Pseudosciaena crocea interleukin 22 recombinant protein and application thereof

Abstract

The invention discloses a larimichthys crocea IL-22 recombinant protein which is a larimichthys crocea IL-22 mature peptide sequence, and the amino acid sequence of the larimichthys crocea IL-22 recombinant protein is as shown in SEQ ID NO. 2. The recombinant protein is obtained by amplifying and coding gene segments of amino acids from 31st to 190th positions of large yellow croaker IL-22 mature peptide through PCR (Polymerase Chain Reaction), and transforming escherichia coli for heterologous expression. The larimichthys crocea IL-22 recombinant protein can activate a transcription factor STAT3 in a larimichthys crocea head kidney cell line (LYCK) and also can induce expression of inflammatory factors (IL-1beta, IL-6 and IL-8) in immune organs of larimichthys crocea, so that the larimichthys crocea IL-22 recombinant protein has an obvious immunoregulation effect and shows a good application prospect in research and development of aquatic immunomodulators.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to large yellow croaker interleukin 22 (IL-22), and in particular to large yellow croaker IL-22 recombinant protein and application thereof. Background Art

[0002] IL-22 belongs to the IL-10 family of cytokines and is mainly produced by lymphoid cells, including helper T cells, natural killer T cells, CD8 +T cells, etc. The main function of IL-22 is to provide a protective response against pathogens on the barrier surface, playing an important role in cell proliferation and survival (Sonnenberg GF, Fouser LA, Artis D. Border patrol: regulation of immunity, inflammation and tissue homeostasis at barriers surfaces by IL-22[J]. Nat Immunol, 2011, 12(5): 383-390.). IL-22 is a key regulator of epithelial homeostasis, inducing the production of antimicrobial peptides such as β-defensin-2 (β-BD-2) and β-BD-3, enhancing the expression of proinflammatory cytokines (such as IL-6 and IL-8), and protecting the host from invasion by various pathogens (Wolk K, Sabat R. Interleukin-22: novel T- and NK-cell derived cytokine that regulates the biology of tissue cells[J]. Cytokine Growth Factor Rev, 2006, 17(5): 367-380.). IL-22 can also upregulate the expression of pro-survival genes Bcl-2, Bcl-XL and Mcl-1, as well as proliferation factors c-Myc, CyclinD1, Rb2 and CDK4, and participate in epithelial cell regeneration, permeability regulation and the production of mucus and complement (Lindemans CA, Calafiore M, Mertelsmann AM, et al. Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration [J]. Nature, 2015, 528 (7583): 560-564.). Fish IL-22 is mainly expressed in mucosal tissues and systemic lymphoid tissues, is upregulated after pathogen infection, and can also induce the expression of various inflammatory factors in tissues. This patent cloned the IL-22 gene of large yellow croaker, expressed and purified the recombinant IL-22 protein of large yellow croaker, and detected the changes in the expression levels of inflammatory factors in the leukocytes of the head kidney and spleen of large yellow croaker after stimulation with the IL-22 protein of large yellow croaker. The recombinant protein IL-22 from large yellow croaker can activate the transcription factor STAT3, significantly upregulate the expression of inflammatory factors IL-1β, IL-6 and IL-8 in the white blood cells of the head kidney and spleen of large yellow croaker, exert an immunomodulatory effect, and can be developed as an immunomodulator with good application prospects. Summary of the Invention

[0003] One of the objectives of the present invention is to provide a large yellow croaker IL-22 recombinant protein, wherein the recombinant protein is a large yellow croaker IL-22 mature peptide sequence, and its amino acid sequence is shown in SEQ ID NO.2.

[0004] Furthermore, the present invention provides a gene encoding the above-mentioned large yellow croaker IL-22 recombinant protein, the nucleotide sequence of which is shown in SEQ ID NO.1, wherein positions 3-93 are signal peptides.

[0005] A second object of the present invention is to provide an application of large yellow croaker IL-22 recombinant protein.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] Using large yellow croaker head kidney cDNA as a template, primers IL-22-F and IL-22-R were used to amplify the gene fragment encoding amino acids 31 to 190 of the large yellow croaker IL-22 mature peptide by PCR technology, and then ligated into the Escherichia coli expression vector pET-28a to obtain a recombinant expression vector pET-28a-IL-22 containing the large yellow croaker IL-22 mature peptide gene fragment. The recombinant expression vector was transformed into the Escherichia coli BL21 strain. After ampicillin selection and sequencing identification, the Escherichia coli engineered strain BL21 / pET-28a-IL-22 capable of expressing the large yellow croaker IL-22 recombinant protein was obtained. The primer sequences are as follows:

[0008] IL-22-F: 5'-ATGGACTACAAAGACGATGACGACAAGCTGCCCGTCGACCGGCCGCTC-3',

[0009] IL-22-R: 5'-CTACTGTTTGGCGGCGTCGG-3'.

