed on a single vector, so that the expression of the resistance screening gene-Hyg is coupled with the expression of the target gene-CrHirudin, and the acquisition rate of positive transformants is improved. Finally, through the self-cleavage mechanism between the glycine and the proline at the end of the 2A peptide, the Hyg+ resistance target protein is separated from the resistance protein after translation, thereby eliminating the interference of the resistance protein in the same reading frame, and then facilitating the study of the structure and function of the target protein.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0006] A method for constructing an algae expression vector for expressing recombinant hirudin. The vector comprises an endogenous promoter beta2-tubulin and an RBCS2 intron sequence as a key promoter element, which is linked to a hygromycin resistance gene. A 2A peptide self-cleavage sequence is added between the recombinant hirudin gene and the hygromycin resistance gene, and an HA tag is connected to the end.

[0007] Furthermore, the algae expression vector is constructed by Escherichia coli and checked by first generation sequencing.

[0008] Furthermore, the algae is Chlamydomonas reinhardtii.

[0009] Furthermore, the coding sequence of the recombinant hirudin gene is SEQ ID No.1.

[0010] Furthermore, the nucleotide sequence of the hygromycin resistance gene is SEQ ID No.2.

[0011] Furthermore, the 2A peptide self-cleavage sequence 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: In order to achieve efficient expression of recombinant hirudin, the present invention designs a method for constructing an algae expression vector for expressing recombinant hirudin. After multiple attempts, it was found that the expression vector can obtain the target protein, i.e., recombinant hirudin, more efficiently than other expression vectors. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] 1. Figure 1 Design, modification, assembly and testing of Chlamydomonas reinhardtii expression system components

[0016] 2. Figure 2 Phyg3-FMDV2A-hirudin-HA plasmid map

[0017] 3. Figure 3 Screening of pHyg3-FMDV2A-Hirudin-HA positive transformed algae strains Specific implementation methods

[0019] The terms used in the present invention, unless otherwise specified, generally have the meanings that are commonly understood by those of ordinary skill in the art.

[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] In order to increase the synthetic yield of recombinant hirudin, reduce production costs, and produce recombinant protein products with background source activity, the present invention provides a method for constructing an efficient microalgae recombinant protein drug expression vector using a single-cell eukaryotic organism, Chlamydomonas reinhardtii, as a chassis cell.

[0023] Amplification of Hirudin target fragment

[0024] 1. Using the artificially synthesized CrHirudin fragment plasmid SEQ ID No.1 as a template, PCR amplification was performed using the high-fidelity enzyme (Cat. No.: P505) of Nanjing Novozyme Biotechnology Co., Ltd. The amplification primer sequence Primer-S is SEQ ID No.5, Primer-AS is SEQ ID No.6, and the fragment length is 267bp;

[0025] 2. Add the PCR amplification system in the following order:

[0026]

[0027] 3. The PCR program was set as follows:

[0028]

[0029] 4. After 1% agarose gel electrophoresis, cut out the gel block of target size and use the gel recovery kit (B518131-0100) of Sangon Biotechnology (Shanghai) Co., Ltd. for gel recovery.

[0030] Homologous recombination to obtain expression vector

[0031] according to Figure 2 As shown, using vector pHyg3 as a template, the fragment was inserted into the vector by homologous recombination, located 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, and the first generation sequencing was performed for confirmation.

[0032] 1. Linearize the vector using restriction endonucleases from Takara and add the following reagents in order:

[0033]

[0034] 2. After 2% agarose gel electrophoresis, the gel block with a target size of 4.3 kb was cut out and gel recovery was performed using the gel recovery kit (B518131-0100) of Sangon Biotechnology (Shanghai) Co., Ltd.

[0035] 3. Perform homologous recombination using the Nanjing Novozyme Biotech Co., Ltd. kit (C112-02) and add the following reagents in the following order:

[0036]

[0037] Maintain a constant temperature of 4.37℃ for 30 minutes.

