Recombinant expression plasmid vector for expressing TNFSF11 protein as well as construction method and application of recombinant expression plasmid vector

Abstract

The invention belongs to the field of gene engineering, and particularly discloses a recombinant expression plasmid vector for expressing TNFSF11 protein and a construction method and application of the recombinant expression plasmid vector, a nucleotide sequence contained in the plasmid vector is optimized according to host cell preferred codons under the condition that an amino acid coding sequence is not changed, and the nucleotide sequence is shown as SEQ ID NO: 1. Furthermore, an expression vector of the nucleotide sequence is constructed, and the recombinant TNFSF11 protein is expressed in an HEK293 cell; the concentration of the expressed recombinant TNFSF11 protein is 3.04 mg / mL, the activity is 2.47 * 10 < 6 > U / mg, and the purity is 99%. The expression quantity and biological activity of the soluble protein are improved, and conditions are created for large-scale production of the soluble recombinant TNFSF11 protein.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and specifically relates to a recombinant expression plasmid vector for expressing TNFSF11 protein, and a construction method and application thereof. Background Art

[0002] TNFSF11 is a member of the tumor necrosis factor superfamily (TNFSF), also known as RANKL, a type II transmembrane protein and a ligand for the receptor activator of NF-κB (RANK). When bound to RANK, it induces differentiation of monocyte / macrophage lineage cells into osteoclasts and further leads to maturation of osteoclast precursors. Therefore, RANKL is essential for osteoclast maturation, bone modeling and bone remodeling, and the development of lymph nodes (LN). In bone tissue, RANKL is expressed by osteoblasts, osteocytes, and immune cells, especially in osteoblasts and osteocytes. RANKL is also expressed by T cells and can promote the proliferation and survival of dendritic cells. In mice, RANKL / RANK signaling reduces inflammation in the ischemic brain through the Toll-like receptor signaling pathway. RANKL consists of a cytoplasmic domain (1-47), a helical domain (48-68), and an extracellular domain (69-317). When cleaved by enzymes such as matrix metalloproteinases (MMP3 or 7) and ADAM, the soluble chains (140-317) are released.

[0003] CN102443594B discloses the construction of a prokaryotic expression vector for a recombinant RANKL protein and the process of expression and purification of the product. Although the expressed protein is in a soluble form, the concentration is extremely low, only 56.72 μg / ml. KR101764538B1 discloses a method for expressing RANKL in Escherichia coli, using a codon optimization strategy and a response surface analysis method to optimize the production of soluble mRANKL protein expressed in Escherichia coli, but there is no data on the specific expression effect. The use of a prokaryotic expression system has its own limitations, for example: there are endotoxin residues in the recombinant protein, and additional treatment is required to remove the endotoxin; the structure of the expressed protein is different from that of the natural protein, and the protein is difficult to fold, modify or locate correctly; some complex proteins may form insoluble inclusion bodies in Escherichia coli, resulting in inability to express correctly; Escherichia coli cannot glycosylate proteins, which limits the expression of proteins that require glycosylation in some cases. Summary of the invention

[0004] In view of the problems of low expression, low activity, low purity, unstable expression, etc. of TNFSF11 protein in the prior art, the present application provides a codon-optimized TNFSF11 gene having a nucleotide sequence shown in SEQ ID NO: 1. The present application also provides a TNFSF11 protein and a preparation method and application thereof, which can efficiently express recombinant TNFSF11 protein in cells, and the preparation and purification operations are simpler, saving time, reducing production costs, and facilitating industrial applications.

[0005] The first aspect of the present invention provides a recombinant TNFSF11 protein gene, which is a nucleotide sequence having at least 95% identity with the nucleotide sequence shown in SEQ ID NO:1, and the gene always encodes an amino acid sequence corresponding to SEQ ID NO:3.

[0006] Furthermore, the nucleotide sequence of the gene has at least 98% identity with the nucleotide sequence shown in SEQ ID NO:1, and more preferably the nucleotide sequence of the gene is as shown in SEQ ID NO:1.

[0007] The second aspect of the present invention provides a nucleic acid construct comprising the gene, a signal peptide sequence, a histidine tag sequence and a stop codon.

[0008] Furthermore, the nucleic acid construct has a nucleotide sequence shown in SEQ ID NO:4.

[0009] The third aspect of the present invention provides a recombinant TNFSF11 protein having an amino acid sequence as shown in SEQ ID NO: 3, wherein the amino acid sequence is encoded by the recombinant TNFSF11 protein gene.

