Preparation method and application of recombinant human long-acting interleukin-22 binding protein

Abstract

The present invention relates to the field of biotechnology, and in particular to a preparation method and application of a recombinant human long-acting interleukin-22 binding protein. The amino acid sequence of the recombinant human long-acting interleukin-22 binding protein is shown in SEQ ID No.1. The present invention utilizes an Escherichia coli (E.coli) prokaryotic expression system to prepare a method for IL-22BP recombinant protein with high biological activity, by designing a novel fusion protein, so that the sequence encoding human IL-22BP is connected to the sequence encoding ABD, constructed as an IL-22BP-ABD fusion gene, induced to be expressed in Escherichia coli, after separating the inclusion body protein, the target protein is obtained by denaturation, purification, and renaturation, and the biological activity of IL-22BP-ABD is verified by in vivo and in vitro experiments.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing and applying a recombinant human long-acting interleukin-22 binding protein. Background Technology

[0002] Interleukin-22 (IL-22) is a member of the interleukin-10 (IL-10)-associated cytokine family, which includes IL-19, IL-20, IL-24, and IL-26. These cytokines share the same amino acid sequence and structural similarity as IL-10. For signal transduction, their heterodimeric receptors share several distinct receptor chains, which are differentiated to produce different receptors with varying cytokine specificities. All members of this cytokine family are involved to some extent in tissue-mediated responses during inflammation. IL-22 is the most extensively studied member, promoting both innate and adaptive immune responses. The primary source of IL-22 is CD4+. + T cells and type 3 innate lymphocytes (ILC3) can also be produced by other immune cells, such as γδ T cells, natural killer (NK) cells, T cells, and mucosa-associated inertial T cells (MAIT). Like many other cytokines, IL-22 is produced upon activation of immune cells, which may be related to CD4+. + T cells exhibit MHCII antigen-specific responses to peptides or innate cytokines (such as IL-23 or IL-1β of ILC3).

[0003] IL-22 has both protective and pathogenic effects in inflammation. This dual role is best elucidated in gene-deficient mice or experimental mouse models of neutralizing antibodies. This dual nature of activity is thought to depend on the inflammatory environment, including the levels of affected tissues, other cytokines, oxygen and metabolites and their derivatives, and other inflammatory mediators such as leukotrienes and prostaglandins. Generally, IL-22 is pathogenic in skin inflammation. For example, in psoriasis, IL-22 promotes the proliferation of keratinocytes, thereby inhibiting normal differentiation as cells rapidly migrate from the dermis to the epidermis. Anti-IL-22 antibodies have shown promise in treating patients with atopic dermatitis. In contrast, IL-22 has been shown to be both protective and pathogenic in various gastrointestinal inflammatory models; it is generally protective in acute models and pathogenic in chronic inflammatory models. Therefore, based on the negative regulatory role of IL-22 in certain diseases, IL-22 holds promise as an important therapeutic target.

[0004] Interleukin-22 binding protein (IL-22BP; IL-22RA2) has been identified as a soluble receptor homolog of the interleukin-22 receptor (IL-22R). The gene encoding IL-22BP (IL22RA2 in humans) is encoded as IL22ra2 in mice, located on human chromosome 6 and mouse chromosome 10. IL-22 has a 10,000-fold higher affinity for IL-22BP than for IL-22R, and both IL-22BP and IL-22R share the same binding site with IL-22. Therefore, IL-22BP competitively binds to IL-22R, thereby blocking downstream signal transduction. Like IL-22, IL-22BP is primarily produced by immune cells. Unlike many immune effector molecules, IL-22BP is constitutively expressed by activated immune cells at higher levels than by resting immune cells. IL-22BP has been shown to be primarily produced by dendritic cells (DCs), although other immune cells, such as eosinophils and CD4 T cells, also produce IL-22BP.

[0005] To date, searches on databases such as the National Patent Network and PubMed have revealed very few instances of using prokaryotic cells to express recombinant IL-22BP or to prepare IL-22BP fusion proteins.

[0006] ABD is a small triple-helix protein domain that can bind to human serum albumin (HSA). Studies have reported that fusion of exogenous adherent molecules, antibody fragments, and peptides with ABD can significantly prolong their in vivo half-life. ABD can effectively increase the plasma half-life of fusion conjugates to approximately the same level as endogenous albumin (approximately 30 hours in mice and 12-17 days in humans) without causing immunogenicity issues.

[0007] Currently, most recombinant proteins are prepared using eukaryotic systems (such as monkey COS7 cells, Chinese hamster CHO cells, and SF9 insect cells). Eukaryotic systems have drawbacks such as low expression levels, high production costs, long culture cycles, and susceptibility to contamination. In contrast, prokaryotic expression systems, represented by *E. coli*, offer advantages such as low cost, short production cycles, and high protein expression levels. However, no products using prokaryotic systems to prepare IL-22BP have yet emerged. Furthermore, IL-22BP, as a structurally complex secreted protein, forms inactive inclusion bodies entirely when expressed in *E. coli*, and the refolding rate is extremely low (below 4M urea, inclusion bodies re-form). Summary of the Invention

[0008] To address the aforementioned problems, this invention provides a method for preparing and applying recombinant human long-acting interleukin-22 binding protein. This invention utilizes an E. coli prokaryotic expression system to prepare a recombinant IL-22BP protein with high biological activity. A novel fusion protein is designed, linking the sequence encoding human IL-22BP with the sequence encoding ABD to construct the IL-22BP-ABD fusion gene. This gene is then induced to express in E. coli. After isolating inclusion body proteins, the target protein is obtained through denaturation, purification, and renaturation. The biological activity of IL-22BP-ABD is verified through in vivo and in vitro experiments.

[0009] In order to achieve the above object, the present invention provides the following technical solutions:

[0010] The present invention provides a recombinant human long-acting interleukin-22 binding protein, the amino acid sequence of which is shown in SEQ ID No. 1.

[0011] Preferably, the coding sequence of the recombinant human long-acting interleukin-22 binding protein is shown in SEQ ID No. 2.

[0012] This invention also provides a method for preparing the recombinant human long-acting interleukin-22 binding protein described in the above technical solution, comprising the following steps:

[0013] 1) The coding sequence of the recombinant human long-acting interleukin-22 binding protein was cloned into the prokaryotic expression plasmid pET-20b to obtain the recombinant expression plasmid;

[0014] 2) Transform the recombinant expression plasmid obtained in step 1) into Escherichia coli to obtain recombinant Escherichia coli;

[0015] 3) The recombinant E. coli obtained in step 2) is induced with IPTG to obtain induced bacteria;

[0016] 4) The induced bacteria obtained in step 3) are sequentially broken and centrifuged to obtain bacterial cells;

[0017] 5) The bacterial cells obtained in step 4) are washed and denatured with inclusion body washing solution to obtain denatured supernatant;

[0018] 6) The denatured supernatant obtained in step 5) is refolded and purified to obtain recombinant human long-acting interleukin-22 binding protein.

