CN114516910B

CN114516910B - Protein for treating obesity-related fatty liver

Info

Publication number: CN114516910B
Application number: CN202011300266.XA
Authority: CN
Inventors: 陆炎; 周冰; 卞华; 李小英
Original assignee: Shanghai Inpson Biomedical Technology Co ltd
Current assignee: Shanghai Inpson Biomedical Technology Co ltd
Priority date: 2020-11-19
Filing date: 2020-11-19
Publication date: 2023-11-03
Anticipated expiration: 2040-11-19
Also published as: CN114516910A

Abstract

The invention discloses a protein for treating obesity-related fatty liver, the amino acid sequence of which is SEQ ID NO. 1, and an over-expression recombinant adenovirus thereof can be used for preparing a medicament for treating obesity-related fatty liver, and have development and application prospects.

Description

Protein for treating obesity-related fatty liver

Technical Field

The invention belongs to the field of medicines, and relates to a protein for treating obesity-related fatty liver and application thereof in preparing medicines for treating obesity-related fatty liver.

Background

With modern diet and lifestyle changes, obesity is prevalent, and nonalcoholic fatty liver disease (Non-alcoholic fatty liver disease, NAFLD) presents a rapidly increasing and globalized trend of onset. Global Prevalence is 24% and chinese Prevalence is also as high as 29.2% and is particularly pronounced in obese people (Garber k. The new lever epidemic innovation.nat biotechnology.2019, 37 (3): 209-214.Li J,Zou B,Yeo YH,et al, prevalice, incoedence, and outcome of non-alcoholic fatty liver disease in Asia,1999-2019:a systematic review and meta-analysis.Lancet gastro-ol 2019,4 (5): 389-398.). NAFLD is also closely related to the development of extrahepatic diseases such as cardiovascular and cerebrovascular diseases, type 2 diabetes, chronic kidney disease and malignant tumors (Younossi Z, anstee QM, marietti M, hardy T, henry L, eslam M, george J, bugianesi E.Global burden of NAFLD and NASH: products, predictors, risk factors and precursors.Nat Rev Gastroenterol hepatol 2018,15 (1): 11-20.Gluchowski NL, becure M, walter TC, farese RV Jr.Lipid droplets and liver disease: from basic biology to clinical imaging, nat Rev Gastroenterol Hepatol.2017,14 (6): 343-355.). NAFLD is mainly characterized by excessive accumulation of triglycerides in hepatocytes. However, there is a lack of effective and specific therapeutic agents. At present, the clinical treatment is mainly based on life style education, improving insulin resistance and antioxidant stress, and no officially approved specific medicine for reducing liver triglyceride deposition exists (Rotman Y, sanyal AJ. Current and upcoming pharmacotherapy for non-alcoholic fatty liver disease. Gut.2017,66 (1): 180-190).

A large number of documents have been reported: in obese conditions, increased triglyceride synthesis (i.e., adipogenesis) is an important cause of NAFLD formation (Friedman SL, neuschwander-Tetri BA, rinella M, sanyal AJ. Mechanisms of NAFLD development and therapeutic strategies. Nat. Med.2018,24 (7): 908-922.). In this process, numerous hormones and cytokines promote the excessive synthesis and accumulation of triglycerides, such as insulin, glucocorticoids, fetuin a, tumor necrosis factor, etc. Based on this phenomenon, we conceived that there may be a feedback inhibitor in the liver to reduce or block the synthesis of triglycerides, thereby protecting the development of NAFLD.

Disclosure of Invention

To verify this assumption, we performed differential proteomic screening of liver tissue of obese mice, and found a polypeptide that was significantly reduced in liver tissue of obese mice, whose amino acid sequence was determined to be SEQ ID NO. 1 by sequencing. Further proved by functional experiments, the polypeptide can obviously reduce the content of liver triglyceride of obese mice and improve the course of fatty liver. On molecular mechanism, the polypeptide is remarkableDown-regulating expression of triglyceride synthesis related gene, it is predicted to have development and application prospect in treating obesity related fatty liver. Therefore, we named it "Inpolin" ("Inpolin")Inhibitor of lipogenesis), chinese is named "hypolipidemic".

Based on this finding, the first aspect of the present invention is to provide a protein for treating obesity-related fatty liver, i.e., inpolin, having the amino acid sequence of SEQ ID NO. 1.