[0010] The fourth aspect of the present invention provides a recombinant plasmid vector for expressing TNFSF11 protein, which comprises a eukaryotic expression vector and the recombinant TNFSF11 protein gene or the nucleic acid construct.

[0011] Furthermore, the eukaryotic expression vector is selected from one of pcDNA series vectors, pGAPZαA, pEGFP-N1 and pPIC9K, preferably pcDNA3.4.

[0012] Furthermore, the construction method of the above-mentioned plasmid vector is as follows: the recombinant TNFSF11 protein gene or nucleic acid construct is introduced into a eukaryotic expression vector to obtain a pcDNA3.4-TNFSF11 plasmid vector; the introduction method is a conventional cloning method in the art.

[0013] The fifth aspect of the present invention provides a recombinant genetically engineered cell, which contains the recombinant TNFSF11 protein gene or nucleic acid construct or recombinant plasmid vector; the host cell of the recombinant genetically engineered bacteria is a mammalian cell, preferably a CHO cell or a HEK293 cell.

[0014] A sixth aspect of the present invention provides a method for preparing a recombinant TNFSF11 protein, comprising culturing the recombinant genetically engineered cells, expressing the recombinant TNFSF11 protein, and isolating and purifying the recombinant TNFSF11 protein.

[0015] Furthermore, the method for preparing the protein comprises the following steps:

[0016] (1) preparing a gene encoding the amino acid sequence of a recombinant TNFSF11 protein, wherein the nucleotide sequence of the gene has at least 95% identity with the sequence shown in SEQ ID NO: 1, preferably at least 98% identity, more preferably the nucleotide sequence shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 3;

[0017] (2) introducing the recombinant TNFSF11 protein gene or nucleic acid construct into a eukaryotic expression vector to obtain the desired recombinant plasmid;

[0018] (3) transfecting the recombinant plasmid into mammalian cells to obtain recombinant genetically engineered cells;

[0019] (4) Recombinant genetically engineered cells were expressed and recombinant TNFSF11 protein was obtained after purification.

[0020] Preferably, the preparation method satisfies one or more of the following conditions:

[0021] The gene of the recombinant TNFSF11 protein described in step (1) is obtained by optimizing the codon preference of the host cell and further screening and replacing;

[0022] The introduction method in step (2) is a conventional cloning method;

[0023] The eukaryotic expression vector in step (2) is selected from one of the pcDNA series vectors, pGAPZαA, pEGFP-N1 and pPIC9K, preferably pcDNA3.4;

[0024] In step (3), the transfection agent is PEI; the mass ratio of the recombinant plasmid to the transfection agent is 1:1-6; preferably, the mass ratio of the recombinant plasmid to the transfection agent is 1:3;

[0025] In step (3), the transfection buffer is KPM; the volume ratio of the recombinant plasmid to the transfection buffer is 5 to 15:1; preferably, the volume ratio of the recombinant plasmid to the transfection buffer is 10:1;

[0026] The purification method in step (4) is selected from one or more combinations of affinity chromatography, ion exchange chromatography, size exclusion chromatography, hydrophobic chromatography, and reverse chromatography, preferably Ni column affinity chromatography and size exclusion chromatography;

[0027] A seventh aspect of the present invention provides an application of a recombinant TNFSF11 protein, namely, application of the gene, the recombinant plasmid or the recombinant TNFSF11 protein in the preparation of a diagnostic kit for degenerative bone diseases.

[0028] Beneficial technical effects of the present invention:

[0029] (1) The nucleotide sequence and nucleic acid construct designed by codon optimization of the present invention can effectively increase the production of TNFSF11 protein.

[0030] (2) The prior art has not reported the technical solution of the mammalian cell expression of the present invention, especially the technical solution of eukaryotic expression using HEK293 cells.

[0031] (3) The optimized nucleotide sequence of the present invention is simple in terms of process preparation and purification, and the purified purity is 99%.

