Codon-optimized calprotectin, nucleic acid molecule and preparation method and application thereof

Abstract

The invention belongs to the technical field of genetic engineering, and relates to codon-optimized calprotectin, a nucleic acid molecule and a preparation method and application of the codon-optimized calprotectin and the nucleic acid molecule. Aiming at the technical problem that in the prior art, when calprotectin is produced by utilizing engineering bacteria, a double-vector expression strategy is generally adopted for S100A8 and S100A9, so that the biological activity and stability of expression are poor, the invention provides a codon-optimized calprotectin nucleic acid molecule, and a nucleic acid molecule for coding S100A8 has a nucleotide sequence shown as SEQ ID NO: 3; the nucleic acid molecule for coding the S100A9 has a nucleotide sequence as shown in SEQ ID NO: 4, and the purity, concentration, yield, activity and ED50 of the prepared calprotectin are improved compared with those in the prior art. The invention further provides a preparation method of the calprotectin, target protein is expressed and purified by using the calprotectin nucleic acid molecule, the recombinant expression vector containing the calprotectin nucleic acid molecule and / or the recombinant genetic engineering cell, the cost is low, the method is suitable for large-scale production, and the protein is high in biological activity and good in stability.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene engineering, and in particular, relates to codon-optimized calprotectin, nucleic acid molecules, and preparation methods and applications thereof. Background Art

[0002] S100A8 (S100 calcium-binding protein A8) and S100A9 (S100 calcium-bindingprotein A9) are two calcium-binding proteins belonging to the S100 protein family. S100A8 and S100A9 usually exist as heterodimers, and this complex is also called calprotectin. Calprotectin is mainly used in the diagnosis and monitoring of inflammatory bowel disease (IBD) in clinical practice, as well as to distinguish IBD from other intestinal diseases such as irritable bowel syndrome (IBS). Calprotectin levels in stool can reflect the degree of intestinal inflammation, and as a non-invasive biomarker, it helps reduce reliance on colonoscopy. Research on calprotectin is still ongoing, including its role in tumor development and as a potential anti-inflammatory and anti-cancer therapeutic target.

[0003] There are mainly the following methods for producing calprotectin: (1) Monoclonal antibody production. For example, the Chinese patent application publication number is CN117430701A, the application date is December 20, 2023, and the invention name is "Monoclonal antibody composition and application of anti-human calprotectin". The disclosed preparation method is to mix and emulsify the affinity-purified human calprotectin S100A8 / A9 complex with Freund's adjuvant in equal volumes, screen mice with good immunity for hybridoma fusion experiments, and use the affinity-purified human calprotectin S100A8 subunit, S100A9 subunit and S100A8 / A9 complex to screen antibodies specific to human calprotectin S100A8 subunit, S100A9 subunit and S100A8 / A9 complex, clone monoclonal cell lines, culture and ferment cell lines in serum-free conditions, and collect culture supernatants. This method can produce calprotectin with strong specificity and high purity, which is suitable for studying the biological functions of calprotectin and as a diagnostic reagent. However, the production cost is high, the production cycle is long, and it is difficult to produce on a large scale. (2) Production using Escherichia coli. For example, Qin Fengjin et al., In vitro construction of heterodimers of calcium-binding proteins S100A8 and S100A9, Shandong Medicine, Vol. 51, No. 19, 2011, pp. 37-38. The use of prokaryotic expression vectors of S100A8 and S100A9 was disclosed. This method is low-cost and suitable for large-scale production. However, prokaryotic expression systems such as Escherichia coli may not be able to perform correct post-translational modifications, such as glycosylation, which may affect the biological activity and stability of the protein. (3) Recombinant protein production. It can be produced in different host cells, including yeast, insect cells or mammalian cells, through genetic engineering technology to obtain properties closer to natural proteins. However, the production process is complicated, requires fine process control, and the cost is relatively high. (4) Natural extraction. Calprotectin with complete biological activity can be obtained by direct extraction from natural sources such as neutrophils. However, the extraction process is complicated, the yield is limited, and it may contain other impurities.

[0004] When calprotectin is produced in E. coli, complex formation between S100A8 and S100A9 depends to some extent on the thermodynamic stability of the proteins during expression. Complexes spontaneously form during co-expression of stable S100A8 / A9 heterodimers and are purified to be biologically active. Since protein assembly occurs rapidly after protein synthesis, co-expression of related proteins is essential for the design of multi-component recombinant protein expression systems. Improving expression levels in prokaryotic systems, such as purity, concentration, yield, activity, and ED50, through codon optimization of S100A8 and S100A9 nucleic acid molecules, is an urgent problem to be solved. Summary of the invention

[0005] 1. Technical problem to be solved by the invention

[0006] In view of the technical problem that when calprotectin is produced by engineering bacteria in the prior art, a dual-vector expression strategy is usually adopted for S100A8 and S100A9, resulting in poor biological activity and stability of the expression, the present application provides a codon-optimized calprotectin nucleic acid molecule, and the purity, concentration, yield, activity and ED50 of the prepared calprotectin are all improved compared with the prior art. The present application also provides a method for preparing calprotectin, which has low cost, is suitable for large-scale production, and has high biological activity and good stability of the protein.