[0010] The large yellow croaker IL-22 recombinant protein was obtained by inducing, expressing and purifying the BL21 / pET-28a-IL-22 engineering bacteria in LB medium with 0.1 mM IPTG.

[0011] The beneficial effect of the present invention is that the recombinant large yellow croaker IL-22 protein can activate the transcription factor STAT3 in the large yellow croaker head kidney cell line (LYCK). After stimulation with the recombinant large yellow croaker IL-22 protein, the phosphorylation level of STAT3 in the large yellow croaker head kidney cells increased significantly, reaching a peak at 15 minutes, which is twice the level before stimulation.

[0012] The large yellow croaker IL-22 recombinant protein of the present invention can induce the expression of inflammatory factors (IL-1β, IL-6 and IL-8) in the immune organs of large yellow croaker. After stimulation with the large yellow croaker IL-22 recombinant protein, the expression levels of the inflammatory factors IL-1β, IL-6 and IL-8 in the spleen leukocytes of primary large yellow croaker are significantly increased within 24 hours, by 3.66 times, 4.27 times and 2.09 times, respectively; in the primary cells of the head kidney of large yellow croaker, IL-1β and IL-8 are significantly increased within 24 hours by 1.76 times and 1.49 times, respectively, and the expression level of IL-6 is significantly increased within 12 hours and 24 hours, by 2.84 times and 2.62 times that of the control group, respectively; this indicates that the large yellow croaker IL-22 recombinant protein has obvious immunoregulatory effects, showing its good application prospects in the research and development of aquatic immunomodulators. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The purpose is to express and purify the recombinant protein of IL-22 from large yellow croaker. Figure 1 A is Western blot to determine the expression of control protein and large yellow croaker IL-22 recombinant protein, M lane is the protein molecular weight standard; lanes 1 and 2 are purified large yellow croaker IL-22 recombinant protein; lanes 3 and 4 are purified Trx recombinant protein as control protein. Figure 1 B is the purified large yellow croaker IL-22 recombinant protein analyzed by SDS-PAGE, and the arrow points to the large yellow croaker IL-22 recombinant protein.

[0014] Figure 2 Figure 2: Activation of the transcription factor STAT3 by recombinant IL-22 in a yellow croaker head kidney cell line. Figures A and B show changes in STAT3 phosphorylation levels in yellow croaker head kidney cells after stimulation with recombinant IL-22 for 0, 5, 10, 15, 25, and 40 minutes. Figure C shows confocal microscopy observation of STAT3 phosphorylation and intracellular localization in yellow croaker head kidney cells after stimulation with recombinant IL-22 and Trx for 15 minutes, respectively.

[0015] Figure 3 Figure 2 shows the regulatory effect of recombinant IL-22 protein on the expression of inflammatory factors in leukocytes of the head kidney and spleen of large yellow croaker. A is IL-1β, B is IL-6, and C is IL-8. DETAILED DESCRIPTION

[0016] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific embodiments. The embodiments are intended to illustrate the present invention and are not intended to limit the present invention in any form.

[0017] Example 1 Construction of Escherichia coli engineered bacteria that efficiently express large yellow croaker IL-22 recombinant protein

[0018] The large yellow croaker IL-22 recombinant protein of the present invention is the large yellow croaker IL-22 mature peptide sequence (amino acids 32-190, as shown in SEQ ID NO.2). The sequence of large yellow croaker IL-22 has been published (Genbank: XP_027139193.1). The primers IL-22-F and IL-22-R were used using the TransGen FastPfu Fly DNA Polymerase was used to perform PCR to amplify the gene fragment encoding amino acids 31 to 190 of the mature peptide of large yellow croaker IL-22 (the sequence is shown in SEQ ID NO.1). The PCR product was ligated with the linearized pET-28a vector using the Basic Seamless Cloning and Assembly Kit to generate the recombinant expression vector pET-28a-IL-22, containing the large yellow croaker IL-22 mature peptide gene fragment. The recombinant expression vector was transformed into the Escherichia coli BL21 strain. After ampicillin selection and sequencing, the engineered E. coli strain BL21 / pET-28a-IL-22 expressing the large yellow croaker IL-22 recombinant protein was obtained.