[0019] Preferably, in step 3), the recombinant E. coli is induced by IPTG in the form of a bacterial suspension, and the OD of the bacterial suspension is... 600 The value is 0.6 to 0.8;

[0020] The conditions for IPTG induction include: a final IPTG concentration of 0.3–1 mmol / L, a temperature of 20–37 °C, and a time of 3–8 h.

[0021] Preferably, step 4) involves ultrasonic crushing with a power of 500W, a duration of 2 seconds of ultrasonic treatment followed by a 3-second pause, and a duration of 20–30 minutes.

[0022] The centrifugation conditions include: a rotation speed of 12,000 rpm and a time of 10 min.

[0023] Preferably, the components of the inclusion body washing solution in step 5) include: 20 mmol / L Tris-HCl, 50 mmol / L NaCl, 2 mol / L urea and 2% Triton X100 by mass, pH=8.0;

[0024] The denaturing solution used for denaturation comprises: 20 mmol / L Tris-HCl, 50 mmol / L NaCl, 10 mmol / L β-mercaptoethanol, 8 mol / L urea, and 1 mmol / L EDTA, with a pH of 8.0.

[0025] Preferably, in step 6), the refolding is performed sequentially using a gradient refolding solution, wherein the concentrations of urea in the refolding solution are 6 mol / L, 4 mol / L, 2 mol / L, 1 mol / L and 0 mol / L, and the dialysis refolding is performed in descending order of concentration.

[0026] The dialysis bags used for dialysis refolding have a molecular weight cutoff of 3500D, and the dialysis time for each gradient is 12–16 hours.

[0027] Preferably, the refolding solution further comprises 20 mmol / L Tris-HCl, 50 mmol / L NaCl, 0.5 mmol / L GSSG (oxidized glutathione), 1 mmol / L GSH (reduced glutathione), and 1 mmol / L EDTA, with pH = 8.0.

[0028] The present invention also provides the application of the recombinant human long-acting interleukin-22 binding protein described in the above technical solution in improving the refolding rate of interleukin-22 binding protein.

[0029] The present invention also provides the application of the recombinant human long-acting interleukin-22 binding protein described in the above technical solution in improving the biological activity of interleukin-22 binding protein.

[0030] This invention also provides an in vitro cell activity detection method for the recombinant human long-acting interleukin-22 binding protein described in the above technical solution, comprising the following steps:

[0031] A. Spread 100 μL of HepG2 cell solution into each well of a 96-well plate. After the cells adhere, discard the old culture medium and add 100 μL of complete culture medium containing human IL-22 to each well. Incubate at 37°C and 5% CO2 for 12 h. Then add 10 μL of CK-8 solution to each well and incubate for 2 h.

[0032] The cell content of the HepG2 cell solution was 3 × 10⁻⁶. 5 The concentration of the human IL-22 solution is 10 ng / mL;

[0033] B. Add 100 μL of complete culture medium containing human IL-22 and recombinant human long-acting interleukin-22 binding protein to each well, and incubate at 37℃ and 5% CO2 for 12 h. Then add 10 μL of CCK-8 solution to each well and incubate for 2 h. Detect the ability of recombinant human long-acting interleukin-22 binding protein to inhibit the proliferation of HepG2 cells promoted by human IL-22.

[0034] The concentration of the solution containing recombinant human long-acting interleukin-22 binding protein is 50 μg / mL.

[0035] The present invention also provides a formulation protectant for the recombinant human long-acting interleukin-22 binding protein described in the above technical solution, characterized in that the formulation protectant contains 2% mannitol and 3% trehalose by mass.

[0036] The beneficial effects of this invention are:

[0037] 1. This invention overcomes the shortcomings of IL-22BP protein in eukaryotic systems, such as low expression levels, high production costs, long culture cycles, and susceptibility to contamination.

[0038] 2. This invention provides a method for preparing recombinant human IL-22BP protein by fusing the ABD domain. This method not only provides stability and half-life, but also significantly improves the refolding rate (even after all urea is removed by dialysis, a high content of soluble protein is still maintained). This overcomes the shortcomings of IL-22BP protein, which forms inactive inclusion bodies when expressed in E. coli and has an extremely low refolding rate (inclusion bodies are formed again when the urea content is less than 4M). Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0040] Figure 1This diagram shows the specific expression of recombinant IL-22BP-ABD and the screening of the optimal expression strain. In section A, M represents the marker; 1: total protein before PET20b induction; 2: total protein after PET20b induction; 3: total protein before IL-22BP-ABD induction; 4: total protein after IL-22BP-ABD induction; 5: supernatant of cell lysis after induction; 6: precipitate of cell lysis after induction; 7: supernatant after inclusion body denaturation; 8: precipitate of inclusion body denaturation. In section B, M represents the marker; 1: total protein before induction of recombinant strain ①; 2: total protein after induction of recombinant strain ①; 3: total protein before induction of recombinant strain ②; 4: total protein after induction of recombinant strain ②; 5: total protein before induction of recombinant strain ③; 6: total protein after induction of recombinant strain ③.

[0041] Figure 2 This diagram shows the optimization of induction conditions for recombinant protein IL-22BP-ABD. In section A, M represents the marker; 1 represents the total bacterial protein before IL-22BP-ABD induction; and 2-6 represent the total bacterial protein after induction at 0, 0.3, 0.5, 0.7, and 1 mmol / L IPTG. In section B, M represents the marker; 1 represents the total bacterial protein before IL-22BP-ABD induction; and 2-4 represent the total bacterial protein after induction at 20, 30, and 37 degrees Celsius. In section C, M represents the marker; 1 represents the total bacterial protein before IL-22BP-ABD induction; and 2-7 represent the total bacterial protein after induction at 3, 4, 5, 6, 7, and 8 hours.

[0042] Figure 3 This is a comparison diagram of the refolding conditions of recombinant protein IL-22BP-ABD and protein IL-22BP. In A, M represents the marker; 1 represents the supernatant of denatured IL-22BP-ABD protein; 2 represents the supernatant of IL-22BP-ABD protein folded with 4M urea; 3 represents the supernatant of IL-22BP-ABD protein folded with 2M urea; 4 represents the supernatant of IL-22BP-ABD protein folded with 1M urea; and 5 represents the supernatant of IL-22BP-ABD protein folded with 0M urea. In B, M represents the marker; 1 represents the supernatant of denatured IL-22BP protein; 2 represents the supernatant of IL-22BP protein folded with 4M urea; 3 represents the supernatant of IL-22BP protein folded with 2M urea; 4 represents the supernatant of IL-22BP protein folded with 1M urea; and 5 represents the supernatant of IL-22BP protein folded with 0M urea.

[0043] Figure 4 Figure 1-3 shows the results of Western Blot detection of the target protein IL-22BP-ABD.

[0044] Figure 5The image shows the purification results of recombinant IL-22BP-ABD. M is the marker; 1 is the unpurified protein; 2-4 are 20 mmol imidazole washing buffer; 5-8 are 250 mmol imidazole elution buffer.