QNPEHVNITIGDPITNETLSWLSDKWFFIGAAVLNPDYRQEIQKTQMVFFNLTPNL INDTMELREYHTIDDHCVYNSTHLGIQRENGTLSKYVGGVKIFADLIVLKMHGAFMLAFDLKDEKKRGLSLNAKRPDITPELREVFQKAVTHVGMDESEIIFVDWKKDRCSQQE KQQLELEKETKKDPEEGQA(SEQ ID NO:1)。

The protein SEQ ID NO. 1 can be easily prepared by a polypeptide synthesis method, such as a full-automatic polypeptide synthesizer by a conventional solid phase synthesis method; the expression can also be achieved by constructing genetically engineered bacteria and fermenting.

A second aspect of the present invention provides a gene encoding the protein SEQ ID NO. 1. Preferably, the nucleotide sequence of the coding gene is SEQ ID NO. 2.

gccaccatggcactgcacatgattcttgtcatggtgagcctcctgccgctgttggaagctcagaacccagaacatgtcaacatca ccataggcgaccctatcaccaatgagaccctgagctggctctctgacaaatggtttttcattggtgcggctgtcctaaaccctgattaccggcaggaaattcaaaagacgcagatggtattttttaaccttacccccaacttgataaatgacacgatggagcttcgagagtatcacaccata gatgaccactgtgtctataactccactcatctaggaatccagagagagaatgggaccctctccaagtatgtaggaggagtaaaaatctttgcagacctgatagtcttgaagatgcatggggccttcatgcttgcctttgacttgaaggatgagaagaaacggggactgtccctcaatgca aaaaggccagatatcaccccggagctgcgggaagtattccagaaggctgtcacacacgtgggcatggatgaatcagaaatcatatttgtcgactggaaaaaggacaggtgcagtcagcaggagaagcagcagcttgagctggagaaggagaccaagaaagatcctgaggaag gccaggcatacccctacgacgtgcccgactacgcctga(SEQ ID NO:2)。

In a third aspect, the present invention provides a recombinant adenovirus for expressing the above protein SEQ ID NO. 1 or comprising a gene encoding the same (e.g. SEQ ID NO. 2), for use in gene therapy.

The fourth aspect of the invention is to provide the application of the protein SEQ ID NO. 1, the coding gene thereof, such as SEQ ID NO. 2, or the recombinant adenovirus containing the protein coding gene in the preparation of the obesity-related fatty liver treatment drug.

In one embodiment, the medicament is a pharmaceutical composition comprising the protein SEQ ID NO. 1, the polynucleic acid SEQ ID NO. 2 or a recombinant adenovirus for expressing the protein SEQ ID NO. 1, and a pharmaceutically acceptable carrier.

The vector should be able to properly retain the activity of the protein or nucleic acid. Such carriers include, but are not limited to, solvents (e.g., water, buffered solutions), dispersion media, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, and the like.

Preferably, when the protein SEQ ID NO. 1 is the active ingredient of the medicament, the pharmaceutical composition also contains a protein stabilizer.

The protein stabilizing agent may be selected from stabilizing agents commonly used in protein pharmaceuticals for maintaining protein activity, for example, including but not limited to at least one of the following groups, or a combination of two or more of the following: (1) buffer solution: such as sodium citrate-citric acid buffer; (2) and (2) a surfactant: such as nonionic surfactants polysorbate; (3) sugar and polyol: such as sucrose, glucose, trehalose, maltose, glycerol, mannitol, sorbitol, PEG, inositol, and the like; (4) salts: such as sodium chloride; (5) polyethylene glycols; (6) macromolecular compound: such as 2-hydroxypropyl-beta-cyclodextrin, albumin, serum protein (HAS), and the like; (7) histidine, glycine, glutamic acid and lysine hydrochloride, and the like.

In another embodiment, when the polynucleotide SEQ ID NO. 2 or a recombinant adenovirus comprising the polynucleotide SEQ ID NO. 2 is the active ingredient of a medicament, the pharmaceutical composition further comprises a nucleic acid stabilizer.

The nucleic acid stabilizer may be selected from stabilizers commonly used in nucleic acid pharmaceuticals for maintaining nucleic acid activity, including, but not limited to, the Biomatrica nucleic acid stabilizer product SampleMatrix series.

In another embodiment, the medicament is a pharmaceutical composition comprising, in addition to the pharmaceutically active ingredient,

optionally, the medicament is a pharmaceutical composition, and comprises at least one auxiliary therapeutic agent in addition to the pharmaceutically active ingredient proteins SEQ ID NO. 1 and polynucleic acid SEQ ID NO. 2 or recombinant adenovirus for expressing the proteins SEQ ID NO. 1, thereby forming a compound medicament. Preferably, the adjuvant therapeutic agent is other pharmaceutical ingredient having the effect of lowering the triglyceride content of the liver.