[0032] (4) The optimized nucleotide sequence of the present invention has a homology of 75.9% with the nucleotide sequence of natural mouse TNFSF11 protein, and the encoded amino acid sequence is the same. The recombinant TNFSF11 protein expressed by the optimized nucleotide sequence in HEK293 cells has a concentration of 3.04 mg / mL and an activity of 2.47×10 6 Compared with the prior art, the concentration and activity of the recombinant TNFSF11 protein are significantly improved, and it is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a comparison diagram of the gene sequence of natural TNFSF11 before and after codon optimization in Example 1;

[0034] Figure 2 is the pcDNA3.4 recombinant plasmid map in Example 2;

[0035] Figure 3The SDS-PAGE gel electrophoresis diagram of the target protein after purification by Ni column in Example 3.1; wherein, M, pre-stained protein loading maker; Ft, flow-through recovery solution; W1, eluate after elution with Buffer A; E1-12, eluate after elution with Buffer C;

[0036] Figure 4 is the SDS-PAGE gel electrophoresis diagram after purification by size exclusion chromatography in Example 3.2; wherein, M is the pre-stained protein loading maker; X1-8 is the solution after purification by size exclusion chromatography;

[0037] Figure 5 is the SDS-PAGE gel electrophoresis diagram of the recombinant protein purity detection in Example 5; wherein, M, pre-stained protein loading maker; R, target protein solution;

[0038] Figure 6 The recombinant protein concentration and OD in Example 6 450 relationship diagram. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0040] the term

[0041] Unless otherwise defined, all terms, annotations and other scientific terms used in the art used herein are intended to have the meanings commonly understood by those skilled in the art in the field of the present invention. In some cases, terms with commonly understood meanings are defined in this document for the purpose of clarity and / or quick reference, and the inclusion of such definitions in this document should not necessarily be interpreted as representing a basic difference relative to what is generally understood in the prior art.

[0042] The techniques and procedures described or referred to in this document are generally well understood by those skilled in the art and are employed using routine methods. Where appropriate, processes involving the use of commercially available kits and reagents are generally performed according to protocols and / or parameters defined by the manufacturers, unless otherwise stated.

[0043] It is worth mentioning that, where appropriate, the present invention is not limited to the methods, protocols, cell lines, constructs and specific reagents as described, which can obviously vary. In addition, the terms used in this document are only used for the purpose of describing the examples of its specific embodiments and are not intended to limit the scope of the present invention.

[0044] Throughout this document, the singular forms "a", "an", and "the" or any term or expression in the singular include references to the plural unless the context clearly indicates otherwise.

[0045] Throughout this document, the word "comprise" and any variations thereof such as "include" or "comprising" should be interpreted as "open terms" that may imply the inclusion of additional elements or groups of elements not expressly mentioned without limiting the nature.

[0046] Throughout this document, where an exact value or an exact range of values ​​is provided for a particular factor, amount, concentration or a particular preference, it should be interpreted that the corresponding value or range also provides approximate values, for example by the expression "about".

[0047] Throughout this document, words and expressions such as "preferably", "particularly", "for example", "such as", "as", "more particularly" etc. and variations thereof must be interpreted as purely optional features, preferred embodiments or possible non-exhaustive examples, without limiting the scope of the invention.

[0048] Throughout this document, words and expressions such as "nucleic acid", "nucleotide", etc. should be interpreted as naturally occurring, synthetic or artificial nucleic acids or nucleotides. They include single-stranded or double-stranded deoxyribonucleotides (DNA) or ribonucleotides (RNA) in sense or antisense configuration, or any nucleotide analogs and polymers or hybrids thereof. Unless otherwise indicated, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly indicated sequences. The term "nucleic acid" is used interchangeably with the terms "gene", "cDNA", "mRNA", "oligonucleotide", "nucleic acid molecule" or "primer" in this document.

[0049] The expressions "nucleic acid molecule", "nucleic acid sequence" and the like refer to a polymer of single-stranded or double-stranded DNA or RNA bases read from the 5' end to the 3' end. It includes in particular chromosomal DNA, self-replicating plasmids, infectious DNA or RNA polymers that play a major structural role. They also refer to a continuous list of abbreviations, letters, characters or words representing nucleotides or genes, as commonly used in the technical field of the present invention.

[0050] Throughout this document, when referring to a molecule or sequence of the invention, the expression "codon optimized" should be interpreted as a nucleotide molecule or sequence that has undergone a process of adapting its nucleotide composition (C+G and A+T content) to the composition of the host or recipient organism, so that it can more efficiently express heterologous proteins. The codon optimization process is known to those skilled in the art.

[0051] Throughout this document, words and expressions such as "sequence similarity", "identity" and the like, with respect to another sequence, should be interpreted as the percentage of nucleotides in that sequence that are identical to the nucleotides in the other sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity.