[0045] Figure 6 The chart shows the screening results of protective agents for recombinant protein IL-22BP-ABD. In the chart, A represents the grayscale statistics of each group of samples after being stored at 37℃ for one day; B represents the grayscale statistics of each group of samples after being stored at 37℃ for eight days (n=4, *p<0.05, **p<0.01); C represents the grayscale analysis results of each group of samples with the combined protective agent formulation after being stored at 37℃ for one day; and D represents the grayscale analysis results of each group of samples with the combined protective agent formulation after being stored at 37℃ for eight days.

[0046] Figure 7 The image shows the in vitro activity assay results of recombinant IL-22BP-ABD, n=4, *p<0.05, **p<0.01;

[0047] Figure 8 The image shows the in vivo activity assay of recombinant IL-22BP-ABD. In this image, A represents the PASI scores of mice in each group after 7 consecutive days of modeling; B represents the spleen index of mice after dissection on day 8 (n=4, *p<0.05, **p<0.01); and C represents the protein expression of the specified gene determined by Western blotting after mice were sacrificed on day 8.

[0048] Figure 9 Figure 1 shows the results of the in vivo activity assay of recombinant IL-22BP-ABD and the administration method. Figure A shows the PASI scores of mice in each group after 6 consecutive days of modeling; Figure B shows the spleen index after dissection of mice on day 7; Figure C shows the protein expression of the specified gene determined by Western blotting after mice were sacrificed on day 7.

[0049] Figure 10 The pharmacokinetic diagrams are for recombinant protein IL-22BP-ABD and recombinant IL-22BP protein without ABD, where n = 4. Detailed Implementation

[0050] This invention provides a fusion protein (IL-22BP-ABD) of recombinant human long-acting interleukin-22 binding protein and ABD, wherein the amino acid sequence of IL-22BP-ABD is shown in SEQ ID No. 1, and is as follows:

[0051] MTQSTHESLKPQRVQFQSRNFHNILQWQPGRALTGNSSVYFVQYKIYGQRQWKNKEDCWGTQELSCDLTSETSDIQEPYYGRVRAASAGSYSEWSMTPRFTPWWETKIDPPVMNITQVNGSLLVILHAPNLPYRYQKEK NVSIEDYYELLYRVFIINNSLEKEQKVYEGAHRAVEIEALTPHSSYCVVAEIYQPMLDRRSQRSEERCVEIPGGGGSGGGGSGGGGSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPHHHHHH.

[0052] In this invention, the encoding sequence of IL-22BP-ABD is shown in SEQ ID No. 2, and is as follows:

[0053] ATGACTCAATCTACCCATGAATCTCTGAAGCCGCAACGTGTACAGTTTCAGTCTCGTAACTTCCACAACATCCTGCAATGGCAGCCGGGTCGTGCACTGACTGGTAACTCTTCCGTATATTTCGTTCAATACAAAATCTACGGTCAGCGTCAATGGAAGAACAAAGAAGATTGTTGGGGTACCCAGGAACTGTCCTGCGACCTGACCAGCGAAACTTCTGATATCCAGGAACCGTATTACGGCCGTGTACGCGCAGCCAGCGCGGGTTCTTATAGCGAATGGTCCATGACTCCACGTTTCACTCCGTGGTGGGAAACTAAAATCGACCCACCAGTAATGAACATCACTCAAGTTAACGGTTCCCTGCTGGTAATCCTGCACGCGCCAAACCTGCCATATCGCTATCAGAAAGAAAAGAATGTGTCCATCGAAGATTATTATGAGCTGCTGTACCGTGTGTTCATCATCAACAACAGCCTGGAGAAAGAACAGAAAGTGTACGAAGGCGCACACCGTGCAGTTGAAATCGAGGCGCTGACTCCGCACAGCTCCTATTGTGTGGTAGCCGAAATCTATCAGCCGATGCTGGACCGCCGTTCTCAGCGCTCTGAGGAACGTTGCGTAGAGATCCCAGGTGGCGGTGGCAGCGGTGGCGGCGGTAGCGGTGGCGGTGGTTCTCTGGCGGAAGCCAAGGTGCTGGCAAACCGTGAGCTGGATAAGTACGGTGTATCCGACTATTACAAGAACCTGATTAACAATGCTAAAACCGTAGAAGGCGTGAAGGCACTGATTGACGAAATCCTGGCAGCTCTGCCGCACCACCACCATCACCACTAA。

[0054] In this invention, the coding sequence of the recombinant human long-acting interleukin-22 binding protein is obtained by fusing the IL-22BP sequence and the ABD sequence. The nucleotide sequence of the IL-22BP sequence is shown in SEQ ID No. 3, and the nucleotide sequence of the ABD sequence is shown in SEQ ID No. 4. When the IL-22BP sequence is expressed alone in Escherichia coli, it forms inclusion bodies, has low refolding efficiency, and exhibits poor half-life and stability in vivo. After fusing the IL-22BP sequence with the ABD sequence, not only can the in vivo half-life be significantly prolonged, but it was also unexpectedly found to significantly improve the refolding rate of inclusion bodies, laying a solid foundation for the preparation and production of the protein.

[0055] SEQ ID No. 3:

[0056] ;

[0057] SEQ ID No.4:

[0058] CTGGCGGAAGCCAAGGTGCTGGCAAACCGTGAGCTGGATAAGTACGGTGTATCCGACTATTACAAGAACCTGATTAACAATGCTAAAACCGTAGAAGGCGTGAAGGCACTGATTGACGAAATCCTGGCAGCTCTGCCGCACCACCACCATCACCACTAA.

[0059] This invention also provides a method for preparing the recombinant human long-acting interleukin-22 binding protein described in the above technical solution, comprising the following steps:

[0060] 1) The coding sequence of the recombinant human long-acting interleukin-22 binding protein was cloned into the prokaryotic expression plasmid pET-20b to obtain the recombinant expression plasmid;

[0061] 2) Transform the recombinant expression plasmid obtained in step 1) into Escherichia coli to obtain recombinant Escherichia coli;

[0062] 3) The recombinant E. coli obtained in step 2) is induced with IPTG to obtain induced bacteria;

[0063] 4) The induced bacteria obtained in step 3) are sequentially broken and centrifuged to obtain bacterial cells;

[0064] 5) The bacterial cells obtained in step 4) are washed and denatured with inclusion body washing solution to obtain denatured supernatant;

[0065] 6) The denatured supernatant obtained in step 5) is refolded and purified to obtain recombinant human long-acting interleukin-22 binding protein.

[0066] This invention clones the coding sequence of the recombinant human long-acting interleukin-22 binding protein into the prokaryotic expression plasmid pET-20b to obtain the recombinant expression plasmid. This invention does not impose any particular limitation on the method for constructing the recombinant expression plasmid; those skilled in the art can use conventional methods.

