Method for the preparation of interleukin-6 truncates and uses thereof

Abstract

This application discloses a method for preparing and applying a truncated form of interleukin-6. In this application, the present invention develops a method for preparing a truncated form of interleukin-6 based on genetic engineering technology. A polynucleotide encoding the truncated form of interleukin-6, optimized for synonymous codon bias, is introduced into a vector, successfully constructing a prokaryotic expression plasmid. Compared to untruncated interleukin-6, the expression level is significantly increased.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to the preparation method and application of interleukin-6 truncated bodies. Background Technology

[0002] Interleukin-6 (IL-6) is a cytokine with complex biological functions produced by various cells in the body. Besides its immunomodulatory, anti-tumor, and anti-infective functions, IL-6 also has a significant regulatory effect on the hematopoietic system, particularly in the recovery of platelets and the reconstruction of bone marrow hematopoietic function, which has certain clinical application value. IL-6 plays a central role in acute inflammatory responses, rapidly reflecting the effectiveness of antibiotic treatment and predicting prognosis; it is an excellent indicator for early inflammation, the severity of sepsis, and prognostic assessment.

[0003] The level of IL-6 in normal individuals is generally below 10 pg / ml. The preparation of IL-6 diagnostic kits requires highly specific and sensitive anti-IL-6 monoclonal antibodies, which in turn necessitates obtaining high-purity IL-6 protein. Previously, IL-6 was primarily extracted from tissue cells, resulting in extremely low yields and high costs. Producing IL-6 using recombinant gene therapy can solve these problems.

[0004] Traditional methods for extracting natural IL-6 from tissues and cells suffer from low efficiency, low purity, and high purification costs, and also involve unknown potential risks. Furthermore, the purification process is complex, making it difficult to obtain high-purity protein. The polyclonal antibody serum prepared from this natural protein also exhibits significant cross-contamination with other components in human serum, posing considerable challenges to product performance optimization. Currently, some laboratories in China have studied the expression of human IL-6 using an E. coli expression system. However, because human IL-6 expression in E. coli is heterologous, it is reported to be in inclusion body form. Inclusion body expression products require complex denaturation and renaturation treatments to restore their biological activity. Changing the induction temperature and IPTG concentration has not significantly altered the product's solubility. Although Dr. Liu Hongyan obtained inclusion bodies using the pBV220 expression vector in E. coli, the resulting product is also an inclusion body. Other researchers have used the 293F cell system for expression, but this method is costly and yields low expression levels, hindering the industrial production of IL-6. Furthermore, Monique C.J. et al. confirmed that glycosylation or non-glycosylation has no effect on its biological activity. Therefore, the recombinant IL-6 expressed in prokaryotic cells is no different from the natural product, and its use can be met by expressing it in the E. coli system. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing interleukin-6 truncated bodies.

[0006] Another object of the present invention is to provide a polynucleotide sequence encoding a truncated form of interleukin-6.

[0007] Another object of the present invention is to provide a vector adapted to a polynucleotide sequence encoding a truncated form of interleukin-6.

[0008] Another object of the present invention is to provide a kit containing a polynucleotide sequence encoding a truncated form of interleukin-6.

[0009] To address the aforementioned technical problems, in a first aspect, the present invention provides a polynucleotide encoding an interleukin-6 truncated form, wherein the polynucleotide is codon-optimized and is selected from any of the following:

[0010] (i) A polynucleotide having the sequence shown in SEQ ID NO.4;

[0011] (ii) a polynucleotide having greater than 95% homology to the sequence shown in SEQ ID NO. 4; and

[0012] (iii) A polynucleotide having a sequence complementary to the polynucleotide sequence described in (i) or (ii).

[0013] In some preferred embodiments, the 5' end (N-terminus) of the polynucleotide is also linked to a polynucleotide sequence encoding a Sumo tag.

[0014] In a second aspect, the present invention provides an expression vector comprising the polynucleotide provided in the first aspect of the present invention.

[0015] In some preferred embodiments, the expression vector is an Escherichia coli expression vector, more preferably pET-32a(+).

[0016] In some preferred embodiments, the vector comprises a polynucleotide sequence expressing a His×6 tag. More preferably, in the vector, the 3' end of the polynucleotide is linked to a polynucleotide sequence expressing a His×6 tag;

[0017] In some preferred embodiments, the vector comprises a polynucleotide sequence expressing a TrxA tag. More preferably, in the vector, the 5' end of the polynucleotide is linked to a polynucleotide sequence expressing a TrxA tag;

[0018] In a third aspect, the present invention provides a host cell comprising the expression vector provided in the second aspect of the present invention; or

[0019] The host cell genome integrates polynucleotides as provided in the first aspect of the present invention.

