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

Abstract

The invention relates to the technical field of biology, 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 as shown in SEQ ID No. 1. According to the method for preparing the IL-22BP recombinant protein with high biological activity by utilizing an Escherichia coli prokaryotic expression system, a novel fusion protein is designed, a sequence for coding human IL-22BP is connected with a sequence for coding ABD to construct an IL-22BP-ABD fusion gene, the IL-22BP-ABD fusion gene is induced to be expressed in Escherichia coli, inclusion body protein is separated, and the IL-22BP recombinant protein with high biological activity is obtained. The target protein is obtained through denaturation, purification and renaturation, and the biological activity of the IL-22BP-ABD is verified through in-vivo and in-vitro experiments.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for preparing a recombinant human long-acting interleukin-22 binding protein and its application. Background Art

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

[0003] IL-22 has a protective or pathogenic role in inflammation. This dual role has been best demonstrated in experimental mouse models of gene-deficient mice or neutralizing antibodies. This dual-natured activity is thought to depend on the inflammatory environment, including the affected tissue, the levels of 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, preventing normal differentiation as the cells reach the epidermis from the dermis more quickly. 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 different inflammatory models of the gastrointestinal tract; it generally has a protective role in acute models and a pathogenic role in chronic inflammatory models. Therefore, based on the negative regulatory role of IL-22 in certain diseases, IL-22 is expected to become 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 and is 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 IL-22BP and IL-22R share the same binding site for IL-22. Therefore, IL-22BP inhibits the binding of IL-22 to IL-22R through competitive binding, thereby blocking subsequent downstream signal transduction. Similar to IL-22, IL-22BP is mainly produced by immune cells. Different from many immune effector molecules, which are expressed at higher levels in activated immune cells than in resting immune cells, IL-22BP is constitutively expressed. IL-22BP has been shown to be mainly produced by dendritic cells (DCs), and other immune cells such as eosinophils and CD4 T cells also produce IL-22BP.

[0005] To date, through searches in databases such as the national patent network and PubMed, there are few reports on expressing recombinant IL-22BP or preparing IL-22BP fusion proteins using prokaryotic cells.

[0006] ABD is a small three-helix protein domain that can bind to human serum albumin (HSA). Previous studies have reported that after fusion with exogenous adhesin molecules, antibody fragments, and polypeptides, ABD can significantly extend the in vivo half-life. ABD can effectively increase the plasma half-life of the fusion conjugate to approximately the half-life of endogenous albumin (i.e., about 30 hours in mice and about 12 - 17 days in humans), without 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 disadvantages such as low expression levels, high production costs, long culture cycles, and easy contamination. In contrast, prokaryotic expression systems represented by Escherichia coli have advantages such as low cost, short cycle, and high protein expression levels. However, there are currently no products using prokaryotic systems to express and prepare IL-22BP. Moreover, as a secreted protein with a relatively complex structure, IL-22BP forms inactive inclusion bodies when expressed in Escherichia coli, and the refolding rate is extremely low (inclusion bodies reform below 4M urea). Summary of the Invention

[0008] To solve the above problems, the present invention provides a method for preparing and applying a recombinant human long-acting interleukin-22 binding protein. The present invention uses the Escherichia coli (E. coli) prokaryotic expression system to prepare an IL-22BP recombinant protein with high biological activity. By designing a novel fusion protein, the sequence encoding human IL-22BP is linked to the sequence encoding ABD to construct an IL-22BP-ABD fusion gene. After inducing its expression in E. coli, separating the inclusion body protein, and through denaturation, purification, and renaturation, the target protein is obtained. Through in vivo and in vitro experiments, the biological activity of IL-22BP-ABD is verified.

[0009] 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, and the amino acid sequence of the recombinant human long-acting interleukin-22 binding protein is shown as SEQ ID No.1.

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

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

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

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

[0015] 3) Induce the recombinant E. coli obtained in step 2) with IPTG to obtain an induced bacterium;

[0016] 4) Subject the induced bacterium obtained in step 3) to disruption and centrifugation in sequence to obtain bacterial cells;

[0017] 5) Wash and denature the bacterial cells obtained in step 4) with an inclusion body washing solution to obtain a denatured supernatant;

[0018] 6) Renature and purify the denatured supernatant obtained in step 5) to obtain a recombinant human long-acting interleukin-22 binding protein.

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

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

[0021] Preferably, in step 4), ultrasonic disruption is used, with a power of 500 W, ultrasonic treatment for 2 s, pause for 3 s, and continuous treatment for 20 - 30 min.

[0022] The centrifugation conditions include: a rotation speed of 12000 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% (by mass) Triton X100, pH = 8.0.

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

[0025] Preferably, in step 6), gradient renaturation solution is used for sequential renaturation. The concentrations of urea in the renaturation solution are 6 mol / L, 4 mol / L, 2 mol / L, 1 mol / L, and 0 mol / L respectively, and dialysis renaturation is carried out in descending order of concentration.

[0026] The cut-off molecular weight of the dialysis bag used for dialysis renaturation is 3500 D, and the dialysis time for each gradient is 12 - 16 h.

[0027] Preferably, the renaturation solution also includes 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, 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 renaturation 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] The present 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, including the following steps:

[0031] A. Pipette 100 μL of the HepG2 cell solution into each well of a 96-well plate. After the cells adhere to the well, discard the old culture medium, and add 100 μL of complete 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.

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

[0033] B. Add 100 μL of complete medium containing human IL-22 and recombinant human long-acting interleukin-22 binding protein 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 to 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 preparation protectant for the recombinant human long-acting interleukin-22 binding protein described in the above technical solution, characterized in that the mass percentage content of mannitol in the preparation protectant is 2%, and the mass percentage content of trehalose is 3%.

