Adiponectin mutant with high anti-inflammatory activity as well as preparation method and application of adiponectin mutant

Abstract

The invention relates to an adiponectin mutant with high anti-inflammatory activity as well as a preparation method and application of the adiponectin mutant. The adiponectin mutant is characterized in that lysine at the 172nd site of adiponectin is mutated into proline, or lysine at the 169th site of adiponectin is mutated into proline. Wherein the amino acid sequence of the adiponectin mutant mADPNK172P is as shown in SEQ ID NO. 5, and the nucleotide sequence of the adiponectin mutant mADPNK172P is as shown in SEQ ID NO. 6. The amino acid sequence of the adiponectin mutant hADPNK169P is as shown in SEQ ID NO.7, and the nucleotide sequence of the adiponectin mutant hADPNK169P is as shown in SEQ ID NO.8. Compared with the conventional wild-type adiponectin, the adiponectin mutant mADPNK172P or the adiponectin mutant hADPNK169P disclosed by the invention has the advantages that the anti-inflammatory activity is remarkably improved, the problem of low anti-inflammatory activity level of the wild adiponectin is solved, the anti-inflammatory effect of the adiponectin is remarkably improved, and the application prospect is wide. The compound has a good application prospect in the production of biological medicines for resisting metabolic diseases such as diabetes, inflammation and atherosclerosis.

Description

Technical Field

[0001] The invention relates to an adiponectin mutant with high anti-inflammatory activity and a preparation method and application thereof, belonging to the technical field of biological medicines. Background Art

[0002] Adiponectin (ADPN) is an adipokine secreted by adipose tissue. As one of the most abundant adipokines in the blood, adiponectin plays an important role in a variety of biological processes, including enhancing insulin sensitivity, preventing atherosclerosis, and anti-inflammation. Current studies on humans and animal models have shown that adiponectin has great potential in treating metabolic diseases such as obesity, diabetes, and atherosclerosis.

[0003] The full-length adiponectin of mice and humans consists of 247 and 244 amino acids, respectively, and both contain four major domains: an N-terminal signal peptide domain, a highly variable domain, a collagen domain, and a C-terminal globular domain. In the blood circulation, ADPN exists mainly in three subtypes, including low molecular weight (LMW) trimers, medium molecular weight (MMW) hexamers, and high molecular weight (HMW) aggregates consisting of 12 to 18 subunits. These different polymers show different binding affinities to adiponectin receptors, and the effects of ADPN on its target tissues depend on the expression of specific receptors and the polymer form to which they bind. Under different metabolic conditions, the absolute level of HMWADPN and its ratio to the total ADPN level will change. There is a stronger correlation between HMWADPN and insulin sensitivity. Studies have shown that the level of HMWADPN in the blood is significantly reduced in people with diabetes, obesity, and cardiovascular disease. Experiments have shown that intraperitoneal injection of HMW ADPN into mice can produce a significant hypoglycemic effect.

[0004] Due to the influence of obesity and diabetes-related diseases, the production of HMW ADPN in the body is impaired. Therefore, exploring strategies to increase the level of HMW ADPN is expected to become a potential innovative approach for the treatment of metabolic diseases. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides an adiponectin mutant with high anti-inflammatory activity and a preparation method and application thereof.

[0006] The technical solution of the present invention is as follows:

[0007] A mutant of adiponectin with high anti-inflammatory activity, wherein the lysine at position 172 of adiponectin is mutated to proline, or the lysine at position 169 is mutated to proline.

[0008] According to the present invention, preferably, the mutant is adiponectin mutant mADP K172P The amino acid sequence is shown in SEQ ID NO.5, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.6; it is mouse adiponectin (mADPN wt ) mutated to proline at position 172, mouse adiponectin (mADPN wt ) The amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0009] According to the present invention, the mutant is preferably adiponectin mutant hADPN K169P The amino acid sequence is shown in SEQ ID NO.7, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.8; it is human adiponectin (hADPN wt ) mutated to proline at position 169, human adiponectin (hADPN wt ) The amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.4.

