Adiponectin mutant with high anti-inflammatory activity and preparation method and application thereof

Abstract

The present application relates to a kind of high anti-inflammatory activity adiponectin mutant and its preparation method and application.The adiponectin mutant is the 172th lysine of adiponectin is mutated into proline, or the 169th lysine is mutated into proline.The amino acid sequence of adiponectin mutant mADPN K172P As shown in SEQ ID NO.5, nucleotide sequence as shown in SEQ ID NO.6.The amino acid sequence of adiponectin mutant hADPN K169P As shown in SEQ ID NO.7, nucleotide sequence as shown in SEQ ID NO.8.Compared with conventional existing wild type adiponectin, the anti-inflammatory activity of adiponectin mutant mADPN K172P Or adiponectin mutant hADPN K169P Significantly improved, solve the problem of low level of wild adiponectin anti-inflammatory activity, significantly improve the anti-inflammatory effect of adiponectin, in the biological medicine production of anti-diabetic, anti-inflammatory and anti-atherosclerosis and other metabolic diseases has good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of high anti-inflammatory activity adiponectin mutant and its preparation method and application, belong to biological medicine technical field. BACKGROUND

[0002] Adiponectin (ADPN) is a fat tissue-secreted adipocyte factor. As one of the most abundant adipokines in blood, adiponectin plays an important role in various biological processes, including enhancing insulin sensitivity, preventing atherosclerosis and anti-inflammatory, etc. Current studies on humans and animal models show 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 blood circulation, ADPN mainly exists 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 multimers show different binding affinities to adiponectin receptors, and the influence of ADPN on its target tissues depends on the expression of specific receptors and the multimeric form they bind to. Under different metabolic conditions, the absolute level of HMW ADPN and its proportion to the total ADPN level change. There is a stronger correlation between HMW ADPN and insulin sensitivity. Studies have shown that the level of HMW ADPN in 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 related diseases such as obesity and diabetes, the generation of HMW ADPN in the body is impaired. Therefore, exploring strategies to improve the level of HMW ADPN is expected to become a potential innovative method for treating metabolic diseases. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a kind of high anti-inflammatory activity adiponectin mutant and its preparation method and application.

[0006] The technical scheme of the present application is as follows:

[0007] A kind of high anti-inflammatory activity adiponectin mutant, the adiponectin mutant is the 172th lysine of adiponectin is mutated to proline, or the 169th lysine is mutated to proline.

[0008] According to the present application, preferably, the mutant is the adiponectin mutant mADPN K172P , the amino acid sequence of which is shown as SEQ ID NO. 5, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 6; which is a mutant of mouse adiponectin (mADPN wt ) in which the lysine at position 172 is mutated to proline. wt The amino acid sequence of the mouse adiponectin (mADPN K169P ) is shown as SEQ ID NO. 1, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 2.

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

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

[0011] According to the present application, preferably, the plasmid vector is pCMV-C-Flag.

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

[0013] According to the present application, preferably, the host cell is human embryonic kidney cell HEK293.

[0014] A method for preparing the adiponectin mutant with high anti-inflammatory activity, comprising the following steps:

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

[0016] (2) Designing site-directed mutation primers, and using the recombinant plasmid vectors pCMV-mADPN wt and pCMV-hADPN wtThe PCR product is transformed into DH5a competent cells, and sequencing verification is performed to obtain the mutant plasmid vector pCMV-mADPN K172P and pCMV-hADPN K169P ;

[0017] The sequence of the site-directed mutation primer is as follows:

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

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

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

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

[0022] (3) The recombinant plasmid vectors pCMV-mADPN K172P and pCMV-hADPN K169P are respectively transformed into human embryonic kidney cells HEK293 for expression, cell culture medium is collected, cell debris is removed by centrifugal filtration, and after concentration and purification, the mutant adiponectin mADPN K172P and hADPN K169P are obtained.

[0023] The high-anti-inflammatory-activity adiponectin mutant can be applied to the preparation of obesity treatment drugs, anti-inflammatory drugs, diabetes treatment drugs and atherosclerosis treatment drugs.

[0024] The present application has the following advantages:

[0025] The present application is based on mouse-derived adiponectin (mADPN) and selects the 172th amino acid for site-directed mutation to mutate lysine into proline, or is based on human-derived adiponectin (hADPN) and selects the 169th amino acid for site-directed mutation to mutate lysine into proline, and a high-anti-inflammatory-activity adiponectin mutant is obtained. Compared with conventional existing wild-type adiponectin, the adiponectin mutant mADPN K172Por adiponectin mutant hADPN K169P The anti-inflammatory activity of the adiponectin mutant is significantly improved, and the problem of low anti-inflammatory activity of wild-type adiponectin is solved, and the anti-inflammatory effect of adiponectin is significantly improved. In addition, supplementing the adiponectin mutant with high anti-inflammatory activity can significantly reduce body inflammation and inflammation-induced insulin resistance, and has good application prospects in the production of biological drugs for anti-diabetic, anti-inflammatory and anti-atherosclerosis metabolic diseases. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 mADPN K172P and human hADPN K169P protein concentration and western-blot detection results;

[0027] In the figure, A is the concentration detection results of purified mouse mADPN wt and mADPN K172P protein and human hADPN wt and hADPN K169P protein; B is the western-blot detection results of mouse mADPN wt and mADPN K172P protein and human hADPN wt and hADPN K169P protein.