[0036] Advantages of the present invention:

[0037] 1. The present invention overcomes the disadvantages of low expression level, high production cost, long culture cycle, and easy contamination of IL-22BP protein in the eukaryotic system.

[0038] 2. The present invention provides a method for preparing recombinant human IL-22BP protein by fusing the ABD domain, which can not only provide stability and half-life, but also significantly improve the renaturation rate (urea can be completely dialyzed off, and a high content of soluble protein can still be maintained), overcoming the deficiencies that IL-22BP protein forms inactive inclusion bodies when expressed in Escherichia coli and the renaturation rate is extremely low (inclusion bodies are formed again when the urea concentration is lower than 4 M). BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0040] Figure 1Figure for the specific expression of recombinant IL-22BP-ABD and screening of the optimal expression strain. In A, M: marker; 1: total protein before induction of PET20b; 2: total protein after induction of PET20b; 3: total protein before induction of IL-22BP-ABD; 4: total protein after induction of IL-22BP-ABD; 5: supernatant of broken bacteria after induction; 6: precipitate of broken bacteria after induction; 7: supernatant after denaturation of inclusion bodies; 8: precipitate after denaturation of inclusion bodies. In B, M: marker; 1: total protein before induction of recombinant bacterium No. ①; 2: total protein after induction of recombinant bacterium No. ①; 3: total protein before induction of recombinant bacterium No. ②; 4: total protein after induction of recombinant bacterium No. ②; 5: total protein before induction of recombinant bacterium No. ③; 6: total protein after induction of recombinant bacterium No. ③.

[0041] Figure 2 Figure for the optimization of induction conditions of recombinant protein IL-22BP-ABD. In A, M: marker; 1: total protein of bacteria before induction of IL-22BP-ABD; 2 - 6: total protein of bacteria after induction with 0, 0.3, 0.5, 0.7, 1 mmol / L IPTG. In B, M: marker; 1: total protein of bacteria before induction of IL-22BP-ABD; 2 - 4: total protein of bacteria after induction at 20, 30, 37 °C. In C, M: marker; 1: total protein of bacteria before induction of IL-22BP-ABD; 2 - 7: total protein of bacteria after induction for 3, 4, 5, 6, 7, 8 h.

[0042] Figure 3 Figure for the comparison of renaturation conditions between recombinant protein IL-22BP-ABD and protein IL-22BP. In A, M: marker; 1: supernatant of denatured IL-22BP-ABD protein; 2: supernatant of IL-22BP-ABD renatured with 4 M urea; 3: supernatant of IL-22BP-ABD protein renatured with 2 M urea; 4: supernatant of IL-22BP-ABD protein renatured with 1 M urea; 5: supernatant of IL-22BP-ABD protein renatured with 0 M urea. In B, M: marker; 1: supernatant of denatured IL-22BP protein; 2: supernatant of IL-22BP renatured with 4 M urea; 3: supernatant of IL-22BP protein renatured with 2 M urea; 4: supernatant of IL-22BP protein renatured with 1 M urea; 5: supernatant of IL-22BP protein renatured with 0 M urea.

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

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

[0045] Figure 6 Figure for the screening of the protectant for recombinant protein IL-22BP-ABD. Among them, A is the gray scale statistics of each group of samples after standing at 37°C for one day; B is the gray scale statistics of each group of samples after standing at 37°C for eight days (n = 4, *p < 0.05, **p < 0.01); C is the gray scale analysis result of each group of samples of the combined protectant formula after standing at 37°C for one day; D is the gray scale analysis result of each group of samples of the combined protectant formula after standing at 37°C for eight days.

[0046] Figure 7 Figure of the in vitro activity detection result of recombinant IL-22BP-ABD, n = 4, *p < 0.05, **p < 0.01;

[0047] Figure 8 Figure of the in vivo activity detection of recombinant IL-22BP-ABD. Among them, A is the PASI score result of each group of mice after continuous modeling for 7 days; B is the spleen index of mice after dissection on the 8th day, (n = 4, *p < 0.05, **p < 0.01); C is to sacrifice the mice on the 8th day, extract proteins from the skin tissue, and measure the protein expression of the specified gene by Western blot.

[0048] Figure 9 Figure of the exploration result of the administration method for the in vivo activity detection of recombinant IL-22BP-ABD. Among them, A is the PASI score result of each group of mice after continuous modeling for 6 days; B is the spleen index of mice after dissection on the 7th day; Figure C is to sacrifice the mice on the 7th day, extract proteins from the skin tissue, and measure the protein expression of the specified gene by Western blot.

[0049] Figure 10 Figure of the pharmacokinetics of recombinant protein IL-22BP-ABD and recombinant IL-22BP protein without ABD, where n = 4. Detailed implementation mode

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

[0051] MTQSTHESLKPQRVQFQSRNFHNILQWQPGRALTGNSSVYFVQYKIYGQRQWKNKEDCWGTQELSCDLTSETSDIQEPYYGRVRAASAGSYSEWSMTPRFTPWWETKIDPPVMNITQVNGSLLVILHAPNLPYRYQKEKNVSIEDYYELLYRVFIINNSLEKEQKVYEGAHRAVEIEALTPHSSYCVVAEIYQPMLDRRSQRSEERCVEIPGGGGSGGGGSGGGGSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPHHHHHH。

