CN117205299A

CN117205299A - Application of recombinant apolipoprotein J in preparation of medicine for treating ophthalmic diseases

Info

Publication number: CN117205299A
Application number: CN202310680018.XA
Authority: CN
Inventors: 陈翔; 杜佩云
Original assignee: Dexinwonder Medical Technology Co ltd
Current assignee: Dexinwonder Medical Technology Co ltd
Priority date: 2022-06-09
Filing date: 2023-06-09
Publication date: 2023-12-12

Abstract

The application provides an application of recombinant apolipoprotein J in preparing a medicament for treating ophthalmic diseases, wherein the recombinant apolipoprotein J comprises an amino acid primary sequence for encoding the apolipoprotein J, two cysteines are arranged in the amino acid primary sequence, and a tertiary structure formed by the amino acid primary sequence is provided with a pair of disulfide bonds. The application optimizes the amino acid sequence of the recombinant apolipoprotein J so that the recombinant apolipoprotein J can be industrially prepared and has good and even better treatment effect on ophthalmic diseases, especially dry eye, glaucoma and conjunctival injury symptoms.

Description

Application of recombinant apolipoprotein J in preparation of medicine for treating ophthalmic diseases

Technical Field

The application relates to the technical field of biology, in particular to application of recombinant apolipoprotein J in preparation of a medicament for treating ophthalmic diseases.

Background

The surface of the mammalian eyeball is transparent vascular-free cornea tissue with a diameter of about 1-12mm and a thickness of about 520 μm, and various physiological and biochemical phenomena on the tissue mainly play a role in maintaining the transparency, which has an important effect on vision. Since the eyeball is directly exposed to the outside, a tear layer consisting of three layers of an oil layer, a water layer and a mucin layer is formed on the outer side of the cornea to prevent invasion of microorganisms and foreign substances, wherein the mucin layer covers the surface of the hydrophobic corneal epithelial cells and converts them into hydrophilicity, which plays an important role in maintaining the normal structure of the eyeball. Corneal epithelial defects caused by dry eye, corneal epithelial loss, keratitis, corneal ulcers and the like are usually rapidly and naturally repaired within a few days; however, if delayed or not repaired for some reason, the transparency thereof is deteriorated to form various ophthalmic diseases.

Wherein insufficient tear supply can lead to long-term changes or imbalance in the composition of the surface tear layer and trigger stress pathways in the epithelial cells to alter the homeostasis of the ocular surface, leading to "Dry Eye Syndrome (DES)". DES causes eye injury, dizziness, headache and affects sleep, is very painful and weak, and can lead to blindness in severe cases. In all forms of dry eye, decreased tear flow and/or increased evaporation results in hyperosmolar tears, triggering the vicious circle of dry eye pathology. The hyperosmotic induced inflammatory cascade activates, promotes apoptosis, and stimulates the expression and activity of Matrix Metalloproteinases (MMPs), resulting in disruption of the ocular surface barrier.

Apolipoprotein J (Clusterin), also known as an aggregation factor or Clusterin, after mRNA directed synthesis of the precursor polypeptide chain, enzymatically removes the secretion signal peptide and breaks down to form alpha and beta chains, which are antiparallel joined by 5 disulfide bonds, which are glycosylated to form a heterodimeric sulfated glycoprotein comprising 449 amino acids, including glycosyl groups, having a molecular weight of about 75-80kDa. Apolipoprotein J is the first secreted macromolecular chaperone found in the human body, is widely present in body fluids such as tears, is the main protein in the minor protein component of tears, and has a protective effect on ocular surface tissues. In human inflammatory diseases manifested by severe dry eye, the expression of apolipoprotein J in ocular surface epithelium is significantly reduced; whereas, in the dry eye mouse model, ocular surface apolipoprotein J protein and mRNA levels were reduced by about 30% when induced by dry stress, indicating that downregulation of ocular surface apolipoprotein J expression under dry stress is due to activation of the inflammatory cascade.