[0052] Throughout this document, the expression "nucleic acid construct" should be interpreted as a single-stranded or double-stranded, linear or circular DNA construct that can cause the expression of the target protein. Generally, it contains a promoter sequence and a coding sequence.

[0053] Throughout this document, words and expressions such as "promoter", "promoter sequence" and the like should be interpreted as a DNA sequence that, once operably linked to a target nucleotide sequence, is capable of controlling the transcription of the target nucleotide sequence into RNA. The promoter is located 5' (or upstream) of the transcription start site of the target nucleotide sequence whose mRNA is transcribed, and provides specific binding sites for RNA polymerase and other transcription factors for transcription initiation. It may include other regulatory sequences known to those skilled in the art. According to the present invention, the promoter may be heterologous or homologous to the respective cell or host. If the nucleic acid sequence is derived from a different species, or if it is derived from the same species, it is modified from its original form, then it is "heterologous" to the organism or second nucleic acid sequence.

[0054] Throughout this document, the term "transformation" and the like should be interpreted as a process for introducing heterologous DNA into cells. It can be carried out in eukaryotic host cells under natural or artificial conditions, for example using several methods well known in the art.

[0055] One of the embodiments of the present invention relates to a method for cell transformation. Any cell transformation method is included within the scope of the present invention and has no particular relevance for implementing the embodiments of the present invention as long as it involves means known to those skilled in the art.

[0056] Throughout this document, the term "vector" should be interpreted as a construct containing a DNA sequence that is operably linked to one or more suitable control sequences that can lead to the expression of the DNA sequence in a suitable host. Such control sequences include, for example, a promoter that performs transcription, an optional operator sequence that is used to control such transcription, a sequence encoding a suitable mRNA binding site for the ribosome, and a sequence that controls the termination of transcription and translation.

[0057] Throughout this document, the expressions "host cell", "host organism" and the like should be interpreted as a specific host organism or a specific target cell, but should also be interpreted as the offspring or potential offspring of these organisms or cells. Because certain modifications may occur in successive generations due to mutations or environmental effects, these offspring are not necessarily identical to the parent cell. However, they are still included in the scope of protection of the present invention.

[0058] Throughout this document, all headings and sub-headings are used for convenience only and should not be construed as limiting the invention.

[0059] Genetic optimization

[0060] Optimal gene expression can be achieved by redesigning and synthesizing genes, such as eliminating rare codons and optimizing codon sequences. There are 64 genetic codes, but most organisms tend to use a subset of these codons. Those that are used most frequently are called optimal codons, and those that are not used frequently are called rare or low-usage codons. Virtually every organism used for protein expression or production (including E. coli, yeast, mammalian cells, Pichia, plant cells, and insect cells) exhibits some degree of difference or preference in codon usage. Each organism, such as E. coli, yeast, fruit flies, and primates, has a unique set of 8 codons that are rarely used. Genes encoding proteins with high abundance in E. coli, yeast, and fruit flies clearly avoid codons with low utilization. Therefore, the expression of recombinant proteins may be affected by codon usage. The redesign of genes that synthesize genes using preferred codons and avoid codons with low or rare utilization is called codon optimization.

[0061] However, some other factors besides codons can also affect protein expression, including sequences that destabilize mRNA, minimize DNA secondary structures, adjust the GC content of DNA, gene translation start frame, translation termination sequence frame, etc. Redesigning synthetic genes can remove or change these sequences, resulting in high-level expression, making exogenous protein production more efficient and economical.

[0062] Example 1 Codon Optimization of Recombinant TNFSF11 Protein Nucleotide Sequence and Construction of Recombinant Plasmid

[0063] The recombinant TNFSF11 nucleotide sequence was optimized and designed based on NCBI (LOCUS: NM_011613.4). While ensuring the amino acid sequence remained unchanged, the free energy and stability of the mRNA secondary structure, the motifs affecting expression, etc. were taken into consideration. The sequence was optimized and designed according to the codon preference of the HEK293 host cells. The sequence consistency was 75.9%, and then the whole gene was synthesized. The optimized TNFSF11 nucleotide sequence is shown in SEQ ID NO: 1, and the natural TNFSF11 nucleotide sequence is shown in SEQ ID NO: 2.

[0064] (SEQ ID NO:1)

[0065] (SEQ ID NO:2)

[0066] The whole gene was synthesized, and the amino acid sequence of the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 3. The comparison chart of the optimized TNFSF11 nucleotide sequence and the natural TNFSF11 nucleotide sequence (as shown in Figure 1 shown).