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

[0053] ATGACTCAATCTACCCATGAATCTCTGAAGCCGCAACGTGTACAGTTTCAGTCTCGTAACTTCCACAACATCCTGCAATGGCAGCCGGGTCGTGCACTGACTGGTAACTCTTCCGTATATTTCGTTCAATACAAAATCTACGGTCAGCGTCAATGGAAGAACAAAGAAGATTGTTGGGGTACCCAGGAACTGTCCTGCGACCTGACCAGCGAAACTTCTGATATCCAGGAACCGTATTACGGCCGTGTACGCGCAGCCAGCGCGGGTTCTTATAGCGAATGGTCCATGACTCCACGTTTCACTCCGTGGTGGGAAACTAAAATCGACCCACCAGTAATGAACATCACTCAAGTTAACGGTTCCCTGCTGGTAATCCTGCACGCGCCAAACCTGCCATATCGCTATCAGAAAGAAAAGAATGTGTCCATCGAAGATTATTATGAGCTGCTGTACCGTGTGTTCATCATCAACAACAGCCTGGAGAAAGAACAGAAAGTGTACGAAGGCGCACACCGTGCAGTTGAAATCGAGGCGCTGACTCCGCACAGCTCCTATTGTGTGGTAGCCGAAATCTATCAGCCGATGCTGGACCGCCGTTCTCAGCGCTCTGAGGAACGTTGCGTAGAGATCCCAGGTGGCGGTGGCAGCGGTGGCGGCGGTAGCGGTGGCGGTGGTTCTCTGGCGGAAGCCAAGGTGCTGGCAAACCGTGAGCTGGATAAGTACGGTGTATCCGACTATTACAAGAACCTGATTAACAATGCTAAAACCGTAGAAGGCGTGAAGGCACTGATTGACGAAATCCTGGCAGCTCTGCCGCACCACCACCATCACCACTAA。

[0054] In the present 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 as shown in SEQ ID No.3, and the nucleotide sequence of the ABD sequence is as shown in SEQ ID No.4. When the IL-22BP sequence is expressed alone in Escherichia coli, inclusion bodies are formed, the renaturation efficiency is low, and the half-life and stability of the protein in vivo are poor. After the IL-22BP sequence is fused with the ABD sequence, not only can the in vivo half-life be significantly prolonged, but it is also unexpectedly found that the inclusion body renaturation rate can be significantly increased, laying a solid foundation for the preparation and production of the protein.

[0055] SEQ ID No.3:

[0056] ATGACTCAATCTACCCATGAATCTCTGAAGCCGCAACGTGTACAGTTTCAGTCTCGTAACTTCCACAACATCCTGCAATGGCAGCCGGGTCGTGCACTGACTGGTAACTCTTCCGTATATTTCGTTCAATACAAAATCTACGGTCAGCGTCAATGGAAGAACAAAGAAGATTGTTGGGGTACCCAGGAACTGTCCTGCGACCTGACCAGCGAAACTTCTGATATCCAGGAACCGTATTACGGCCGTGTACGCGCAGCCAGCGCGGGTTCTTATAGCGAATGGTCCATGACTCCACGTTTCACTCCGTGGTGGGAAACTAAAATCGACCCACCAGTAATGAACATCACTCAAGTTAACGGTTCCCTGCTGGTAATCCTGCACGCGCCAAACCTGCCATATCGCTATCAGAAAGAAAAGAATGTGTCCATCGAAGATTATTATGAGCTGCTGTACCGTGTGTTCATCATCAACAACAGCCTGGAGAAAGAACAGAAAGTGTACGAAGGCGCACACCGTGCAGTTGAAATCGAGGCGCTGACTCCGCACAGCTCCTATTGTGTGGTAGCCGAAATCTATCAGCCGATGCTGGACCGCCGTTCTCAGCGCTCTGAGGAACGTTGCGTAGAGATCCCA;

[0057] SEQ ID No.4:

[0058] CTGGCGGAAGCCAAGGTGCTGGCAAACCGTGAGCTGGATAAGTACGGTGTATCCGACTATTACAAGAACCTGATTAACAATGCTAAAACCGTAGAAGGCGTGAAGGCACTGATTGACGAAATCCTGGCAGCTCTGCCGCACCACCACCATCACCACTAA。

[0059] The present 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) Cloning the coding sequence of the recombinant human long-acting interleukin-22 binding protein into the prokaryotic expression plasmid pET-20b to obtain a recombinant expression plasmid;

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

[0062] 3) Inducing the recombinant Escherichia coli obtained in step 2) with IPTG to obtain an induced bacterium;

[0063] 4) Sequentially disrupting and centrifuging the induced bacterium obtained in step 3) to obtain bacterial cells;

[0064] 5) Washing and denaturing the bacterial cells obtained in step 4) with an inclusion body washing solution to obtain a denatured supernatant;

[0065] 6) Renaturing and purifying the denatured supernatant obtained in step 5) to obtain the recombinant human long-acting interleukin-22 binding protein.

[0066] The present invention clones the coding sequence of the recombinant human long-acting interleukin-22 binding protein into the prokaryotic expression plasmid pET-20b to obtain a recombinant expression plasmid. The present invention has no special limitation on the method for constructing the recombinant expression plasmid, and those skilled in the art can adopt conventional methods.

[0067] The present invention transforms the obtained recombinant expression plasmid into Escherichia coli to obtain recombinant Escherichia coli. The present invention has no special limitation on the method for transforming the recombinant expression plasmid into Escherichia coli, and those skilled in the art can adopt conventional methods.