Certain ophthalmic diseases, such as open angle glaucoma and dry eye fundus maculopathy, which result from protein deposition and catheter blockage due to protein metabolism disorders, may be exacerbated by insufficient or malfunctioning apolipoprotein J that balances protein metabolism, and by supplementation with recombinant apolipoprotein J, it is possible to ameliorate and treat these ophthalmic diseases.

Although apolipoprotein J is widely present in humans, the cost of extraction to pharmaceutical grade is extremely high. Even if the indexes such as purity and the like can be qualified, only a single medicinal dose can be extracted, and thousands of liters of human blood can be needed, and obviously, the large-scale production can not be realized, so that the recombination and in-vitro expression of the apolipoprotein J become the research key points of the scientific community at home and abroad. In order to ensure that the recombinant apolipoprotein J has biological activity, the existing recombinant apolipoprotein J can form 5 pairs of disulfide bonds, such as polypeptide sequences disclosed in application numbers 202010644415.8 and 201580074616.6, mismatch and reduction are easy to occur during purification to form impurities, the renaturation difficulty is high, and the final purification yield is extremely low; if protein tags are used for separation and purification, the finally obtained recombinant protein with the tags is difficult to meet the requirements of pharmaceutical regulations and cannot be prepared, which is one of the biggest barriers for realizing industrial production.

In view of this, the present application has been made.

Disclosure of Invention

In order to solve the problems, the application also provides an application of the recombinant apolipoprotein J in preparing medicines for treating ophthalmic diseases, wherein the recombinant apolipoprotein J comprises an amino acid primary sequence for encoding the apolipoprotein J, two cysteines are arranged in the amino acid primary sequence, and a tertiary structure formed by the two cysteines is provided with a pair of disulfide bonds.

The insufficient apolipoprotein J in tears can lead to the decrease of the protective capability of tears on ocular surface tissues, the damage of the conjunctival epithelial cells of the cornea, and the occurrence of ophthalmic diseases; the recombinant apolipoprotein J is supplemented to ocular surface tissues of ocular diseases such as xerophthalmia and the like in an eye drop mode, so that the ocular disease symptoms such as xerophthalmia and the like can be improved and treated, and the damage of the ocular surface tissues can be repaired. The recombinant apolipoprotein J only retains a pair of disulfide bonds, so that the separation and purification process is simple and reliable, the repeatability is strong, and the final yield is high; the product has no label, and can be purified to reach the pharmaceutical purity specified by the country.

Preferably, at least 95% of the primary amino acid sequence is identical to SEQ ID NO. 1. Preferably, the primary amino acid sequence is any one of SEQ ID NOs 4-28.

Preferably, the cysteines are located at amino acid positions 107 and 263, or at amino acid positions 99 and 273, or at amino acid positions 94 and 280, or at amino acid positions 91 and 283, or at amino acid positions 80 and 291 of SEQ ID NO. 1.

Preferably, the amino acid positions at positions 80, 91, 94, 99, 273, 280, 283 and 291 of the SEQ ID NO. 1 are glycine or alanine.

Preferably, the recombinant apolipoprotein J further comprises a signal peptide sequence connected with the N end of SEQ ID NO. 1, and at least 95% of the signal peptide sequence is identical with SEQ ID NO. 2 or SEQ ID NO. 3. Preferably, the signal peptide is SEQ ID NO. 2 or SEQ ID NO. 3. By optimizing disulfide bond positions and signal peptide sequences, renaturation effect and expression quantity can be further improved.

Preferably, the medicament is in the form of eye drops or eye ointments.