[0067] This invention involves transforming the obtained recombinant expression plasmid into *E. coli* to obtain recombinant *E. coli*. This invention does not specifically limit the method for transforming the recombinant expression plasmid into *E. coli*; conventional methods can be used by those skilled in the art.

[0068] In this invention, recombinant *E. coli* is induced by IPTG to obtain induced bacteria. In this invention, the recombinant *E. coli* is preferably induced by IPTG (isopropyl-β-D-thiogalactoside) in the form of a bacterial suspension. The OD of the bacterial suspension... 600The preferred value is 0.6–0.8. This invention does not impose any particular limitation on the preparation method of the bacterial culture; those skilled in the art can obtain it using conventional culture methods. In this invention, the preferred conditions for IPTG induction include: a final IPTG concentration of 0.3–1 mmol / L, a temperature of 20–37°C, and a time of 3–8 h. More preferably, the conditions for IPTG induction include: a final IPTG concentration of 0.5 mol / L, a temperature of 37°C, and a time of 6 h.

[0069] In this invention, the induced bacteria are sequentially disrupted and centrifuged to obtain bacterial cells. Preferably, the disruption is performed using ultrasonic disruption at a power of 500W, with a 2-second sonication period followed by a 3-second pause, lasting 20-30 minutes. Preferably, the centrifugation conditions include a rotation speed of 12000 rpm and a centrifugation time of 10 minutes.

[0070] In this invention, the obtained bacterial cells are washed and denatured with an inclusion body washing solution to obtain a denatured supernatant. In this invention, the inclusion body washing solution preferably comprises: 20 mmol / L Tris-HCl, 50 mmol / L NaCl, 2 mol / L urea, and 2% (w / w) Triton X100, with a pH of 8.0. In this invention, the denaturing solution used for denaturation preferably comprises: 20 mmol / L Tris-HCl, 50 mmol / L NaCl, 10 mmol / L β-mercaptoethanol, 8 mol / L urea, and 1 mmol / L EDTA, with a pH of 8.0.

[0071] This invention involves refolding and purifying the denatured supernatant to obtain recombinant human long-acting interleukin-22 binding protein. In this invention, the refolding is preferably performed using a gradient refolding solution, with the urea concentrations in the refolding solution preferably being 6 mol / L, 4 mol / L, 2 mol / L, 1 mol / L, and 0 mol / L, respectively, followed by dialysis refolding according to the concentration from high to low. In this invention, the dialysis bag used for dialysis refolding preferably has a molecular weight cutoff of 3500D, and the dialysis time for each gradient is preferably 12–16 hours. In this invention, the refolding solution also preferably comprises 20 mmol / L Tris-HCl, 50 mmol / L NaCl, 0.5 mmol / L GSSG, 1 mmol / L LSH, and 1 mmol / L EDTA, with a pH of 8.0. Figure 3As can be seen, by optimizing the formulation and ratio of the refolding solution, a refolding solution formulation that can restore denatured IL-22BP-ABD to its normal structure was obtained through gradient refolding at 6M, 4M, 2M, 1M, and 0M. Gray-scale analysis showed that this formulation could restore 50% of the denatured protein to its normal conformation. For IL-22-BP, only about 10% was restored to its normal spatial conformation.

[0072] The present invention does not impose any particular limitation on the purification method; any conventional method may be used.

[0073] This invention also provides the application of the recombinant human long-acting interleukin-22 binding protein described in the above-mentioned technical solution in improving the refolding rate of interleukin-22 binding protein. Improving the refolding rate of recombinant human IL-22BP protein involves fusing the ABD domain to the C-terminus of IL-22BP. Improving the refolding rate of recombinant human IL-22BP protein can enhance the half-life of IL-22BP in vivo.

[0074] The present invention also provides the application of the recombinant human long-acting interleukin-22 binding protein described in the above technical solution in improving the biological activity of interleukin-22 binding protein.

[0075] This invention also provides an in vitro cell activity detection method for the recombinant human long-acting interleukin-22 binding protein described in the above technical solution, comprising the following steps:

[0076] A. Spread 100 μL of HepG2 cell solution into each well of a 96-well plate. After the cells adhere, discard the old culture medium and add 100 μL of complete culture medium containing human IL-22 to each well. Incubate at 37°C and 5% CO2 for 12 h. Then add 10 μL of CK-8 solution to each well and incubate for 2 h.

[0077] The cell content of the HepG2 cell solution was 3 × 10⁻⁶. 5 The concentration of the human IL-22 solution is 10 ng / mL;

[0078] B. Add 100 μL of complete culture medium containing human IL-22 and recombinant human long-acting interleukin-22 binding protein to each well, and incubate at 37℃ and 5% CO2 for 12 h. Then add 10 μL of CCK-8 solution to each well and incubate for 2 h. Detect the ability of recombinant human long-acting interleukin-22 binding protein to inhibit the proliferation of HepG2 cells promoted by human IL-22.

[0079] The concentration of the solution containing recombinant human long-acting interleukin-22 binding protein is 50 μg / mL.

[0080] The present invention also provides a formulation protectant for the recombinant human long-acting interleukin-22 binding protein described in the above technical solution, characterized in that the formulation protectant contains 2% mannitol and 3% trehalose by mass.

[0081] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0082] Example 1

[0083] Engineered bacteria for constructing fusion proteins and recombinant human long-acting interleukin-22 binding proteins

[0084] 1. Constructing an expression carrier

[0085] Based on the wild-type human IL-22BP and ABD sequences in GenBank, the fusion gene sequence was modified and optimized using E. coli preferred codons to construct the fusion protein IL-22BP-ABD (SEQ ID No. 1).

[0086] The recombinant pET-20b-IL-22BP-ABD expression plasmid was synthesized and constructed by Guangzhou Aiji Biotechnology Co., Ltd.

[0087] 2. Transformation and amplification of recombinant plasmids

[0088] 1) Remove E. coli DH5α competent cells from the -80℃ freezer, thaw them on ice, add 5 μL of recombinant plasmid to 100 μL of DH5α competent cells, incubate on ice for 30 min, then place in a 42℃ water bath for 90 s, and then incubate on ice for 2 min.

[0089] 2) Add 400 μL of LB liquid culture medium to competent cells in a clean bench and shake at 37°C and 200 rpm for 1 h.

[0090] 3) Centrifuge at 4000 rpm for 4 min, discard the supernatant, and keep 200 μL of bacterial culture.

[0091] Mix the bacterial culture thoroughly and spread it evenly on LB solid medium (Amp+). Incubate at 37°C for 12-16 hours.

[0092] 3. Plasmid mini-extraction

[0093] Plasmids for recombinant bacteria were extracted according to the instructions of the YEASEN Biotechnology Plasmid Mini-Prep Kit.

[0094] 1) Column equilibration: Add 200 μL of Buffer AC to the adsorption column that has been placed in the collection tube, centrifuge at 12000 rpm for 1 min, and pour out the waste liquid in the collection tube.

[0095] 2) Take 2-3 mL of bacterial culture, centrifuge at 12000 rpm for 1 min, and discard the supernatant.