[0068] The present invention induces the obtained recombinant Escherichia coli with IPTG to obtain an induced bacterium. In the present invention, the recombinant Escherichia coli is preferably induced with IPTG (isopropyl-β-D-thiogalactoside) in the form of a bacterial solution, and the OD of the bacterial solution 600The value is preferably 0.6 to 0.8. The present invention does not particularly limit the method for preparing the bacterial solution, and those skilled in the art can obtain it by using a conventional culture method. In the present invention, the conditions for IPTG induction preferably include: the final concentration of IPTG is 0.3 to 1 mmol / L, the temperature is 20 to 37 °C, and the time is 3 to 8 h. In the present invention, the conditions for IPTG induction are more preferably: the final concentration of IPTG is 0.5 mol / L, the temperature is 37 °C, and the time is 6 h.

[0069] In the present invention, the induced bacteria obtained are successively broken and centrifuged to obtain bacterial cells. In the present invention, the breaking is preferably carried out by ultrasonic breaking, with a power of 500 W, ultrasonic treatment for 2 s, pause for 3 s, and continuous treatment for 20 to 30 min. In the present invention, the conditions for centrifugation preferably include: the rotation speed is 12,000 rpm and the time is 10 min.

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

[0071] In the present invention, the denatured supernatant obtained is renatured and purified to obtain recombinant human long-acting interleukin-22 binding protein. In the present invention, the renaturation is preferably carried out by successively renaturing with a gradient renaturation solution. The concentrations of urea in the renaturation solution are preferably 6 mol / L, 4 mol / L, 2 mol / L, 1 mol / L, and 0 mol / L, respectively, and dialysis renaturation is carried out in descending order of concentration. In the present invention, the cut-off molecular weight of the dialysis bag used for dialysis renaturation is preferably 3500 D, and the dialysis time for each gradient is preferably 12 to 16 h. In the present invention, the renaturation solution also preferably includes 20 mmol / L Tris-HCl, 50 mmol / L NaCl, 0.5 mmol / L GSSG, 1 mmol / L GSH, and 1 mmol / L EDTA, pH = 8.0. From Figure 3It can be seen that through optimizing the formulation and ratio of the refolding solution, the recombinant protein IL-22BP-ABD is refolded through a gradient of 6M, 4M, 2M, 1M, and 0M to obtain a refolding solution formulation that can restore the denatured IL-22BP-ABD to its normal structure. Through grayscale analysis, it is found that 50% of the denatured protein can be restored to its normal conformation. For IL-22-BP, only about 10% is restored to its normal spatial conformation.

[0072] The present invention has no special limitation on the purification method, and conventional methods can be adopted.

[0073] 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. Improving the refolding rate of the recombinant human IL-22BP protein is achieved by fusing the ABD domain to the C-terminus of IL-22BP. Improving the refolding rate of the 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] The present 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 in each well of a 96-well plate. After the cells adhere, discard the old culture medium, add 100 μL of complete medium containing human IL-22 to each well, culture at 37°C and 5% CO2 for 12 h, and then add 10 μL of CCK-8 solution to each well and incubate for 2 h;

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

[0078] B. Add 100 μL of complete medium containing human IL-22 and the recombinant human long-acting interleukin-22 binding protein to each well, culture at 37°C and 5% CO2 for 12 h, and then add 10 μL of CCK-8 solution to each well and incubate for 2 h to detect the ability of the 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 the 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 mass percentage content of mannitol in the formulation protectant is 2%, and the mass percentage content of trehalose is 3%.

[0081] To further illustrate the present invention, the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0082] Example 1

[0083] Construct engineering bacteria of the fusion protein and recombinant human long-acting interleukin-22 binding protein

[0084] 1. Construct an expression vector

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

[0086] Entrust Guangzhou Aiji Biotechnology Co., Ltd. to synthesize and construct the recombinant pET-20b-IL-22BP-ABD expression plasmid.

[0087] 2. Recombinant plasmid transformation and amplification

[0088] 1) Take out the E. coli DH5α competent cells from the -80°C refrigerator, dissolve them on ice, take 5 μL of the recombinant plasmid and add it to 100 μL of DH5α competent cells, incubate on ice for 30 min, then place it in a 42°C water bath for 90 s, and then place it on ice for 2 min.

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

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

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

[0092] 3. Small-scale plasmid extraction

[0093] Extract the plasmid of the recombinant bacteria according to the instruction manual of the plasmid small-scale extraction kit of YEASEN Biotech Co., Ltd.

[0094] 1) Column equilibration: Add 200 μL of Buffer AC to the adsorption column already 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 the bacterial solution, centrifuge at 12,000 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 the tube up and down to mix well, and lyse the bacterial cells thoroughly.

[0098] 5) Add 350 μL of Buffer P3 to the centrifuge tube, gently invert the tube up and down 4 - 6 times. At this time, 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 12,000 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 12,000 rpm for 1 min, discard the waste liquid, and repeat this step.

[0101] 8) Place the adsorption column in the collection tube, centrifuge at 12,000 rpm for 2 min, and let it stand at room temperature for 2 min.

[0102] 9) Place the adsorption column in a clean centrifuge tube, add 50 μL of ddH2O in the middle of the adsorption column, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 2 min, and collect the plasmid in the centrifuge tube.