[0067] The amino acid sequence is shown in SEQ ID NO:3:

[0068] RAQMDPNRISEDSTHCFYRILRLHENADLQDSTLESEDTLPDSCRRMKQAFQGAVQKELQHIVGPQRFSGAPAMMEGSWLDVAQRGKPEAQPFAHLTINAASIPSGSHKVTLSSWYHDRGWAKI SEQ ID NO:3)

[0069] Example 2 Expression process of recombinant TNFSF11 protein

[0070] The recombinant plasmid was constructed, which included introducing the codon-optimized SEQ ID NO: 1 nucleotide sequence obtained in Example 1 into the pcDNA3.4 eukaryotic expression vector (eg Figure 2 The sequencing result was correct, and the next step of the experiment was carried out (completed by Suzhou Genewise Biotechnology Co., Ltd.).

[0071] The nucleotide sequence of the introduced vector is shown in SEQ ID NO:4, and the corresponding amino acid sequence is shown in SEQ ID NO:5:

[0072] ATGGAGACAGATACACTGCTCCTGTGGGTGCTCCTGCTGTGGGTCCCCGGGAGCACCGGCCACCATCACCATCACCACAGAGCTCAGATGGACCCCAACAGAATCAGCGAGGATAGCACCCACTGCTTCTACAGAATCCTGAGACTGCACGAGAACGCCGACCTGCAAGACAGCACCCTGGAGAGCGAGGACACCCTGCCCGACAGCTGCAGAAGAATGAAGCAAGCCTTCCAAGGCGCCGTGCAGAAGGAGCTGCAGCACATCGTGGGCCCTCAGAGATTCAGCGGCGCCCCCGCCATGATGGAGGGCAGCTGGCTGGACGTGGCTCAGAGAGGCAAGCCCGAGGCTCAGCCCTTCGCCCACCTGACCATCAACGCCGCCTCCATCCCTAGCGGCAGCCACAAGGTGACCCTGAGCAGCTGGTACCACGACAGAGGCTGGGCCAAGATCAGCAACATGACCCTCAGCAACGGCAAGCTGAGAGTGAACCAAGACGGCTTCTACTACCTGTACGCCAACATCTGCTTCAGACACCACGAGACAAGCGGCAGCGTGCCCACCGACTACCTGCAGCTGATGGTGTACGTGGTGAAGACAAGCATCAAGATCCCTAGCAGCCACAACCTGATGAAGGGCGGCAGCACCAAGAACTGGAGCGGCAACAGCGAGTTCCACTTCTACAGCATCAACGTGGGCGGCTTCTTCAAGCTGAGAGCCGGCGAGGAGATCAGCATCCAAGTGAGCAACCCTAGCCTGCTGGACCCCGACCAAGACGCCACCTACTTCGGCGCCTTCAAGGTGCAAGACATCGACTGA(SEQ ID NO:4)

[0073] METDTLLLWVLLLWVPGSTGHHHHHHRAQMDPNRISEDSTHCFYRILRLHENADLQDSTLESEDTLPDSCRRMKQAFQGAVQKELQHIVGPQRFSGAPAMMEGSWLDVAQRGKPEAQPFAHLTINAASIPSGSHKVTL SSWYHDRGWAKISNMTLSNGKLRVNQDGFYYLYANICFRHHETSGSVPTDYLQLMVYVVKTSIKIPSSHNLMKGGSTKNWSGNSEFHFYSINVGGFFKLRAGEEISIQVSNPSLLDPDQDATYFGAFKVQDID*(SEQ ID NO:5) (where * indicates stop codon)

[0074] 2.1 Conversion

[0075] (1) Thaw the DH5α competent cells from -80°C on ice and gently blow the DH5α competent cells evenly;

[0076] (2) Take 2 μL of plasmid and add it into DH5α competent cells;

[0077] (3) Place on ice for 30 min, activate at 42°C for 90 s, place back on ice for 2-5 min, and add 100-500 μL of antibody-free LB medium;

[0078] (4) The shaking temperature was 37°C and the speed was 220 rpm. After shaking for about 1 hour, the OD 600 The value is between 0.6-0.8;

[0079] (5) Take 100 μL of the transformed bacterial solution and spread it evenly on an LB plate containing Ampicilin antibiotics, and incubate it upside down in a 37°C constant temperature incubator overnight.

