Recombinant cluster and application thereof

Abstract

The invention provides a recombinant cluster and application, the recombinant cluster comprises an amino acid primary sequence for coding the recombinant cluster, the amino acid primary sequence is at least 95% homologous with any one of SEQ ID NO.1-17, and a tertiary structure formed by the amino acid primary sequence does not have a disulfide bond. The recombinant cluster provided by the invention can greatly improve the yield of purification after escherichia coli expression, simplifies the production process, and realizes industrial large-scale production; the space structure of the recombinant cluster is free of artificially added sequences or structures, so that immunogenicity reaction is not easily induced, no other toxic or side effect exists, and the druggability is strong; in animal experiments, heat aggregation of protein can be inhibited, an obvious protection effect on oxidative stress of HCE-T cells is achieved, a new choice is provided for treatment of various animal diseases, and the good clinical application value is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant clusterin and an application thereof. Background Art

[0002] Clusterin was first isolated from the testicles of rams. Because it can promote the aggregation of Sertoli cells and support the maturation and development of sperm, it is also named agglutination factor or apolipoprotein J (Clusterin). It is widely expressed in tissues and is the first secretory macromolecular chaperone discovered in the human body. It is closely related to various physiological and pathological processes such as inhibiting cell apoptosis, inactivating complement factors, recycling and transporting lipids, protecting cell membranes, and maintaining connections between cells or between cells and the matrix.

[0003] After clusterin is synthesized by precursor polypeptide chain guided by mRNA, the secretory signal peptide is removed by enzymatic hydrolysis and broken into α and β chains. The two chains are antiparallel and connected by 5 disulfide bonds. After glycosylation modification, a heterodimeric sulfated glycoprotein is formed, including 449 amino acids, including sugar groups, with a molecular weight of about 75-80kDa. As a macromolecular chaperone, clusterin can non-covalently bind to denatured proteins in the body through its own amphiphilic groups, so that the denatured proteins remain dissolved and degraded and cleared through normal metabolic channels, maintaining the dynamic balance of protein metabolism in the body. At the same time, it can bind to specific proteins and have anti-inflammatory and immune response inhibition functions, and may bind to cell receptors to participate in regulating cell apoptosis and proliferation.

[0004] Due to its multifunctional properties, clusterin participates in a variety of pathophysiological processes in the body and has an inhibitory effect on the development of many early and middle-stage diseases. For example, it has a relieving and therapeutic effect on a variety of early and middle-stage protein amyloid diseases, cardiovascular diseases, eye diseases, etc. It is considered by mainstream scientists at home and abroad to be a potential therapeutic drug.

[0005] Although clusterin is widely present in the human body, the cost of extracting it to a pharmaceutical grade is extremely high. Even without considering whether indicators such as purity can meet the requirements, it may take thousands of liters of human blood to extract a single medicinal dose, which is obviously impossible to achieve large-scale production. Therefore, how to achieve large-scale production has become a research focus of the scientific community at home and abroad. In order to ensure its biological activity, the existing recombinant apolipoprotein J retains the original 5 pairs of disulfide bonds, such as the polypeptide sequences disclosed in application numbers 202010644415.8 and 201580074616.6. It is easy to mismatch and be reduced to form impurities during purification, and the renaturation is difficult and the final purification yield is extremely low; if the protein tag is used for separation and purification, the recombinant protein with the tag is difficult to meet the requirements of pharmaceutical regulations and cannot be made into a drug. This is one of the biggest obstacles to its industrial production. Therefore, it is of great significance to develop recombinant clusterin with high yield, large-scale production and biological activity. Summary of the invention

[0006] The technical problems solved by the present invention are the low yield of existing recombinant clusterin, the complex preparation process, and the difficulty in realizing industrial large-scale production. It is generally believed that the disulfide bonds contained in protein molecules are necessary to maintain their spatial structure, which is a prerequisite for them to exert their biological activity. According to previous discoveries, when the disulfide bonds of clusterin are connected to a pair by mutation of amino acid sites, its biological activity does not decrease and the yield is greatly improved, which is completely beyond conventional cognition. However, compared with other biological products, its yield is still not ideal, and the high cost of industrial production limits its commercial application. Further research in this application found that when we knocked out all the disulfide bonds of Clusterin, its biological activity was still well retained, and the total activity yield of the extraction was greatly improved, which belongs to the family of disordered proteins.