[0096] 3) Add 250 μL of Buffer P1 to the precipitate and resuspend the bacteria with a pipette.

[0097] 4) Add 250 μL of Buffer P2 to the centrifuge tube, gently invert to mix, and allow the cells to fully lyse.

[0098] 5) Add 350 μL of Buffer P3 to the centrifuge tube and gently invert it 4-6 times. A white flocculent precipitate will appear. Centrifuge at 12,000 rpm for 10 min.

[0099] 6) Transfer the supernatant to the adsorption column placed in the collection tube, centrifuge at 12000 rpm for 1 min, and discard the waste liquid in the collection tube.

[0100] 7) Add 600 μL of Buffer PW to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and repeat this step.

[0101] 8) Place the adsorption column into the collection tube, centrifuge at 12000 rpm for 2 min, and let stand at room temperature for 2 min.

[0102] 9) Place the adsorption column into a clean centrifuge tube, add 50 μL of ddH2O to the middle of the adsorption column, let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 2 min, and collect the plasmid in the centrifuge tube.

[0103] 5. The recombinant plasmid was transformed into E. coli BL21(DE3).

[0104] The specific steps are the same as those used in the recombinant plasmid transformation and amplification method described above.

[0105] 6. Detection of IL-22BP-ABD recombinant bacterial specific expression and screening of optimal expression strains

[0106] 1) Pick a single transformed colony and an empty vector and place them separately into 5 mL of LB liquid medium containing Amp, and incubate overnight at 37°C and 220 rpm with shaking. After incubation, take 1 mL of each and inoculate into 100 mL of LB liquid medium containing Amp, and incubate at 37°C and 220 rpm with shaking. When the bacterial culture OD... 600When the pH value was 0.6–0.8, IPTG with a final concentration of 0.5 mmol / L was added to induce expression, and the culture was carried out at 37℃ and 180 rpm for 5 h. After induction, the bacterial culture was centrifuged at 12000 rpm for 10 min, the supernatant was discarded, and the precipitate was resuspended in 20 mL of 20 mM Tris-HCl (pH 8.0). The precipitate was then sonicated at 500 W for 2 s, paused for 3 s, and continued for 20–30 min. The cytosed bacterial culture was centrifuged, the precipitate was collected, washed with inclusion bodies, and denatured with 8 M urea denaturing solution to obtain the denatured supernatant of IL-22BP-ABD.

[0107] The total protein before pET20b induction, the total protein after pET20b induction, the total protein before IL-22BP-ABD induction, the total protein after IL-22BP-ABD induction, the supernatant of lysed bacterial cells after induction, the supernatant of lysed bacterial cells after induction, the supernatant after denaturation of inclusion bodies, and the denaturation precipitate of inclusion bodies were analyzed by electrophoresis to determine the specific expression of recombinant IL-22BP-ABD. The results are as follows: Figure 1 As shown in Figure A, the recombinant plasmid can specifically express the target protein, and the target protein is mostly present in the form of inclusion bodies.

[0108] Three recombinant strains were randomly selected from the successfully transformed strains and induced to express the protein under the same conditions. The total protein levels of strains ① before and after induction, ② before and after induction, and ③ before and after induction were analyzed by electrophoresis. The expression levels of the target protein after induction were analyzed using ImageJ software. Strain ②, which showed the best results, was selected as the seed strain for subsequent protein preparation. Figure 1 As shown in B.

[0109] Example 2

[0110] Recombinant protein expression, denaturation and refolding condition optimization and purification

[0111] 1. Exploration of recombinant protein induction expression conditions

[0112] 1) Optimization of IPTG inducer concentration

[0113] Recombinant strain ② was selected and induced to express protein at the same temperature (37℃) for the same duration (6 h) using 0, 0.3, 0.5, 0.7, and 1 mmol / L IPTG, respectively. The expression levels were then measured. The results showed that at 37℃, the highest recombinant protein expression level was observed with an IPTG concentration of 0.5 mmol / L. Figure 2 As shown.

[0114] 2) Optimization of induction temperature

[0115] For the same strain, IPTG was added at a concentration of 0.5 mmol / L, and expression was induced at 20℃, 30℃, and 37℃ for the same duration (6-8 h). The results showed that the expression level of the target protein IL-22BP-ABD was highest under the induction at 0.5 mmol / L IPTG and 37℃. Figure 2 As shown.

[0116] 3) Optimization of induction time

[0117] For the same strain, expression was induced at 37°C for 3, 4, 5, 6, 7, and 8 hours using the same IPTG concentration (0.5 mmol / L). Figure 2 As shown, the target protein IL-22BP-ABD showed the highest expression level after 6 hours of induction at 0.5 mmol / L IPTG and 37℃.

[0118] 2. Exploration of refolding conditions for recombinant proteins

[0119] Take 1 ml of the denatured IL-22BP-ABD and IL-22BP protein solutions and dilute them according to the ratio to a final protein concentration of 0.3 mg / ml;

[0120] The refolding urea gradients of the refolding solution were set to 6M, 4M, 2M, 1M and 0M, with the specific formula as follows;

[0121] ①6M urea gradient refolding solution: 20mmol / L Tris-HCl, 50mmol / L NaCl, 0.5mmol / L GSSSG, 1mmol / L GSH, 6mol / L urea, pH=8.0;

[0122] ② 4M urea gradient refolding solution: 20 mmol / L Tris-HCl, 50 mmol / L NaCl, 0.5 mmol / L GSSSG, 1 mmol / L GSH, 4 mol / L urea, pH=8.0;

[0123] ③ 2M urea gradient refolding solution: 20 mmol / L Tris-HCl, 50 mmol / L NaCl, 0.5 mmol / L GSSSG, 1 mmol / L GSH, 2 mol / L urea, pH=8.0;

[0124] ④ 1M urea gradient refolding solution: 20mmol / L Tris-HCl, 50mmol / L NaCl, 0.5mmol / L GSSSG, 1mmol / L GSH, 1mol / L urea, pH=8.0;

[0125] ⑤ 0M urea gradient refolding solution: 20 mmol / L Tris-HCl, 50 mmol / L NaCl, 0.5 mmol / L GSSG, 1 mmol / L GSH, pH=8.0.

[0126] The diluted protein solution was placed in a dialysis bag with a flow rate of 3500D and dialyzed stepwise according to the urea gradient from high to low, with each gradient dialyzed for 14 hours.

[0127] Protein solutions before and after dialysis were subjected to 12% SDS-PAGE electrophoresis, and the recovery rates of the two proteins were compared by grayscale analysis using ImageJ.

[0128] The results are as follows Figure 3 As shown in Figure A, by optimizing the formulation and ratio of the refolding solution, a refolding solution formulation was obtained that can restore denatured IL-22BP-ABD to its normal structure through gradient refolding at 6M, 4M, 2M, 1M, and 0M. Gray-scale analysis showed that it can restore 50% of the denatured protein to its normal conformation. Figure 3 As shown in Figure B, only about 10% of IL-22-BPs recovered their normal spatial conformation.