[0103] 5. Transformation of the recombinant plasmid into Escherichia coli BL21(DE3)

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

[0105] 6. Detection of specific expression of the IL - 22BP - ABD recombinant bacteria and screening of the optimal expression strain

[0106] 1) Respectively pick single transformed colonies and the empty vector into 5 mL of LB liquid medium containing Amp, culture overnight at 37 °C with shaking at 220 rpm on a shaker; after culturing, respectively take 1 mL and inoculate it into 100 mL of LB liquid medium containing Amp, culture with shaking at 37 °C and 220 rpm on a shaker; when the OD of the bacterial solution 600When the value is 0.6 - 0.8, IPTG with a final concentration of 0.5 mmol / L was added for induced expression, and cultured at 37°C with shaking at 180 rpm for 5 h. The induced bacterial solution was centrifuged at 12000 rpm for 10 min, the supernatant was discarded, the precipitate was resuspended with 20 mL of 20 mM Tris-HCl (pH 8.0), and sonicated using a sonicator with a power of 500 w, sonication for 2 s, pause for 3 s, lasting for 20 - 30 min. After centrifuging the sonicated bacterial solution respectively, the precipitate was collected. After the inclusion body was washed and denatured with 8 M urea denaturing solution, the denatured supernatant of IL-22BP-ABD was obtained.

[0107] The total protein before induction of pET20b, the total protein after induction of PET20b, the total protein before induction of IL-22BP-ABD, the total protein after induction of IL-22BP-ABD, the supernatant of the broken bacteria after induction, the precipitate of the broken bacteria after induction, the supernatant after inclusion body denaturation, and the precipitate after inclusion body denaturation were detected by electrophoresis respectively to analyze the specific expression of recombinant IL-22BP-ABD. The results are as Figure 1 shown in A. The recombinant plasmid can specifically express the target protein and most of the target protein exists in the form of inclusion bodies.

[0108] Three randomly selected recombinant strains with successful transformation were induced to express under the same conditions. The total protein before induction of recombinant bacteria No. ①, the total protein after induction of recombinant bacteria No. ①, the total protein before induction of recombinant bacteria No. ②, the total protein after induction of recombinant bacteria No. ②, the total protein before induction of recombinant bacteria No. ③, and the total protein after induction of recombinant bacteria No. ③ were detected by electrophoresis. The expression level of the target protein after induction was analyzed by imageJ software, and the optimal No. ② strain was selected as the seed for subsequent protein preparation, as Figure 1 shown in B.

[0109] Example 2

[0110] Optimization and purification of recombinant protein expression, renaturation and refolding conditions

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

[0112] 1) Optimization of IPTG inducer concentration

[0113] Recombinant strain No. ② was selected and induced to express for the same time (6 h) at the same temperature (37°C) using 0, 0.3, 0.5, 0.7, 1 mmol / L IPTG respectively, and the expression level was detected. After detection, at 37°C, the addition amount of 0.5 mmol / L IPTG had the highest recombinant protein expression level. As Figure 2 shown.

[0114] 2) Optimization of induction temperature

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

[0116] 3) Optimization of induction time

[0117] For the same strain under the condition of 37 °C, inductions were carried out for 3 h, 4 h, 5 h, 6 h, 7 h, and 8 h respectively using the same IPTG concentration (0.5 mmol / L). As Figure 2 shown, the target protein IL-22BP-ABD had the highest expression level under the induction of 0.5 mmol / L IPTG at 37 °C for 6 h.

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

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

[0120] Set the renaturation urea gradients of the renaturation solution to 6 M, 4 M, 2 M, 1 M, and 0 M respectively, and the specific formulations are as follows;

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

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

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

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

[0125] ⑤ 0M Urea Gradient Renaturation Buffer: 20 mmol / L Tris-HCl, 50 mmol / L NaCl, 0.5 mmol / L GSSG, 1 mmol / L GSH, pH = 8.0.

[0126] Load the diluted protein solution into a dialysis bag with a cut-off molecular weight of 3500 D and perform stepwise dialysis according to the decreasing urea gradient. Dialyze for 14 h at each gradient.

[0127] Take the protein solutions before and after dialysis and perform 12% SDS-PAGE electrophoresis. Use Image J for grayscale analysis to compare the recovery rates of the two proteins after successful renaturation.

[0128] The results are as Figure 3 shown in A. By optimizing the formulation and ratio of the renaturation buffer, the recombinant protein IL-22BP-ABD was renatured through 6M, 4M, 2M, 1M, 0M gradient renaturation to obtain a renaturation buffer formulation that can restore the denatured IL-22BP-ABD to its normal structure. Through grayscale analysis, it was found that 50% of the denatured protein could be restored to its normal conformation. As Figure 3 shown in B, for IL-22-BP, only about 10% was restored to its normal spatial conformation.

[0129] Detect the target protein IL-22BP-ABD by Western Blot. After detection and exposure with the anti-His tag antibody, the results show that the expression of the target protein IL-22BP-ABD is good. The results are as Figure 4 .

[0130] 3. Purification of the Recombinant Protein

[0131] Centrifuge the denatured protein supernatant at 12000 rpm and 4 °C for 30 min, collect the supernatant, filter it through a 0.45 μm filter membrane to sterilize, and store the sample in aliquots at -80 °C after subpackaging.

[0132] The specific steps of denaturation are as follows: Centrifuge the induced-expressed bacteria at 12000 rpm for 10 min using a centrifuge. Use an ultrasonic disruptor to disrupt the bacteria at 500 w, disrupt for 2 s, with a 3 s interval, for a total of 30 minutes. After disruption, centrifuge at 12000 rpm for 30 min and discard the supernatant. Resuspend the precipitate with an appropriate amount of inclusion body washing buffer A, stir and wash for 20 min at 4 °C, centrifuge at 12000 rpm for 15 min, discard the supernatant, and repeat 3 - 4 times. Then wash once with an appropriate amount of 20 mmol / L Tris-HCl buffer (pH = 8.0), centrifuge at 12000 rpm for 30 min at 4 °C, and discard the supernatant.