The obesity-related fatty liver may be nonalcoholic fatty liver disease NAFLD.

Preferably, the above pharmaceutical dosage forms may be selected from lyophilisates or injections.

When the protein Inpolin is verified to inhibit the functions of obesity-related fatty liver, two methods are adopted: (1) Recombinant adenovirus and control virus carrying Inpolin gene are constructed, and the Inpolin is specifically and highly expressed in db/db mouse liver tissue through tail vein injection. (2) Inpolin recombinant purified protein (SEQ ID NO: 3) was constructed, which had a histidine tag "HHHHHH" added to the N-terminus of SEQ ID NO:1 and a Flag tag "DYKDDDK" added to the C-terminus, and was injected into db/db mice via the abdominal cavity using PBS buffer as a control. The two methods are consistent in conclusion that Inpolin can obviously reduce the deposition of liver triglyceride and inhibit the expression of triglyceride synthesis genes, so that the disease course of obesity-related fatty liver is improved. Thus, the Inpolin protein is expected to become a novel therapeutic target of NAFLD.

Drawings

FIG. 1 is a schematic representation of differential proteomic screening data. Wherein A is the detection result of leptin receptor deficient obese mice and control mice; b is the test result of high fat diet induced obese mice and normal diet mice.

FIG. 2 is a Western Blotting photograph of Inpolin in liver tissue of leptin receptor deficient mice and controls, high fat diet mice and controls.

FIG. 3 is a block diagram of a plasmid used to express an Inpolin recombinant protein.

FIG. 4 is a SDS-PAGE coomassie blue stained electropherogram of recombinant protein Inpolin (i.e., RN 057-Inpolin). Wherein FT is Flow through (Flow through) after column loading, i.e. liquid from which the sample flows out after passing through the chromatographic column; elu-1 is eluting the received first tube liquid; elu-2 is eluting the received first tube liquid; m is a protein Marker.

FIG. 5 is a graph showing isoelectric point and molecular weight analysis of recombinant protein Inpolin.

FIG. 6 is a graph of the extinction coefficient analysis and identification of recombinant protein Inpolin.

FIG. 7 is a block diagram of adenovirus vector GV 269.

FIG. 8 is a flow chart for detecting adenovirus expressed Inpolin.

FIG. 9 shows the results of liver changes and triglyceride levels in the liver after mice were injected with Inpolin recombinant protein.

FIG. 10 is a graph showing the results of detection of liver changes and triglyceride levels in the liver after mice are injected with an over-expressed Inpolin recombinant adenovirus.

FIG. 11 is a statistical bar graph of the expression of the genes involved in triglyceride synthesis inhibition by protein Inpolin. Srebf1, fasn, scd1 and Acc1 in the figure are all related genes responsible for triglyceride synthesis, where Srebf1: sterol regulatory element-binding transcription factor, sterol response element binding protein 1; fasn: fatty acid synthase, fatty acid synthase; scd1: stearoyl-CoA desaturase-1, stearoyl-CoA desaturase; acc1: acetyl-Coenzyme A carboxylase 1, acetyl-CoA carboxylase.

Detailed Description

The inventors found that the content of the Inpolin protein was significantly down-regulated in liver tissue of both obese mice by differential proteomic screening of liver tissue in two obese mouse models and corresponding control mice, including leptin receptor deficient obese mice (db/db), high fat diet induced obese mice (HFD). This result was further verified by Western Blot experiments (Western Blot, WB), which found that: the content of Inpolin protein in liver tissue was significantly reduced in obese mice compared to control normal mice. By comparing the gene sequence and the protein sequence, the Inpolin protein belongs to a cytokine family, has a signal peptide at the N end and belongs to a secretory protein. Experiments prove that the Inpolin has the function of inhibiting the fatty liver related to obesity and has potential to be developed into a medicament for treating the fatty liver related to obesity, so the Inpolin is called as 'lipid-lowering agent' for short.

The protein Inpolin of the invention can be easily prepared by a polypeptide synthesis method, such as a conventional solid phase synthesis method by using a full-automatic polypeptide synthesizer; the expression can also be achieved by constructing genetically engineered bacteria and fermenting.

The protein of the invention SEQ ID NO. 1 has only 189 amino acids in number and is well-defined in structure, so that the person skilled in the art will readily be able to obtain the genes encoding it, expression cassettes and plasmids comprising these genes, and transformants comprising the plasmids when it is desired to produce the protein by microbial fermentation.

These genes, expression cassettes, plasmids, transformants can be obtained by genetic engineering construction methods well known to those skilled in the art. The transformant host can be any microorganism suitable for expressing the protein Inpolin, including bacteria and fungi. For example, the microorganism may be E.coli, B.subtilis, pichia pastoris, baker's yeast, saccharomyces cerevisiae, etc.