[0020] In some preferred embodiments, the host cell is *Escherichia coli*.

[0021] In some preferred embodiments, the host cell is Escherichia coli Rosetta (DE3) strain.

[0022] A fourth aspect of the present invention provides a method for preparing interleukin-6 truncated molecules, the method comprising the steps of: culturing the host cells described in the third aspect of the present invention to express the target protein; and

[0023] The target protein is isolated to obtain the interleukin-6 truncated form;

[0024] The target protein has an amino acid sequence as shown in SEQ ID NO:3.

[0025] In some preferred embodiments, the host cell is obtained by transforming Escherichia coli with a plasmid containing the polynucleotide described in the first aspect of the invention.

[0026] In some preferred embodiments, the host cells are cultured in SB, TB, or SOC media. In a more preferred embodiment, to obtain high levels of soluble expression, the host cells are cultured in TB media.

[0027] In some preferred embodiments, the host cells are cultured in an oscillating environment.

[0028] In some preferred embodiments, the host cells are cultured at a temperature of 36 to 38°C.

[0029] In some preferred embodiments, culturing the host cells in TB medium at a temperature of 36 to 38°C can yield higher expression of the soluble target protein.

[0030] In some preferred embodiments, the culture medium used to culture the host cells contains a kanamycin resistance gene.

[0031] In some preferred embodiments, IPTG is used to induce the expression of the target protein when culturing the host cells.

[0032] In some preferred embodiments, the host cells are cultured until the OD600 is between 0.6 and 0.8, and then induced with IPTG to express the target protein.

[0033] In some preferred embodiments, the step of isolating the target protein includes:

[0034] The supernatant of the lysed target protein is passed through a chromatography column for elution, and the eluent is collected.

[0035] In some preferred embodiments, the chromatography column is a Ni-column affinity chromatography column.

[0036] A fifth aspect of the present invention provides a kit comprising: a polynucleotide as provided in the first aspect of the present invention; or

[0037] Such as the expression vector provided in the second aspect of the present invention; or

[0038] The host cell as described in the third aspect of the present invention; or

[0039] Or a truncated form of interleukin-6 prepared by the method according to the fourth aspect of the present invention.

[0040] Compared with the prior art, the present invention has at least the following advantages:

[0041] (1) This invention develops a method for preparing interleukin-6 truncated polynucleotides based on genetic engineering technology. The polynucleotide encoding the interleukin-6 truncated polynucleotides optimized by synonymous codon preference is introduced into a vector, and a prokaryotic expression plasmid is successfully constructed. Compared with the untruncated interleukin-6, the expression level is greatly improved.

[0042] (2) The interleukin-6 truncated body prepared by the preferred embodiment of the present invention is expressed with IL-6 fused with a TrxA-sumo tandem tag at the N-terminus to achieve soluble expression in the Escherichia coli system, and the obtained protein has high stability and immunological activity.

[0043] (3) The method for preparing interleukin-6 truncated bodies provided by the present invention has the advantages of high yield, short production cycle, easy purification of expression products and low cost, and can realize the industrial production of IL-6 protein.

[0044] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0045] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

[0046] Figure 1 This is an SDS-PAGE identification result of interleukin-6 and its truncated form prepared according to the embodiments of the present invention;

[0047] Figure 2This is an image showing the SDS-PAGE identification results of the truncated form of interleukin-6 induced at 18℃ according to an embodiment of the present invention.

[0048] Figure 3 This is an electrophoresis image of interleukin-6 induced at 25°C according to an embodiment of the present invention. Detailed Implementation

[0049] Through extensive and in-depth research, the inventors modified interleukin-6 by truncating it, obtaining the truncated form of interleukin-6 (SEQ ID NO:3), and developed an expression system for the truncated form of interleukin-6 based on a prokaryotic expression system.

[0050] Furthermore, through synonymous codon preference optimization, a polynucleotide sequence (SEQ ID NO:4) encoding the interleukin-6 truncated form was obtained that can express the target protein in large quantities in the E. coli expression system. The expressed target protein has high activity and good stability.

[0051] The inventors further improved the soluble expression of the target protein by fusing a polynucleotide sequence expressing a TrxA tag and a polynucleotide sequence expressing a sumo tag to the N-terminus of the polynucleotide sequence encoding the interleukin-6 truncated form, thereby reducing purification steps and achieving an expression level of more than 50% of the total bacterial protein.

[0052] Obtain the nucleic acid sequence related to the target gene / protein.

[0053] In this invention, the full-length nucleotide sequence or fragments of the target protein or its elements can typically be obtained using PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on publicly available nucleotide sequences, especially open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared using conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order.