[0007] The present invention provides a recombinant clusterin, comprising an amino acid primary sequence encoding the recombinant clusterin, wherein the amino acid primary sequence is at least 95% homologous to any one of SEQ ID NO.1-17, and the tertiary structure formed by the amino acid primary sequence does not have a disulfide bond.

[0008] The recombinant clusterin of the present invention does not contain a disulfide bond, which can greatly improve the final yield after expression in E. coli, and simplifies the production process, so that industrial large-scale production can be achieved; related experiments have proved that it has considerable in vitro activity, cell biological activity and immunological activity, and can be internalized after binding to the corresponding receptors (Lrp2 or megalin, VLDLR, APOER2). The spatial structure of the recombinant clusterin is a tertiary structure, including 2-4 domains, 2-5 β folds and 2-5 α helices.

[0009] Preferably, the amino acid primary sequence has 0-1 cysteine. Preferably, the amino acid primary sequence of the recombinant clusterin is any one of SEQ ID NO. 1-17.

[0010] The present invention also provides a method for preparing recombinant clusterin, including bacterial cell crushing, inclusion body washing and dissolution, anion chromatography, desalting and fluid replacement, cation chromatography, ultrafiltration and molecular exclusion chromatography, etc. After the above sequence is expressed in a prokaryotic cell host, the recombinant clusterin can be directly extracted in the culture medium or the supernatant of the crushed cells; with specific optimal procedures and parameters, including the selection and concentration optimization of the buffer, the selection of pH, the selection and concentration optimization of additives, after protein precipitation and fractionation, the recombinant clusterin is purified by ion exchange column chromatography, affinity column chromatography, and molecular exclusion column chromatography; ultrafiltration, dialysis and column chromatography methods are used for desalting. The separation and purification method includes one or more of isoelectric precipitation, ammonium sulfate precipitation / fractionation, ion exchange column chromatography, hydrophobic column chromatography, affinity column chromatography, molecular exclusion gel column chromatography, ultrafiltration, and dialysis.

[0011] Preferably, the separation and purification method comprises: bacterial cell crushing; inclusion body washing; anion exchange chromatography; cation exchange chromatography; ultrafiltration; molecular sieve purification. Preferably, the ultrafiltration is TFF ultrafiltration, and the molecular sieve purification is SEC chromatography. The filler used in the column chromatography method comprises one or more of apolipoprotein J antibody coupling, heparin coupling and metal chelate filler. The column chromatography method comprises specific step gradient, continuous gradient and multiple gradient elution.

[0012] The present invention also provides a pharmaceutical composition, comprising the above-mentioned recombinant clusterin and pharmaceutically acceptable excipients. Preferably, the pharmaceutical composition is in the form of a lyophilized powder injection or an injection.

[0013] The present invention also provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the recombinant clusterin as described above.

[0014] Preferably, the nucleotide sequence encoding clusterin is operably linked to a promoter, which is a gene promoter that functions in eukaryotic cells or prokaryotic cells.

[0015] The use of the recombinant clusterin in the preparation of a drug for preventing and / or treating a disease. Preferably, the disease is a cardiovascular disease, an ophthalmic disease or an amyloidosis.

[0016] The diseases include cardiovascular diseases, hypertension, hyperlipidemia, hyperglycemia, atherosclerosis, amyloidosis, eye diseases, Alzheimer's disease, Alzheimer's disease, stroke, renal failure, autoimmune diseases, skin diseases, traumatic wounds, male infertility, alcoholism, cervical squamous metaplasia, longevity drugs, health care drugs, skin diseases, wounds, reduced immunity and genetic defect diseases. Further, the diseases include cardiovascular diseases, amyloidosis, dry eyes, Alzheimer's disease, alcoholism, male infertility, etc. The cardiovascular diseases include atherosclerosis, ischemic heart disease, coronary heart disease, myocardial infarction, hyperlipidemia, hypercholesterolemia, hypertension, hyperglycemia, chronic heart failure, ischemic heart failure, coronary heart failure, intravascular plaque deposition, etc. The protein amyloidosis diseases include primary and secondary protein amyloidosis diseases of light chain (AL), heavy chain (AA), serum amyloid protein (SAA / AA), thyroxine transporter (TTR), and eye diseases.