The polynucleotide SEQ ID NO. 2 can be easily prepared by synthesis, and the recombinant adenovirus for expressing the protein Inpolin can be easily constructed by conventional technical means.

The protein SEQ ID NO. 1, the polynucleotide SEQ ID NO. 2 or the recombinant adenovirus for expressing the protein SEQ ID NO. 1 can be used as an active ingredient to prepare various dosage forms of medicaments, such as freeze-drying agents or injection. The dosage forms may be formulated using adjuvants commonly used in the pharmaceutical arts, and the preparation process is well known to those skilled in the art.

When the drug is formulated as a lyophilized formulation, it may be prepared as a ready-to-use formulation, dissolved in a predetermined solvent such as water, and then infused.

The present invention will be described in further detail with reference to specific examples. It is to be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

In the examples of the present invention, if no specific explanation is made for the experimental operating temperature, this temperature is generally referred to as the room temperature (10-30 ℃).

The amounts, amounts and concentrations of various substances are referred to herein, wherein the percentages refer to percentages by mass unless otherwise specified.

Statistical analysis: in the study, the numerical variables are all expressed by mean ± standard error, the comparison between two groups adopts a double-tail Student t test, and the comparison between three groups adopts an ANOVA test. Statistical differences were considered when P < 0.05.

Example 1 differential proteomic screening

To find important proteins that affect the development of NAFLD, we performed protein extraction and proteomic screening on liver tissue of two groups of mice: the first group is leptin receptor deficient obese mice (db/db) and littermate control mice (Lean); the second group is High fat Diet induced obese mice (HFD) and Normal Diet mice (ND). The specific operation comprises the following steps:

1.1 8 week old C57BL/6, db/db and ob/ob male mice were selected and purchased from Jiangsu Jieqiao Kangyaku Corp.

Constructing a high-fat diet feeding animal model: the male mice were fed with high-fat feed or normal feed for 12 weeks at 8 weeks of age C57BL/6, respectively. High fat feeds were purchased from Research Diet, usa (cat No. D12492) and normal feeds were purchased from Shanghai Style laboratory animals Co.

The steps of treating, separating and extracting proteins from the liver of the mice are briefly described as follows: mice were sacrificed for cervical dislocation, 75% ethanol was rubbed against the skin, the abdominal cavity was cut off, liver tissue was sheared, and the mice were frozen in liquid nitrogen. When protein was extracted, about 0.1g of liver tissue was weighed out from liquid nitrogen, and the tissue was minced in a plate and transferred to a homogenizer. PMSF (10. Mu.g/ml) was added to the pre-chilled lysate. Taking 1.0ml of precooled lysis buffer, rapidly adding into a homogenizer, and fully grinding under ice bath condition. The tissue milling solution was transferred to a 1.5ml centrifuge tube. Centrifuge at 4℃at 15000rpm for 10min. Taking the crude extract of the tissue protein of the supernatant to a new 1.5ml centrifuge tube, and collecting the total protein. Protein quantification was performed and stored at-80 ℃.

1.2 differential proteomic detection (liquid chromatography-Mass Spectrometry combination, LC-MS/MS)

Protein digestion: the protein was digested using the modified filter assisted cleavage (FASP) method. Briefly, total protein was loaded onto the filters and washed 2 to 3 times with a buffer containing 8M urea to completely replace Sodium Dodecyl Sulfate (SDS). The protein was then alkylated with iodoacetamide and the excess reagent was washed through a filter. The reduced alkylated protein was digested with trypsin overnight at an enzyme to protein ratio of 1:75 (w/w).

LC-MS/MS analysis and data acquisition: liquid chromatographic separations were performed on an Easy nLC 1000 system (ThermoFisher Scientific). The column (15 cm long with an inner diameter of 75 μm) was loaded with ReproSil-Pur C18-AQ 3 μm resin (Dr. Maisch GmbH). To obtain high quality data, a Q-exact mass spectrometer (ThermoFisher Scientific) was used and equipped with a nanoelectrospray ion source (Proxeon Biosystems, now ThermoFisher Scientific). The protein was loaded onto a chromatographic column and separated with a linear gradient of 6-35% buffer B (ACN and 0.1% formic acid) at a flow rate of 300nL/min over 300 minutes. The total time for the reverse phase analysis was 60 minutes. Data were acquired in a data-dependent "top12" mode, in which the twelve most abundant parent ions were selected for HCD cleavage at high resolution (70 000@m/z 200) from a full scan (400-1500 m/z). Precursor ions having both single charge and unassigned charge information are excluded. The resolution of the MS/MS spectrum was set to 17500@m/z 200, the target value was 1E5 (AGC control was enabled), the isolation window was set to 2.0m/z, and the normalized collision energy was 30.