[0080] 2.2 Plasmid extraction

[0081] The plasmid was extracted using the MN Endotoxin-Free Plasmid Extraction Kit, and its concentration and A260 / A280 purity ratio were determined using NanoDrop. The concentration was >200 μg / μL, and the A260 / A280 ratio was 1.8.

[0082] 2.3 Recombinant protein expression

[0083] 2.3.1 Cell preparation before transfection

[0084] (1) Cell status detection: Take out the cells from the incubator and perform counting and viability detection. Status description: The cells are in good condition.

[0085] (2) Cell treatment: One day before transfection, dilute the cells to a density of approximately 1×10 6 / mL for transfection the next day.

[0086] 2.3.2 Transfection and expression

[0087] (1) Ensure that the cell density for expression is approximately 2×10 6 / mL, high cell viability (>95%);

[0088] (2) 400 μg of plasmid (1 μg / mL) and 1200 μg of transfection reagent (PEI) were added to 20 mL of transfection buffer KPM (purchased from Zhuhai Kairui Biotechnology Co., Ltd.), and then the transfection reagent and plasmid were mixed, allowed to stand at room temperature for 10 min, and then added to 400 mL of cells (gently invert to mix);

[0089] (3) 24 hours after transfection, add cell protein expression enhancer (HEK293 0.6%) and 1× transient transfection nutrient additive, transfer to 32°C and culture for 7 days.

[0090] Example 3 Purification of recombinant TNFSF11 protein

[0091] 3.1 Nickel column affinity purification

[0092] 3.1.1 Sample preparation

[0093] (1) Centrifuge at 5000 rpm for 10 min and collect the supernatant;

[0094] (2) Filter the supernatant through a 0.45 μM filter to remove solid particles and transfer to a clean centrifuge tube.

[0095] 3.1.2 Nickel column affinity purification

[0096] Buffer A: 20mM PB, 150mM NaCl (pH7.8)

[0097] Buffer B: 20mM PB, 150mM NaCl, 20mM imidazole (pH 7.8)

[0098] Buffer C: 20mM PB, 150mM NaCl, 250mM imidazole (pH 7.8)

[0099] (1) Take 1 mL of nickel column and balance the gravity column with 50 mL of buffer A;

[0100] (2) Filter the supernatant after impurities removal, place it on ice, load 200 mL of the sample onto a gravity column, and recover the flow-through, which is recorded as Ft;

[0101] (3) Wash with 50 mL of buffer A and collect the wash solution for subsequent analysis and record it as W1;

[0102] (4) Elute with 50 mL of buffer B and collect the elution solution for subsequent analysis and record it as W2;

[0103] (5) Elute with 50 mL of buffer C, 4 mL per tube, and collect 12 tubes in total. Collect the eluate for subsequent analysis and record it as E1-E12;

[0104] (6) After collection, continue to elute with 3 column volumes of buffer A to remove all proteins;

[0105] (7) washing the medium with 3 column volumes of ultrapure water at the gravity column flow rate to remove the eluent in the medium;

[0106] (8) Using 3 column volumes of 20% ethanol at the gravity column flow rate to wash the medium, seal the column, and store the nickel column at 4°C;

[0107] (9) Take 20 μL of each sample (Ft, W1, W2, E1-12) and add 5 μL of 5× protein loading buffer and mix well;

[0108] (10) SDS-PAGE analysis was performed; the results were as follows: Figure 3 As shown, E1-E12 were combined and subjected to subsequent purification.

[0109] 3.2 Size Exclusion Chromatography

[0110] Sample: Concentrate the sample to 2 mL;

[0111] Buffer: 50mM PB, 150mM NaCl;

[0112] Column equilibration:

[0113] (1) Rinse port A1 with endotoxin-free water and add 500 mL of buffer;

[0114] (2) Turn on the computer instrument, open the software, and click Connect;

[0115] (3) Manual control, constant pressure control;

[0116] (4) Manual control, detector, UV zeroing, insert execution;

[0117] (5) Select the positive flow setting of column 1 in the lower right corner of the valve;

[0118] (6) Manual control, others, timed end, volume 120 mL Buffer, flow rate 1 mL / min, column equilibration for about 2 h, equilibrated to UV280 baseline.

[0119] Loading:

[0120] (1) Use a 5 mL syringe to draw 2 mL of concentrated protein and remove the bubbles;

[0121] (2) Insert the syringe into the injection hole and inject the sample;

[0122] (3) Manual control, flow path, injection valve, injection, manual filling, insert execution; (display inject, buffer into column);

[0123] (4) inject shows about 2 ring volumes (3 mL ring volume, 6 mL wash);

[0124] (5) Manual control, flow path, injection valve, injection changed to manual filling, insert execution;

[0125] (6) Manual control, detector, UV zero, insert execution.