[0010] A recombinant vector is a plasmid vector into which the coding gene of the adiponectin mutant with high anti-inflammatory activity is inserted.

[0011] Preferably according to the present invention, the plasmid vector is pCMV-C-Flag.

[0012] A recombinant cell is obtained by transforming the above recombinant vector into a host cell.

[0013] Preferably according to the present invention, the host cell is a human embryonic kidney cell HEK293.

[0014] The method for preparing the above-mentioned adiponectin mutant with high anti-inflammatory activity comprises the following steps:

[0015] (1) Artificially synthesizing the mouse adiponectin gene and the human adiponectin gene according to the sequence information shown in SEQ ID NO.2 and SEQ ID NO.4, and then recombining them into the plasmid vector pCMV-C-Flag with a C-terminal fusion expression Flag tag to obtain the recombinant plasmid vector pCMV-mADPN wt and pCMV-hADPN wt ;

[0016] (2) Design site-directed mutagenesis primers and use the recombinant plasmid vector pCMV-mADPN obtained in step (1) to wt and pCMV-hADPN wtPCR amplification was performed using 50 μM PCR product as template, and the obtained PCR product was transformed into DH5α competent medium and sequenced to obtain the mutant plasmid vector pCMV-mADPN. K172P and pCMV-hADPN K169P ;

[0017] The site-directed mutagenesis primer sequences are as follows:

[0018] mADPN mut -F:5'-gaggctcaccttcacatctggcatgtacaccgtgatgtgg-3',

[0019] mADPN mut -R: 5'-ccacatcacggtgtacatgccagatgtgaaggtgagcctc-3';

[0020] hADP mut -F:5'-gaggctgaccttcacatccggcatatagactgtgatgtgg-3',

[0021] hADP mut -R: 5'-ccacatcacagtctatatgccggatgtgaaggtcagcctc-3';

[0022] (3) The recombinant plasmid vector pCMV-mADPN obtained in step (2) K172P and pCMV-hADPN K169P The mutant adiponectin mADPN was obtained by transferring the adiponectin into human embryonic kidney cells HEK293 for expression. The cell culture medium was collected and centrifuged to remove cell debris. The mutant adiponectin mADPN was obtained after concentration and purification. K172P and hADPN K169P .

[0023] The adiponectin mutant with high anti-inflammatory activity is used in the preparation of drugs for treating obesity, anti-inflammatory drugs, drugs for treating diabetes and drugs for treating atherosclerosis.

[0024] The beneficial effects of the present invention are:

[0025] The present invention is based on mouse-derived adiponectin (mADPN), and the amino acid at position 172 is selected for site-directed mutagenesis to mutate lysine to proline, or based on human-derived adiponectin (hADPN), the amino acid at position 169 is selected for site-directed mutagenesis to mutate lysine to proline, thereby obtaining an adiponectin mutant with high anti-inflammatory activity. Compared with conventional existing wild-type adiponectin, the adiponectin mutant mADPN of the present invention has a high anti-inflammatory activity. K172Por adiponectin mutant hADPN K169P The anti-inflammatory activity of adiponectin is significantly improved, which solves the problem of low anti-inflammatory activity of wild adiponectin and significantly improves the anti-inflammatory effect of adiponectin. In addition, the adiponectin mutant with high anti-inflammatory activity of the present invention can significantly reduce inflammation and insulin resistance induced by inflammation, and has good application prospects in the production of biological drugs for metabolic diseases such as anti-diabetes, anti-inflammatory and anti-atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Mouse adiponectin mADP-N K172P hADPN K169P Protein concentration and western-blot test results;

[0027] In the figure, A is the purified mouse mADP-N wt and mADP-N K172P hADPN wt and hADPN K169P Protein concentration test results; B is mouse mADPN wt and mADP-N K172P hADPN wt and hADPN K169P Western-blot detection results of proteins.