1.3 data analysis: mass spectrometry and subsequent bioinformatics analysis were performed by Shanghai cloud order biosciences, inc. MS original files were analyzed by MaxQuant software version 1.5.2.8 and Uniprot FASTA database searched for a list of peptide chains. Database searches were configured with cysteine carbamoylmethylation as the fixed modification, N-terminal acetylation and oxidation of the amino acid as the variable modification. The enzyme specificity was set to the C-terminal ends of arginine and lysine using trypsin as protease. At most two incomplete enzymatic fragments are allowed. When polypeptide identification is carried out, the initial precursor mass deviation is 7ppm at maximum, and the fragment mass deviation is 20ppm. The False Discovery Rate (FDR) at the protein and polypeptide level was set to 0.01, and the minimum length for polypeptide identification was 7 amino acids. The minimum ratio count for label free protein quantification (LFQ) was 2.

By the detection of the liquid chromatography-mass spectrometry (LC-MS/MS), the differentially expressed polypeptide is searched by taking the change multiple of >2 and the P value of <0.05 as the cutting point. Next, intersection analysis was performed on the two sets of differential proteins to identify polypeptides that were co-up and down-regulated in two obese mice, including the Inpolin protein of the present invention. The specific data are shown in FIG. 1.

The results showed that in the comparison of leptin receptor deficient obese mice and control mice, the number of up-regulated proteins was 74 and the number of down-regulated proteins was 170. In the comparison of fat diet-induced obese mice and normal diet mice, the amount of up-regulated protein was 238, and the amount of down-regulated protein was 277. In the two groups of obese mice, the number of the co-up-regulated proteins is 10, the number of the co-down-regulated proteins is 15, one polypeptide is obviously down-regulated, protein sequencing is carried out by Shanghai cloud sequence biotechnology Co-Ltd, the amino acid sequence of the polypeptide is determined to be SEQ ID NO:1, and the polypeptide is named as Inpolin.

1.4 protein expression of Inpolin was verified by immunoblotting (Western Blot) in leptin receptor-deficient obese mice (db/db), leptin-deficient obese mice (ob/ob), and High fat diet-induced obese mice (HFD), respectively. The results confirm that: the protein content of Inpolin in liver tissue was significantly reduced in all three groups of obese mice compared to the respective control mice (P < 0.001). Referring to the photograph of fig. 2, (a) expression of Inpolin in liver tissue of leptin receptor deficient mice was significantly reduced compared to control mice. (B) The expression of Inpolin in liver tissue of leptin-deficient mice was significantly reduced compared to control mice. (C) The expression of Inpolin in liver tissue was significantly reduced in high fat diet mice compared to normal diet mice.

Example 2 preparation of protein Inpolin

2.1 in order to investigate whether Inpolin inhibits the function of obesity-related fatty liver, an expression strain and cells thereof were constructed by genetic engineering. Inpolin recombinant protein (SEQ ID NO: 3) was designed by adding a histidine tag "HHHHHHH" at the N-terminus of SEQ ID NO:1 and a Flag tag "DYKDDDK" at the C-terminus:

HHHHHHHHQNPEHVNITIGDPITNETLSWLSDKWFFIGAAVLNPDYRQEIQKTQ MVFFNLTPNLINDTMELREYHTIDDHCVYNSTHLGIQRENGTLSKYVGGVKIFADLIVLKMHGAFMLAFDLKDEKKRGLSLNAKRPDITPELREVFQKAVTHVGMDESEIIFVDW KKDRCSQQEKQQLELEKETKKDPEEGQADYKDDDDK(SEQ ID NO:3)。

in order to express the Inpolin recombinant protein SEQ ID NO. 3 in an Expi293E cell, the nucleotide sequence SEQ ID NO. 4 of the expressed gene is optimally designed as a template:

gccaccatggcactgcacatgattcttgtcatggtgagcctcctgccgctgttggaagctcagaacccagaacatgtcaacatca ccataggcgaccctatcaccaatgagaccctgagctggctctctgacaaatggtttttcattggtgcggctgtcctaaaccctgattaccggcaggaaattcaaaagacgcagatggtattttttaaccttacccccaacttgataaatgacacgatggagcttcgagagtatcacaccata gatgaccactgtgtctataactccactcatctaggaatccagagagagaatgggaccctctccaagtatgtaggaggagtaaaaatctttgcagacctgatagtcttgaagatgcatggggccttcatgcttgcctttgacttgaaggatgagaagaaacggggactgtccctcaatgca aaaaggccagatatcaccccggagctgcgggaagtattccagaaggctgtcacacacgtgggcatggatgaatcagaaatcatatttgtcgactggaaaaaggacaggtgcagtcagcaggagaagcagcagcttgagctggagaaggagaccaagaaagatcctgaggaag gccaggcatacccctacgacgtgcccgactacgcctga(SEQ ID NO:4)。

