Preparation method of high-activity recombinant human TGF-beta1

Abstract

The invention discloses a preparation method of high-activity recombinant human TGF-beta1, belongs to the technical field of biology, provides a preparation method of high-activity recombinant human TGF-beta1 protein and the prepared recombinant human TGF-beta1 protein, and further provides polynucleotide for coding the recombinant human TGF-beta1 protein. According to the preparation method, the recombinant human TGF-beta1 protein with high activity and high purity can be obtained. The recombinant human TGF-beta1 protein can be used for preparing the recombinant human TGF-beta1 protein.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for preparing highly active recombinant human TGF-β1. Background Art

[0002] Transforming growth factor beta 1 (TGF-β1) is a multifunctional cytokine that belongs to the transforming growth factor superfamily, which includes three different mammalian isoforms, TGF-β1, β2, β3, and many other signaling proteins. TGF-β protein can be produced by a variety of white blood cells, including macrophages, which can regulate the inflammatory response in the intestine and play a vital role in stem cell differentiation. TGF-β1 can promote the proliferation of T-helper 17 cells (Th17) cells and regulatory T-cells (Treg). TGF-β1 can promote the proliferation of rodent fibroblasts. It can inhibit cells from staying in the G1 phase, control cell apoptosis, and promote fibrosis.

[0003] After TGF-β protein synthesis, the homodimer of TGF-β interacts with a LAP to form a small complex called small latent complex (SLC). The SLC complex binds to the LTBP (latent TGF-β-binding protein) protein in the cell to form a large complex called large latent complex (LLC). LLC can be secreted into the cell matrix. Integrins can bind to the bowtie-shaped tail of LAP, causing the LAP polypeptide chain to bend and release TGF-β1. Summary of the invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions.

[0005] A method for preparing a highly active recombinant human TGF-β1 protein, the preparation method comprising the following steps: 1) HEK293 serum-free basal medium (Beijing Tongli Haiyuan, AS-11) and sterile plasmid DNA were mixed at a ratio of 25 ml:1 mg, wherein the amino acid sequence expressed by the sterile plasmid DNA is shown in SEQ ID NO:1; 2) adding the mixed solution in step 1) to the premixed transfection solution, wherein the premixed transfection solution comprises HEK293 serum-free basal medium and TA-293 transfection reagent (Zhuhai Kairui, K20001), and the ratio of HEK293 serum-free basal medium to TA-293 transfection reagent is 25 ml:5 ml, to obtain a plasmid-vector complex; 3) The plasmid-vector complex was allowed to stand at room temperature for 10 minutes; 4) Take the cells for transfection and add the prepared plasmid-vector complex while shaking to complete the transfection. Put the transfected cells back into the cell culture constant temperature shaker for shaking culture. The current day is Day 0. 5) On transfection day 1, add the initial volume of 0.6% KE-293 (Zhuhai Kairui, K30001) and 5% feed medium (Tongli Haiyuan, AS-18) (i.e., add 6 mL KE-293 and 50 mL feed medium to 1 L cell culture medium); 6) Add 5% of the initial volume of feed medium on transfection day 3; 7) On Day 5, centrifuge at 10,000 rpm for 10 min to harvest the cell solution; 8) In step 7), the cell feed solution was purified by Ni, using 5 mL Ni-NTA Purose 6 FastFlow, with 4%, 10%, and 50% equilibration-loading-equilibration-elution processes, the equilibration solution was 50 mM Tris 0.1 M NaCl pH 8.0, and the elution solution was 50 mM Tris 0.1 M NaCl 0.5 M imidazole pH 8.0; 9) The product of step 8) was further purified by cationic chromatography, specifically using 5 mL SP SepharoseTMFast Flow, adjusted to cond: 4.8 mS / cm with injection water, adjusted to pH 4.5 with 6 M HCl, and carried out a process of equilibrium-loading-equilibrium-step elution, wherein the equilibrium solution components were 20 mM NaAc, 150 mM NaCl, 20 mM ammonium acetate, 0.5 M urea 1% glacial acetic acid, pH 4.5; the eluent components were 20 mM NaAc, 1 M NaCl, 0.5 M urea, 1% glacial acetic acid, pH 4.5, to obtain purified highly active recombinant human TGF-β1 protein.