[0019] SEQ ID NO.2:

[0020] LPVDRPLSQPLRNPETYQAVREVSQHAQNLQLHNESSTRLMPKVDTTLDTQDHM KICCLHANILDFYLNNILRHRDHTHPRMPRLKIDLSRVSEDLQTHGCNVTHYHDHQHA VEFRRKLTKMGGSDGMNKAVGEIDILFTYLQDFCVQPRN STDADAAKQ.

[0021] The PCR primers are as follows:

[0022] IL-22-F:

[0023] 5'-ATGGACTACAAAGACGATGACGACAAGCTGCCCGTCGACCGGCCGCTC-3'

[0024] IL-22-R: 5'-CTACTGTTTGGCGGCGTCGG-3'.

[0025] The LB solid culture medium formula is: 1% NaCl, 1% tryptone, 0.5% yeast extract and 1.5% agar.

[0026] SEQ ID NO.1 is as follows, wherein the underlined portion is the signal peptide;

[0027] ATG CAGCCCGCCACCATCGCCTCCTTCCTCCGTCTGGCCGCAGTCGTGCTGCTGCCGCTGCTCCTGAT TGGCTGGGCTGAAGAGGCGGTGGCG CTGCCCGTCGACCGGCCGCTCAGCCAGCCGCTACGAAACCCGGAGACATACCAGGCCGTCCGAGAGGTGTCGCAACACGCTCAGAATCTGCAGCTGCACAACGAGTCCAGCACCAGACTGATGCCCAAAGTCGACACTTTGGACACCCAGGACCACATGAAGATCTGCTGCCTCCACGCCAACATCCTCGACTTCTACCTGAACAACATCCTCCGTCACCGCGACCACACGCATCCTCGC ATGCCGAGGCTGAAGATCGACCTGAGCCGGGTCAGCGAGGACCTGCAGACCCACGGCTGCAATGTGACTCACTACCACGACCATCAGCACGCTGTGGAGTTTCGCAGGAAGCTCACCAAAATGGGCGGCAGTGACGGGATGAACAAGGCGGTGGGAGATCGACATCCTGTTCACGTACCTGCAGGACTTCTGCGTCCAGCCGAGAAACTCCACCGACGCCGACGCCGCCAAACAGTGA.

[0028] Example 2 Expression and purification of large yellow croaker IL-22 recombinant protein

[0029] The E. coli BL21 / pET-28a-IL-22 engineered bacteria obtained in Example 1 were inoculated into 5 mL of LB liquid culture medium containing ampicillin and cultured at 37°C with shaking at 200 rpm. 600 When the value is about 0.4, IPTG (final concentration 0.5 mmol / L) was added and induced at 37°C for 4 h. The bacterial solution was centrifuged at 8000 g at 4°C for 5 min, and the cells were collected and subjected to SDS-PAGE electrophoresis. Figure 1Figure A shows a band of approximately 20 kDa in the lysate of the control strain BL21 / pET-28a, representing the recombinant protein Trx expressed by pET-28a. In contrast, a band of approximately 35 kDa appeared in the lysate of the E. coli BL21 / pET-28a-IL-22 engineered strain, representing the recombinant protein of large yellow croaker IL-22. After confirming expression, the induced BL21 / pET-28a-IL-22 engineered strain was harvested by centrifugation. Protease inhibitors were added at a 1:1000 ratio, and the cells were disrupted using a high-pressure disrupter. The supernatant was collected by centrifugation at 10,000 g for 10 minutes at 4°C, and filtered through a 0.45 μm filter. The filtered supernatant was slowly transferred to a chromatography column containing 4 mL of Ni-NTA and allowed to bind at 4°C for 2 hours. The column was then washed five times with 2 mL of protein wash buffer containing varying concentrations of imidazole, followed by a protein stripping solution to collect the target protein. Transfer the target protein solution into a dialysis bag, dialyze it three times with PBS buffer at 4°C, and then centrifuge it at 12000 rpm for 10 minutes to remove the precipitate. Take a small amount of sample for SDS-PAGE electrophoresis and stain it with Coomassie Brilliant Blue. The results are as follows: Figure 1 B shows that a high-purity recombinant protein of large yellow croaker IL-22 was obtained. The dialyzed protein was aliquoted and stored at -80°C.

[0030] The LB liquid culture medium formula: 1% NaCl, 1% tryptone and 0.5% yeast extract.

[0031] The formula of the protein washing solution is: 20 mM NaH2PO4, 500 mM NaCl, 20 mM imidazole (imidazole concentration gradient is 40 mM, 60 mM, 80 mM and 100 mM), pH=7.4.