[0129] The target protein IL-22BP-ABD was detected by Western blotting. After exposure with anti-His tag antibody, the results showed that the expression of the target protein IL-22BP-ABD was good. Figure 4 .

[0130] 3. Purification of recombinant proteins

[0131] The denatured protein supernatant was centrifuged at 12,000 rpm and 4°C for 30 min. The supernatant was collected and sterilized by filtration through a 0.45 μm filter membrane. The samples were aliquoted and stored at -80°C.

[0132] The denaturation process is as follows: Collect the induced bacterial cells by centrifugation at 12000 rpm for 10 minutes. Disrupt the cells using an ultrasonic homogenizer at 500 W for 2 seconds, with a 3-second interval, for a total of 30 minutes. After disruption, centrifuge at 12000 rpm for 30 minutes, discard the supernatant, and resuspend the precipitate in an appropriate amount of inclusion body washing buffer A. Wash with agitation for 20 minutes, centrifuge at 12000 rpm for 15 minutes at 4°C, discard the supernatant, and repeat 3-4 times. Wash once more with an appropriate amount of 20 mmol / L Tris-HCl buffer (pH = 8.0), centrifuge at 12000 rpm for 30 minutes at 4°C, and discard the supernatant.

[0133] The inclusion bodies obtained above were denatured and dissolved in a denaturing buffer (containing 20 mmol / L Tris-HCl, 50 mmol / L NaCl, 10 mmol / L β-mercaptoethanol, 8 mol / L urea, pH 8.0) at a ratio of 10 ml / g inclusion bodies. The inclusion bodies were resuspended by repeated pipetting and denaturation at 4°C for 12 h. The mixture was then centrifuged at 12000 rpm for 30 min, and the supernatant was collected. The supernatant protein was diluted with denaturing buffer to approximately 0.3 mg / ml.

[0134] The purified recombinant protein, after filtration and sterilization, is collected and purified as follows:

[0135] 1) Turn on the UV detector, recorder, and peristaltic pump;

[0136] 2) Column equilibration: Equilibrate the pre-packed Ni-NTA affinity chromatography column with 20mM Tris-HCl buffer at pH 8.0 until the absorbance reading of the detector is zero;

[0137] 3) Sample loading: Load the aliquoted protein sample IL-22BP-ABD onto a Ni-NTA affinity chromatography column at a flow rate of 2 mL / min. After the sample loading is complete, allow the sample to bind in the chromatography column for 60 min.

[0138] 4) Washing: Wash with 20mM Tris-HCl buffer containing 20mM imidazole at a flow rate of 3mL / min, and collect the washing peak liquid;

[0139] 5) Elution: Elute with 20mM Tris-HCl buffer containing 250mM imidazole at a flow rate of 3mL / min, and collect the elution peak.

[0140] 6) Column preservation: Elute the column with 20mM Tris-HCl buffer at pH 8.0 at a flow rate of 4mL / min, then wash the column with 20% ethanol, and store the column in a chromatography cabinet at 4℃.

[0141] Purity assay of recombinant IL-22BP-ABD protein: The molecular weight and purity of the recombinant protein were determined by 12% SDS-PAGE electrophoresis of the sample loading, washing, and elution buffers. Figure 5 As shown, the recombinant IL-22BP-ABD was purified by Ni-NTA affinity chromatography using a gradient elution of 40 mm imidazole and 250 mm imidazole, and its purity was calculated to be 90%.

[0142] Example 3

[0143] Screening of protective agents for recombinant protein IL-22BP-ABD

[0144] A double-concentration alternative protective agent was dissolved in 20 mm Tris-HCl (pH = 8.0) for sterilization. After sterilization, it was mixed with 0.5 mg / mL IL-22BP-ABD (stored in 20 mm Tris-HCl) at a volume ratio of 1:1 and added to 1.5 mL Eppendorf tubes, 1 mL per tube. The tubes were sealed and placed at 37°C (75% relative humidity) for accelerated testing. Samples were taken for electrophoresis on days 1 and 8 to initially screen for suitable single protective agents. The selected protective agents were then combined and added to the protein using the same method to further observe the protective effect of the combined formulation. Based on literature review, six protective agents were initially selected, with three different concentrations for each agent: 1% arginine, 2% arginine, 3% arginine, 1% glucose, 2% glucose, 3% glucose, 1% glycine, 2% glycine, 3% glycine, 1% trehalose, 2% trehalose, 3% trehalose, 1% mannitol, 2% mannitol, and 3% mannitol. Each protective agent was mixed with protein at a 1:1 volume ratio and stored at 37°C. On day eight, 1% arginine, 2% mannitol, and 3% trehalose showed a certain protective effect. Therefore, four combinations were further developed: 1% arginine, 2% mannitol, and 3% trehalose. These combinations were compared with single-component protective agents of 1% arginine, 2% mannitol, and 3% trehalose to select the optimal combination protective agent formulation.

[0145] The results showed that the grayscale analysis results of SDS-PAGE detection on the first day of storage at 37℃, the grayscale analysis results of SDS-PAGE detection on the eighth day of storage at 37℃, the grayscale analysis results of SDA-PAGE electrophoresis results of the combined protective agent formulation sample on the first day of storage at 37℃, and the grayscale analysis results of SDA-PAGE electrophoresis results of the combined protective agent formulation sample on the eighth day of storage at 37℃ were as follows: Figure 6 The results above demonstrate that a protective agent formulation of 2% mannitol + 3% trehalose provides the best protein protection.

[0146] Example 4

[0147] Assay of recombinant IL-22BP-ABD protein activity

[0148] 1. In vitro activity assay of recombinant IL-22BP-ABD protein

[0149] The recombinant protein IL-22BP-ABD was co-incubated with HepG2 cells and human IL-22. Specifically, HepG2 cells were stimulated with 10 ng / ml human IL-22. The biological activity of IL-22BP-ABD was evaluated by detecting the ability of the recombinant IL-22BP-ABD protein to inhibit the proliferative effect of IL-22. Specific steps:

[0150] Reviving HepG2 cells: Add 8 mL of DMEM culture medium to a 10 mL centrifuge tube; remove the frozen HepG2 cells and thaw them rapidly in a 37°C water bath; immediately add the thawed HepG2 cells to a pre-prepared centrifuge tube and centrifuge at 1000 rpm for 5 min; discard the supernatant, resuspend the pellet in 1 mL of complete culture medium and add it to a culture dish containing 6 mL of culture medium, gently shake to mix, and incubate at 37°C in a 5% CO2 incubator.

[0151] Observe the HepG2 cells under a microscope. Once the cells are in good growth condition, they can be seeded into 96-well plates.

[0152] Plating: Collect cells into sterile centrifuge tubes using a pipette, centrifuge at 1000 rpm for 5 min; discard the supernatant, resuspend in 1 mL of complete culture medium and mix well. Take 1 μL and dilute it 10-fold with complete culture medium before counting cells under a microscope; 100 μL of HepG2 cells (3 × 10⁻⁶ cells) per well. 5 (cell / mL).