[0006] Furthermore, the cell density and viability of cells need to be determined before transient transfection.

[0007] Furthermore, the cells were in the exponential growth phase and the cell density was about 2-4×10 6 Cells were transfected at a survival rate of more than 95%.

[0008] Further, the cells do not need to be centrifuged.

[0009] Furthermore, the cell density is 3.0×10 6 When the concentration is 100 μg / ml, directly transfect.

[0010] Furthermore, the cell density is >3.0×10 6 / ml, add HEK293 serum-free basal medium, and dilute the cell density to 3×10 6 / ml, and then transfect.

[0011] Furthermore, the cells were cultured in a 5% CO2 constant temperature shaker at 37°C and 120 rpm for transfection.

[0012] Furthermore, 1 ml of the cell suspension was taken on Day 1 and Day 3 for counting, recording the cell density and viability, and centrifuging to retain the supernatant.

[0013] Furthermore, on Day 4, 1 ml of the cell suspension was taken for counting, the cell density and viability were recorded, and the cell suspension was centrifuged to retain the supernatant.

[0014] Further, the initial volume of 0.6% KE-293 is 6 ml KE-293.

[0015] Further, the supplement volume of the 5% feed medium is 50 mL.

[0016] In some embodiments, in the preparation method, HEK293 serum-free basal medium, TA-293 transfection reagent, and KE-293 are from HEK-293 transient expression kit of Zhuhai Kairui.

[0017] The present invention provides a high-purity and high-activity recombinant human TGF-β1 protein, wherein the recombinant human TGF-β1 protein is prepared by the above-mentioned preparation method.

[0018] The present invention provides a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1.

[0019] The present invention provides a vector, which comprises the polynucleotide described above.

[0020] Furthermore, the vector includes a plasmid vector and a viral vector.

[0021] Furthermore, the viral vector includes a lentiviral vector, an influenza virus vector, a hepatitis virus vector, an alpha virus vector, a filovirus vector, an adenovirus vector, an adeno-associated virus vector and / or a flavivirus vector.

[0022] Furthermore, the adeno-associated viral vector includes ssAAV, scAAV and / or hybrid AAV subtypes.

[0023] Furthermore, the adeno-associated virus vector includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13.

[0024] Furthermore, the vector is a plasmid vector.

[0025] In some embodiments, the function of transporting nucleic acid molecules possessed by the vector used in the present invention is well known to the public, and the technologies, methods and materials required for transporting nucleic acid molecules are publicly available in technical documents in the corresponding fields. The technologies include but are not limited to vector construction, connection between the vector and the target gene, transport of the vector, and cell culture after transport.

[0026] In some embodiments, the vector may also contain other appropriate "regulatory elements" or "regulatory sequences", including but not limited to enhancers; transcription factors; transcription terminators; efficient RNA processing signals, such as splicing and polyadenylation signals (polyA); sequences that stabilize cytoplasmic mRNA, such as woodchuck hepatitis virus (WHV) post-transcriptional regulatory elements (WPRE); sequences that enhance translation efficiency (i.e., kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance secretion of the encoded product when necessary. In certain embodiments, examples of polyA include SV40, bovine growth hormone (bGH) and TKpolyA. In certain embodiments, examples of enhancers include alpha fetoprotein enhancer, TTR minimal promoter / enhancer, LSP (TH binding globulin promoter / alpha 1-microglobulin / bikunin enhancer) and other enhancers.

[0027] In some embodiments, the viral vector is an enveloped virus or a non-enveloped virus. In some embodiments, examples of enveloped viruses are selected from the family in the group including: herpesvirus, poxvirus, hepadnavirus, flavivirus, togavirus, coronavirus, hepatitis delta, orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, filovirus and retrovirus. In some embodiments, examples of non-enveloped viruses are selected from the family in the group including: adenovirus, reovirus, papillomavirus, picornavirus, calicivirus.