[0054] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0055] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0056] The method of amplifying DNA / RNA using PCR technology is preferred for obtaining the gene of the present invention. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0057] In one embodiment of the present invention, the amino acid sequence (SEQ ID NO: 1) of the target protein is analyzed using the NCBI database to obtain the target gene sequence information.

[0058] Synonymous codon preference optimization

[0059] To overcome the potential problem of reduced yield when expressing heterologous proteins in *E. coli*, this invention relates to a polynucleotide sequence optimized by synonymous codon preference. The obtained target gene sequence is optimized by synonymous codon preference. The optimized target gene sequence (SEQ ID NO:2) can express the same amino acid sequence as the target protein, but the stability and efficiency of the expression process are improved, and the final target protein maintains high activity.

[0060] The present invention also relates to polynucleotides with greater than 95% homology to the sequence shown in SEQ ID NO:2; and polynucleotides complementary to the sequence shown in SEQ ID NO:2.

[0061] To improve the solubility of the expressed protein, this invention also modifies the amino acid sequence of the target protein (SEQ ID NO:1), truncating it to obtain an amino acid sequence (SEQ ID NO:3). The corresponding gene sequence is then obtained through analysis and optimized for synonymous codon bias to obtain the gene sequence encoding the truncated version of the target protein (SEQ ID NO:4). This truncated version not only possesses immunomodulatory activity comparable to the target protein but also improves the solubility of the resulting protein.

[0062] The present invention also relates to polynucleotides with greater than 95% homology to the sequence shown in SEQ ID NO:4; and polynucleotides complementary to the sequence shown in SEQ ID NO:4.

[0063] Vector of the target gene

[0064] This invention also relates to vectors comprising the polynucleotides of this invention. In this invention, "vector" refers to a linear or circular DNA molecule containing a fragment encoding a target protein, said target protein being operatively linked to other fragments that provide for its transcription. Such additional fragments may include promoter and terminator sequences and may optionally include one or more origins of replication, one or more optional markers, enhancers, polyadenylation signals, vectors, etc. The vector fragment may be derived from a host organism, another organism, plasmid or viral DNA, or may be synthetic. The vector may be any expression vector, either synthetic or readily manipulated with recombinant DNA, and the choice of vector generally depends on the host cell to which the vector is to be introduced. Thus, the vector may be a self-replicating vector, i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid. Alternatively, the vector may be a vector that integrates into the host cell genome upon introduction into a host cell and replicates along with the chromosome into which it is integrated. In one embodiment, the vector of this invention is an expression vector. In one embodiment of this invention, pET-32a(+) is selected as the vector to obtain more efficient expression.

[0065] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence of the protein of the present invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Exemplarily, the vector DNA molecule is cleaved into linear molecules that can be linked to a foreign gene using a DNA endonuclease, and then a codon-optimized target gene fragment is ligated into the vector. The insertion of the foreign DNA fragment can be achieved by using sticky-end ligation at a single restriction enzyme site, directed cloning of double-restricted fragments, sticky-end ligation at different restriction enzyme sites, blunt-end ligation, artificial adapter ligation, or ligation to oligonucleotide ends.

[0066] Transformation of host cells using a vector containing the target gene

[0067] This invention also relates to host cells genetically engineered using the vector or fusion protein coding sequence of this invention. A vector containing a codon-optimized target gene can be inserted into a host cell by known methods, transfected, or otherwise transformed to obtain a transformant containing the codon-optimized target gene of this invention and capable of expressing the target protein. In this invention, "host cell" refers to a cell incorporating exogenous polynucleotides and / or a vector. The host cell can be a eukaryotic or prokaryotic host cell, preferably a bacterium, and more preferably *Escherichia coli* Rosetta(DE3) strain.

[0068] Methods for preparing target proteins

[0069] This invention also relates to a method for preparing the target protein, which can be used to express or produce recombinant proteins using the polynucleotide sequence of this invention. Generally, the method includes the following steps:

[0070] (1) Transform or transduce suitable host cells using the polynucleotide (or variant) encoding the protein of the present invention, or using a recombinant expression vector containing the polynucleotide;

[0071] (2) Host cells cultured in a suitable culture medium;

[0072] (3) Isolate and purify proteins from culture media or cells.

[0073] In step (1), the recombinant expression vector containing the polynucleotide is transformed or transduced into a suitable host cell by conventional techniques known to those skilled in the art. When the host is Escherichia coli, heat shock and electroconversion methods can be used.