[0007] 2. Technical solution

[0008] In order to achieve the above purpose, the technical solution provided is:

[0009] The codon-optimized calprotectin nucleic acid molecule of the present invention comprises S100 calcium-binding protein A8 and S100 calcium-binding protein A9: the nucleic acid molecule encoding the S100 calcium-binding protein A8 has the nucleotide sequence shown in SEQ ID NO:3; the nucleic acid molecule encoding the S100 calcium-binding protein A9 has the nucleotide sequence shown in SEQ ID NO:4.

[0010] Calprotectin comprises S100 calcium-binding protein A8 and S100 calcium-binding protein A9; the S100 calcium-binding protein A8 is encoded by the nucleotide sequence shown in SEQ ID NO:3 and has the amino acid sequence shown in SEQ ID NO:5; the S100 calcium-binding protein A9 is encoded by the nucleotide sequence shown in SEQ ID NO:4 and has the amino acid sequence shown in SEQ ID NO:6.

[0011] The recombinant expression vector comprises the nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO:3 and the nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO:4.

[0012] The recombinant genetically engineered cell contains the recombinant expression vector; the host cell of the recombinant genetically engineered cell is Escherichia coli.

[0013] The method for preparing the calprotectin protein comprises using the calprotectin protein nucleic acid molecule, the recombinant expression vector and / or the recombinant genetic engineering cell.

[0014] Furthermore, the recombinant genetically engineered cells are cultured to express the calprotectin; and the calprotectin is isolated.

[0015] Furthermore, the steps of culturing the recombinant genetically engineered cells are: picking a single clone and culturing it in LB medium until the OD600 is 0.6-0.8, adding IPTG, and inducing at a preset temperature; and collecting the bacteria by centrifugation.

[0016] Furthermore, the purification method for isolating the calprotectin protein is to sequentially perform Ni-NTA affinity chromatography, concentration, and anion purification.

[0017] Furthermore, the process of Ni-NTA affinity chromatography is as follows: after washing with Buffer C, eluting with Buffer A and Buffer B prepared into 20mM, 50mM, 250mM, 500mM imidazole, and during the elution process, detecting with Coomassie Brilliant Blue method until the color does not turn blue;

[0018] The anion purification process is as follows: before using the ion column, wash it with 0.5M sodium hydroxide for 2 column volumes; wash it with endotoxin-free water for 2 column volumes; wash it with Buffer E for 2 column volumes; wash it with Buffer D for 2 column volumes; load the sample; wash it with Buffer D for 5 column volumes; until the UV absorption reaches a stable baseline; gradient elution, linear gradient elution uses 15 column volumes;

[0019] Buffer A: 50 mM HEPES, 200 mM NaCl, pH 8.0;

[0020] Buffer B: 50 mM HEPES, 200 mM NaCl, 500 mM imidazole, pH 8.0;

[0021] Buffer C: 50 mM HEPES, 200 mM NaCl, pH 8.0, 0.1% Triton 114;

[0022] Buffer D: 50 mM HEPES, 30 mM NaCl, pH 7.5;

[0023] The Buffer E includes 50 mM HEPES, 1 M NaCl, pH 7.5.

[0024] Use of calprotectin nucleic acid molecules or calprotectin in preparing a kit: the kit is used for diagnosing inflammation.

[0025] 3. Beneficial effects

[0026] Compared with the existing known technology, the technical solution provided by the present invention has the following beneficial effects:

[0027] The codon-optimized calprotectin nucleic acid molecule of the present invention, the nucleic acid molecule encoding S100 calcium-binding protein A8 has the nucleotide sequence shown in SEQ ID NO:3, and the nucleic acid molecule encoding S100 calcium-binding protein A9 has the nucleotide sequence shown in SEQ ID NO:4. The present application proposes a co-expression single vector strategy, and the expression system is constructed using the double codon-optimized S100A8 / A9 nucleic acid molecule. Compared with the synthetic inclusion bodies in the prior art, the obtained calprotectin is a soluble protein with a purity of 99%, a concentration of 15.805 mg / mL, a yield of 236 mg / L, an ED50 of 1.472 μg / mL, and an activity of 679.348 U / mg. The preparation of recombinant proteins with biological activity is of great significance for basic research and the production of biological preparations. Using Escherichia coli as a host is considered to be a very good initial choice for co-expressing a sufficient number of protein complexes. This simplified method can obtain a large amount of biologically active heterodimeric proteins from Escherichia coli and then purify them.

[0028] The preparation method of calprotectin of the present invention uses the calprotectin nucleic acid molecule, or a recombinant expression vector and / or a recombinant genetic engineering cell containing the calprotectin nucleic acid molecule, cultivates the recombinant genetic engineering cell, expresses calprotectin, and separates the calprotectin. In the recombinant protein expression system, Ni-NTA affinity chromatography, concentration, and anion purification are sequentially performed, the cost is low, it is suitable for large-scale production, and the protein has high biological activity and good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the gel electrophoresis diagram of the purified calprotectin in the example.

[0030] Figure 2 This is the gel electrophoresis diagram of calprotectin after concentration and fluid replacement in the example.