[0032] The protein eluent formula is: 20 mM NaH2PO4, 500 mM NaCl, 500 mM imidazole, pH=7.4.

[0033] Example 3 Large yellow croaker IL-22 recombinant protein activates transcription factor STAT3 in head kidney cell line

[0034] In a 6-well cell culture plate, 1 × 10 6Large yellow croaker head kidney (LYCK) cells were cultured for at least 12 hours. Recombinant large yellow croaker IL-22 protein was stimulated with a final concentration of 200 ng / mL for 0, 5, 10, 15, 25, and 40 minutes. The cells were harvested and lysed on ice for 10 minutes using RIPA lysis buffer (Biyuntian). The cells were then incubated overnight with rabbit anti-STAT3 (phospho Y705) and mouse anti-tubulin. After washing, goat anti-mouse IgG (Abcam) and goat anti-rabbit IgG (Abcam) were added, and the cells were visualized using a colorimetric developer. At the same time, rabbit anti-STAT3 (phospho Y705) and mouse anti-Tubulin were used to directly label the yellow croaker head kidney cells stimulated with yellow croaker IL-22 recombinant protein (200 ng / mL), and then incubated with goat anti-rabbit IgG (Abcam) coupled with Alexa Fluor 647 and goat anti-mouse IgG (Abcam) coupled with Alexa Fluor 488, and the cell nuclei were stained with DAPI dye. Finally, the fluorescence was observed under a laser scanning confocal microscope (Leica). Figure 2 As shown in Figure 2, after stimulation with IL-22 recombinant protein, the phosphorylation level of STAT3 in the head kidney cells of yellow croaker increased significantly. The phosphorylation level reached the highest value at 15 minutes, which was 2 times that before stimulation. Then it began to decline and returned to normal level at 40 minutes ( Figure 2 A. Figure 2 B); similar results were obtained by laser confocal microscopy observation. After stimulation with large yellow croaker IL-22 recombinant protein, phosphorylated STAT3 accumulated in the cell nucleus. This indicates that large yellow croaker IL-22 recombinant protein can activate the transcription factor STAT3 in large yellow croaker immune cells ( Figure 2 C).

[0035] Example 4 Large yellow croaker IL-22 recombinant protein promotes the expression of inflammatory factors

[0036] Primary leukocytes from the head kidney and spleen of yellow croaker were stimulated with 20 ng / mL recombinant protein of yellow croaker IL-22, and the same concentration of Trx-tagged protein was used as a control. Cells were collected at 12 h and 24 h, and the expression changes of inflammatory factors (IL-1β, IL-6, and IL-8) were detected by real-time fluorescence quantitative PCR. The gene primers are shown in Table 1. Figure 3As shown in the data, after stimulation with large yellow croaker IL-22 recombinant protein, the expression levels of inflammatory factors IL-1β, IL-6 and IL-8 in primary large yellow croaker spleen leukocytes were significantly upregulated at 24 hours, by 3.66 times, 4.27 times and 2.09 times, respectively; in large yellow croaker head kidney primary cells, IL-1β and IL-8 were significantly upregulated by 1.76 times and 1.49 times, respectively, at 24 hours, and the expression level of IL-6 was significantly upregulated at 12 hours and 24 hours, by 2.84 times and 2.62 times that of the control group, respectively; this indicates that large yellow croaker IL-22 recombinant protein induces the expression of inflammatory factors in large yellow croaker immune organs and has an immunoregulatory effect.

[0037] Table 1

[0038]

[0039]

[0040] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A large yellow croaker IL-22 recombinant protein, characterized in that The recombinant protein is the mature peptide sequence of large yellow croaker IL-22, and its amino acid sequence is shown in SEQ ID NO.

2.

2. A large yellow croaker IL-22 gene, characterized in that: Encoding the large yellow croaker IL-22 recombinant protein as claimed in claim 1, the nucleotide sequence of the large yellow croaker IL-22 gene is shown in SEQ ID NO.1, wherein positions 3-93 are signal peptides.

3. Recombinant plasmid pET-28a-IL-22, characterized in that: Contains the 94th to 573rd nucleotide sequence of the large yellow croaker IL-22 gene as claimed in claim 2.

4. Use of the large yellow croaker IL-22 recombinant protein as claimed in claim 1 in activating the transcription factor STAT3 of the large yellow croaker head kidney cell line.

5. The effect of the large yellow croaker IL-22 recombinant protein in inducing the expression of inflammatory factors as described in claim 1, wherein the inflammatory factors include IL-1β, IL-6 and IL-8.

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