[0028] The present invention provides a cell, wherein the cell comprises the above-mentioned vector.

[0029] Furthermore, the cell includes a prokaryotic cell or a eukaryotic cell.

[0030] Furthermore, the prokaryotic cells include bacteria.

[0031] Furthermore, the bacteria include Escherichia coli.

[0032] Furthermore, the cells include human or non-human mammalian cells.

[0033] Furthermore, the cells are HEK293 cells.

[0034] The present invention provides the use of the aforementioned preparation method, the aforementioned polynucleotide, the aforementioned vector, and the aforementioned cell in the preparation of high-purity and high-activity recombinant human TGF-β1 protein.

[0035] In the present invention, vector refers to a medium into which a polynucleotide encoding a protein can be operably inserted to cause the expression of the protein. The vector can be used for transformation, transduction or transfection of a host cell so that it expresses the genetic elements carried in the host cell. The vector can contain a variety of elements for controlling expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element and a reporter gene. In addition, the vector can contain a replication origin. The term replication origin refers to a sequence that initiates replication when present in a vector. The replication origin can be recognized by a replication initiation factor or alternatively by a DNA helicase. The vector can also include materials that help it enter the cell, including but not limited to virus particles, liposomes or protein envelopes.

[0036] Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phage and M13 phage, plasmids. Specific examples of vectors include, but are not limited to, pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, and pEF-Bos.

[0037] In the present invention, the cell is a cell for receiving, maintaining, replicating and amplifying the vector. The cell can also be used to express the polypeptide encoded by the vector. When the cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. In one embodiment, the cell is a genetic package that can be induced to express a variant polypeptide on its surface. In another embodiment, the cell is infected with a genetic package.

[0038] The cell can be virtually any cell for which an expression vector is available. This includes prokaryotic cells, eukaryotic cells, including but not limited to true bacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, such as Escherichia (DH5α, BL21DE3, BL21DE3pLysS, JM109, TOP10, HB101, SCS110, E. coli JM110); Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, such as Salmonella typhimurium; Serratia, such as Serratia marcescens. marcescans); and Shigella; as well as Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as P. aeruginosa; and Streptomyces.

[0039] Eukaryotic cells include, but are not limited to, protist cells, animal cells, plant cells, or fungal cells, wherein the animal cells include mammalian cells, avian cells, and insect cells. Among them, mammalian cells include, but are not limited to, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, 293T cells, 293F cells, and HEK293 cells.

[0040] The use of the terms "any," "optional," or "either" in this disclosure means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.

[0041] The terms "polynucleotide" or "nucleic acid" used in the present invention are used interchangeably herein and refer to nucleotide polymers of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. Thus, for example, the polynucleotides defined herein include but are not limited to single-stranded and double-stranded DNA, DNA comprising single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA comprising single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA, which can be single-stranded, or more typically double-stranded, or comprising single-stranded and double-stranded regions. In addition, the term "polynucleotide" when used herein refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The chains in such regions can come from the same molecule or from different molecules. The region can include the entirety of one or more molecules, but more typically only includes a region of some molecules. One of the molecules in the triple helix region is often an oligonucleotide.

[0042] The nucleotides may also be modified at the level of the nucleotide bond (e.g., phosphorothioate, H-phosphate, alkyl phosphate), at the level of the backbone (e.g., α-oligonucleotides) or PNA or 2′-O-alkyl ribose). Each of these modifications may occur in combination, as long as at least one phosphate is present in the nucleotide. The nucleotides may be natural or synthetic, oligonucleotides, polynucleotides, nucleic acid fragments, ribosomal RNA, messenger RNA, transfer RNA, nucleotides obtained by enzymatic amplification techniques. The enzymatic amplification techniques are, for example, PCR (polymerase chain reaction) and its RT-PCR derivatives, PCR (repair chain reaction), 3SR (self-maintaining sequence amplification), NASBA (nucleic acid sequence-dependent amplification), TMA (transcription-mediated amplification).