[0028] Figure 2 The anti-inflammatory activity test results of the wild-type adiponectin and adiponectin mutants in the present invention;

[0029] In the figure, A is mouse adiponectin mADP N wt and mADP-N K172P Resistance to LPS-induced cell inflammation; B is human adiponectin hADPN wt and hADPN K169P Resistance to LPS-induced cellular inflammation; "-" represents no addition of LPS or mutant adiponectin, and "+" represents addition of LPS or adiponectin; the vertical axis "MFI of TNF-α" represents the mean fluorescence intensity of intracellular fluorescein PE-coupled TNF-α antibody, which is positively correlated with the intracellular TNF-α protein level; data are expressed as mean ± standard error, and statistical analysis was performed using unpaired t-test. Statistically significant results are marked with asterisks, where "*" indicates p<0.05 and "**" indicates p<0.01. DETAILED DESCRIPTION

[0030] The present invention is not limited to the following specific embodiments. A person skilled in the art can adopt other various specific embodiments to implement the present invention based on the contents disclosed in the present invention, or any design structure and concept of the present invention with simple changes or modifications, which fall within the protection scope of the present invention.

[0031] The plasmid vector pCMV-C-Flag for expressing the Flag tag at the C-terminus of the present invention is available from Addgene.

[0032] Mouse mononuclear macrophage leukemia cells RAW 264.7 and human acute myeloid leukemia cells THP-1 were purchased from the National Biomedical Experimental Cell Resource Bank.

[0033] Example 1: Mutant recombinant plasmid vector pCMV-mADPN K172P and pCMV-hADPN K169P Construction

[0034] 1. Beijing Qingke Biotechnology Co., Ltd. artificially synthesized the mouse adiponectin gene and the human adiponectin gene according to the sequence information shown in SEQ ID NO.2 and SEQ ID NO.4, and then connected and recombined them into the plasmid vector pCMV-C-Flag with a C-terminal fusion expression Flag tag to obtain the recombinant plasmid vector pCMV-mADPN wt and pCMV-hADPN wt .

[0035] 2. Design site-directed mutagenesis primers on the QuickChange website, including mouse adiponectin site-directed mutagenesis primers (mADPNmut-F / R) and human adiponectin site-directed mutagenesis primers (hADPNmut-F / R). wt and pCMV-hADPN wt PCR amplification was performed using PCR as a template to obtain the adiponectin mutant mADP K172P Sequences and mutants of adiponectin hADPN K169P Sequences are shown in SEQ ID NO.7 and SEQ ID NO.8 respectively;

[0036] The site-directed mutagenesis primers are specifically as follows:

[0037] mADPN mut -F:5'-gaggctcaccttcacatctggcatgtacaccgtgatgtgg-3',

[0038] mADPN mut-R: 5'-ccacatcacggtgtacatgccagatgtgaaggtgagcctc-3';

[0039] hADP mut -F:5'-gaggctgaccttcacatccggcatatagactgtgatgtgg-3',

[0040] hADP mut -R: 5'-ccacatcacagtctatatgccggatgtgaaggtcagcctc-3';

[0041] PCR amplification system: 10 ng of template plasmid, 2 μL of each 10 μM primer, 25 μL of 2× PrimeSTAR Max Premix, and fill up to 50 μL with ddH2O.

[0042] PCR amplification program: pre-denaturation, 95℃ for 5 min; denaturation, 95℃ for 10 sec; annealing, 55℃ for 30 sec; extension, 72℃ for 1 min, 30 cycles; termination of extension, 72℃ for 5 min; insulation at 16℃.

[0043] After the PCR program was completed, 1 μL DpnI restriction enzyme and 5.5 μL 10× Fast Digest Green Buffer were added to the PCR product and incubated at 37°C for 30 min. The PCR product was recovered by column.

[0044] 3. The PCR product recovered from the column was transformed into DH5α competent cells, spread on a resistant LB agar plate containing ampicillin, and cultured in a 37°C incubator overnight. A single colony was picked from the resistant plate and inoculated into LB liquid culture medium containing ampicillin. A small amount of plasmid was extracted and sequenced for verification. After verification, the mutant recombinant plasmid vector pCMV-mADPN was obtained. K172P and pCMV-hADPN K169P .