Preferably, the ophthalmic disease is a corneal disorder or a keratoconjunctival lesion. Preferably, the corneal disorder is any one of dry eye, corneal epithelial loss, keratitis, or corneal ulcer. The ophthalmic disease is any of various types of dry eye, glaucoma, ocular keratoconjunctival damage, meibomian gland dysfunction. The dry eye may be caused by decreased tear secretion and damaged conjunctiva of the cornea. Preferably, the ophthalmic diseases are open angle glaucoma and dry fundus macular degeneration. Preferably, the corneal disorder is caused by insufficient levels of native apolipoprotein J (Clu) in tear fluid. Preferably, the ophthalmic disease is an ocular disease caused by deposition of denatured proteins.

The application optimizes the molecular structure of the apolipoprotein J to realize the industrial batch production of the recombinant apolipoprotein J which meets the requirements of pharmaceutical regulations and has therapeutic effect on eye diseases.

The application also provides a pharmaceutical formulation comprising recombinant apolipoprotein J comprising an amino acid primary sequence encoding apolipoprotein J having two cysteines in the amino acid primary sequence such that the spatial structure thereof forms a pair of disulfide bonds. The pharmaceutical preparation can fill the blank of the medicines which are lack in the current domestic and foreign markets and have the anti-inflammatory effect and are specially used for treating the cornea and conjunctiva injury.

Preferably, the pharmaceutical preparation is an eye drop or an eye ointment, and the eye drop is an artificial tear or an anti-inflammatory agent. The eye drop is artificial tear with anti-inflammatory effect, and can be used as anti-inflammatory agent for long-term repeated use.

The application also provides a method for preparing the high-expression recombinant apolipoprotein J, which comprises the following steps: step 1), expressing a polynucleotide molecule encoding a recombinant apolipoprotein J expressed in high levels as described above; step 2), extracting and purifying the expressed polypeptide from the cells.

Preferably, step 2) comprises: s21, crushing thalli and separating inclusion bodies; s22, washing and dissolving inclusion bodies; s23, ion exchange chromatography; s23, diluting and renaturation; s24, ultrafiltration concentration and liquid exchange or ammonium sulfate precipitation and heat exchange; s25, molecular sieve chromatography.

The application provides a recombinant apolipoprotein J, comprising an apolipoprotein J amino acid primary sequence which is at least 95% homologous to SEQ ID NO. 1 and has two cysteines so that the tertiary structure formed by the amino acid primary sequence has a pair of disulfide bonds.

Preferably, one of the two cysteines is located at one of the 80 th, 91 st, 94 th, 99 th and 107 th amino acid positions of SEQ ID NO. 1, and the other cysteine is located at one of the 263 rd, 273 th, 280 th, 283 th and 291 th amino acid positions of SEQ ID NO. 1.

Preferably, the rest amino acid in the 80 th, 91 st, 94 th, 99 th, 107 th, 263 rd, 273 th, 280 th, 283 th and 291 th amino acid locus of the SEQ ID NO. 1 is glycine or alanine.

Preferably, the two cysteines are located at amino acid positions 107 and 263 of SEQ ID NO. 1, respectively. Preferably, the amino acid positions at positions 91, 94, 99, 107, 263, 273, 280 and 283 of the SEQ ID NO. 1 are glycine or alanine. Preferably, the primary amino acid sequence comprises SEQ ID NOS.4-28.

Preferably, the primary amino acid sequence further comprises a signal peptide sequence connected with the N end of SEQ ID NO. 1, wherein the signal peptide sequence is SEQ ID NO. 2 or SEQ ID NO. 3. Preferably, the signal peptide sequence is SEQ ID NO. 2.

Drawings

FIG. 1 is an electrophoresis chart of recombinant apolipoprotein J of example 2 of the present application before and after dilution and renaturation;

FIG. 2 is an electrophoretogram of different recombinant apolipoprotein J samples purified in example 2 according to the present application;

FIG. 3 is an SDS-Page electrophoresis of example 4 of the present application after addition of human serum to various samples;

FIG. 4 is a graph showing the statistics of tear secretion after treatment with different samples in example 5 of the present application;

FIG. 5 is a chart showing HE staining of cornea corresponding to different samples in example 6 of the present application;

FIG. 6 is a chart showing the staining of conjunctival PAS corresponding to the different samples in example 6 of the present application;

FIG. 7 is a graph showing statistics of the healing rate of corneal epithelium corresponding to the different samples of example 7 of the present application;

FIG. 8 is a chart of HE staining of the cornea corresponding to the different samples of example 8 of the present application;

FIG. 9 is a graph showing the immunofluorescence staining and statistics of Ki67, E-cadherin, PMN, F4/80 after treatment with different samples in example 9 of the present application.