[0043] The carrier of the present invention can also contain a selection marker gene, which helps to select cells transformed or transfected with the recombinant molecule of the present invention. Examples of selection marker genes are genes encoding proteins that confer resistance to certain drugs, such as neomycin and hygromycin, beta-galactosidase, chloramphenicol acetyltransferase, firefly luciferase or immunoglobulin or its portion, such as the Fc portion of an immunoglobulin, preferably IgG. The transcription of the selection marker gene is monitored by changes in the concentration of a selection marker protein such as beta-galactosidase, chloramphenicol acetyltransferase or firefly luciferase.

[0044] The present invention uses the terms "transformation", "transduction" and "transfection" to include the introduction of nucleic acids (e.g., vectors) into cells by one of many possible techniques known in the art. As used herein, the term "transformed cells" or "transfected cells" is also intended to include cells that have been transformed with the vectors of the present invention and are capable of producing recombinant human TGF-β1 protein. Prokaryotic cells can be transformed with nucleic acids by, for example, electroporation or calcium chloride-mediated transformation. For example, methods for introducing nucleic acids into mammalian cells by conventional techniques, such as calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, electroporation or microinjection can be found in the prior art.

[0045] Throughout the present invention, the term "and / or" is a grammatical conjunction and should be understood as its connection covering one or more situations that may occur. Furthermore, throughout the present invention, the term "comprising" should be understood as covering all the specifically mentioned features as well as optional, additional, undescribed ones. As used herein, the use of the term "comprising" also discloses an embodiment in which there are no other features than the specifically mentioned features (i.e. "consisting of..."). Furthermore, the indefinite article "a" or "an" does not exclude a plurality. The fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a diagram showing the results of Ni purification in cell fermentation liquid.

[0047] Figure 2 It is the Ni2+ metal chelation chromatography spectrum.

[0048] Figure 3 This is a diagram of the purification results of cationic chromatography.

[0049] Figure 4 It is a cation chromatography spectrum.

[0050] Figure 5 It is the SEC-HPLC purity analysis chart.

[0051] Figure 6 This is a statistical chart of the activity test results of different batches of recombinant human TGF-β1 protein products. DETAILED DESCRIPTION

[0052] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0053] Example 1. Design of expression plasmid According to the differences in signal peptide sequences, tag positions, latent peptide sources, etc., we designed the following target proteins, outsourced the corresponding expression gene synthesis, and constructed the expression genes on pCDNA3.4 to construct 5 expression plasmids.

[0054] JDS-22A (SEQ ID NO:1): MKWVTFLLLLFISGSAFSHHHHHHHHLSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPE WLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS.

[0055] JDS-22B (SEQ ID NO: 2): METDTLLLWVLLLWVPGSTGDAAHHHHHHHHALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS.

[0056] JDS-22C(SEQ ID NO:3):METDTLLLWVLLLWVPGSTGDAAHHHHHHHHLSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS。

[0057] JDS-22D(SEQ ID NO:4):MKWVTFLLLLFISGSAFSHHHHHHHHALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS。

[0058] JDS-22E (SEQ ID NO:5):METDTLLLWVLLLWVPGSTGDAAHHHHHHHHLSTSKTIDMELVKRKRIEAIRGQILSKLRLASPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFQTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSQNSWRYLSNRLLAPSD SPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS.

[0059] 2. Plasmid extraction and transient expression test The expression plasmid was transformed into E. coli DH5α competent cells to maximize the plasmid.

[0060] Use Zhuhai Kairui's HEK-293 transient expression kit (transfection reagent and enhancer) to carry out target protein expression. The detailed steps are as follows:

[0061] a) Before transient transfection of cells, the cell density and viability must be determined. To ensure the transfection effect, it is recommended to use cells in the exponential growth phase (density of about 2~4×10 6 / ml), and the cell viability was greater than 95%.

[0062] b) Cells do not need to be centrifuged. If the cell density is 3.0×10 6 If the cell density is >3.0×10 6 cells / ml, then add HEK293 serum-free basal medium (Beijing Tongli Haiyuan, AS-11) and dilute the cell density to 3×10 6 Pieces / ml.