[0028] Figure 2 are the anti-inflammatory activity detection results of wild-type adiponectin and adiponectin mutants in the present application;

[0029] In the figure, A is the resistance of mouse mADPN wt and mADPN K172P to LPS-induced cell inflammation; B is the resistance of human hADPN wt and hADPN K169P to LPS-induced cell inflammation; "-" represents no addition of LPS or mutant adiponectin, and "+" represents addition of LPS or adiponectin; the vertical coordinate "MFI of TNF-α" represents the average fluorescence intensity of the intracellular fluorescein PE-coupled TNF-α antibody, which is positively correlated with the intracellular TNF-α protein level; the data are expressed as mean ± standard error, and the statistical analysis uses unpaired t test, and the results with statistically significant difference are marked with asterisk, wherein "*" represents p<0.05, and "**" represents p<0.01. DETAILED DESCRIPTION

[0030] The present application is not limited to the following specific embodiments, and those skilled in the art can implement the present application in other various embodiments according to the disclosure of the present application, or any simple changes or modifications made by using the design structure and ideas of the present application, all fall within the protection scope of the present application.

[0031] The plasmid vector pCMV-C-Flag for C-terminal fusion expression of Flag tag in the present application is commercially available from Addgene company.

[0032] The mouse monocyte macrophage leukemia cell RAW 264.7 and the human acute myeloid leukemia cell THP-1 are commercially available from the National Biomedical Experimental Cell Resource Library.

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

[0034] 1. The murine adiponectin gene and the human adiponectin gene were artificially synthesized by Beijing Qianke Biotechnology Co., Ltd. according to the sequence information shown in SEQ ID NO. 2 and SEQ ID NO. 4, and then were respectively linked to the plasmid vector pCMV-C-Flag for C-terminal fusion expression of Flag tag to obtain the recombinant plasmid vectors pCMV-mADPN wt and pCMV-hADPN wt .

[0035] 2. The site-directed mutation primers were designed on the QuickChange website, including the murine adiponectin site-directed mutation primers (mADPNmut-F / R) and the human adiponectin site-directed mutation primers (hADPNmut-F / R), and the recombinant plasmid vectors pCMV-mADPN wt and pCMV-hADPN wt obtained in the above steps were respectively used as templates for PCR amplification to obtain the adiponectin mutant mADPN K172P sequence and the adiponectin mutant hADPN K169P sequence, which are respectively shown in SEQ ID NO. 7 and SEQ ID NO. 8.

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

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

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

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

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

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

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

[0043] After the PCR program, 1 μL of DpnI restriction endonuclease and 5.5 μL of 10x FastDigest Green Buffer were added to the PCR product, and incubated at 37°C for 30 min. The PCR product was column-recovered.

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

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

[0046] The recombinant plasmid vectors pCMV-mADPN wt and pCMV-hADPN wt , and the mutant recombinant plasmid vectors pCMV-mADPN K172P and pCMV-hADPN K169PHEK293 cells were transfected respectively, and 24 h later, the cell culture medium was replaced with serum-free DMEM medium containing 0.1 g / L vitamin C and 0.2% BSA, and the cells were cultured for another 48 h.

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

[0048] The four proteins were subjected to protein concentration measurement and western-blot immunoblotting detection using a BCA protein concentration assay kit, and the results are shown in Figure 1 .

[0049] As can be seen from Figure 1 , the concentrations and molecular weights of mADPN K172P protein and hADPN K169P protein are close to those of the corresponding wild-type adiponectin proteins, indicating that the heterologous expression and purification of mADPN K172P protein and hADPN K169P protein are successful. The results of non-reducing western-blot experiment show that the HMW levels of mADPN 172P protein and hADPN K169P protein are higher than those of the corresponding wild-type adiponectin proteins.

[0050] Example 3, Anti-inflammatory activity detection of adiponectin protein

[0051] 1. Mouse monocyte 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. After culturing, RAW 264.7 cells were evenly seeded into 24-well plates containing complete culture medium and cultured overnight in a cell culture incubator. Then, the complete culture medium was replaced with serum-free DMEM, and the cells were cultured for another 6 hours. Finally, mADPN was added to each well to a final concentration of 20 ng / mL. wt and mADPN K172P The protein was cultured for another 2 hours. Finally, 100 ng / mL of lipopolysaccharide (LPS) was added to the well plate and induced for 3 hours to obtain an inflammation verification model of RAW264.7 induced by LPS. RAW264.7 without LPS and adiponectin protein was used as the blank group, and RAW264.7 with LPS but without adiponectin protein was used as the control group. The inflammation verification model, blank group and control group together constituted experimental group 1.