[0153] After the cells adhered to the culture medium, the old culture medium was discarded, and human recombinant IL-22 protein was mixed into the culture medium at different concentrations. 100 μL of mixed culture medium was added to each well, and the cells were cultured at 37°C in a 5% CO2 incubator for 12 h.

[0154] Add 10 μl of CCK-8 solution to each well of the plate.

[0155] Incubate the culture plate in an incubator for 2 hours.

[0156] The absorbance at 450 nm was measured using an ELISA reader to determine the optimal concentration and duration of IL-22 for stimulating cell proliferation.

[0157] After reviving HepG2, perform the same plate-laying operation as above.

[0158] After the cells adhered to the culture medium, the old culture medium was discarded. Human recombinant IL-22 protein (10 ng / ml) was mixed with IL-22BP-ABD at different concentration gradients in the culture medium. 100 μL of the mixture was added to each well and cultured at 37°C in a 5% CO2 incubator for 12 h.

[0159] Add 10 μl of CCK-8 solution to each well of the plate.

[0160] Incubate the culture plate in an incubator for 2 hours.

[0161] The absorbance at 450 nm was measured using a microplate reader to determine the efficacy of IL-22-ABD and the concentration gradient effect of its efficacy. Figure 7 As shown, the protein IL-22BP-ABD can significantly inhibit the proliferation of IL-22-stimulated HepG2 cells by binding to IL-22.

[0162] 2. In vivo activity assay of recombinant IL-22BP-ABD protein

[0163] The in vivo activity of recombinant protein IL-22BP-ABD was detected using a mouse psoriasis model. The specific methods and results are as follows:

[0164] 1) Female Balb / c mice were induced by applying 60 mg / mouse of 5% imiquimod cream to their backs for 7 consecutive days. Simultaneously, starting from the first day of model induction, mice were injected intraperitoneally with different doses of IL-22BP-ABD formulation for 7 consecutive days. The skin lesions on the backs of the mice and changes in body weight were monitored daily.

[0165] 2) Conduct group experiments: (4 mice per group)

[0166] ① Control group: 60 μL of 20 mM Tris-HCl (pH 8.0) was applied to the back;

[0167] ② Model group: 60mg / animal 5% imiquimod cream was applied to the back, and PBS (total volume 200μL) was injected intraperitoneally;

[0168] ③ High-dose experimental group: 60mg / animal 5% imiquimod cream was applied to the back, and recombinant IL-22BP-ABD preparation (total volume 200μL, containing 5mg / kg recombinant IL-22BP-ABD) was injected intraperitoneally.

[0169] ④ Low-dose experimental group: 60 mg / animal 5% imiquimod cream was applied to the back, and recombinant IL-22BP-ABD preparation (total volume 200 μL, containing 2.5 mg / kg recombinant IL-22BP-ABD) was injected intraperitoneally.

[0170] 3) After continuous intraperitoneal injection of recombinant protein IL-22BP-ABD for 7 days, mice were sacrificed on day 8, and diseased skin tissue was collected. Total protein was extracted from the tissue, and the expression levels of IL-22 and protein were detected by Western blotting. At the same time, the size of splenic lesions was compared, and the spleen index was calculated.

[0171] The results are as follows Figure 8 As shown, a mouse psoriasis model was induced by applying 5% imiquimod cream to the dorsal skin of mice for 7 consecutive days. The in vivo activity of recombinant IL-22BP-ABD protein was detected. The PASI scores of the skin lesions were monitored daily after imiquimod cream administration. Figure 8 As shown, the 5 mg / kg recombinant IL-22BP-ABD formulation significantly inhibited skin lesions after day four. Figure 8 As shown in Figure B, mice were continuously injected with different concentrations of IL-22BP-ABD for 7 days while applying 5% imiquimod cream for 7 consecutive days. On the 8th day, the mice were sacrificed and the spleen index of each group was compared. It was found that high-dose IL-22BP-ABD injection could reduce the spleen index of mice. Figure 8 As shown in Figure C, after the mice were sacrificed on day 8, proteins were extracted from the skin tissue homogenate. The protein expression of IL-22 was measured by Western blotting. The results showed that after injection of IL-22BP-ABD, the protein expression of IL-22 in the high-dose experimental group mice decreased significantly.

[0172] Example 5

[0173] Investigation of in vivo activity assay and administration route for recombinant IL-22BP-ABD protein

[0174] 1. Methods: Female Balb / c mice were used to establish a skin lesion by applying 60 mg / mouse of 5% imiquimod cream to their backs for 6 consecutive days. Simultaneously, starting on the first day of lesion establishment, mice were administered the same concentration and volume of IL-22BP-ABD via different administration routes for 6 consecutive days. Skin lesions on the backs of the mice and changes in body weight were monitored daily.

[0175] 2. The experimental groups are as follows: (4 mice per group)

[0176] ① Blank group: Apply 60μL of 20mM Tris-HCl (pH 8.0) to the back.

[0177] ② Model group: 60 mg / animal 5% imiquimod cream was applied to the back, and PBS (total volume 200 μL) was injected intraperitoneally.

[0178] ③ Subcutaneous injection experimental group: 60mg / animal 5% imiquimod cream was applied to the back, and recombinant IL-22BP-ABD preparation (total volume 200μL, containing 5mg / kg recombinant IL-22BP-ABD) was injected subcutaneously.

[0179] ④ Intraperitoneal injection experimental group: 60mg / animal 5% imiquimod cream was applied to the back, and recombinant IL-22BP-ABD preparation (total volume 200μL, containing 5mg / kg recombinant IL-22BP-ABD) was injected intraperitoneally.

[0180] 3. After administering the recombinant protein IL-22BP-ABD preparation to each group for 6-7 days, the mice were sacrificed every other day, and the lesioned skin tissue was collected. Total protein was extracted from the tissue, and the expression levels of IL-22 and protein were detected by Western blotting. At the same time, the size of the splenic lesions was compared, and the spleen index was calculated.

[0181] 4. Results

[0182] The results are as follows Figure 9 As shown in Figure A, the in vivo activity assay of recombinant IL-22BP-ABD and the administration method were investigated. For six consecutive days, 5% imiquimod cream was applied to the back skin of mice to induce psoriasis. By constructing a mouse psoriasis model and administering the same concentration of IL-22BP-ABD via different methods, the optimal administration method was determined. The PASI score for monitoring skin lesions daily after imiquimod cream administration is shown in Figure A. B: While applying 5% imiquimod cream for six consecutive days, the same dose of IL-22BP-ABD was injected via different methods for six consecutive days. On day 7, mice were sacrificed, and the spleen index of each group was compared. Intraperitoneal injection of IL-22BP-ABD significantly reduced the spleen index. C: After sacrifice on day 7, protein was extracted from skin tissue homogenates, and IL-22 protein expression was measured by Western blotting. The results showed that after both methods of IL-22BP-ABD injection, the intraperitoneal injection group showed a higher decrease in IL-22 expression than the subcutaneous injection group.