[0031] Figure 3 The figure is the SDS-PAGE gel electrophoresis diagram of the purity detection of calprotectin in the embodiment; wherein: M, pre-stained protein loading maker; R, calprotectin solution.

[0032] Figure 4 This is a diagram for detecting the activity of calprotectin in the examples. DETAILED DESCRIPTION

[0033] 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.

[0034] Abbreviations or glossaries

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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".

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] pETDuet-1 is a commonly used Escherichia coli protein dual expression vector. This vector is designed to express two exogenous genes simultaneously. It contains two multiple cloning sites (MCS1 and MCS2), each of which consists of a T7 promoter / Lac operator and a ribosome binding site (RBS) to drive gene expression.

[0053] IPTG (Isopropyl-beta-D-thiogalactopyranoside) is a chemical inducer commonly used in molecular biology experiments, especially for inducing protein expression in Escherichia coli. IPTG binds to the repressor protein and changes its conformation by simulating the metabolite of lactose, allolactose, thereby preventing the inhibition of the β-galactosidase encoding gene lacZ, thereby activating the lac operon and inducing the expression of the target protein.

[0054] Ni-NTA (Nitrilotriacetic acid-Nickel) is a nickel nitrilotriacetic acid. It is commonly used as a medium for biomolecule affinity chromatography, especially when purifying proteins with histidine (His) tags. Ni-NTA chelates nickel ions (Ni 2+ ) forms a stable coordination bond with the histidine residues on the protein, thereby achieving selective adsorption and purification of the target protein. This technology is widely used in the field of protein expression and purification, especially in molecular biology and bioengineering research.

[0055] Triton X-114, a nonionic surfactant, has a low turbidity point (23°C) and is often used in biochemical experiments to separate aqueous proteins and lipoproteins, such as membrane protein extraction. This substance is miscible with water below the turbidity point, and separates into an aqueous phase and a detergent phase above the turbidity point.

[0056] Imidazole, a five-membered heterocyclic compound, contains two nitrogen atoms and has a molecular formula of C3H4N2. It is a white crystalline substance with weak alkalinity and can form salts with acids. It is used as a catalyst in organic synthesis and is also used to synthesize imidazole antifungal drugs, such as diclofenac, econazole, ketoconazole, clotrimazole, etc.

[0057] SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is a commonly used protein separation technique that can separate proteins according to their molecular weight by electrophoresis in polyacrylamide gel.

[0058] DMEM (Dulbecco's Modified Eagle Medium) is a widely used medium for cell culture, especially suitable for the growth of a variety of adherent cells, including mammalian cells, insect cells and plant cells. DMEM contains higher levels of amino acids and vitamins than the original Eagle medium, and usually requires the addition of serum and other supplements to provide a complete cell growth environment.

[0059] Trizol (TRIzol), also known as total RNA extraction reagent, is a widely used molecular biology reagent used to extract total RNA from cell and tissue samples, including mRNA, rRNA, tRNA, and small molecule RNA.

[0060] DEPC (Diethyl Pyrocarbonate), also known as diethyl pyrocarbonate or diethyl pyrocarbonate. A commonly used molecular biology reagent, it is mainly used to treat water and other solutions by inhibiting the activity of nucleases to ensure that RNA degradation is avoided during RNA extraction and related experiments. DEPC-treated water (DEPC H2O) is often used in RNA experiments to ensure that the experimental water does not contain nucleases. When used, DEPC water needs to be sterilized by high pressure after treatment to remove DEPC, because DEPC itself is a potential carcinogen and toxic to the human body. It needs to be operated in a well-ventilated environment and take appropriate protective measures.

[0061] Throughout this document, all headings and sub-headings are used for convenience only and should not be construed as limiting the invention.

[0062] Example

[0063] 1. Codon optimization of recombinant protein nucleotide sequence and construction of recombinant plasmid

[0064] This embodiment provides a codon-optimized S100A8 / S100A9 nucleic acid molecule, which is based on the original sequence in NCBI (shown in SEQ ID NO:1 and SEQ ID NO:2), and the sequence is optimized according to the preference of Escherichia coli while ensuring that the amino acid sequence remains unchanged, to obtain a codon-optimized S100A8 / S100A9 nucleic acid molecule sequence. The optimized sequence is shown in SEQ ID NO:3 and SEQ ID NO:4.

[0065] The nucleotide sequence of SEQ ID NO:1 is:

[0066] ATGTTGACCGAGCTGGAGAAAGCCTTGAACTCTATCATCGACGTCTACCACAAGTACTC

[0067] CCTGATAAAGGGGAATTTCCATGCCGTCTACAGGGATGACCTGAAGAAATTGCTAGAGA

[0068] CCGAGTGTCCTCAGTATATCAGGAAAAAGGGTGCAGACGTCTGGTTCAAAGAGTTGGAT

[0069] ATCAACACTGATGGTGCAGTTAACTTCCAGGAGTTCCTCATTCTGGTGATAAAGATGGGC

[0070] GTGGCAGCCCACAAAAAAAGCCATGAAGAAAGCCACAAAGAG

[0071] The nucleotide sequence of SEQ ID NO:2 is as follows:

[0072] ACTTGCAAAATGTCGCAGCTGGAACGCAACATAGAGACCATCATCAACACCTTCCACCA

[0073] ATACTCTGTGAAGCTGGGGCACCCAGACACCCTGAACCAGGGGGAATTCAAAGAGCTG

[0074] GTGCGAAAAGATCTGCAAAATTTTCTCAAGAAGGAGAATAAGAATGAAAAGGTCATAG

[0075] AACACATCATGGAGGACCTGGACACAAATGCAGACAAGCAGCTGAGCTTCGAGGAGTT

[0076] CATCATGCTGATGGCGAGGCTAACCTGGGCCTCCCACGAGAAGATGCACGAGGGTGAC

[0077] GAGGGCCCTGGCCACCACCATAAGCCAGGCCTCGGGGAGGGCACCCCC

[0078] The nucleotide sequence of SEQ ID NO:3 is as follows:

[0079] ATGCTAACAGAATTGGAGAAAGCTTTAAATTCCATCATCGACGTGTACCATAAGTATAGC

[0080] CTGATCAAGGGCAACTTCCACGCTGTGTACCGCGATGACCTGAAAAAGCTGTTGGAAAC

[0081] GGAGTGCCCGCAATATATTCGTAAAAAGGGCGCCGATGTTTGGTTTAAAGAGCTGGACA

[0082] TCAACACCGATGGTGCGGTGAATTTTCAGGAGTTCTTGATTTTAGTTATTAAGATGGGTG

[0083] TCGCGGCACATAAGAAAAGCCATGAAGAGTCTCACAAAGAACATCACCACCACCATCACTAA

[0084] The nucleotide sequence of SEQ ID NO:4 is:

[0085] ACATGTAAAATGTCACAACTAGAAAGGAATATCGAAACCATTATCAACACCTTTCATCAG

[0086] TACAGCGTTAAACTGGGTCATCCGGATACCTTGAATCAGGGTGAGTTCAAAGAGCTGGT

[0087] CCGCAAGGACTTACAAAACTTCCTGAAGAAAGAAAATAAGAACGAAAAGGTGATTGAG

[0088] CACATTATGGAAGATTTGGACACGAACGCGGATAAACAACTGTCCTTTGAAGAGTTCAT

[0089] CATGCTGATGGCACGTCTGACCTGGGCTAGCCATGAGAAGATGCATGAGGGCGACGAGG

[0090] GTCCGGGCCATCACCACAAACCGGGTCTCGGCGAGGGCACTCCACATCACCACCACCA

[0091] CCACTAA

[0092] The amino acid sequences encoded by SEQ ID NO:3 and SEQ ID NO:4 are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0093] The amino acid sequence of SEQ ID NO:5 is:

[0094] MLTELEKALNSIIDVYHKYSLIKGNFHAVYRDDLKKLLETECPQYIRKKGADVWFKELDIN

[0095] TDGAVNFQEFLILVIKMGVAAHKKSHEESHKEHHHHHH

[0096] The amino acid sequence of SEQ ID NO:6 is:

[0097] TCKMSQLERNIETIINTFHQYSVKLGHPDTLNQGEFKELVRKDLQNFLKKENKNEKVIEHIM

[0098] EDLDTNADKQLSFEEFIMLMARLTWASHEKMHEGDEGPGHHHKPGLGEGTPHHHHHH

[0099] The two nucleotide sequences were co-transfected into the vector pETDuet-1 to construct a recombinant expression vector, the amino acid sequence of which is shown in SEQ ID NO:7.

[0100] The nucleotide sequence of SEQ ID NO:7 is:

[0101] GGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAG

[0102] GAGATATACCATGCTAACAGAATTGGAGAAAGCTTTAAATTCCATCATCGACGTGTACCA

[0103] TAAGTATAGCCTGATCAAGGGCAACTTCCACGCTGTGTACCGCGATGACCTGAAAAAGC

[0104] TGTTGGAAACGGAGTGCCCGCAATATATTCGTAAAAAGGGCGCCGATGTTTGGTTTAAA

[0105] GAGCTGGACATCAACACCGATGGTGCGGTGATTTTCAGGAGTTCTTGATTTTAGTTATT

[0106] AAGATGGGTGTCGCGGCACATAAGAAAGCCATGAAGAGTCTCACAAAGAACATCACC

[0107] ACCACCATCACTAAGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCTTGCGGCCGC

[0108] ATAATGCTTAAGTCGAACAGAAGTAATCGTATTGTACACGGCCGCATAATCGAAATTAA

[0109] TACGACTCACTATAGGGAATTGTGAGCGGATAACAATTCCCCATCTTAGTATATTAGTTA

[0110] AGTATAAGAAGGAGATATACATATGACATGTAAAATGTCACAACTAGAAAGGAATATCGA

[0111] AACCATTATCACACCTTTCATCAGTACAGCGTTAAACTGGGTCATCCGGATACCTTGAA

[0112] TCAGGGTGAGTTCAAAGAGCTGGTCCGCAAGGACTTACAAAACTTCCTGAAGAAAGAA

[0113] AATAAGAACGAAAAGGTGATTGAGCACATTATGGAAGATTTGGACACGAACGCGGATAA

[0114] ACAACTGTCCTTTGAAGAGTTCATCATGCTGATGGCACGTCTGACCTGGGCTAGCCATG

[0115] AGAAGATGCATGAGGGCGACGAGGGTCCGGGCCATCACCACAAACCGGGTCTCGGCGA

[0116] GGGCACTCCACATCACCACCACCACACTACTCGAGTCTGGTAAAGAAAACCGCTGCTG

[0117] CGAAATTTGAACGCCAGCACATGGACTCGTCTACTAGCGCAGCTTAATTAACCTAGGCT

[0118] GCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAG

[0119] GGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAG

[0120] CGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCA

[0121] GCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCT

[0122] TTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGC

[0123] ACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGA

[0124] TAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTC

[0125] CAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGC

[0126] CGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTA

[0127] ACAAAATATTAACGTTTACAATTTCTGGCGGCACGATGGCATGAGATTATCAAAAAGGAT

[0128] CTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAG

[0129] TAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGT

[0130] CTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGG

[0131] GCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA

[0132] GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAA

[0133] CTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCC

[0134] AGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTC

[0135] GTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCC

[0136] CATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGT

[0137] TGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCC

[0138] ATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTG

[0139] TATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAG

[0140] CAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGA

[0141] TCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAG

[0142] CATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCA

[0143] AAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATCA

[0144] TGATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAG

[0145] AAAAATAAACAAATAGGTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAG

[0146] CGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAA

[0147] TCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAA

[0148] GAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATAC

[0149] TGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTAC

[0150] ATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCT

[0151] TACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG

[0152] GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACC

[0153] TACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTA

[0154] TCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAA

[0155] CGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTG

[0156] TGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTAC

[0157] GGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTC

[0158] TGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGA

[0159] CCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCT

[0160] CCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCT

[0161] CTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGC

[0162] TGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCG

[0163] GCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTC

[0164] ACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGA

[0165] AGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGC

[0166] GTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTC

[0167] ACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGA

[0168] GAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTG

[0169] TGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGT

[0170] CAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCT

[0171] GCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTA

[0172] CGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGC

[0173] AGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAAC

[0174] CCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCTAGTCATGCCCCGC

[0175] GCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCG

[0176] GTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGT

[0177] CGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGG

[0178] TTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGA

[0179] TTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCC

[0180] CAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTC

[0181] GGTATCGTCGTATCCCACTACCGAGATGTCCGCACCAACGCGCAGCCCGGACTCGGTAA

[0182] TGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACG

[0183] ATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCT

[0184] TCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGA

[0185] CGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGAC

[0186] CCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATA

[0187] CTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGC

[0188] AGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGAC

[0189] GCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTA

[0190] CCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACA

[0191] ATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACT

[0192] GTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCG

[0193] CTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAA

[0194] ACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACA

[0195] TTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTG

[0196] CGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAA

[0197] GCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGC

[0198] AAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGA

[0199] AACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCG

[0200] ATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGC

[0201] GTAGAGGATCGAGATCGATCTCGATCCCGCGAAATTAATACGACTCACTATA

[0202] 2. Recombinant protein expression process

[0203] step:

[0204] (1) Pick a single clone from the plate and place it in 50 mL of LB medium and culture it at 37°C for 16 h.

[0205] (2) Transfer 20 mL / bottle to the culture medium and culture at 37°C for 6 h, or until the OD600 is approximately 0.6-0.8, add IPTG and induce at the preset temperature for the preset time.

[0206] (3) Centrifuge at 8000 rpm, 4°C for 3 min to collect the cells.

[0207] Specific:

[0208] 2.1. Plasmid lysis

[0209] (1) The freeze-dried plasmid powder (i.e., the recombinant expression vector constructed above) was first centrifuged at 12,000 rpm for 1 min to allow the plasmid on the tube wall to settle to the bottom of the tube.

[0210] (2) Observe the mass of the labeled plasmid on the tube wall and add sterile ultrapure water at a plasmid to body ratio of 1:25 (1 μg plasmid: 25 μL water) to dissolve the plasmid.

[0211] 2.2. Conversion

[0212] 2.2.1. Take 1 μL of plasmid and add it to 50 μL of competent cells and place on ice for 30 minutes.

[0213] 2.2.2.42℃, heat shock for 90s (strictly control the time), take out and put in ice bath for 3min.

[0214] 2.2.3. Add 600 μL of resistance-free LB liquid culture medium and culture at 37°C, 220 rpm for 1 h.

[0215] 2.2.4. Pipette 100 μL of activated bacteria and spread evenly on the amp LB plate. Incubate upright at 37°C for 15 min. After the liquid on the surface of the culture medium is absorbed, invert and incubate in a 37°C incubator overnight.

[0216] 2.3. Preparation for scale-up culture

[0217] 2.3.1.10x Phosphate stock solution: 0.17M KH2PO4, 0.72M K2HPO4.

[0218] 2.3.2.Aminobenzyl stock solution: 100ug / mL.