[0017] Furthermore, the protein amyloidosis diseases include primary and secondary protein amyloidosis diseases of light chain (AL), heavy chain (AA), serum amyloid protein (SAA / AA), thyroxine transporter (TTR), and eye diseases.

[0018] Furthermore, the eye diseases include dry eye, glaucoma, diabetic eye disease, meibomian gland dysfunction, various types of corneal and conjunctival damage, and fundus macular lesions. The recombinant APO-J protein composed of each sequence of SEQ ID.NO 1-52 of the present invention can increase tear secretion, reduce fluorescence staining score, reduce MMP9 activity, and inhibit corneal and conjunctival keratinization and squamous metaplasia by inhibiting the expression of K10 and Sprr1B. At the same time, the aforementioned recombinant APO-J can treat a variety of inflammation-related eye diseases with MMP9 as a target.

[0019] Furthermore, the patients with the gene defect disease include patients whose apolipoprotein J is lower than normal; patients with genetic variation of apolipoprotein J; patients with apolipoprotein J dysfunction; patients with high or normal high-density lipoprotein (HDL); patients with high or normal low-density lipoprotein (LDL); patients with high or normal ApoA; patients with high or normal ApoA and high or normal ApoB; patients with high or normal ApoA and high or normal cholesterol; patients with high or normal ApoA, high or normal ApoB and high or normal cholesterol; patients with low high-density lipoprotein or ApoA; and patients with low Patients with high or normal density lipoprotein and / or high or normal ApoB and / or high or normal cholesterol and / or high or normal triglycerides; patients with high blood sugar; patients with hypertension; patients with atherosclerosis; patients with arteriosclerosis and arteriolar sclerosis; patients with symptoms of myocardial infarction; patients after myocardial infarction; patients with symptoms of cerebrovascular infarction; patients after cerebrovascular infarction; patients with coronary heart disease or symptoms of coronary heart disease; patients with chronic heart failure; patients with symptoms of heart failure; patients with Alzheimer's disease; patients with alcoholism; patients with clinical diseases caused by low expression levels and / or functional loss of alcohol dehydrogenase and / or acetaldehyde dehydrogenase; patients with fertility problems.

[0020] Furthermore, the diseases include skin diseases, wounds, and low immunity.

[0021] Furthermore, as an external medicine and / or injection, the recombinant APO-J protein composed of SEQ No 1-17 can effectively treat cervical squamous metaplasia.

[0022] Furthermore, the targets of the recombinant clusterin in the human body are apolipoprotein A1, apolipoprotein B100, apolipoprotein B58, various in vivo proteins susceptible to amyloidosis, corneal mucin layer, and cell receptors related to apolipoprotein J; the clinical administration method can be intravenous injection, intravenous drip or intramuscular injection; the drug preparation form can be a water injection solution injection or a lyophilized powder injection. The clinical dosage is 5-100 mg of the above protein per injection; the medication interval is one injection per day, every two days, every three days, every four days, every five days, every six days, every seven days, every eight days, every nine days, and every ten days of the dosage. When used to treat dry eye, the drug protein concentration is 0.5-50 micrograms / ml, the drug dosage is 1-2 drops / eye, 1-4 times / day, and the course of treatment is more than 3 days.

[0023] Compared with the prior art, the recombinant clusterin and its structure, extraction method and application described in the present invention have the following advantages:

[0024] The amino acid primary sequences of the recombinant clusterin described in the present invention are all or based on the natural sequences of the human body, which are not easy to induce immunogenic reactions, have no other toxic side effects, and have strong drugability; the amino acid primary sequences of the recombinant clusterin described in the present invention do not contain disulfide bonds, which can greatly improve the yield of purification after expression in Escherichia coli, simplify the production process, and enable industrial large-scale production. At the same time, the purified product has obvious immunological activity, in vitro activity and cell biological activity, which is higher than similar products obtained by existing technologies, can effectively treat diseases of test animals, and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the SEC-HPLC chromatogram of the recombinant clusterin sample prepared in Example 1 of the present invention;

[0026] Figure 2 The SDS-PAGE image of the recombinant clusterin prepared in Example 6 of the present invention and the control after purification;

[0027] Figure 3 This is a graph showing the inhibition results of the thermal aggregation of different proteins by the recombinant clusterin samples and controls prepared in Example 6 of the present invention;

[0028] Figure 4 The recombinant clusterin sample without disulfide bonds in Example 7 of the present invention is 2 O 2 The protective result of causing HCE-T cell apoptosis;

[0029] Figure 5 The natural clusterin containing five pairs of disulfide bonds in Example 7 of the present invention is 2 O 2 The protective effect of inducing apoptosis of HCE-T cells. DETAILED DESCRIPTION

[0030] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. The data in the following experimental examples are obtained by the inventor through a large number of experiments. Due to space limitations, only a part of them is shown in the specification. Ordinary technicians in this field can understand and implement the present invention based on these data.