[0053] 3. Spread the cultured THP-1 cells evenly in a 24-well plate containing complete culture medium, then add phorbol ester (PMA) to a final concentration of 100 ng / mL and incubate for 24 h to induce macrophage transformation. Replace the complete culture medium with serum-free RPMI 1640 and continue incubation for 6 h. Add hADPN to each well to a final concentration of 20 ng / mL. wt and hADPN K169P Protein was cultured for another 2 hours; finally, 100 ng / mL LPS was added to the well plate and induced for 3 hours to obtain an inflammation verification model of human acute myeloid leukemia cells THP-1 induced by phorbol ester PMA and lipopolysaccharide LPS. At the same time, RAW264.7 cells without LPS and adiponectin protein were used as blank groups, and RAW264.7 cells with LPS but without adiponectin protein were used as control groups. The inflammation verification model, blank group and control group together constituted experimental group 2.

[0054] 4. Cells from experimental groups 1 and 2 were digested separately using 0.25% trypsin, and digestion was terminated with complete culture medium. Intracellular tumor necrosis factor TNF-α protein levels were detected using a fluorescein-pepper-conjugated TNF-α antibody and flow cytometry. The results are as follows: Figure 2 As shown.

[0055] Depend on Figure 2 It was found that the TNF-α protein level in the blank group was significantly lower than that in the control group, indicating that the inflammation validation model was successfully constructed. mADPN was also added. K172P or hADPN K169P The TNF-α protein level in macrophages was significantly lower than that of wild-type adiponectin, indicating that the mutant adiponectin mADPN of this invention... K172P or hADPN K169PThe anti-inflammatory activity of the wild-type adiponectin is significantly improved, the problem of low anti-inflammatory activity of the wild-type adiponectin is solved, and the anti-inflammatory effect of the adiponectin is significantly improved, so that the adiponectin has a good application prospect in the production of biological drugs for treating metabolic diseases such as diabetes, inflammation and atherosclerosis.

[0056] The specific embodiments of the present application are described above. It should be understood that the present application is not limited to the specific embodiments described above, and various modifications or changes can be made by those skilled in the art within the scope of the claims, which do not affect the essential content of the present application.

Claims

1. A mutant adiponectin, characterized in that, The mutant adiponectin is a murine adiponectin with a lysine residue at position 172 changed to proline, and is mADPN. K172P Alternatively, the lysine residue at position 169 of human adiponectin may be mutated to proline, resulting in hADPN. K169P ; Wherein, the mADPN K172P The amino acid sequence of the gene is shown in SEQ ID NO.5, and the nucleotide sequence of the gene encoding the gene is shown in SEQ ID NO.6; the amino acid sequence of the mouse adiponectin is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the gene is shown in SEQ ID NO.

2. The hADPN K169P The amino acid sequence of the gene is shown in SEQ ID NO.7, and the nucleotide sequence of the gene encoding the gene is shown in SEQ ID NO.8; the amino acid sequence of the human adiponectin is shown in SEQ ID NO.3, and the nucleotide sequence of the gene encoding the gene is shown in SEQ ID NO.

4.

2. A recombinant vector, characterized in that, The gene encoding the mutant adiponectin described in claim 1 is inserted into a plasmid vector.

3. The recombinant vector as described in claim 2, characterized in that, The plasmid vector is pCMV-C-Flag.

4. A recombinant cell, characterized in that, It is obtained by converting the recombinant vector described in claim 3 into host cells.

5. The recombinant cell as described in claim 4, characterized in that, The host cell is human embryonic kidney cell HEK293.

6. The method for preparing the mutant adiponectin according to claim 1, characterized in that, The steps include the following: (1) Mouse-derived adiponectin gene and human-derived adiponectin gene were artificially synthesized according to the sequence information shown in SEQ ID NO.2 and SEQ ID NO.4, and then they were recombined into the plasmid vector pCMV-C-Flag with C-terminal fusion expression Flag tag to obtain recombinant plasmid vectors pCMV-mADPN and pCMV-hADPN; (2) Design site-directed mutagenesis primers and perform PCR amplification using the recombinant plasmid vectors pCMV-mADPN and pCMV-hADPN obtained in step (1) as templates to obtain the adiponectin mutant mADPN. K172P Sequence and adiponectin mutant hADPN K169P Sequence, and then the adiponectin mutant mADPN K172P Sequence and adiponectin mutant hADPN K169P The sequences were ligated and recombined into the plasmid vector pCMV-C-Flag, which expresses a Flag tag at 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 cells were transduced into HEK293 human embryonic kidney cells for heterologous expression. Cell culture medium was collected, and cell debris was removed by centrifugation and filtration. After concentration and purification, the mutant adiponectin mADPN was obtained. K172P and hADPN K169P .

7. The use of the mutant adiponectin according to claim 1 in the preparation of anti-inflammatory drugs, characterized in that, The inflammation is LPS-induced; the anti-inflammatory drug treats the inflammation by reducing TNF-α levels in macrophages.

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