[0219] 2.3.3. Cannabidiol mother solution: 50ug / mL.

[0220] 2.3.4.IPTG mother solution: 1 mol / L.

[0221] 2.3.5.LB medium formula: 10.0 g / L Tryptone, 5.0 g / L Yeast Extract, 10.0 g / L NaCl.

[0222] 2.3.6.TB basal medium: 12g / L Tryptone, 24g / L Yeast Extract, 4ml / L Glycerol.

[0223] 2.4. Scale-up

[0224] 2.4.1. Pick a single round and full colony from the plate and place it in 25 mL LB medium at 37°C, 220 rpm, and culture for 16 h.

[0225] 2.4.2. The next day, add 1 mL of the corresponding antibiotic stock solution and 100 mL of 10x phosphate stock solution to 1 L TB basal medium (5 L conical flask), and mix well; add 25 mL of seed solution, culture at 37 ° C, 200 rpm, and then add 0.5 mL of IPTG stock solution after 5 hours, and adjust the shaking temperature to 16 ° C, and continue to induce culture overnight.

[0226] 2.4.3. Centrifuge at 8000rpm, 4℃ for 5min to collect the bacteria.

[0227] 3. Purification of Recombinant Protein

[0228] 3.1. Reagent preparation

[0229] Buffer A: 50mM HEPES, 200mM NaCl, pH 8.0;

[0230] Buffer B: 50 mM HEPES, 200 mM NaCl, 500 mM imidazole, pH 8.0;

[0231] Buffer C: 50 mM HEPES, 200 mM NaCl, pH 8.0, 0.1% Triton 114.

[0232] 3.1.1. Affinity purification: sample purification (gravity column purification)

[0233] 3.1.1.1. Take the empty column tube of the chromatography column that has been soaked overnight and clean the inner wall of the tube with endotoxin-free water.

[0234] 3.1.1.2. Loading the treated Ni-NTA.

[0235] 3.1.1.3. Wash with 50 mL of endotoxin-free water.

[0236] 3.1.1.4. Wash with 50 mL of Buffer C.

[0237] 3.1.1.5. Wash with 50 mL of Buffer A.

[0238] 3.1.1.6. Loading.

[0239] 3.1.1.7. Rinse with 50 mL of Buffer A.

[0240] 3.1.1.8. After washing with 50 mL of Buffer C, elute with Buffer A and Buffer B prepared with 20 mM, 50 mM, 250 mM, and 500 mM imidazole. During the elution process, use Bradford dot until the color is no longer blue.

[0241] 3.1.1.9. Take the original solution, supernatant, precipitate, flow-through, 1-2CV before Triton-114 elution, 20mM, 50mM, 250mM, 500mM collection solution and run SDS-Page.

[0242] 3.1.2. Result analysis: see Figure 1 ,in:

[0243] 1.S100A8-S100A9 Heterodimer Protein: whole bacteria;

[0244] 2. S100A8-S100A9 Heterodimer Protein: supernatant;

[0245] 3.S100A8-S100A9 Heterodimer Protein: precipitation;

[0246] 4.S100A8-S100A9 Heterodimer Protein: Flow-through;

[0247] 5.S100A8-S100A9 Heterodimer Protein: 20mM imidazole elution tube;

[0248] 6.S100A8-S100A9 Heterodimer Protein: 50mM imidazole elution tube;

[0249] 7.S100A8-S100A9 Heterodimer Protein: 100mM imidazole elution tube;

[0250] 8.S100A8-S100A9 Heterodimer Protein: 250mM imidazole elution tube.

[0251] 3.2. Reagent preparation

[0252] Buffer D: 50mM HEPES, 30mM NaCl, pH 7.5;

[0253] Buffer E: 50mM HEPES, 1M NaCl, pH 7.5.

[0254] 3.2.1. Sample concentration:

[0255] 3.2.1.1. Precool the centrifuge to 4°C.

[0256] 3.2.1.2. Before concentrating the protein, the concentration tube must be washed twice with endotoxin-free water and rinsed twice with buffer. The specific process is to pour in Buffer D, centrifuge at 5000 rpm, 4°C, for 5 to 10 minutes, pour out the centrifuged liquid, and repeat twice.

[0257] 3.2.1.3. Protein centrifugation: Add protein collection solution to the rinsed concentrating tubes, about 15 mL per tube, and centrifuge at 5000 rpm and 4°C for 30 min.

[0258] 3.2.1.4. After the sample is concentrated, soak the concentrator tube in 0.1M sodium hydroxide solution.

[0259] 3.2.2. Anion purification

[0260] 3.2.2.1. Before using the ion column, it must be flushed with 0.5M sodium hydroxide for 2 column volumes, about 10mL.

[0261] 3.2.2.2. Rinse 2 column volumes (approximately 10 mL) with endotoxin-free water.

[0262] 3.2.2.3. Rinse with Buffer E for 2 column volumes, approximately 10 mL.

[0263] 3.2.2.4. Rinse with Buffer D for 2 column volumes, approximately 10 mL.

[0264] 3.2.2.5. Sample loading (samples were diluted 10-fold with 50 mM HEPES, pH 7.5 before loading).