experimental materials and reagents: expi293E cells (SMART, 20200616), PEI max (Polysciences, 24765-1), OPM medium (OPM, 13052301), opti-MEM I (Gibco, 2185849), ni Smart (SMART, SA 036025), plasmid Puroficatin MaxiPrep kit (SMART, 2019041301), bamHI (Takara, 1010A), xhoI (Takara, 1094A), anti-His Anti-object (SMART, SLAB 28), anti-HA Anti-object (SMART, SLAB 02).

Molecular biology experiments include plasmid construction, digestion, ligation, competent cell preparation, transformation, medium preparation, etc., and are mainly performed with reference to the "molecular cloning Experimental guidelines" (Molecular Cloning: A Laboratory Manual) (third edition), J.Sam Broker, D.W. Lassel, et al, huang Peitang, et al, science Press, beijing, 2002). The specific experimental conditions can be determined by simple experiments, if necessary.

The PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the plasmid or DNA template suppliers. Can be adjusted if necessary by simple tests.

The whole gene synthesis, primer synthesis and sequencing in the examples were all performed by su Jin Weizhi biotechnology, inc.

2.2 construction of plasmid: the primer was synthesized by su Jin Weizhi biotechnology limited, conventional PCR was performed using KOD FX high fidelity enzyme, the fragment was cut to recover the fragments through the cleavage sites BamHI and XhoI, and the insertional gene sequence was recombined into the RN057 vector using recombinase to obtain insertional gene recombinant plasmid, see fig. 3. Then the recombinant plasmid is transformed into a DH5a competent E.coli expression host to obtain the recombinant E.coli expressing the Inpolin gene.

Plasmid extraction: recombinant E.coli plates were picked as single colonies and inoculated into 200ml ampicillin LB liquid medium and incubated overnight at 37℃and 250 rpm. The RN057 vector carrying the Inpolin gene is extracted by using a plasmid large extraction kit of Dimman and biotechnology limited company, and the endotoxin is less than or equal to 0.25EU/ml.

Cell transfection: plasmid transfection was performed using PEI transfection reagent and into Expi293E cells.

Extraction and purification of Inpolin protein: because the designed Inpolin recombinant protein is added with a structure of 8 XHis at the N-terminal, the supernatant of the Expi293E is purified by Ni Smart, 1 XPBS (pH=7.4) is used as an equilibrium buffer, 250mM imidazole is eluted, after purity and concentration are detected by electrophoresis, buffer replacement is carried out by a centrifugal desalting column, the final protein buffer is 1 XPBS (pH=7.4), and endotoxin is controlled in the whole process of the protein purification. Finally, the purified recombinant protein Inpolin is obtained. Since the recombinant protein Inpolin is obtained by fermenting engineering bacteria transfected by the vector RN057, the recombinant protein Inpolin can be expressed as RN057-Inpolin.

2.3 recombinant protein Inpolin assay

The isoelectric point and molecular weight analysis results of the protein are shown in FIG. 4; the results of the protein extinction coefficient analysis are shown in FIG. 5. The recombinant protein Inpolin has correct sequence and same design.

The RN057-Inpolin protein is purified by Ni Smart, and finally purity verification is carried out by Coomassie brilliant blue staining, see FIG. 6, and purity SDS-PAGE is more than or equal to 95%.

EXAMPLE 3 construction of an over-expressed Inpolin adenovirus vector

The construction of an over-expression Inpolin adenovirus vector was carried out by Shanghai Ji Kai Gene chemical technology Co.

3.1 vector information: the vector is named GV269; the element sequence is CMV-MCS; cloning site is AgeI/EcoRI; control number CON176; the structure of the map is shown in fig. 7.

AgeI/EcoRI digested GV269 vector (available from Shanghai Ji Kai Gene chemical technology Co., ltd.) gave a linearized adenovirus vector.

3.2 the following primer pair Inpolin (57108-1) -P1/Inpolin (57108-1) -P2 is adopted, and the expression gene SEQ ID NO. 4 of the recombinant protein Inpolin is cloned into a linearization adenovirus vector by PCR, so that the recombinant adenovirus is constructed.