[0045] Example 2, wild-type adiponectin protein, mutant adiponectin mADPN K172P and hADPN K169P Protein purification

[0046] The recombinant plasmid vector pCMV-mADPN was transfected into wt and pCMV-hADPN wt , and mutant recombinant plasmid vector pCMV-mADPN K172P and pCMV-hADPN K169PThe cells were transfected into HEK293 cells respectively, and after 24 hours, the cell culture medium was replaced with serum-free DMEM medium containing 0.1 g / L vitamin C and 0.2% BSA and cultured for a further 48 hours.

[0047] Then, the cell culture medium was collected and centrifuged at 300g for 10min to remove cell debris. The supernatant after centrifugation was filtered through a 0.22μm filter membrane and concentrated using a 10kD ultrafiltration tube. FLAG M2 affinity gel was added to the concentrated supernatant and incubated overnight at 4°C to enrich the adiponectin protein secreted into the supernatant. The gel was washed with 5 column volumes of buffer (20mM Tris-HCl, pH 7.5, 150mM NaCl), and finally, the ADPN-FLAG protein was eluted from the affinity gel with 150μg / mL 3×Flag peptide to obtain wild-type mouse adiponectin protein mADPN and mADPN, respectively. wt , wild-type human adiponectin protein hADPN wt , and mutant adiponectin mADP K172P hADPN K169P protein.

[0048] The protein concentration of the above four proteins was measured using the BCA protein concentration assay kit and western-blot immunoblot detection. The results are as follows Figure 1 shown.

[0049] Depend on Figure 1 It can be seen that mADP K172P hADPN K169P The concentration and molecular weight of the protein are close to those of the corresponding wild-type adiponectin protein, indicating that mADN K172P hADPN K169P The protein was successfully expressed and purified. The results of non-reducing western-blot experiments showed that mADPNK 172P hADPN K169P The HMW levels of the proteins were higher than those of their corresponding wild-type adiponectin proteins.

[0050] Example 3: Detection of anti-inflammatory activity of adiponectin protein

[0051] 1. Mouse mononuclear macrophage leukemia cells (RAW 264.7) were placed in DMEM medium containing 10% fetal bovine serum (FBS) and cultured to the logarithmic phase; human acute myeloid leukemia cells THP-1 were placed in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and cultured to the logarithmic phase.

[0052] 2. The cultured RAW 264.7 cells were evenly plated in a 24-well plate containing complete medium and cultured overnight in a cell culture incubator. The complete medium was then replaced with serum-free DMEM and cultured for 6 hours. mADP-N was added to each well at a final concentration of 20 ng / mL. wt and mADP-N K172P protein and continued to culture for 2 hours; finally, 100 ng / mL lipopolysaccharide (LPS) was added to the well plate and induced for 3 hours to obtain the inflammatory verification model of RAW264.7 induced by LPS. At the same time, RAW264.7 without added LPS and adiponectin protein was used as the blank group, and RAW264.7 with added LPS but not added adiponectin protein was used as the control group. The inflammatory verification model, blank group and control group together constituted experimental group 1.

[0053] 3. The cultured THP-1 cells were evenly plated in a 24-well plate containing complete medium, and then phorbol ester PMA was added at a final concentration of 100 ng / ml. The cells were cultured for 24 h to transform into macrophages. The complete medium was replaced with serum-free RPMI1640 and cultured for another 6 h. hADPN was added at a final concentration of 20 ng / mL to each well. wt and hADPN K169P protein and continued to culture for 2 hours; finally, 100 ng / mL LPS was added to the well plate and induced for 3 hours to obtain the inflammation verification model of human acute myeloid leukemia THP-1 cells induced by phorbol ester PMA and lipopolysaccharide LPS. At the same time, RAW264.7 without added LPS and adiponectin protein was used as the blank group, and RAW264.7 with added LPS but not added adiponectin protein was used as the control group. The inflammation verification model, blank group and control group together constituted experimental group 2.

[0054] 4. Use 0.25% trypsin to digest the cells of experimental group 1 and experimental group 2 respectively, and terminate the digestion with complete medium. Fluorescein PE-conjugated TNF-α antibody and flow cytometry were used to detect the protein level of tumor necrosis factor TNF-α in cells. The results are as follows: Figure 2 shown.