[0126] collect:

[0127] (1) Elution 120 mL, flow rate 1 mL / min;

[0128] (2) Start collecting when a peak appears when about 46 mL of buffer has been passed;

[0129] (3) Manual control, collection, fixed volume collection, collection type 15 mL centrifuge tube, collection volume 3 mL, (number: X1-X8);

[0130] (4) After the peak ends, manually control, collect, stop fixed volume collection, end, disconnect, and turn off the computer instrument.

[0131] Detection:

[0132] (1) Select the peak tubes X1-X8 for 15% gel SDS-PAGE detection. The results are as follows Figure 4 As shown;

[0133] (2) Result analysis: Combine X4-X6 and proceed to the next step of testing.

[0134] Example 4 Recombinant protein concentration and endotoxin detection

[0135] The protein concentration was measured by BCA method and was 3.04 mg / mL. The endotoxin test by gel method was qualified, <1 EU / μg.

[0136] Example 5 Purity detection of recombinant TNFSF11 protein

[0137] (1) Purity was tested by SDS-PAGE gel electrophoresis;

[0138] (2) SDS-PAGE gel electrophoresis: It is a denaturing polyacrylamide gel electrophoresis method. The principle of this method for separating proteins is that most proteins can combine with the anionic surfactant sodium dodecyl sulfate (SDS) to form a complex by weight, so that the negative charge carried by the protein molecule far exceeds the net charge of the natural protein molecule, eliminating the charge effect of different protein molecules and separating the proteins according to molecular size.

[0139] (3) Sample treatment: 0.5 μg of sample was pipetted and added with reduced sample buffer at a ratio of 3:1. Mix well, heat in a metal bath at 100°C for 5 min, cool to room temperature, and load on SDS-PAGE.

[0140] (4) Electrophoresis conditions: Constant voltage electrophoresis, initial voltage is 80 V, adjusted to 200 V when entering the separation gel, and electrophoresis is stopped when bromophenol blue migrates to the bottom of the gel;

[0141] (5) Fixation and staining (Coomassie Brilliant Blue method): Take out the electrophoresis film, stain it in staining solution for 15 min, and decolorize it in purified water;

[0142] (6) Gel image processing: After the gel is developed, it is photographed or scanned. Usually, a commercial gel scanning system with data analysis software is used to take pictures and analyze the data to obtain molecular weight and purity information. Molecular weight analysis: Calibrate based on molecular weight standards. Purity analysis: Scan with a gel imager and calculate the results using the peak area normalization method. The results are as follows: Figure 5 As shown, the molecular weight is about 35.13 kDa and the purity is 99%.

[0143] Example 6 Analysis of biological activity of recombinant TNFSF11 protein

[0144] 6.1 Cell culture

[0145] (1) Cell line used for activity measurement: RAW264.7 cells

[0146] (2) Cell culture medium: DMEM medium + 10% FBS + 1% P / S

[0147] 6.2 Experimental procedures

[0148] (1) Cells were seeded in 24-well plates at a density of 30,000 cells / well;

[0149] (2) After adhesion, the culture medium was discarded, and 500 μL of culture medium containing different concentrations of TNFSF11 protein was added to the 24-well plate, so that the final concentration of TNFSF11 protein in each well was 50, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078, 0.039, and 0 ng / mL;

[0150] (3) Cells were cultured for 72 h;

[0151] (4) After the incubation time is reached, lyse the cells with 200 μL of cell lysis buffer (containing PMSF at a final concentration of 1 mM); after 2 min, collect the cell lysate, centrifuge, and take the supernatant;

[0152] (5) Use a 96-well plate to set up blank control wells, standard wells, and sample wells. The amounts of standard wells are 4, 8, 16, 24, 32, and 40 μL, respectively;

[0153] Blank Standard Sample Assay Buffer 40μL (80-x)μL (40-y)μL Chromogenic substrate 40μL - 40μL tartaric acid solution 5μL 5μL 5μL sample - - yμL Standard working solution - xμL -

[0154] (6) Mix by gently pipetting and incubate at 37°C for 10 minutes;

[0155] (7) Add 160 μl of reaction stop solution to each well to terminate the reaction;

[0156] (8) Measure the absorbance at 405 nm.