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

[0134] Collect the recombinant protein filtered and sterilized by bacteria, and its purification steps are as follows:

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

[0136] 2) Column equilibration: Equilibrate the pre-packed Ni-NTA affinity chromatography column with 20 mM 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 the Ni-NTA affinity chromatography column at a flow rate of 2 mL / min, and after complete sample loading, allow the sample to bind in the chromatography column for 60 min;

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

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

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

[0141] Purity detection of recombinant IL-22BP-ABD protein: Perform 12% SDS-PAGE electrophoresis on the above sample loading, impurity washing, and elution liquids to detect the molecular weight and purity of the recombinant protein. As Figure 5 shown, after the recombinant IL-22BP-ABD was purified by Ni-NTA affinity chromatography using gradient elution with 40 mM imidazole and 250 mM imidazole, its purity was calculated to be 90%.

[0142] Example 3

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

[0144] An alternative protectant with a 2-fold concentration was dissolved in 20 mM Tris-HCl (pH = 8.0), sterilized, and mixed with IL-22BP-ABD (stored in 20 mM Tris-HCL) at a concentration of 0.5 mg / mL in a volume ratio of 1:1 and added to 1.5 mL Ep tubes, 1 mL per group. After sealing, they were placed in an environment of 37 °C (relative humidity 75%) for an accelerated experiment, and samples were taken for electrophoresis detection on the 1st and 8th days respectively to preliminarily screen and select suitable single protectants. Then, the preliminarily screened protectants were combined and added to the protein according to the above method to further observe the protection effect of the combined formula. By consulting the literature, a total of 6 protectants were selected for the preliminary screening. Each protectant was selected at 3 different concentrations, namely arginine at 1% mass concentration, arginine at 2% mass concentration, arginine at 3% mass concentration, glucose at 1% mass concentration, glucose at 2% mass concentration, glucose at 3% mass concentration, glycine at 1% mass concentration, glycine at 2% mass concentration, glycine at 3% mass concentration, trehalose at 1% mass concentration, trehalose at 2% mass concentration, trehalose at 3% mass concentration, mannitol at 1% mass concentration, mannitol at 2% mass concentration, and mannitol at 3% mass concentration. The above single protectants were respectively mixed with the protein in a volume ratio of 1:1 and stored at 37 °C. On the eighth day, it was found that 1% arginine, 2% mannitol, and 3% trehalose had a certain protective effect. Therefore, arginine at 1% mass concentration, mannitol at 2% mass concentration, and trehalose at 3% mass concentration were further combined into: arginine at 1% mass concentration + mannitol at 2% mass concentration + trehalose at 3% mass concentration, arginine at 1% mass concentration + mannitol at 2% mass concentration, arginine at 1% mass concentration + trehalose at 3% mass concentration, mannitol at 2% mass concentration + trehalose at 3% mass concentration, four combinations, and at the same time compared with the single protectants of arginine at 1% mass concentration, mannitol at 2% mass concentration, and trehalose at 3% mass concentration to select the best combined protectant formula.

[0145] The results showed that the gray-scale analysis of SDS-PAGE detection for each group on the first day of storage at 37 °C, the gray-scale analysis of SDS-PAGE detection on the eighth day of storage at 37 °C, the gray-scale analysis of the SDA-PAGE electrophoresis results of the combined protectant formula samples on the first day of storage at 37 °C, and the gray-scale analysis of the SDA-PAGE electrophoresis results of the combined protectant formula samples on the eighth day of storage at 37 °C were respectively as Figure 6 shown in A - D below. The above results proved that the protectant formula of 2% mannitol + 3% trehalose had the best protective effect on the protein.

[0146] Example 4

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

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

[0149] Co-incubate recombinant protein IL-22BP-ABD with HepG2 and human IL-22. Specifically, stimulate HepG2 cells with 10 ng / ml human IL-22, and evaluate the biological activity of IL-22BP-ABD by detecting the ability of recombinant IL-22BP-ABD protein to inhibit the proliferation-promoting ability of IL-22. Specific steps:

[0150] Resuscitate HepG2 cells: Add 8 mL of DMEM culture medium to a 10 mL centrifuge tube; Take out the cryopreserved HepG2 cells and quickly thaw them in a 37 °C water bath; Immediately add the thawed HepG2 cells to the pre-prepared centrifuge tube, centrifuge at 1000 rpm for 5 min; Aspirate and discard the supernatant, resuspend the precipitate with 1 mL of complete culture medium and add it to a culture dish containing 6 mL of culture medium, gently shake evenly, and culture in a 37 °C, 5% CO2 incubator.

[0151] Observe HepG2 cells under a microscope. When the cell growth state is good, seed HepG2 cells into a 96-well plate.

[0152] Seeding: Use a pipette to collect the cells into a sterile centrifuge tube, centrifuge at 1000 rpm for 5 min; Discard the supernatant, resuspend and mix well with 1 mL of complete culture medium, take 1 μL and dilute it 10 times with complete culture medium, and then perform cell counting under a microscope; Add 100 μL of HepG2 cells (3×10 5 cell / mL) to each well.

[0153] After the cells adhere to the wall, discard the old culture medium, mix human recombinant IL-22 protein at different concentrations in the culture medium, add 100 μL of the mixed culture medium to each well, and culture in a 37 °C, 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 the incubator for 2 hours.

[0156] Use a microplate reader to measure the absorbance at 450 nm to determine the optimal concentration and stimulation time of IL-22 for stimulating cell proliferation.

[0157] After resuscitating HepG2, perform the seeding operation as described above.