[0063] c) The shake flask was placed in a 5% CO2 constant temperature shaker, cultured at 37°C and 120 rpm, and then used for transfection.

[0064] d) Prepare two 50 ml sterile centrifuge tubes, add 25 ml HEK293 serum-free basal medium and 1 mg sterile plasmid DNA into one of them, and gently pipette to mix; take another centrifuge tube, add 25 ml HEK293 serum-free basal medium and 5 ml TA-293 transfection reagent (Zhuhai Kairui, K20001), and gently pipette to mix.

[0065] e) Transfer all the liquid in the centrifuge tube containing the plasmid to the centrifuge tube containing the transfection reagent and mix by gently pipetting.

[0066] f) Let stand at room temperature for 10 minutes.

[0067] g) Remove the cells for transfection from the constant temperature shaker, add the prepared plasmid-vector complex while shaking, and take 1 ml of the cell suspension for counting after transfection, record the cell density and viability, centrifuge and retain the supernatant, put the transfected cells back into the cell culture constant temperature shaker for shaking culture, and take that day as Day 0.

[0068] h) On transfection day 1, take 1 ml of cell suspension and count, record cell density and viability, centrifuge and keep supernatant. Add the initial volume of 0.6% KE-293 (Zhuhai Kairui, K30001) and 5% feed medium (Tongli Haiyuan, AS-18) (i.e., add 6 mL KE-293 and 50 mL feed medium to 1 L cell culture medium).

[0069] i) On transfection day 3, take 1 ml of cell suspension for counting, record cell density and viability, centrifuge and retain supernatant, and add 5% of the initial volume of feed medium again, i.e. 50 ml of feed medium.

[0070] j) On transfection day 4, take 1 ml of cell suspension and count, record cell density and viability, centrifuge and save supernatant. Day 5 or when cell viability is less than 75% is the end point of harvest.

[0071] k) Centrifuge at 10,000 rpm for 10 min, harvest the cell solution, and carry out purification and detection of the target protein.

[0072] Transfection results: No target protein was expressed in JDS-22B and JDS-22D; the expression level of JDS-22A was higher than that of JDS-22C and JDS-22E.

[0073] 3. Ni purification in cell fermentation liquid The transfected cell fermentation liquid was purified by Ni using Ni-NTA Purose 6 Fast Flow (5 mL) and the process of equilibrium-loading-equilibrium-elution (4%, 10%, 50%) was performed. The equilibrium liquid component was 50 mM Tris 0.1 M NaCl pH 8.0, and the elution liquid component was 50 mM Tris 0.1 M NaCl 0.5 M imidazole pH 8.0. The results are shown in Figure 1 , Figure 2 shown.

[0074] 4. Cationic chromatography purification SP SepharoseTM Fast Flow (5mL) was used for cationic chromatography purification. Sample source: 3. Eluted sample of Ni purification. Sample treatment: adjusted to cond: 4.8mS / cm with injection water, adjusted to 4.5 with 6M HCl, and carried out the process of equilibrium-loading-equilibrium-step elution. Among them, the equilibrium liquid composition was 20mM NaAc, 150mM NaCl, 20mM ammonium acetate, 0.5M urea 1% glacial acetic acid, pH4.5, and the eluent composition was 20mM NaAc, 1M NaCl, 0.5M urea, 1% glacial acetic acid, pH4.5. The results are as follows Figure 3 , Figure 4 shown.

[0075] 5. SEC-HPLC analysis and activity detection The purity of the purified recombinant human TGF-β1 protein was 99.40% as analyzed by SEC-HPLC. Figure 5 shown.

[0076] Activity detection: After repeated testing of multiple batches, the purification process is stable, and the activity of the obtained TGF-β1 protein is significantly better than the products sold by R* Company and Kai* Company. The specific results are shown in Table 1, and the detailed information is shown in Figure 6 .