[0074] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media, preferably SB, TB, or SOC media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature change or chemical induction), and the cells are cultured for a further period. In a preferred embodiment of this invention, to promote the expression of the target protein and increase the expression level of soluble proteins, host cells are cultured in TB media containing a kanamycin resistance gene.

[0075] To further promote the soluble expression of the target protein, in a preferred embodiment of the present invention, host cells are cultured to OD0.05. 600 After reaching a concentration between 0.6 and 0.8, induction was performed using IPTG, and the mixture was further cultured at 36 to 39°C for approximately 8 to 12 hours.

[0076] The proteins described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods. In one embodiment of the invention, affinity chromatography is used to molecularly target the protein.

[0077] In this invention, any exemplary or illustrative terminology (e.g., “”) used with respect to certain embodiments herein is merely for the purpose of better presenting the invention and does not limit the scope of the invention as otherwise claimed. No terminology herein should be construed as indicating an element not described in the claims that is indispensable to the implementation of this invention.

[0078] If the definition or use of a term in a cited reference is inconsistent with or inconsistent with the definition of a term described herein, the definition of the term described herein shall be used instead of the definition of the term in the cited reference.

[0079] The various terms used herein are as follows. If a term used in the claims is not defined below, the broadest definition of that term given by a person skilled in the art should be given, as reflected in the publication printed at the time of application or in the published patent.

[0080] As used herein, the term "isolated" refers to a nucleic acid or polypeptide isolated from at least one other component (e.g., a nucleic acid or polypeptide) present in its natural source. In one embodiment, the nucleic acid or polypeptide is found to be present only (if any) in a solvent, buffer, ion, or other component normally present in its solution. The terms "isolated" and "purified" do not include nucleic acids or polypeptides present in their natural source.

[0081] As used herein, the terms "polynucleotide" and "polynucleotide sequence" can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding.

[0082] This invention also relates to variants of the aforementioned polynucleotides that encode protein fragments, analogs, and derivatives having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be the substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the encoded polypeptide.

[0083] As used in this article, the term "codon optimization" refers to the method of improving gene synthesis efficiency by avoiding the use of low-utilization or rare codons based on the differences in codon utilization exhibited by the actual organisms performing protein expression or production (including E. coli, yeast, mammalian blood cells, plant cells, insect cells, etc.).

[0084] As used herein, the terms “homology” and “identity” are used interchangeably and refer to the percentage of identical (i.e., same) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be measured by arranging the nucleotide or amino acid sequences of the polynucleotide or polypeptide, scoring the number of positions in the arranged polynucleotide or polypeptide containing the same nucleotide or amino acid residues, and comparing this to the number of positions in the arranged polynucleotide or polypeptide containing different nucleotide or amino acid residues. Polynucleotides can differ at one position, for example, by containing different nucleotides (i.e., substitutions or variations) or by the deletion of nucleotides (i.e., the insertion or deletion of one or two nucleotides in the polynucleotide). Polypeptides can differ at one position, for example, by containing amino acids (i.e., substitutions or variations) or by the deletion of amino acids (i.e., the insertion of one or two amino acids in the polypeptide or the deletion of amino acids). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percentage identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide, and then multiplying by 100.

[0085] As used herein, the terms “sequence complement” and “reverse sequence complement” are used interchangeably and refer to a sequence that is in the opposite direction to the original polynucleotide sequence and is complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is ACTGAAC, then its reverse complementary sequence is GTTCAT.

[0086] As used herein, the term "expression" includes any step involved in the production of a polypeptide in a host cell, including but not limited to transcription, translation, post-translational modification, and secretion. Post-expression can be harvested, i.e., the host cell or the expressed product can be recovered.

[0087] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0088] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0089] Example 1: Construction of IL-6 plasmid and transfection of host cells

[0090] (1) The amino acid sequence of human IL-6 (SEQ ID NO:1) was obtained and analyzed to obtain the gene sequence. Synonymous codon bias optimization was performed on the gene sequence to obtain the gene sequence (SEQ ID NO:2) after synonymous codon bias optimization. After E. coli synonymous codon bias optimization, the gene sequence was ligated into the vector pET-28a(+) to synthesize recombinant expression plasmids.

[0091] (2) Obtain the amino acid sequence of human IL-6 SEQ ID NO:1, remove the first 29 amino acids from the N-terminus, determine the amino acid sequence shown in SEQ ID NO:3 (truncated A), and then optimize the gene sequence encoding it by E. coli synonym codon preference to obtain SEQ ID NO:4. The ligation vector is pET-32a(+), the N-terminus is fused with the TrxA tag, and a sumo tag and a C-terminal (His)6 tag are added to the protein to synthesize a recombinant expression plasmid.