[0038] 5. Take 10uL of the recombinant product and add it to the E. coli competent cells DH5α, tap the tube wall gently, and let it stand on ice for 30 minutes.

[0039] 6. Place in a 2.0℃ water bath for 45 seconds, incubate on ice for 2 minutes, then add SOC, mix well, and incubate at 37℃, 200 rpm for 1 hour.

[0040] 7. After 1 hour, spread the liquid in the centrifuge tube onto a solid culture plate containing 100 ng / uLAmp+, blow dry, and culture inverted in a 37°C incubator for 8 hours.

[0041] 8. Pick the positive monoclonal colonies for liquid culture, shake and mix, and then perform first-generation sequencing to confirm.

[0042] Preparation of expression vector DNA

[0043] 1. Inoculate an appropriate amount of bacterial solution into a culture medium containing 100 ng / mL Amp+ and culture at 37°C, 200 rpm for 12 h.

[0044] 2. Centrifuge at 8000xg for 2 min to collect the bacteria.

[0045] 3. Use the column plasmid extraction kit (B515109-0100) of Sangon Biotech (Shanghai) Co., Ltd., add 250uL Buffer P1 suspended bacterial solution, then add 250uL Buffer P2, let stand at room temperature for 4 minutes, and finally add 300uL Buffer P3, invert 5-10 times while adding, centrifuge at 12000xg for 5-10 minutes, and pour the supernatant into the adsorption column.

[0046] 4. Add 500uL wash buffer, centrifuge at 9000xg for 30s, and repeat once.

[0047] 5. Centrifuge the empty adsorption column at 9000xg for 1 min.

[0048] 6. Place the adsorption column in a 1.5mL centrifuge tube, add 50-100uL ddH20, let stand for 1min, and centrifuge for 1min.

[0049] 7. Linearize with KPnⅠ-HF endonuclease (R3142L) from NEB and add reagents in the following order:

[0050]

[0051] 8. The product was purified using a kit (DC301-01) from Nanjing Novozyme Biotech Co., Ltd. and then electroporated into Chlamydomonas reinhardtii cells.

[0052] Screening of positive transformed algae strains

[0053] 1. Spread the electroporated cells evenly onto TAP plates containing Hygromycin resistance and culture under weak light for transformant screening.

[0054] 2. Pick the successfully grown cell transformants to continue culturing on TAP plates containing Hygromycin resistance, then pick some of the cell transformants into 24-well plates (TAP liquid culture medium with Hygromycin resistance), and culture at 24°C with shaking until the stationary phase.

[0055] 3. Collect the algae solution, extract the protein after centrifugation, and screen the cultured Chlamydomonas reinhardtii cells using western blot. The expected size of the recombinant hirudin protein is 20.2 kDa. Figure 3 shown.

[0056] 4. Screen out recombinant hirudin algae strains derived from Chlamydomonas reinhardtii that overexpress hirudin.

[0057] Related recipes

[0058] 1. Prepare TAP medium in the following order:

[0059]

[0060]

[0061] 2.Phosphate Buffer Ⅱ (for 100ml) is prepared in the following order:

[0062]

[0063] 3. Solution A (for 500 ml) is prepared in the following order:

[0064]

[0065] 4. Dissolve TAP trace elements (for 1L) in the following order and mix well:

[0066]

[0067] 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.

[0068] Sequence Listing Information:

[0069] DTD version: V1_3

[0070] File name: A method for constructing an algae expression vector for expressing recombinant hirudin.xml

[0071] Software Name:WIPO Sequence

[0072] Software version: 2.3.0

[0073] Date of creation: 2024-11-07

[0074] Basic Information:

[0075] Current application / IP office: CN

[0076] Current application / applicant profile name:Wuhan Yuanqi He Sheng Technologiae SocietasLimitata

[0077] Name of applicant: Wuhan Yuanqi Hesheng Technology Co., Ltd.