[0031] Example 1 Extraction of recombinant clusterin

[0032] The method for extracting recombinant clusterin comprises:

[0033] Step S1: bacterial cell crushing

[0034] Each 1g of bacterial cells was fully resuspended with 10mL of bacterial suspension solution, centrifuged at 8000rpm at 4°C for 15min, and the supernatant was discarded; the bacterial cells were resuspended with 10mL / g bacterial suspension solution again, and the bacterial suspension was placed under the probe of an ultrasonic crusher, with a power of 400W, ultrasonic for 3s, and an interval of 6s for about 15min until the crushing was complete, and centrifuged at 15000g at 4°C for 30min, and the supernatant was discarded to collect the precipitate. The construction of the bacterial cells is prior art and will not be described here. Preferably, the bacterial suspension solution is 50mM Tris-HCl, pH 8.0. The bacterial cells contain the nucleotide sequence corresponding to SEQID.NO.1, and are cultured to express recombinant clusterin. The bacterial cell construction and culture method are both prior art.

[0035] Step S2: Inclusion body washing

[0036] The precipitate from step S1 was added with washing solution at 10 ml / g, fully resuspended and incubated at 4°C with shaking for 1 h, centrifuged at 15000 g, 4°C for 30 min, and the precipitate was collected again; the precipitate was added with dissolving solution at 10 mL / g, stirred and dissolved, and then centrifuged at 15000 g, 4°C for 30 min, and the supernatant was taken.

[0037] Usually 1g of wet bacteria can obtain 30mg of inclusion bodies; as an example of the present invention, the washing solution includes washing solutions 1 and 2, and the bacteria are washed with washing solutions 1 and 2 respectively, wherein washing solution 1 is 50mM Tris-HCl, 2M NaCl, 2mMEDTA, pH 8.0; washing solution 2 is 50mM Tris-HCl, 1% Triton-100, 2mM EDTA, pH 8.0; the dissolving solution is 50mM Glycine-NaOH, pH 9.0.

[0038] Step S3, anion exchange chromatography

[0039] A POROS HQ column was used for column chromatography purification, and the column was washed with buffer A (50 mM Gly-NaOH, pH 9.0), and then washed with buffer B (50 mM Gly-NaOH, 1 M NaCl, pH 9.0) and collected. The concentration of the collected sample was 3.0 mg / ml, and the yield was 80%. The specific operation method of the anion exchange chromatography is prior art and will not be described in detail here.

[0040] Step S4: desalting and replacing solution

[0041] Rinse the membrane package with buffer (20 mM sodium acetate, pH 5.5) and vent to fill the membrane package with buffer. Place the inlet in the sample, control the transmembrane pressure to less than 10 psi, flow rate 200 LMH, concentrate to 1.5-3.0 mg / mL and then change the liquid for 5CV until the pH of the filtrate is 5.4-5.6, then collect the concentrated sample.

[0042] Step S5, cation exchange chromatography

[0043] Column chromatography purification was performed using a POROS XS column, which was washed with buffer A (20 mM sodium acetate, pH 5.5) and then with buffer B (20 mM sodium acetate, 1 M NaCl, pH 5.5) and the eluate was collected. The final sample concentration was 2.0 mg / ml and the yield was 75%.

[0044] Step S6, SEC chromatography

[0045] The sample was loaded at 3-5% of the SEC column volume and eluted with 20 mM pH 7.4 PB buffer. The highest purity of the recombinant clusterin in the eluted sample was combined, and the yield was 79%. The results were shown in the table below. Figure 1 .