Detailed Description

The application is further described below in conjunction with the detailed description. It should be noted that the data in the following examples are obtained by the inventors through a lot of experiments, and are only shown in some of the descriptions, and those skilled in the art can understand and practice the present application under the data. These examples are only for illustrating the present application and are not intended to limit the scope of the present application. It should be further understood that various changes and modifications to the present application may be made by those skilled in the art after reading the disclosure of the present application, and such changes and modifications are also within the scope of the present application.

The functions of the protein are closely related to the spatial structure of the protein, while five pairs of disulfide bonds are generally considered to be necessary for maintaining the spatial structure of the apolipoprotein J, for example, 10 cysteines are also included in the amino acid sequences claimed in application numbers 202010644415.8 and 201580074616.6, the spatial structures of the five pairs of disulfide bonds form five pairs of disulfide bonds, so that the extraction and purification difficulties are great, mismatch is easy to occur during renaturation during prokaryotic expression, and the disulfide bonds are easy to be reduced during eukaryotic expression, so that the problem of product consistency exists, and industrial production cannot be realized. At present, only high-purity apolipoprotein J with a label exists in the market, but cannot be prepared as a medicine because of being out of compliance with the medicine rule.

The inventors have unexpectedly found that recombinant apolipoprotein J (hereinafter rClu) having only one pair of disulfide bonds also retains the biological activity of natural apolipoprotein J, can be isolated and purified to a high purity pharmaceutical grade (> 95%) and has the characteristics of high yield (> 10%) of industrial production. The rClu with only one pair of disulfide bonds prepared by the application has obvious functions and curative effects in vitro experiments and animal experiments, for example, can inhibit protein denaturation, aggregation and precipitation and MMP9 activity, has obvious curative effects in animal experiments, and has high clinical application value.

Specifically, 10 cysteines in the amino acid sequence SEQ ID NO. 1 are changed into rClu by point mutation, only 2 cysteines are reserved, the number of disulfide bonds correspondingly formed is changed from 5 pairs to 1 pair, the disulfide bonds can be prevented from being reduced to form a mixture during eukaryotic expression, the renaturation rate (> 50%) during prokaryotic expression is obviously improved, and the mismatch rate of the disulfide bonds is reduced. The recombinant apolipoprotein J-rClu of the application also forms a double subunit structure when expressed in eukaryotic cells, but the two subunits are connected by a pair of interchain disulfide bonds; and when expressed in a prokaryotic system, forms a single subunit with a pair of intrachain disulfide bonds forming a cyclic structure.

EXAMPLE 1 expression of recombinant apolipoprotein J

DNA sequence synthesis is carried out after codon optimization according to SEQ ID No. 4-28 and SEQ ID No. 2-3, the DNA sequence is inserted into plasmid pET-30a (+) at the sticky end, the plasmid pET-30a (+) is introduced into host cell BL21 (DE 3), induction expression is carried out by using IPTG, inclusion bodies are collected by centrifugation after bacterial cell disruption, quantitative analysis is carried out by washing, electrophoresis after extraction and gray scanning, and the protein expression quantity is calculated, and the result is shown in Table 1. The related DNA synthesis, recombinant plasmid construction, and introduction into host cells and expression are all prior art and are not described in detail herein.