[0265] 3.2.2.6. Rinse 5 column volumes (about 25 mL) with Buffer D until the UV absorbance reaches a stable baseline.

[0266] 3.2.2.7. Gradient elution: Linear gradient elution uses 15 column volumes, about 75 mL; (i.e. Buffer D 0-100% in 15 column volumes). Regenerate the column after use. When not in use for a long time, store it in 20% ethanol to prevent the column from getting moldy or blocked.

[0267] 3.2.2.8.SDS-Page, the results are as follows Figure 2 As shown, lane 1: before S100A8-S100A9 loading; lane 2: flow-through; lane 3: elution tube.

[0268] 4. Purity testing

[0269] 4.1. Mix the sample with loading buffer at a ratio of 4:1, boil at 100℃ for 10 min, and load 3 μg.

[0270] 4.2. Install vertical glass plates and inject purified water into the gap between the two glass plates to check for leakage.

[0271] 4.3. Sample loading: Fix the gel on the electrophoresis device, fill the electrophoresis tank with 1X electrophoresis buffer, and use a pipette to add the prepared test solution and protein marker into the sample well. The protein loading amount is 3μg and the marker is 5μL.

[0272] 4.4. Electrophoresis Connect the electrode wires to the electrodes correctly, turn on the power supply, and perform electrophoresis at a constant current of 50 mA. When the bromophenol blue moves to the separation gel, adjust the current to 80 mA. When the bromophenol blue moves to the bottom of the gel, turn off the power supply and stop the electrophoresis.

[0273] 4.5. After the staining electrophoresis is completed, remove the glass plate from the electrophoresis device, take out the glass plate with glue, and gently pry open the glass.

[0274] 4.6. Take out the gel from the plate and place it in a container containing staining solution (the staining solution must cover the surface of the gel) and stain on a shaker for at least 30 minutes.

[0275] 4.7. Decolorization: Discard the staining solution, place the gel in the decolorizing solution, and decolorize on a shaker until the background blue color is almost completely faded.

[0276] 4.8. Gel photography: Place the completely destained gel into a gel imaging system to take a photo and save the experimental data.

[0277] 4.9. Result analysis and qualification criteria: Use Image analysis software to analyze the purity of calprotectin.

[0278] 4.10. Result analysis: The results are as follows Figure 3 As shown, after calculation, the purity is 99% and the molecular weight is 12-15KDa.

[0279] 5. Recombinant protein activity test

[0280] Cell culture

[0281] The cells were 1.5×10 4 The cells were seeded at a density of 100 μL / well in a 24-well plate, with a volume of 500 μL per well.

[0282] The protein was prepared with DMEM medium, and 700 μL of complete medium containing different concentrations of protein was added to a 24-well plate so that the final concentration of the protein in each well was 50, 25, 10, 5, 2.5, 0.5, 0.25, 0.1, 0.01, 0.001, and 0 μg / mL.

[0283] The cells were cultured in a 37°C, 5% CO2 incubator for 72 h.

[0284] After the incubation time is reached, the cell pellet is obtained by centrifugation and RNA is extracted for qPCR detection.

[0285] 5.2. RT-qPCR detection of IL-6 secretion

[0286] RNA extraction

[0287] 5.2.1.1. After adding 1 mL of Trizol to the cells, place them at room temperature for 10 min and mix them by pipetting to fully lyse them.

[0288] 5.2.1.2. Add chloroform at a rate of 200 μL chloroform / mL Trizol, vortex to mix, and leave at room temperature for 3 min.

[0289] 5.2.1.3. Centrifuge at 12,000 rpm for 10 min and aspirate the upper aqueous phase into another centrifuge tube.

[0290] 5.2.1.4. Add an equal volume of isopropanol, mix well, and leave at room temperature for 20 minutes.

[0291] 5.2.1.5. Centrifuge at 12,000 rpm for 10 min and discard the supernatant.

[0292] 5.2.1.6. Add 1 mL of 75% ethanol and gently shake the centrifuge tube to resuspend the precipitate.

[0293] 5.2.1.7. Centrifuge at 12,000 rpm for 5 min and discard the supernatant.

[0294] 5.2.1.8. Dry at room temperature or vacuum dry for 5 to 10 minutes.

[0295] 5.2.1.9. Add 50 μL of autoclaved DEPC H2O to dissolve the RNA sample.

[0296] 5.2.1.10. Determine RNA concentration.

[0297] RT-qPCR detection

[0298] Reaction system and procedures.

[0299] Table 1 Reverse transcription reaction system

[0300]

[0301] Table 2 Reverse transcription reaction procedure

[0302]

[0303] Table 3 qPCR reaction system

[0304]

[0305]

[0306] Table 4 qPCR reaction procedure

[0307]

[0308] Prepare Mix according to the above system, make three parallel tubes for each concentration gradient, and three parallel tubes for internal reference. Dilute the extracted RNA to 100 ng / μL, add 2μL, and load onto the machine.

[0309] 5.3. Experimental results and analysis

[0310] Using GraphPad Prism 9 software, the IL-6 secretion value was used as the ordinate and the protein concentration was used as the abscissa to draw a curve and calculate the ED50 value. Figure 4 As shown. After calculation, ED50 = 1.472 μg / mL. According to the enzyme activity conversion calculator: 679.348 U / mg.