[0046] After two-step ion exchange column chromatography, the endotoxin content was less than 5eu / mg protein, and the HCD (host DNA) residue was less than 0.5ng / mg protein, which met the requirements of drug regulations. The total yield of clusterin obtained by the above extraction method in this application is about 80%*75%*75%=45%, while the total yield of clusterin with five pairs of disulfide bonds prepared by the same method is about 0.5%, and the total yield of recombinant clusterin with one pair of disulfide bonds is about 1.5-2.5%. Obviously, the yield of clusterin without disulfide bonds in this application is greatly improved; the bacterial cells containing the corresponding nucleotide sequence of SEQ ID.NO.2-17 have basically the same results when the above experiments are carried out, which will not be repeated here.

[0047] Example 2 Experiment on the protection of recombinant clusterin against ApoB100

[0048] 1. Experimental Materials

[0049] ApoB100: Luoyang Biotech Co., Ltd., concentration 3.0 mg / mL; Sample 1: 1.7 mg / mL of recombinant clusterin sample (1# protein, corresponding to SEQ ID.NO.1); Sample 2: 1.7 mg / mL of recombinant clusterin sample (2# protein, corresponding to SEQID.NO.2); Sample 3: 2.2 mg / mL of commercially available clusterin sample (3# protein, containing 5 pairs of disulfide bonds, purchased from Creative Biomart); BSA: dissolved in 20 mM PB, pH 8.0 buffer, concentration 1.0 mg / mL.

[0050] 2. Experimental methods

[0051] The molecular chaperone activity of recombinant clusterin was used to detect the aggregation inhibition effect of ApoB100. The specific method was as follows: the ApoB100 solution was heated to 70°C, maintained for 5 min and 15 min, and the OD360 was measured to check its thermal aggregation degree as a control; at the same time, ApoB100 was mixed with recombinant clusterin (sample 1, sample 2, sample 3), and after adding the samples according to Table 1, they were heated to 70°C, maintained for 5 and 15 min, and then the OD360 was measured, and the aggregation inhibition rate (%) was calculated. The results are shown in Table 3.

[0052] Table 1 Sample addition table for each group

[0053]

[0054] Table 2 Final concentration of each group after adding samples

[0055]

[0056] Table 3 Absorbance (OD360) and aggregation inhibition rate of samples before and after heating

[0057]

[0058] It can be seen from the above that, compared with BSA, the recombinant clusterin prepared in the present application can also effectively inhibit the thermal aggregation of ApoB100 under heating conditions.

[0059] Example 4 Pharmacodynamics Experiment

[0060] 1. Experimental Materials

[0061] The recombinant clusterin (amino acid sequence: SEQ ID. NO. 1) was prepared by the method of Example 1, the purification level was PAGE level, and after dissolution, it was filtered through a 0.22 bacterial filter membrane.

[0062] 2. Experimental methods

[0063] Forty male C57BL recombinant clusterin knockout atherosclerosis model mice (SPF level, about 25g / mouse) were randomly divided into 4 groups, namely, a low-dose group (5μg / 25g mouse), a medium-dose group (20μg / 25g mouse), a high-dose group (50μg / 25g mouse) and a normal saline control group. After 1 week of adaptive feeding, the drug was intravenously injected after gas anesthesia from 2 to 16 weeks, once a week. Two animals were tested every 4 weeks, and then dissected for pathological section observation.

[0064] It can be seen from the results that compared with the control group, the mice in the medium-dose group (the highest blood drug concentration is 20 μg / ml) showed an increase in left ventricular ejection fraction after 4 weeks of administration, and the increase increased from 10-12% after 4 weeks to 20-25% after 16 weeks; compared with the control group, the mice in each dose group had a significant decrease in intravascular deposits after 4 weeks, from 25-30% in the 4th week to 50-60% in the 16th week; at the same time, the cardiac ultrasound of mice in different dose groups showed significant improvement in cardiac function. In other words: the recombinant clusterin (SEQ ID.NO.1) of the present application shows a clinical effect of treating chronic heart failure in atherosclerosis model mice. It should be pointed out that the recombinant apolipoproteins corresponding to other sequences (SEQ ID.NO 2-17) in the present invention have similar pharmacodynamic experimental results, which will not be repeated here.

[0065] Example 5 Toxicology Experiment

[0066] 1. Experimental Materials

[0067] The recombinant clusterin (amino acid sequence: SEQ ID. NO. 1) was prepared by the method of Example 1, the purification level was PAGE level, and after dissolution, it was filtered through a 0.22 bacterial filter membrane.