[0158] After the cells adhered to the wall, discard the old culture medium, mix human recombinant IL-22 protein (10 ng / ml) with IL-22BP-ABD at different concentration gradients in the culture medium, add 100 μL of the mixture to each well, and culture in an incubator at 37 °C and 5% CO2 for 12 h.

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

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

[0161] Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 450 nm to determine the effect of IL-22-ABD and the concentration gradient effect of the effect. As Figure 7 shown, the protein IL-22BP-ABD can significantly inhibit the proliferation of IL-22-stimulated HepG2 cells by binding to IL-22.

[0162] 2. Detection of the in vivo activity of recombinant IL-22BP-ABD protein

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

[0164] 1) Female balb / c mice were modeled by applying 60 mg / animal of 5% imiquimod cream to the back for 7 consecutive days, and at the same time, different doses of IL-22BP-ABD preparations were intraperitoneally injected for 7 consecutive days starting from the first day of modeling. Monitor the skin lesions on the back of the mice and the body weight changes every day.

[0165] 2) Conduct a grouped experiment: (4 mice in each group)

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

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

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

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

[0170] 3) After 7 consecutive days of intraperitoneal injection of recombinant protein IL-22BP-ABD, the mice were sacrificed on the 8th day, and the diseased skin tissues were taken to extract the total tissue protein. The protein expression levels of IL-22 were detected by Western blot. At the same time, the size of the spleen lesions was compared, and the spleen index was statistically analyzed.

[0171] The results are as Figure 8 shown. Using a mouse psoriasis model, 5% imiquimod cream was continuously applied to the dorsal skin of mice for 7 days to induce psoriasis in mice, and the in vivo activity of recombinant IL-22BP-ABD protein was detected. The PASI score results of monitoring the skin lesion status every day after administration of imiquimod cream are as Figure 8 shown. The 5 mg / kg recombinant IL-22BP-ABD preparation had a significant inhibitory effect on skin lesions after the fourth day. As Figure 8 shown in B, while continuously applying 5% imiquimod cream for 7 days, different concentrations of IL-22BP-ABD were continuously injected for 7 days. On the 8th day, the mice were sacrificed and the spleen indices of each group of mice were compared. It was found that injection of high-dose IL-22BP-ABD could reduce the spleen index of mice. Figure 8 As shown in C, after the mice were sacrificed on the 8th day, proteins were extracted from the skin tissue homogenate, and the protein expression of IL-22 was determined by Western blot. The results showed that after injection of IL-22BP-ABD, the protein expression of IL-22 in the high-dose experimental group of mice decreased significantly.

[0172] Example 5

[0173] Investigation on the administration method for detecting the in vivo activity of recombinant IL-22BP-ABD protein

[0174] 1. Method: Female balb / c mice were modeled by continuously applying 60 mg / 5% imiquimod cream to the back for 6 days, and at the same time, the same concentration and volume of IL-22BP-ABD were given by different administration methods starting from the first day of modeling for 6 consecutive days. The skin lesions on the back of the mice and the body weight changes were monitored every day.

[0175] 2. The experimental groups were as follows: (4 mice in each group)

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

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

[0178] ③ Subcutaneous injection experimental group: Apply 60 mg / rat of 5% imiquimod cream on the back, and subcutaneously inject the recombinant IL-22BP-ABD preparation (total volume 200 μL, containing 5 mg / kg recombinant IL-22BP-ABD).

[0179] ④ Intraperitoneal injection experimental group: Apply 60 mg / rat of 5% imiquimod cream on the back, and intraperitoneally inject the recombinant IL-22BP-ABD preparation (total volume 200 μL, containing 5 mg / kg recombinant IL-22BP-ABD).

[0180] 3. After 6 - 7 days of administering the recombinant protein IL-22BP-ABD preparation according to each group, sacrifice the mice every other day, take the diseased skin tissue, extract the total tissue protein, and detect the IL-22 and protein expression levels by Western blot. At the same time, compare the size of the spleen lesions and calculate the spleen index.

[0181] 4. Results

[0182] The results are as Figure 9 shown. Exploration of the administration method for detecting the in vivo activity of recombinant IL-22BP-ABD: Continuously apply 5% imiquimod cream on the back of mice for 6 days to induce psoriasis in mice. By constructing a mouse psoriasis model and administering the same concentration of IL-22BP-ABD in different ways to determine the optimal administration method, it was found that the PASI score table for monitoring the skin lesion status every day after applying imiquimod cream is shown in Figure A. B: While continuously applying 5% imiquimod cream for 6 days, continuously inject the same dose of IL-22BP-ABD preparation in different ways for 6 days. Sacrifice the mice on the 7th day and compare the spleen indices of each group of mice. It was found that intraperitoneal injection of the IL-22BP-ABD preparation can more significantly reduce the spleen index of mice. C: After sacrificing the mice on the 7th day, extract the protein from the skin tissue homogenate and determine the IL-22 protein expression by Western blot. The results showed that after injecting the IL-22BP-ABD preparation in two ways, the decrease in the expression of the IL-22 factor in the intraperitoneal injection experimental group of mice was higher than that in the subcutaneous group.

[0183] Example 6

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

[0185] Inject the 5 mg / kg recombinant protein IL-22BP-ABD preparation and IL-22BP into female BALB / c mice by single subcutaneous injection respectively, and analyze the IL-22BP-ABD levels in the serum collected at different time points after injection by ELISA. The specific steps are as follows:

[0186] 1) Eight female BAL B / c mice weighing 18 - 20 g were taken and divided into two groups. Recombinant IL-22BP-ABD and IL-22BP proteins were subcutaneously injected once at a dose of 5 mg / kg. All animal experiments were conducted in an SPF-level laboratory.