Inpolin(57108-1)-P1：

GAGGATCCCCGGGTACCGGCGCCACCATGGCACTGCACATGATTCTTG；

Inpolin(57108-1)-P2：

ATAAGCTTGATATCGAATTTCATGCCTGGCCTTCCTCAGGATC。

3.3 the recombinant plasmid constructed was identified by PCR using the following primer pair pDC315-F/pDC 315-R.

pDC315-F：GGTATAAGAGGCGCGACCAG；

pDC315-R：CGATGCTAGACGATCCAGAC。

The PCR identification result shows that the size of the PCR product of the positive transformant is: 848bp; negative transformant PCR product size: 224bp.

3.3 detection of recombinant adenovirus expression Inpolin

The adenovirus expression of Inpolin was detected by Real time PCR, and the flow is shown in FIG. 8.

The target cells were 293T, DMEM medium (10% fetal bovine serum) was used as the medium, and the primer information is shown in Table 1.

TABLE 1 Real time PCR detection information

Experimental results: the results of fluorescence observations of the transfected cells are shown in Table 2.

TABLE 2 fluorescent observations of recombinant adenovirus transfected cells 293T

Experimental grouping	Ct value of internal reference gene	Ct value of target gene	ΔCt	-ΔΔCt	2 ^-ΔΔCt
						CON	13.79	35.00	21.21	0.020	1.014
CON	13.72	35.00	21.28	-0.050	0.966
						CON	13.80	35.00	21.20	0.030	1.021
OE	14.49	11.62	-2.87	24.100	17981374.884
						OE	14.18	11.45	-2.73	23.960	16318442.146
OE	14.64	11.55	-3.09	24.320	20943511.258

The results illustrate:

1) The Real time PCR detailed experimental data is a qPCR data analysis file,includes original Ct value of each group of samples, 2 ^-ΔΔCt Method data analysis, amplification curve, amplification product melting curve and the like.

2) con: for 293T cell samples (control group),

OE: samples were transfected 293T for the gene plasmid of interest.

3) Calculation of 2- ΔΔCt method shows that: delta Ct = objective gene Ct value-reference gene Ct value, -delta Ct = NC group delta Ct average value-each sample delta Ct value. 2 ^-ΔΔCt Reflecting the relative expression level of the target gene of each sample relative to the control sample.

Conclusion: as can be seen from the quantitative PCR result, the expression abundance of the OE group Inpolin in 293T cells is 18414442.763 times (p < 0.05) that of the CON group, which indicates that the recombinant adenovirus over-expressing Inpolin is successfully constructed.

The virus Titer was measured by the end point dilution method, and as a result, the recombinant adenovirus was found to have a Titer (Titer) of 1E+10 PFU/ml.

3.4 Inpolin expression after recombinant adenovirus infection

Further detecting the expression of Inpolin after recombinant adenovirus infection.

Different doses of adenovirus stock solution are taken and added into HEK293 cells, and the growth condition of the cells is observed after 24-48 hours.

And collecting cells, and extracting RNA for use in Real time PCR detection experiments. In particular, the method comprises the steps of,

the target cell was HEK293, DMEM medium (10% fetal bovine serum) was used as the medium, and the primer information is shown in Table 1. The results of the Real time PCR experiments are shown in Table 3.

TABLE 3 Real-time PCR results

Experimental grouping	Ct value of internal reference gene	Ct value of target gene	ΔCt	-ΔΔCt	2 ^-ΔΔCt
						0ul	13.92	34.04	20.12	-0.207	0.867
0ul	13.81	33.54	19.73	0.183	1.136
						0ul	13.76	33.65	19.89	0.023	1.016
2ul-OE1	13.72	13.9	0.18	19.733	871616.108
						2ul-OE1	13.71	13.87	0.16	19.753	883783.416
2ul-OE1	13.78	13.85	0.07	19.843	940672.715
						20ul-OE2	14.1	12.99	-1.11	21.023	2131345.921
20ul-OE2	14.26	12.72	-1.54	21.453	2871420.789
						20ul-OE2	14.19	12.85	-1.34	21.253	2499716.985

Conclusion: as can be seen from the quantitative PCR results, the expression abundance of Inpolin in the 2ul-OE1 group was approximately 898690.746 times that of the 0ul group (p < 0.05) in HEK293 cells. The abundance of Inpolin expression in the 20ul-OE2 group was approximately 2500827.898 times that in the 0ul group (p < 0.05). The recombinant adenovirus constructed by the invention can express Inpolin in eukaryotic cells.