[0068] 2. Experimental methods

[0069] Twenty healthy C57BL / 6J mice (SPF grade, about 25 g / mouse, male) were adaptively fed for 6 days and randomly divided into 5 groups, 4 mice in each group (the mice were numbered by punching holes on their ears), which served as the placebo group, experimental group 1, experimental group 2, experimental group 3 and experimental group 4 respectively.

[0070] In weeks 2-4, general anesthesia was performed by intraperitoneal administration of 2.5% chloral hydrate at a dose of 0.12 mL / 10 g (mouse body weight), followed by jugular vein injection. The dosages of experimental groups 1-4 were 10, 40, 160, and 640 μg target protein / 25 g (mouse body weight), respectively. The drugs were administered once a week, and the daily behavior and activities of the mice, as well as their weight, diet, and feces were observed and recorded. The results are shown in Table 8. In the experiment, whether adverse reactions or immunogenic reactions and tolerance occurred after drug injection were observed.

[0071] Table 8 Mouse weight record

[0072]

[0073] The possible poisoning symptoms and toxic side effects of the test animals were divided into 4 levels: general reaction (including hair erection, slow movement, laziness and loss of appetite), relatively serious reaction (including cyanosis of the extremities, cyanosis of the mouth and nose, and edema of the extremities), severe reaction (dyspnea), and the most serious reaction (death). During the experiment, all mice had normal appearance (shiny hair, rapid movement, no difference in alertness, normal feces), and no adverse reactions of "relatively serious reaction" or above appeared; at the end of the experiment, 14 mice survived, but 6 mice died due to the surgical process; the surviving mice had normal daily behavior and no abnormal appearance.

[0074] In the third week of the experiment (i.e. the second week of injection), the color of the veins of mice in experimental groups 1 and 2 was redder and darker than that of mice in the placebo group, indicating that the oxygen content in the venous blood was higher; in the fourth week of the experiment, the mice in experimental groups 1-4 were more active, exercised more, and weighed more than the placebo group; after the experiment, the remaining 14 mice were dissected, and the appearance of their heart, liver, spleen, lung, kidney and other organs was clear and normal. In the early stage of the experiment, the average weight of mice in each group was experimental group 3> experimental group 2> experimental group 1> placebo group> experimental group 4, while in the later stage of the experiment, the average weight of mice in each group was experimental group 3> experimental group 1> experimental group 2> placebo group> experimental group 4.

[0075] As can be seen from the above, there is no difference in appearance and daily behavior between the mice in each experimental group and the placebo group, and the mice in each experimental group have no obvious discomfort, and the recombinant clusterin has no toxic side effects; the experimental group 4 may cause mild adverse reactions after the injection of super-large doses, which are mainly manifested in the fact that the mice are lighter in weight, more sensitive to anesthetics, and the activity of the mice after the first administration is slightly less than that of the placebo group, but there is no obvious difference in appearance, and there is no obvious discomfort; however, the situation is significantly improved after 2-3 days of administration, and after 6-7 days of administration, the activity of the mice in the experimental group 4 is no different from that of the placebo group. The recombinant clusterins corresponding to other sequences (SEQID.NO 2-17) in the present invention have similar conclusions in the above toxicity experiments, which will not be repeated here.

[0076] Example 6 Protein thermal aggregation inhibition experiment

[0077] 1. Experimental methods

[0078] Step S1: Sample preparation

[0079] The rCLU (after dialysis, without disulfide bonds, stored at -80°C, corresponding to SEQ ID.NO.1) sample was thawed at 4°C, equilibrated to room temperature, 1 ml of the sample was gently shaken and mixed, 20.4 μl of 0.5 M DTT was added and mixed, and incubated at room temperature for 2 h. The Amicon 4 mL ultrafiltration tube (MWCO 10 kD) was rinsed with deionized water, and then the incubated sample was placed in the ultrafiltration tube, and the liquid exchange buffer was added to a total volume of 4 mL, and concentrated to a final volume of about 500 μL; the liquid exchange buffer was added, and ultrafiltration and liquid exchange were continued to maintain a final volume of about 500 μL. The above steps were repeated until the characteristic smell of DTT in the sample disappeared; OD280 was detected and the protein content (mg / mL) was calculated. The sample was diluted with the liquid exchange buffer to a protein content of 1.0 mg / mL to obtain an rCLU-NJ sample without disulfide bonds. The exchange buffer was prepared by 0.0461 g GSH + 3 mL 0.2 M PB pH 8.0, and the volume was made up to 30 mL with deionized water.