[0092] (3) Obtain the amino acid sequence of human IL-6 SEQ ID NO:1, remove the first 29 amino acids from the N-terminus, determine the amino acid sequence shown in SEQ ID NO:3 (truncated A), and then optimize the gene sequence encoding it by E. coli synonym codon preference to obtain SEQ ID NO:5. The ligation vector is pET-32a(+), the N-terminus is fused with the TrxA tag, and a sumo tag and a C-terminal (His)6 tag are added to the protein to synthesize a recombinant expression plasmid.

[0093] (4) Obtain the amino acid sequence of human IL-6 SEQ ID NO:1, remove the first 29 amino acids from the N-terminus, determine the amino acid sequence shown in SEQ ID NO:3 (truncated A), and then optimize the gene sequence encoding it by E. coli synonym codon preference to obtain SEQ ID NO:6. The ligation vector is pET-32a(+), the N-terminus is fused with the TrxA tag, and a sumo tag and a C-terminal (His)6 tag are added to the protein to synthesize a recombinant expression plasmid.

[0094] (5) Introduction of recombinant plasmids into host Escherichia coli

[0095] Take 1 μL of the expression plasmid prepared in steps (1)-(4), add it to 30 μL of competent E. coli Rosetta (DE3) cells under ice bath conditions, place on ice for 20 min, heat shock for 90 s, immediately place on ice for 2 min, add 400 μL of antibiotic-free SOC medium, and culture at 37℃ and 220 rpm for 50 min with shaking. Take 100 μL of bacterial culture and spread it evenly on LB agar plates containing 100 μg / mL kanamycin resistance, and incubate overnight at 37℃.

[0096] The untagged IL-6 protein monoclonal strain was named A2-1;

[0097] The single-clone strain of IL-6 protein truncated A (codon sequence SEQ ID NO:4) coupled with the TrxA-sumo tag was named B2-1;

[0098] Monoclonal strain B2-3 conjugated with TrxA-sumo tag IL-6 protein truncated A (codon sequence SEQ ID NO:5);

[0099] A monoclonal strain B2-2 conjugated with the TrxA-sumo tag truncated A of the IL-6 protein (codon sequence SEQ ID NO:6). Human IL-6 amino acid sequence SEQ ID NO:1

[0100] MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNNMCES

[0101] SKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKN

[0102] LDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALRQM

[0103] Human IL-6 polynucleotide sequence SEQ ID NO:2 optimized by E. coli synonymous codon preference

[0104] ATGAATTCATTTTCTACAAGTGCTTTCGGACCTGTTGCGTTTAGCTTAGGTCTGCTGCTGGTGCTGCCGGCGGCCTTCCC

[0105] GGCGCCGGTACCACCGGGTGAAGATTCCAAGGACGTGGCGGCTCCGCATCGCCAACCGCTGACCAGTTCGGAGCGCATCG

[0106] ATAAGCAGATTCGTTACATCCTCGACGGCATTAGCGCACTTCGTAAGGAGACTTGTAATAAGAGCAATATGTGCGAAAGC

[0107] TCGAAAGAAGCTCTGGCAGAGAACAACCTAAATTTGCCGAAAATGGCAGAAAAAGACGGCTGCTTTCAGAGCGGCTTTAA

[0108] CGAGGAGACGTGCCTGGTTAAAATCATCACGGGTCTGTTGGAATTCGAGGTTTATTTGGAATACCTGCAAAATCGTTTCG

[0109] AGTCTTCCGAAGAACAGGCGCGTGCGGTCCAGATGAGCACCAAGGTGCTGATTCAGTTCCTCCAAAAAAAGGCGAAAAAC

[0110] CTGGACGCCATCACCACTCCGGATCCGACCACCAACGCTAGCCTGTTGACCAAGCTGCAAGCACAAAACCAGTGGCTGCA

[0111] AGATATGACCACCCACCTGATTTTGAGAAGCTTTAAAGAGTTCCTGCAGTCTTCCCTGCGTGCGTTACGCCAGATG Truncated body A amino acid sequence SEQ ID NO:3

[0112] MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPED

[0113] LDMEDNDIIEAHREQIGGATYPVPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAE

[0114] NNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTP

[0115] DPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALRQM

[0116] The polynucleotide sequence SEQ ID NO:4 encoding truncated A optimized by the synonymous codon preference of Escherichia coli