[0183] Example 6

[0184] Pharmacokinetic analysis of recombinant IL-22BP-ABD protein

[0185] 5 mg / kg of recombinant protein IL-22BP-ABD and IL-22BP were injected subcutaneously into female BALB / c mice. The serum IL-22BP-ABD levels were analyzed by ELISA at different time points after injection. The specific steps are as follows:

[0186] 1) Take 8 female BAL B / c mice weighing 18-20g and divide them into 2 groups. They are given a single subcutaneous injection of recombinant IL-22BP-ABD and IL-22BP protein at a dose of 5mg / kg. All animal experiments are conducted in an SPF-grade laboratory.

[0187] 2) After injection, blood was collected from mice at 0, 1, 2, 4, 8, 12, 24, 36, 48 and 60 h by fundus sampling into clean centrifuge tubes, incubated at 4℃ for 1-2 h, centrifuged at 3500 rpm for 15 min, and serum was collected.

[0188] 3) The levels of human IL-22BP-ABD and IL-22BP in mouse serum were detected by ELISA. The half-life of recombinant IL-22BP-ABD protein and IL-22BP protein in mice was calculated by the Microsoft Excel plugin "PKSolver".

[0189] The results are as follows Figure 10 As shown, male BALB / c mice were injected with a single subcutaneous dose of 5 mg / kg recombinant IL-22BP-ABD. Serum IL-22BP-ABD levels peaked at 1 hour using ELISA, decreased rapidly from 1 to 8 hours, and then decreased slowly from 8 to 36 hours. Pharmacokinetic parameters were determined using PKSolver. Key pharmacokinetic parameters were calculated. The time to peak serum concentration of recombinant IL-22BP-ABD protein in mice was 1 hour, with a maximum concentration of 342.25 g / ml and a half-life of 17 hours. In contrast, recombinant IL-22BP without ABD reached its peak concentration 20 minutes after injection and rapidly degraded, with a half-life of 2 hours.

[0190] In summary, the recombinant IL-22BP-ABD protein prepared by this invention has a high refolding rate and stability. The purified recombinant protein has high biological activity both in vivo and in vitro. This invention establishes for the first time a method for detecting the activity of recombinant IL-22BP-ABD protein and a formulation, laying the foundation for subsequent new drug development.

[0191] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A recombinant human long-acting interleukin-22 binding protein, characterized in that, The amino acid sequence of the recombinant human long-acting interleukin-22 binding protein is shown in SEQ ID No.

1.

2. The recombinant human long-acting interleukin-22 binding protein according to claim 1, characterized in that, The coding sequence of the recombinant human long-acting interleukin-22 binding protein is shown in SEQ ID No.

2.

3. A method for preparing the recombinant human long-acting interleukin-22 binding protein according to claim 1 or 2, characterized in that, Includes the following steps: 1) The coding sequence of the recombinant human long-acting interleukin-22 binding protein was cloned into the prokaryotic expression plasmid pET-20b to obtain the recombinant expression plasmid; 2) Transform the recombinant expression plasmid obtained in step 1) into Escherichia coli to obtain recombinant Escherichia coli; 3) The recombinant E. coli obtained in step 2) is induced with IPTG to obtain induced bacteria; 4) The induced bacteria obtained in step 3) are sequentially broken and centrifuged to obtain bacterial cells; 5) The bacterial cells obtained in step 4) are washed and denatured with inclusion body washing solution to obtain denatured supernatant; 6) The denatured supernatant obtained in step 5) is refolded and purified to obtain recombinant human long-acting interleukin-22 binding protein.

4. The preparation method according to claim 3, characterized in that, In step 3), the recombinant Escherichia coli was induced by IPTG in the form of a bacterial suspension, and the OD600 value of the bacterial suspension was 0.6 to 0.

8. The conditions for IPTG induction include: a final IPTG concentration of 0.3–1 mmol / L, a temperature of 20–37 °C, and a time of 3–8 h.

5. The preparation method according to claim 3, characterized in that, Step 4) The crushing is performed using ultrasonic crushing with a power of 500W. The ultrasonic treatment lasts for 2 seconds, followed by a 3-second pause, and continues for 20 to 30 minutes. The centrifugation conditions include: a rotation speed of 12,000 rpm and a time of 10 min; The inclusion body washing solution comprises: 20 mmol / L Tris-HCl, 50 mmol / L NaCl, 2 mol / L urea, and 2% Triton X100 by mass, with a pH of 8.

0. The denaturing solution used for denaturation comprises: 20 mmol / L Tris-HCl, 50 mmol / L NaCl, 10 mmol / L β-mercaptoethanol, 8 mol / L urea, and 1 mmol / L LEDTA, with a pH of 8.

0.

6. The preparation method according to claim 3, characterized in that, Step 6) refolding uses a gradient refolding solution for sequential refolding. The concentrations of urea in the refolding solution are 6 mol / L, 4 mol / L, 2 mol / L, 1 mol / L and 0 mol / L, respectively, and dialysis refolding is performed according to the concentration from high to low. The dialysis bags used for dialysis refolding have a molecular weight cutoff of 3500D, and the dialysis time for each gradient is 12–16 h. The refolding solution also includes 20 mmol / L Tris-HCl, 50 mmol / L NaCl, 0.5 mmol / L GSSG, 1 mmol / L LSH and 1 mmol / L EDTA, pH=8.

0.

7. The use of the recombinant human long-acting interleukin-22 binding protein according to claim 1 or 2 in improving the refolding rate of interleukin-22 binding protein.

8. The use of the recombinant human long-acting interleukin-22 binding protein according to claim 1 or 2 in enhancing the biological activity of interleukin-22 binding protein.

9. The method for detecting the in vitro cell activity of recombinant human long-acting interleukin-22 binding protein according to claim 1 or 2, characterized in that, Includes the following steps: A. Spread 100 μL of HepG2 cell solution into each well of a 96-well plate. After the cells adhere, discard the old culture medium and add 100 μL of complete culture medium containing human IL-22 to each well. Incubate at 37°C and 5% CO2 for 12 h. Then add 10 μL of CCK-8 solution to each well and incubate for 2 h. The cell content of the HepG2 cell solution was 3 × 10⁻⁶. 5 cell / mL; the concentration of human IL-22 in the complete culture medium containing human IL-22 is 10 ng / mL; B. Add 100 μL of complete culture medium containing human IL-22 and recombinant human long-acting interleukin-22 binding protein to each well, and incubate at 37℃ and 5% CO2 for 12 h. Then add 10 μL of CCK-8 solution to each well and incubate for 2 h. Detect the ability of recombinant human long-acting interleukin-22 binding protein to inhibit the proliferation of HepG2 cells promoted by human IL-22. The concentration of the solution containing recombinant human long-acting interleukin-22 binding protein is 50 μg / mL.

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