[0080] Step S2: Effects on thermal aggregation of different protein substrates

[0081] Thaw the storage solutions of GST, IgG, BSA, and rCLU (prepared with PBS) and fibrinogen (prepared with normal saline), equilibrate to room temperature, and gently shake to mix. Dilute IgG and BSA 5-fold with PBS to 2 mg / mL and 1 mg / mL, respectively.

[0082] Add PBS, rCLU sample (or 1mg / mL BSA), 5mM GSH (in PB7.4, i.e., liquid exchange buffer) and different test proteins in a 1.5mL EP centrifuge tube in order. The reaction system is shown in Tables 9-11. Slightly vortex to mix the sample evenly, and place it in a metal bath preheated to the required temperature and heat it for 5 minutes. Take out the heated sample, cool it at room temperature for 5 minutes, and gently vortex to mix it evenly; adjust to zero with PBS, and use a spectrophotometer to detect the OD360 of the heated sample. The results are shown in Figure 3 .

[0083] Table 9 GST thermal aggregation reaction system (120 μL, 60°C heating for 5 min)

[0084]

[0085]

[0086] Table 10 IgG thermal aggregation reaction system (120 μL, 80°C heating for 5 min)

[0087]

[0088]

[0089] Table 11 Reaction system of fibrinogen thermal aggregation (120 μL, heating at 90°C for 5 min)

[0090]

[0091]

[0092] 2. Experimental results

[0093] The rCLU sample without disulfide bonds prepared by the present application and treated with DTT and GSH was compared with the commercially available rCLU sample containing 5 pairs of disulfide bonds (purchased from Creative Biomart) by SDS-PAGE analysis. The results are shown in Figure 2 , the purity is about 90%, wherein SDS-PAGE uses 12% separation gel, and the sample loading amount is 2μg. From left to right in the figure: M. protein marker; a. rCLU (containing 5 pairs of disulfide bonds, non-reducing conditions); b. rCLU (without 5 pairs of disulfide bonds, non-reducing conditions); c. rCLU (containing 5 pairs of disulfide bonds, reducing conditions); d. rCLU (without 5 pairs of disulfide bonds, reducing conditions). It can be seen from the above that the recombinant clusterin without disulfide bonds prepared in this application has an inhibitory effect on the thermal aggregation of different proteins that is basically equivalent to that of wild clusterin.

[0094] Example 7H 2 O 2Protective experiment of inducing oxidative stress in human corneal epithelial cells

[0095] 1. Experimental methods

[0096] 1.1 HEC-T cell culture

[0097] (1) Cell culture medium: HEC-T (human corneal epithelial cells) cells were purchased from Wuhan Pronocell Life Science Co., Ltd. and cultured in DMEM / F12 medium containing 15% FBS, 5 ug / mL insulin, 10 ng / mL human epidermal growth factor (EGF) and 1% double antibody; the cells were cultured in a 37°C, 5% CO2 cell culture incubator.

[0098] (2) Cell passaging and cryopreservation: Observe the cell morphology and growth under a microscope. When the cells grow to 70-80%, perform passaging. Discard the culture medium, rinse the cells with PBS buffer, discard the PBS, add 1 ml of trypsin, and digest at 37°C for about 4 minutes. After observing that the cells are completely digested under a microscope, terminate the digestion with 1 ml of complete culture medium, blow the cells off gently, mix them, transfer them to a 5 ml centrifuge tube, and centrifuge them at 1000 rpm / min for 3 minutes. Discard the supernatant, add 1 ml of complete culture medium, and mix them. Use a cell counter to count the cells. After counting, according to the cell quantity, mix the appropriate amount of cells with the cryopreservation solution and dispense them into cryopreservation tubes and freeze them in a -80°C refrigerator. The next day, transfer them to a liquid nitrogen tank for storage. The passaged cells can be diluted with complete culture medium to an appropriate cell density according to different culture dishes for cell plating, and cultured in a cell culture incubator at 37°C and 5% CO2.