[0117] ATGAGTGATTCAGAAGTAAATCAAGAGGCTAAGCCGGAGGTTAAGCCAGAAGTGAAGCCGGAAACGCACATCAATCTGAA

[0118] GGTGAGCGATGGCTCTTCGGAAATCTTTTTCAAGATCAAAAAGACCACGCCGCTGCGTCGTCTGATGGAGGCGTTCGCCA

[0119] AGCGCCAGGGTAAGGAGATGGATTCCTTACGTTTTCTGTATGACGGCATCCGCATTCAAGCCGATCAGACCCCGGAGGAC

[0120] CTGGACATGGAGGACAACGATATTATCGAAGCGCATCGTGAACAAATTGGTGGCGCTACCTACCCGGTTCCGCCTGGTGA

[0121] AGACAGCAAGGACGTCGCCGCGCCGCACAGACAACCGTTGACCAGCTCGGAGCGTATTGATAAACAAATCCGCTACATTT

[0122] TGGACGGCATTAGCGCACTGCGCAAAGAAACCTGCAACAAAAGCAATATGTGTGAAAGCTCCAAAGAGGCGCTGGCGGAG

[0123] AACAACCTGAATTTGCCGAAAATGGCAGAAAAAGACGGCTGCTTCCAGAGCGGTTTTAATGAAGAGACGTGCTTGGTGAA

[0124] GATCATCACCGGTCTGCTTGAGTTCGAGGTGTATCTTGAATACCTGCAAAACCGTTTCGAGTCCTCTGAGGAACAGGCGC

[0125] GTGCTGTTCAGATGTCTACCAAAGTTCTGATCCAATTTCTGCAGAAAAAAGCTAAGAACTTAGACGCAATTACCACGCCG

[0126] GATCCGACTACCAACGCGAGCTTGTTGACCAAGCTGCAGGCGCAGAACCAGTGGCTGCAAGATATGACCACCCATCTGAT

[0127] CCTGCGTAGCTTTAAAGAGTTCCTGCAGTCCAGCCTGCGCGCTCTCCGTCAGATG

[0128] The polynucleotide sequence of truncated body A, SEQ ID NO:5, optimized according to the synonymous codon preference of Escherichia coli

[0129] ATGTCAGATAGTGAAGTAAATCAAGAGGCTAAGCCTGAGGTGAAGCCGGAAGTGAAGCCGGAGACCCATATCAACCTGAA

[0130] GGTGAGCGATGGTAGCTCGGAGATTTTTTTTAAGATCAAAAAGACCACTCCGCTGCGTCGTCTGATGGAAGCGTTCGCCA

[0131] AACGTCAAGGTAAAGAGATGGATTCTCTTCGCTTCCTGTATGATGGCATCCGTATCCAGGCTGATCAGACCCCGGAGGAT

[0132] CTGGACATGGAAGACAATGACATTATCGAAGCGCATCGTGAACAGATCGGCGGTGCTACGTACCCGGTTCCACCGGGTGA

[0133] GGACAGCAAGGACGTTGCGGCTCCGCACCGCCAACCGTTGACCAGCTCGGAACGAATTGACAAACAGATTCGTTACATCC

[0134] TGGACGGCATTTCCGCACTGCGGAAGGAGACGTGCAACAAAAGCAATATGTGTGAAAGCAGCAAAGAGGCGTTGGCTGAA

[0135] AACAACCTCAATCTACCGAAGATGGCAGAAAAGGACGGCTGCTTCCAGTCCGGTTTTAACGAAGAGACGTGCCTGGTTAA

[0136] GATTATTACCGGTCTGCTGGAGTTCGAGGTGTACTTAGAGTATCTGCAAAACCGTTTTGAGTCTTCAGAAGAGCAGGCCC

[0137] GTGCGGTCCAGATGTCCACCAAAGTTTTGATCCAATTTTTACAAAAAAAGGCGAAAAACCTGGACGCCATCACCACGCCG

[0138] GATCCGACCACCAATGCAAGCTTGCTGACCAAACTGCAAGCGCAGAACCAGTGGCTGCAAGATATGACCACTCACCTGAT

[0139] CCTGAGAAGCTTCAAAGAATTCTTGCAATCTTCCTTGCGCGCGTTGCGCCAGATG

[0140] The polynucleotide sequence of truncated body A encoding optimized by Escherichia coli synonymous codon preference, SEQ ID NO: 6