[0078] Applicant's name / language:zh

[0079] Applicant's name or title / Latin name:Wuhan Yuanqi He Sheng Technologiae SocietasLimitata

[0080] Inventor Name: Tian Aijie

[0081] Inventor Name / Language:zh

[0082] Inventor Name / Latin Name:TianAijie

[0083] Invention Title: A method for constructing an algae expression vector for expressing recombinant hirudin (zh)

[0084] Total number of sequences: 9

[0085] sequence:

[0086] Serial number (ID): 1

[0087] Length:267

[0088] Molecule type: DNA

[0089] Feature Position / Qualifier:

[0090] -source,1..267

[0091] >mol_type,other DNA

[0092] >organism,synthetic construct

[0093] Residue:

[0094]

[0095] Serial Number(ID):2

[0096] Length:909

[0097] Molecule type: DNA

[0098] Feature Position / Qualifier:

[0099] -source,1..909

[0100] >mol_type,other DNA

[0101] >organism,synthetic construct residue:

[0102]

[0103] Serial number (ID): 3

[0104] Length:72

[0105] Molecule type: DNA

[0106] Feature Position / Qualifier:

[0107] -source,1..72

[0108] >mol_type,other DNA

[0109] >organism,synthetic construct

[0110] Residue:

[0111]

[0112] Serial number (ID): 4

[0113] Residue: 000

[0115] Serial number (ID): 5

[0116] Length:22

[0117] Molecule type: DNA

[0118] Feature Position / Qualifier:

[0119] -source,1..22

[0120] >mol_type,other DNA

[0121] >organism,synthetic construct

[0122] Residue:

[0123] atgttttcgc tgaagctctt cc 22Serial number (ID): 6

[0124] Length:23

[0125] Molecule type: DNA

[0126] Feature Position / Qualifier:

[0127] -source,1..23

[0128] >mol_type,other DNA

[0129] >organism,synthetic construct

[0130] Residue:

[0131] cttgtcgtag tcctcgatag gga 23Serial number (ID): 7

[0132] Length:1948

[0133] Molecule type: DNA

[0134] Feature Position / Qualifier:

[0135] -source,1..1948

[0136] >mol_type,other DNA

[0137] >organism,synthetic construct residue:

[0138]

[0139]

[0140] Serial number (ID): 8

[0141] Length:330

[0142] Molecule type: DNA

[0143] Feature Position / Qualifier:

[0144] -source,1..330

[0145] >mol_type,other DNA

[0146] >organism,synthetic construct residue:

[0147]

[0148] Serial Number(ID):9

[0149] Length:145

[0150] Molecule type: DNA

[0151] Feature Position / Qualifier:

[0152] -source,1..145

[0153] >mol_type,other DNA

[0154] >organism,synthetic construct residues:

[0155]

Claims

1. A method for constructing an algae expression vector for expressing recombinant hirudin, characterized in that: The vector contains the endogenous promoter beta2-tubulin and the RBCS2 intron sequence as the key promoter element, which is linked to the hygromycin resistance gene, adds a 2A peptide self-cleavage sequence between the recombinant hirudin gene and the hygromycin resistance gene, and connects the HA tag at the end.

2. The method for constructing an algae expression vector for expressing recombinant hirudin according to claim 1, characterized in that: The algae expression vector was constructed by Escherichia coli and checked by first generation sequencing.

3. The method for constructing an algae expression vector for expressing recombinant hirudin according to claim 1, characterized in that: The algae is Chlamydomonas reinhardtii.

4. The method for constructing an algae expression vector for expressing 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 algae expression vector for expressing 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 algae expression vector for expressing recombinant hirudin according to claim 1, characterized in that: The 2A peptide self-cleavage sequence is SEQ ID No.

3.

7. The method for constructing an algae expression vector for expressing 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 algae expression vector for expressing recombinant hirudin according to claim 1, characterized in that: The RBCS2 intron sequence is SEQ ID No.9.

Images