[0099] (3) Cell recovery: Take out the cryopreserved cell tube from the liquid nitrogen tank, quickly place it in a 37°C water bath, and shake it quickly to thaw it within 1 min. Aspirate the cell suspension and transfer it to a centrifuge tube. Centrifuge at 1000 rpm / min for 3 min, discard the supernatant, add complete culture medium to resuspend the cells, transfer them to a culture dish, and culture them in a 37°C, 5% CO2 cell culture incubator.

[0100] 1.2 Cell Counting Kit-8 (CCK8) to detect cell viability

[0101] (1) When the cells grew to 70-80%, they were passaged. 100 μL of 10,000 cells / well was added to a 96-well plate, 5 replicates / group, and complete medium was added for culture.

[0102] (2) After the cells adhered to the wall, different concentrations of rClu-NJ230713 and rClu-

[0103] The cells were treated with 20240218 and the cells were cultured for another 24 h, wherein rClu-NJ230713 is a recombinant clusterin without a disulfide bond prepared by the method of Example 1, and rClu-20240218 corresponds to a wild clusterin protein.

[0104] (3) According to the experimental design, H is added to a specific group. 2 O 2 , and continue to culture the cells for 24 h.

[0105] (4) The culture medium was discarded, and 100 μL of serum-free culture medium containing 10% CCK-8 solution was added. The wells without cells were used as blank controls. The cells were incubated in the cell culture incubator for 1.5 hours, and the absorbance was measured at 450 nm using an ELISA reader.

[0106] 1.3 Results Analysis and Arrangement

[0107] The formula for calculating cell viability is: Cell viability (%) = [(As-Ab) / (Ac-Ab)]*100%

[0108] GraphpadPrism 9 was used for plotting and statistical analysis. Data were expressed as Mean ± SEM, and the two groups were compared using independent sample t-test, with P < 0.05 as statistically significant. Each group of experiments was repeated at least 3 times.

[0109] 2. Experimental results

[0110] After cell inoculation, cells were treated with 0, 0.5, 1, 2.5, 5, and 10 μg / ml rClu-NJ230713, rClu20240218, and 300 μM H 2 O 2 Stimulate the cells, and detect proliferation with CCK8 24 hours later. Add only 10 μg / ml rClu-NJ230713 and only 300 μM H 2 O 2 The group without either of them was used as the control. The results are shown in Figure 4 , 5 .

[0111] It can be seen from the above that compared with the control group without drug treatment, the cell proliferation was accelerated after 24h treatment with 10μg / ml rClu-NJ230713. 2 O 2 rClu20240218 has a significant protective effect on HCE-T (human corneal epithelial cells) induced by oxidative stress; 2 O 2It has a significant protective effect on the oxidative stress of HCE-T cells caused by cytokines.

[0112] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. A recombinant clusterin, characterized in that It comprises an amino acid primary sequence encoding a recombinant clusterin, wherein the amino acid primary sequence is at least 95% homologous to any one of SEQ ID NO. 1-17, and the tertiary structure formed by the amino acid primary sequence does not have a disulfide bond.

2. The recombinant clusterin according to claim 1, characterized in that The amino acid primary sequence has 0-1 cysteine.

3. The recombinant clusterin according to claim 2, characterized in that The amino acid primary sequence of the recombinant clusterin is any one of SEQ ID NO.1-17.

4. A pharmaceutical composition, characterized in that The invention comprises the recombinant clusterin according to any one of claims 1 to 3, and pharmaceutically acceptable excipients thereof.

5. The pharmaceutical composition according to claim 4, characterized in that The dosage form of the pharmaceutical composition is freeze-dried powder injection or injection.

6. An isolated polynucleotide molecule, characterized in that The method comprises a nucleotide sequence encoding the recombinant clusterin according to any one of claims 1 to 3.

7. The polynucleotide molecule according to claim 6, characterized in that The nucleotide sequence encoding clusterin is operably linked to a promoter, which is a gene promoter that functions in eukaryotic cells or prokaryotic cells.

8. Use of the recombinant clusterin according to any one of claims 1 to 3 in the preparation of a drug for preventing and / or treating a disease.

9. The use according to claim 8, characterized in that: The disease is a cardiovascular disease, an ophthalmic disease or a protein amyloidosis disease.

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