[0141] ATGTCTGACTCTGAAGTTAACCAGGAAGCTAAACCGGAAGTAAAACCGGAAGTTAAGCCGGAAACCCACATTAATCTGAA

[0142] AGTGTCTGATGGCTCCTCCGAAATCTTTTTCAAAATTAAAAAAACTACTCCACTGCGTCGTCTGATGGAAGCGTTCGCCA

[0143] AACGTCAGGGCAAGGAGATGGATTCCCTGCGCTTCCTGTACGACGGCATCCGTATCCAGGCTGATCAGACTCCGGAAGAC

[0144] CTGGATATGGAAGATAACGACATCATCGAAGCCCACCGCGAACAGATCGGTGGCGCTACTTACCCGGTGCCGCCAGGCGA

[0145] AGACTCTAAGGACGTGGCAGCTCCACACCGTCAGCCGCTGACTTCTAGCGAACGCATCGACAAACAGATTCGCTACATTC

[0146] TGGACGGCATCTCTGCTCTGCGCAAAGAAACTTGTAACAAATCTAACATGTGCGAAAGCAGCAAAGAAGCGCTGGCGGAA

[0147] AACAACCTGAACCTGCCTAAAATGGCTGAAAAAGACGGCTGCTTTCAGTCCGGTTTTAACGAAGAGACTTGCCTGGTCAA

[0148] AATCATCACCGGCCTGCTGGAATTCGAAGTTTACCTGGAATACCTGCAGAACCGTTTCGAATCTAGCGAAGAACAGGCAC

[0149] GTGCAGTTCAGATGTCTACCAAAGTTCTGATCCAGTTCCTGCAGAAAAAGGCTAAAAACCTGGATGCAATCACTACCCCG

[0150] GATCCGACCACCAACGCCAGCCTGCTGACCAAACTGCAGGCTCAAAACCAGTGGCTGCAGGACATGACCACCCACCTGAT

[0151] CCTGCGTTCCTTCAAAGAATTCCTGCAGTCCAGCCTGCGCGCGCTGCGCCAGATG

[0152] Example 2: Expression of the target gene

[0153] Unlabeled IL-6 protein A2-1 monoclonal strains from Example 1 were aseptically inoculated into TB medium containing 100 μg / mL kanamycin resistance and cultured at 37°C with shaking at 220 rpm until the OD600 was between 0.6 and 0.8. They were then induced with IPTG and incubated overnight at 37°C with shaking.

[0154] Select the B2-1 monoclonal strain of IL-6 protein truncated A conjugated with the TrxA-sumo tag from Example 1, aseptically inoculate it into TB medium containing 100 μg / mL kanamycin, and culture at 37°C with shaking at 220 rpm until the OD600 is between 0.6 and 0.8. Induce with IPTG and incubate overnight at 37°C with shaking.

[0155] Select the monoclonal strain B2-3 of IL-6 protein truncated A conjugated with the TrxA-sumo tag from Example 1, aseptically inoculate it into TB medium containing 100 μg / mL kanamycin, and culture at 37°C with shaking at 220 rpm until the OD600 is between 0.6 and 0.8. Induce with IPTG and incubate overnight at 37°C with shaking.

[0156] Select the monoclonal strain B2-2 of the TrxA-sumo tag IL-6 protein truncated form A from Example 1, aseptically inoculate it into TB medium containing 100 μg / mL kanamycin, and culture at 37°C with shaking at 220 rpm until the OD600 is between 0.6 and 0.8. Induce with IPTG and incubate overnight at 37°C with shaking.

[0157] Four different samples were ultrasonically disrupted and then analyzed by SDS-PAGE. The results are shown below. Figure 1 and Figure 2 .

[0158] The results are as follows Figure 1 As shown, at 37℃, no soluble expression of IL-6 protein was observed in the culture medium in the untagged IL-6 protein A2-1 monoclonal strain, while only trace amounts of soluble IL-6 protein were expressed in the culture medium in the monoclonal strains B2-3 and B2-2, which were coupled with the TrxA-sumo tag truncated IL-6 protein A. Figure 2In this study, the monoclonal strain B2-1, which was conjugated with the TrxA-sumo tag and was a truncated form of IL-6 protein A, could be expressed in large quantities in the supernatant of TB medium, with the expression level accounting for more than 50% of the total bacterial protein.

[0159] Example 3: Purification of the expression product

[0160] B2-1 single-clone strain was cultured in 1.5 L of shake flask culture. The wet weight of the bacterial cells was collected by centrifugation: 14 g. Approximately 4 g of the bacterial cells were weighed and resuspended in 35 mL of Lysis Buffer on ice. After sonication and centrifugation at 20,000 rpm, 4 °C for 30 min, the supernatant was collected and filtered through a 0.22 μm syringe filter to obtain the filtered bacterial solution. The filtered solution was then subjected to Ni-column affinity chromatography. The protein eluted with 50 mM Tris-HCl, 50 mM NaCl, and 200 mM imidazole at pH 7.0 was the target protein. 12 mL of protein was obtained, with a concentration of 1.69 mg / mL. The electrophoresis result is shown below. Figure 3 As shown in the figure, the calculated expression level of the target protein is 47.32 mg / L, with a purity exceeding 95%.