[0055] Depend on Figure 2 It can be seen that the TNF-α protein level in the blank group was significantly lower than that in the control group, indicating that the inflammation verification model was successfully constructed. K172P hADPN K169P The TNF-α protein level in macrophages of the present invention was significantly lower than that of wild-type adiponectin, indicating that the mutant adiponectin mADP- K172P hADPN K169PThe anti-inflammatory activity of adiponectin has been significantly improved, which solves the problem of low anti-inflammatory activity level of wild adiponectin and significantly improves the anti-inflammatory effect of adiponectin. It has good application prospects in the production of biological drugs for metabolic diseases such as anti-diabetes, anti-inflammatory and anti-atherosclerosis.

[0056] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A mutant adiponectin, characterized in that The adiponectin is a mouse adiponectin in which the lysine at position 172 is mutated to proline, or a human adiponectin in which the lysine at position 169 is mutated to proline.

2. The mutant adiponectin according to claim 1, characterized in that The adiponectin is mADN K172P , its amino acid sequence is shown in SEQ ID NO.5, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.6; the 172nd lysine of mouse-derived adiponectin is mutated to proline, the amino acid sequence of mouse-derived adiponectin is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

3. The mutant adiponectin according to claim 1, characterized in that The adiponectin is hADPN K169P , its amino acid sequence is shown in SEQ ID NO.7, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.8; the 169th lysine of human-derived adiponectin is mutated to proline, the amino acid sequence of human-derived adiponectin is shown in SEQ ID NO.3, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

4.

4. A recombinant vector, characterized in that: Inserted into a plasmid vector A gene encoding the mutant adiponectin according to claim 2 or claim 3.

5. The recombinant vector according to claim 1, characterized in that The plasmid vector is pCMV-C-Flag.

6. A recombinant cell, characterized in that The method is obtained by transforming the recombinant vector according to claim 4 into a host cell.

7. The recombinant cell according to claim 6, characterized in that The host cell is human embryonic kidney cell HEK293.

8. The method for preparing mutant adiponectin according to claim 1, characterized in that: The steps include: (1) Artificially synthesizing mouse adiponectin gene and human adiponectin gene according to the sequence information shown in SEQ ID NO.2 and SEQ ID NO.4, and then ligating and recombining them into the plasmid vector pCMV-C-Flag with a C-terminal fusion expression Flag tag to obtain recombinant plasmid vectors pCMV-mADPN and pCMV-hADPN; (2) Designing site-directed mutagenesis primers, using the recombinant plasmid vectors pCMV-mADPN and pCMV-hADPN obtained in step (1) as templates for PCR amplification to obtain the adiponectin mutant mADPN K172P Sequences and mutants of adiponectin hADPN K169P adiponectin mutant mADP K172P Sequences and mutants of adiponectin hADPN K169P The sequences were connected and recombined into the plasmid vector pCMV-C-Flag with a Flag tag fused to the C-terminus to obtain the mutant recombinant plasmid vector pCMV-mADPN K172P and pCMV-hADPN K169P ; The site-directed mutagenesis primer sequences are as follows: mADPN mut -F:5’-gaggctcaccttcacatctggcatgtacaccgtgatgtgg-3’, mADPN mut -R:5’-ccacatcacggtgtacatgccagatgtgaaggtgagcctc-3’; hADPN mut -F:5’-gaggctgaccttcacatccggcatatagactgtgatgtgg-3’, hADPN mut -R:5’-ccacatcacagtctatatgccggatgtgaaggtcagcctc-3’; (3) The mutant recombinant plasmid vector pCMV-mADPN obtained in step (2) K172P and pCMV-hADPN K169P The mutant adiponectin mADPN was obtained by transferring the adiponectin into human embryonic kidney cells HEK293 for heterologous expression, collecting the cell culture medium, and removing the cell debris by centrifugation. K172P and hADPN K169P .

9. Use of the mutant adiponectin according to claim 1 in the preparation of drugs for treating obesity, anti-inflammatory drugs, drugs for treating diabetes and drugs for treating atherosclerosis.

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