[0157] 6.3 Data processing

[0158] GraphPad Prism 9 software was used to draw a curve with OD405nm value as the ordinate and protein concentration as the abscissa to calculate ED 50 The result is Figure 6 As shown, ED 50 =0.4042ng / mL, according to the enzyme activity conversion formula: The specific activity of the recombinant protein was calculated to be 2.47×10 6 U / mg.

Claims

1. A recombinant TNFSF11 protein gene, characterized in that: The gene has a nucleotide sequence that is at least 95% identical to the nucleotide sequence shown in SEQ ID NO:

1.

2. The recombinant gene according to claim 1, characterized in that: The nucleotide sequence of the gene has at least 98% identity with the nucleotide sequence shown in SEQ ID NO:1; more preferably, the nucleotide sequence of the gene is as shown in SEQ ID NO:

1.

3. A nucleic acid construct, characterized in that A gene comprising any one of claims 1 or 2, further comprising a signal peptide sequence, a histidine tag sequence and a stop codon.

4. The nucleic acid construct according to claim 3, characterized in that It has the nucleotide sequence shown in SEQ ID NO:

4.

5. A recombinant TNFSF11 protein, characterized in that: Encoded by the gene according to any one of claims 1 to 2; having an amino acid sequence shown in SEQ ID NO:

3.

6. A recombinant plasmid vector expressing TNFSF11 protein, characterized in that: It comprises a eukaryotic expression vector and the recombinant TNFSF11 protein gene according to any one of claims 1 to 2 or the nucleic acid construct according to any one of claims 3 to 4.

7. The recombinant plasmid vector according to claim 6, characterized in that: The eukaryotic expression vector is selected from one of pcDNA series vectors, pGAPZαA, pEGFP-N1 and pPIC9K, preferably pcDNA3.

4.

8. A recombinant genetically engineered cell, characterized in that: It comprises the recombinant TNFSF11 protein gene described in any one of claims 1 to 2, or the nucleic acid construct described in any one of claims 3 to 4, or the recombinant plasmid vector described in any one of claims 6 to 7; the host cell of the recombinant genetic engineering cell is a mammalian cell, preferably a CHO cell or a HEK293 cell.

9. A method for preparing a recombinant TNFSF11 protein, characterized in that: Cultivating the recombinant genetically engineered cell according to claim 8, expressing the recombinant TNFSF11 protein according to claim 5, and isolating and purifying the recombinant TNFSF11 protein; Furthermore, the method for preparing the protein comprises the following steps: (1) preparing a gene encoding the amino acid sequence of a recombinant TNFSF11 protein, wherein the nucleotide sequence of the gene has at least 95% identity with the sequence shown in SEQ ID NO: 1, preferably at least 98% identity, more preferably the nucleotide sequence shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 3; (2) introducing the recombinant TNFSF11 protein gene or nucleic acid construct into a eukaryotic expression vector to obtain the desired recombinant plasmid vector; (3) transfecting the recombinant plasmid vector into mammalian cells to obtain recombinant genetically engineered cells; (4) expressing in recombinant genetically engineered cells and obtaining recombinant TNFSF11 protein after purification; Preferably, the preparation method satisfies one or more of the following conditions: The gene of the recombinant TNFSF11 protein described in step (1) is obtained by optimizing the codon preference of the host cell and further screening and replacing; The introduction method in step (2) is a conventional cloning method; The eukaryotic expression vector in step (2) is selected from one of the pcDNA series vectors, pGAPZαA, pEGFP-N1 and pPIC9K, preferably pcDNA3.4; In step (3), the transfection agent is PEI; the mass ratio of the recombinant plasmid to the transfection agent is 1:1-6; preferably, the mass ratio of the recombinant plasmid to the transfection agent is 1:3; In step (3), the transfection buffer is KPM; the volume ratio of the recombinant plasmid to the transfection buffer is 5 to 15:1; preferably, the volume ratio of the recombinant plasmid to the transfection buffer is 10:1; The purification method in step (4) is selected from one or more combinations of affinity chromatography, ion exchange chromatography, size exclusion chromatography, hydrophobic chromatography, and reverse chromatography, preferably Ni column affinity chromatography and size exclusion chromatography.

10. Use of the gene according to any one of claims 1 to 2, the nucleic acid construct according to claims 3 to 4, or the recombinant TNFSF11 protein according to claim 5 in a kit, characterized in that: The kit is used for diagnosing degenerative bone diseases.

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