[0161] Example 4: Chemiluminescence method for identifying the activity of the target protein

[0162] According to the Darui reagent kit instructions, the activity was detected using a double-antibody sandwich method:

[0163] (1) Preparation of antigen-antibody sandwich complex

[0164] Samples of B2-1 monoclonal strain expressed on TB were mixed with magnetic beads coated with IL-6 protein monoclonal antibody. After washing, another IL-6 monoclonal antibody labeled with acrylonitrile salt was added and reacted under incubation conditions. The antibody captured the antigen in the sample, forming an antigen-antibody sandwich complex.

[0165] (2) Test reading:

[0166] After incubation, the supernatant was removed, the precipitated complex was washed with washing solution, and the waste liquid was aspirated to remove any substances not bound to the magnetic particles. Two excitation solutions were added to induce chemiluminescence signals in the complex, and the luminescence intensity was measured. The results of chemiluminescence detection are shown in Table 1. The highest luminescence value of IL-6 truncated protein reached 4.68 million, indicating good antigen performance. The linear regression equation between concentration and RLU value is: y = 864.5x + 67271, R... 2 =0.9959, indicating good linearity (R² = 0.9959). 2 >0.99).

[0167] Table 1

[0168]

[0169] Example 5: Chemiluminescence assay for protein stability

[0170] Before testing, samples of B2-1 monoclonal strain expressing on TB were stored at 37°C for 3 days and 7 days, respectively. Activity was detected using a double-antibody sandwich assay according to the Darui kit instructions.

[0171] (1) Preparation of antigen-antibody sandwich complex

[0172] The sample and magnetic beads coated with IL-6 monoclonal antibody were mixed and washed. Then, another IL-6 monoclonal antibody labeled with acrylonitrile salt was added and reacted under incubation conditions. The antibody captured the antigen in the sample and formed an antigen-antibody sandwich complex.

[0173] (2) Test reading:

[0174] After incubation, the supernatant was removed, the precipitated complex was washed with a washing solution, and the waste liquid was dried to remove any substances that had not bound to the magnetic particles. Two excitation solutions were added to induce the complex to produce a chemiluminescent signal, and the luminescence intensity was measured.

[0175] As shown in Table 2, the relative deviation of the luminescence value of the IL-6 protein raw material at a concentration of 0-5000 pg / ml after storage at 37℃ for 3 days was within ±20%, and the relative deviation of the luminescence value at a concentration of 20-5000 pg / ml after storage at 37℃ for 7 days was within ±30%. This indicates that the IL-6 protein prepared by this invention did not show a significant decrease in protein activity after storage at 37℃ for 3 days and 7 days, and its stability is good.

[0176] Table 2

[0177]

[0178]

[0179] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.

Claims

1. An isolated polynucleotide encoding interleukin-6, characterized in that, The polynucleotide is codon-optimized and is a polynucleotide with the sequence shown in SEQ ID NO.

4.

2. The polynucleotide according to claim 1, characterized in that, The 5' end of the polynucleotide is also attached to a polynucleotide sequence encoding a Sumo tag.

3. An expression carrier, characterized in that, The expression vector is an Escherichia coli expression vector, comprising the polynucleotides as described in any one of claims 1 or 2.

4. The expression vector according to claim 3, characterized in that, The expression vector is pET-32a(+).

5. The expression vector according to claim 4, characterized in that, The vector includes a polynucleotide sequence expressing a His×6 tag.

6. The expression vector according to claim 4, characterized in that, The vector includes a polynucleotide sequence expressing a TrxA tag.

7. A host cell, characterized in that, The host cell is Escherichia coli, and the host cell includes the expression vector as described in any one of claims 3 to 6.

8. A method for preparing interleukin-6, characterized in that, The method includes the following steps: Transform a host cell containing a polynucleotide vector as described in any one of claims 1 or 2, wherein the host cell is Escherichia coli; The host cells are cultured to express the interleukin-6.

9. The method according to claim 8, characterized in that, The host cells were cultured in SB, TB, or SOC media. And / or, when culturing the host cells, the expression of the target protein is induced by IPTG; And / or, when culturing the host cells, the host cells are cultured at a temperature of 37 to 39°C; And / or, when culturing the host cells, culture them until the OD600 is between 0.6 and 0.8, and then induce expression of the target protein using IPTG.

10. A reagent kit, characterized in that, The kit comprises: a polynucleotide as described in any one of claims 1 or 2; or The expression vector as described in any one of claims 3 to 6; or The host cell as described in claim 7.

Images