[0311] S100 proteins form homodimers and heterodimers as well as higher-order oligomers in the homologous protein family. During the preparation of recombinant S100 proteins, a variety of intermolecular interactions may occur. The intermolecular association to form dimers or oligomers is a spontaneous process that minimizes free energy. Studies have reported that the natural occurrence of S100A8 / A9 heterodimers in vivo indicates that the heterodimers are more stable than related homodimers. However, the heterodimerization of semi-stable homodimers is not a spontaneous process because it requires external energy to dissociate the homodimers into monomers. Due to this property, cofolding or co-expressing heterodimeric S100 proteins is a reasonable strategy to produce stable heterodimers. This application proposes a co-expression single vector strategy, which uses a double codon-optimized S100A8 / A9 nucleic acid molecule to construct an expression system. The preparation of heterologous oligomeric recombinant proteins with biological activity is of great significance for both basic research and the production of biological preparations. The use of Escherichia coli as a host is considered to be a very good initial choice for co-expressing sufficient amounts of protein complexes. This simplified method can obtain a large amount of biologically active heterodimeric protein from Escherichia coli, and then purify it by Ni-NTA. Compared with the synthetic inclusion bodies in the prior art, the calprotectin obtained in this application is a soluble protein with a purity of 99%, a concentration of 15.805 mg / mL, a yield of 236 mg / L, an ED50 of 1.472 μg / mL, and an activity of 679.348 U / mg.

Claims

1. A codon-optimized calprotectin nucleic acid molecule, wherein the calprotectin comprises S100 calcium-binding protein A8 and S100 calcium-binding protein A9, characterized in that: The nucleic acid molecule encoding the S100 calcium binding protein A8 has the nucleotide sequence shown in SEQ ID NO:3; the nucleic acid molecule encoding the S100 calcium binding protein A9 has the nucleotide sequence shown in SEQ ID NO:

4.

2. Calprotectin, characterized in that: The invention comprises S100 calcium binding protein A8 and S100 calcium binding protein A9; the S100 calcium binding protein A8 is encoded by the nucleotide sequence shown in SEQ ID NO:3 described in claim 1, and has the amino acid sequence shown in SEQ ID NO:5; the S100 calcium binding protein A9 is encoded by the nucleotide sequence shown in SEQ ID NO:4 described in claim 1, and has the amino acid sequence shown in SEQ ID NO:

6.

3. A recombinant expression vector, characterized in that: The invention comprises the nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 3 as described in claim 1 and the nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO:

4.

4. A recombinant genetically engineered cell, characterized in that: The recombinant genetically engineered cell contains the recombinant expression vector according to claim 3; and the host cell of the recombinant genetically engineered cell is Escherichia coli.

5. The method for preparing calprotectin according to claim 2, characterized in that: The method comprises using the calprotectin nucleic acid molecule according to claim 1, the recombinant expression vector according to claim 3 and / or the recombinant genetic engineering cell according to claim 4.

6. The method for preparing calprotectin according to claim 5, characterized in that: Cultivating the recombinant genetically engineered cell of claim 4 to express the calprotectin of claim 2; and isolating the calprotectin.

7. The method for preparing calprotectin according to claim 6, characterized in that: The steps of culturing the recombinant genetically engineered cells according to claim 4 are: picking a single clone and culturing it in LB medium until the OD600 is 0.6-0.8, adding IPTG, inducing at a preset temperature; and collecting the bacteria by centrifugation.

8. The method for preparing calprotectin according to claim 7, characterized in that: The purification method for isolating the calprotectin protein is to sequentially perform Ni-NTA affinity chromatography, concentration, and anion purification.

9. The method for preparing calprotectin according to claim 8, characterized in that: The process of Ni-NTA affinity chromatography is as follows: after washing with Buffer C, eluting with Buffer A and Buffer B prepared with 20mM, 50mM, 250mM, and 500mM imidazole, and during the elution process, detecting with the Coomassie brilliant blue method until the color does not turn blue; The anion purification process is as follows: before using the ion column, wash it with 0.5M sodium hydroxide for 2 column volumes; wash it with endotoxin-free water for 2 column volumes; wash it with Buffer E for 2 column volumes; wash it with Buffer D for 2 column volumes; load the sample; wash it with Buffer D for 5 column volumes; until the UV absorption reaches a stable baseline; gradient elution, linear gradient elution uses 15 column volumes; Buffer A: 50 mM HEPES, 200 mM NaCl, pH 8.0; Buffer B: 50 mM HEPES, 200 mM NaCl, 500 mM imidazole, pH 8.0; Buffer C: 50 mM HEPES, 200 mM NaCl, pH 8.0, 0.1% Triton 114; Buffer D: 50 mM HEPES, 30 mM NaCl, pH 7.5; The Buffer E includes 50 mM HEPES, 1 M NaCl, pH 7.

5.

10. Use of the calprotectin nucleic acid molecule according to claim 1 or the calprotectin according to claim 2 in preparing a kit, characterized in that: The kit is used for diagnosing inflammation.

Images