Example 4 mice experiments dosed with Inpolin protein

Inpolin recombinant protein (0.1 mg/kg, 1.0 mg/kg) and control solvent (PBS buffer) were administered intraperitoneally to 6-week-old db/db male mice twice daily (9 am, 16 pm once) and the experiment ended after 10 consecutive days of injection. Mice were sacrificed and livers were removed and analyzed for triglyceride content in the livers according to the procedure in example 1. The results are shown in FIG. 9.

As can be seen from fig. 9, the (a) nipolin purified protein treatment significantly improved the general morphology of fatty liver and the liver became ruddy. (B) liver weight is reduced and dose dependent. (C) Liver triglyceride content is reduced and dose dependent. (C) Plasma triglyceride levels are reduced and dose dependent.

Triglyceride measurement: the triglyceride quantitative detection kit was completed by Biovision corporation, usa, cat No. K622.

EXAMPLE 5 mouse experiments with recombinant adenovirus

Inpolin adenovirus was given intravenously to the tail of 8-week-old db/db mice, and green fluorescent protein adenovirus (Ad-GFP) was given intravenously to the tail of control db/db mice (1X 10) ⁹ pfu/each mouse), mice were sacrificed according to the method in example 1 12 days after injection, livers were removed, and triglyceride content in livers was analyzed.

The results are shown in FIG. 10. As can be seen in FIG. 10, (A) liver tissue immunoblotting experiments confirmed high expression of Inpolin. (B) comparison of liver weight/weight ratio: the Inpolin group drops significantly. (C) comparison of liver triglyceride content: the Inpolin group drops significantly. Comparison of (D) H & E staining and oil red O staining: liver triglyceride deposition was significantly improved in the Inpolin group. * P <0.01.

EXAMPLE 6 inhibition of expression of the protein Inpolin on the triglyceride Synthesis-related Gene

In order to investigate the action mechanism of Inpolin on the molecular mechanism, we examined the gene expression related to triglyceride metabolism, focused on examining the related genes Srebf1, fasn, scd1 and Acc1 responsible for triglyceride synthesis, and carried out real-time quantitative PCR detection, the results of which are shown in FIG. 11. The research finds that: (A) Compared with the control group, the triglyceride synthesis genes of the Inpolin adenovirus group mice are significantly down-regulated. (B) Compared with the control group, the glycerol triester synthesis gene of the mice in the Inpolin protein treatment group is significantly down-regulated and has dose dependency.

The results show that: compared with the control group, the expression of the genes related to the synthesis of the triglyceride in the Inpolin treatment group (adenovirus high expression and purified protein injection) is obviously down-regulated. It was thus suggested that Inpolin might improve the progress of obesity-related fatty liver by inhibiting triglyceride synthesis, reducing its excessive accumulation.

The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the present invention and to implement it, but not limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit and the substance of the present invention should be covered by the scope of the present invention.

Sequence listing

<110> auxiliary Zhongshan Hospital at double denier university

<120> a protein for the treatment of obesity-related fatty liver

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Claims

1. The application of the protein with the amino acid sequence of SEQ ID NO. 1, the encoding gene thereof or the recombinant adenovirus containing the encoding gene of the protein in the preparation of the obesity-related fatty liver treatment medicine.

2. The use according to claim 1, wherein the nucleotide sequence of the gene encoding the protein is SEQ ID NO. 2.

3. The use according to claim 1, wherein the medicament is a pharmaceutical composition comprising a protein as defined in claim 1 and a pharmaceutically acceptable carrier.

4. The use according to claim 1 or 2, wherein the medicament is a pharmaceutical composition comprising a nucleic acid molecule having the nucleotide sequence SEQ ID No. 2 or a recombinant adenovirus comprising the nucleotide sequence SEQ ID No. 2 and a pharmaceutically acceptable carrier.

5. The use according to claim 1, wherein when the protein is the active ingredient of a medicament, the pharmaceutical composition further comprises a protein stabilizer.

6. The use according to claim 4, wherein when said nucleic acid molecule or said recombinant adenovirus is the active ingredient of a medicament, the pharmaceutical composition further comprises a nucleic acid stabilizer.

7. The use according to claim 1, wherein the medicament is a pharmaceutical composition comprising, as a pharmaceutically active ingredient, at least one adjuvant therapeutic agent in addition to the protein, the nucleic acid molecule comprising the gene encoding the protein, or the recombinant adenovirus comprising the gene encoding the protein.

8. The use according to claim 1, wherein the obesity-related fatty liver is a non-alcoholic fatty liver disease.

9. The use according to claim 1, wherein the pharmaceutical is in the form of a lyophilisate or an injection.