[0187] 2) After injection, blood samples of the mice were collected into clean centrifuge tubes by taking blood from the fundus of the eye at each time point of 0, 1, 2, 4, 8, 12, 24, 36, 48, and 60 h. They were left standing at 4°C for 1 - 2 h and then centrifuged at 3500 rpm for 15 min to collect the serum.

[0188] 3) The contents of human IL-22BP-ABD and IL-22BP in the mouse serum were detected by ELISA method, and the half-lives of recombinant IL-22BP-ABD protein and IL-22BP protein in the mice were calculated using the Microsoft Excel add-in "PKSolver".

[0189] The results are as Figure 10 shown. The 5 mg / kg recombinant IL-22BP-ABD preparation was injected into male BALB / c mice by single subcutaneous injection. The serum IL-22BP-ABD level reached the peak at 1 h by the Elisa method, decreased rapidly from 1 - 8 h, and decreased slowly from 8 - 36 h. Pharmacokinetic parameters were measured using PKSolver to calculate important pharmacokinetic parameters. The time to maximum concentration in the serum of the mice after injection of the recombinant IL-22BP-ABD protein preparation was 1 h, the maximum concentration was 342.25 μg / ml, and the half-life in vivo was 17 h; while the recombinant IL-22BP without ABD reached the peak 20 min after injection and degraded rapidly, with a half-life in vivo of 2 h.

[0190] In summary, the recombinant IL-22BP-ABD protein prepared by the present invention has a high renaturation rate and stability. The purified recombinant protein has high biological activity both in vivo and in vitro. The present invention has established for the first time an activity detection method and a formulation for the recombinant IL-22BP-ABD protein, laying a foundation for subsequent new drug research and development.

[0191] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments without creative work based on these embodiments, and these embodiments all belong to 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, wherein The coding sequence of the recombinant human long-acting interleukin-22 binding protein is shown in SEQ ID No.

2.

3. The preparation method of the recombinant human long-acting interleukin-22 binding protein according to claim 1 or 2, characterized in that, It includes the following steps: 1) Clone the coding sequence of the recombinant human long-acting interleukin-22 binding protein into the prokaryotic expression plasmid pET-20b to obtain a recombinant expression plasmid; 2) Transform the recombinant expression plasmid obtained in step 1) into Escherichia coli to obtain recombinant Escherichia coli; 3) Induce the recombinant Escherichia coli obtained in step 2) with IPTG to obtain an induced bacterium; 4) Subject the induced bacterium obtained in step 3) to disruption and centrifugation in sequence to obtain bacterial cells; 5) Wash and denature the bacterial cells obtained in step 4) with an inclusion body washing solution to obtain a denatured supernatant; 6) Renature and purify the denatured supernatant obtained in step 5) to obtain the 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 is induced by IPTG in the form of a bacterial liquid, and the OD 600 value of the bacterial liquid is 0.6-0.8; The conditions for IPTG induction include: the final concentration of IPTG is 0.3 - 1 mmol / L, the temperature is 20 - 37 °C, and the time is 3 - 8 h.

5. The preparation method according to claim 3, characterized in that, In step 4), ultrasonic disruption is used, with a power of 500 W, ultrasonic treatment for 2 s, pause for 3 s, and continuous treatment for 20 - 30 min; The conditions for centrifugation include: the rotation speed is 12000 rpm and the time is 10 min; The components of the inclusion body washing solution include: 20 mmol / L Tris-HCl, 50 mmol / L NaCl, 2 mol / L urea, and 2% (mass percentage) Triton X100, pH = 8.0; The components of the denaturing solution used for denaturation include: 20 mmol / L Tris-HCl, 50 mmol / L NaCl, 10 mmol / L β-mercaptoethanol, 8 mol / L urea, and 1 mmol / L EDTA, pH = 8.

0.

6. The preparation method according to claim 3, characterized in that, In step 6), gradient renaturation solution is used for sequential renaturation. The concentrations of urea in the renaturation solution are 6 mol / L, 4 mol / L, 2 mol / L, 1 mol / L, and 0 mol / L respectively, and dialysis renaturation is carried out in descending order of concentration; The cut-off molecular weight of the dialysis bag used for dialysis renaturation is 3500 D, and the dialysis time for each gradient is 12 - 16 h; The renaturation solution also includes 20 mmol / L Tris-HCl, 50 mmol / L NaCl, 0.5 mmol / L GSSG, 1 mmol / L GSH, and 1 mmol / L EDTA, pH = 8.

0.

7. Use of the recombinant human long-acting interleukin-22 binding protein according to claim 1 or 2 in increasing the renaturation rate of interleukin-22 binding protein.

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

9. The in vitro cell activity detection method of the recombinant human long-acting interleukin-22 binding protein according to claim 1 or 2, characterized in that, It includes the following steps: A. Pipette 100 μL of HepG2 cell solution into each well of a 96-well plate. After the cells adhere to the wall, discard the old culture medium, and add 100 μL of complete 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 is 3×10 5 cell / mL; the concentration of human IL-22 in the complete medium containing human IL-22 is 10 ng / mL; B. Add 100 μL of complete medium containing human IL-22 and recombinant human long-acting interleukin-22 binding protein 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 to 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.

10. A preparation protectant for the recombinant human long-acting interleukin-22 binding protein according to claim 1 or 2, characterized in that, The mass percentage content of mannitol in the formulation protectant is 2%, and the mass percentage content of trehalose is 3%.

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