[0077] Table 1

[0078]

[0079] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for preparing a highly active recombinant human TGF-β1 protein, the preparation method comprising the following steps: 1) HEK293 serum-free basal medium and sterile plasmid DNA were mixed at a ratio of 25 ml:1 mg, wherein the amino acid sequence expressed by the sterile plasmid DNA is shown in SEQ ID NO:1; 2) adding the mixed solution in step 1) to the premixed transfection solution, wherein the premixed transfection solution comprises HEK293 serum-free basal medium and TA-293 transfection reagent, and the ratio of HEK293 serum-free basal medium to TA-293 transfection reagent is 25 ml:5 ml, to obtain a plasmid-vector complex; 3) The plasmid-vector complex was allowed to stand at room temperature for 10 minutes; 4) Take the cells for transfection and add the prepared plasmid-vector complex while shaking to complete the transfection. Put the transfected cells back into the cell culture constant temperature shaker for shaking culture. The current day is Day 0. 5) On transfection day 1, add 0.6% KE-293 and 5% feed medium in the initial volume; 6) Add 5% of the initial volume of feed medium on transfection day 3; 7) On Day 5, centrifuge at 10,000 rpm for 10 min to harvest the cell solution; 8) In step 7), the cell feed solution was purified by Ni, using 5 mL Ni-NTA Purose 6 Fast Flow, with 4%, 10%, and 50% equilibration-loading-equilibration-elution processes, the equilibration solution was 50 mM Tris 0.1 M NaCl pH 8.0, and the elution solution was 50 mM Tris 0.1 M NaCl 0.5 M imidazole pH 8.0; 9) The product of step 8) was further purified by cationic chromatography, specifically using 5 mL SP SepharoseTM FastFlow, adjusted to cond: 4.8 mS / cm with injection water, adjusted to pH 4.5 with 6 M HCl, and performed a process of equilibrium-loading-equilibrium-step elution, wherein: The components of the equilibrium solution are 20mM NaAc, 150mM NaCl, 20mM ammonium acetate, 0.5M urea, 1% acetic acid, pH 4.5; the components of the eluent are 20mM NaAc, 1M NaCl, 0.5M urea, 1% acetic acid, pH 4.5, and purified high-activity recombinant human TGF-β1 protein is obtained.

2. The preparation method according to claim 1, wherein the cell density and viability of the cells need to be determined before transient transfection of the cells; Preferably, the cells are in the exponential growth phase and the cell density is about 2-4×10 6 Cells were transfected at a survival rate of more than 95%.

3. The preparation method according to claim 1, wherein the cells do not need to be centrifuged; Preferably, the cell density is 3.0×10 6 When the number of cells / ml is high, direct transfection is performed; Preferably, the cell density is >3.0×10 6 / ml, add HEK293 serum-free basal medium, and dilute the cell density to 3×10 6 / ml, and then transfect.

4. The preparation method according to claim 1, wherein the cells are cultured in a 5% CO2 constant temperature shaker at 37°C and 120 rpm for transfection.

5. The preparation method according to claim 1, wherein 1 ml of the cell suspension is taken on Day 1 and Day 3 for counting, recording the cell density and viability, and centrifuging to retain the supernatant; Preferably, 1 ml of cell suspension is taken on Day 4 for counting, cell density and viability are recorded, and the cell suspension is centrifuged to retain the supernatant.

6. A high-purity and high-activity recombinant human TGF-β1 protein, wherein the recombinant human TGF-β1 protein is prepared by the preparation method according to any one of claims 1 to 5.

7. A polynucleotide comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:

1.

8. A vector comprising the polynucleotide according to claim 7; Preferably, the vector comprises a plasmid vector or a viral vector; Preferably, the vector is a plasmid vector.

9. A cell, comprising the vector according to claim 8; Preferably, the cell comprises a prokaryotic cell or a eukaryotic cell; Preferably, the prokaryotic cell comprises a bacterium; Preferably, the bacteria comprises Escherichia coli; Preferably, the cell comprises a human or non-human mammalian cell; Preferably, the cells are HEK293 cells.

10. Use of the preparation method according to any one of claims 1 to 5, the polynucleotide according to claim 7, the vector according to claim 8, and the cell according to claim 9 in the preparation of high-purity and high-activity recombinant human TGF-β1 protein.

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