TABLE 1 results of the expression levels of different sequences

Sequence number	Sequence(s)	Protein expression level mg/L
			1	SEQ ID NO:24	1176.9
2	SEQ ID NO:2+SEQ ID NO:24	3980.6
			3	SEQ ID NO:3+SEQ ID NO:24	2854.7
4	SEQ ID NO:4	1151.2
			5	SEQ ID NO:2+SEQ ID NO:4	3865.3
6	SEQ ID NO:3+SEQ ID NO:4	2783.4
			7	SEQ ID NO:1	1317.6

As shown in the table, the expression level of the recombinant plasmid constructed after adding SEQ ID NO. 2 and SEQ ID NO. 3 to the N terminal of SEQ ID NO. 4-28 is obviously improved, and the final protein expression level is highest by the recombinant plasmid which contains SEQ ID NO. 24 and the N terminal of which is connected with SEQ ID NO. 2; the recombinant plasmids constructed according to the other sequences have similar results when introduced into host cells for expression.

EXAMPLE 2 preparation of recombinant apolipoprotein J

A method for preparing recombinant apolipoprotein J comprising:

s1, crushing thalli and separating inclusion bodies

(1) Fully re-suspending thallus (SEQ ID NO:2+SEQ ID NO: 24) according to 1g/10mL of 50mM Tris-HCl solution with pH of 8.0, centrifuging at 8000rpm for 15min under a low-temperature (such as 4 ℃) environment, and discarding the supernatant; (2) And adding 1g/10mL of the solution again for resuspension operation, and then placing the bacterial suspension into an ultrasonic breaker probe for breaking operation for 15min, wherein the operation parameters are as follows: the pellet was collected by centrifugation at 15000rpm for 30min at 400W power, 3s with sonication, 6s batch, and low temperature (e.g., 4 ℃ C.).

S2, washing and dissolving inclusion bodies;

(1) To the collected precipitate, a washing solution was added at 1g/10ml, which washing solution was: 50mM Tris-HCl,2M NaCl,2mM EDTA,pH 8.0, incubating for 1h at low temperature (e.g. 4deg.C) with shaking, centrifuging at 15000rpm for 30min, and collecting precipitate again; (2) A dissolution solution of 8M urea+20 mM dithiothreitol was added at 1g/10ml for dissolution denaturation for 40min, followed by centrifugation at 15000rpm for 20min, and the supernatant was collected.

S3, ion exchange chromatography

Column chromatography was performed using column POROS HQ, washed with buffer A (50 mM Gly-NaOH,8M urea, 10mM DT, pH 9.0), followed by column washing with buffer B (50 mM Gly-NaOH,8M urea, 10mM DDT,1MNaCl,pH 9.0) and collecting the effluent; the collected solution was desalted and subjected to column chromatography by using column POROS XS, washed with buffer C (20 mM sodium acetate, 8M urea, 10mM DDT, pH 5.5), washed with buffer D (20 mM sodium acetate, 8M urea, 10mM DDT,1M NaCl,pH 5.5) and the effluent was collected.

S4, diluting and renaturation;

taking the collecting liquid obtained in the step S3, adding 15 times (V/V) dilution buffer, and incubating at 4 DEG C24 hours. The dilution buffer was 1%CHAPS+500mM NaCl+500mMArg+0.5% sorbitol+ 0.01mM GSSH+50mM Tris-HCl pH 9.0, or 500mM arg+0.5% sorbitol+0.00001 mM CuCl ₂ +20mM PB pH7.4, or 1%CHAPS+500mM NaCl+0.0001mM CuCl ₂ +20mM PB pH7.4。

And (3) after renaturation of the prokaryotic expression protein, forming a polymer and a monomer, primarily judging the renaturation rate by using a protein electrophoresis gray scanning monomer, and finally determining the renaturation rate by using disulfide bond positioning analysis and free sulfhydryl analysis results of a third party. As shown in FIG. 1, the renaturation rate of the rClu-sample rAJ-1 with five pairs of disulfide bonds through dilution is about 8%, the renaturation rate of the rClu-sample rAJ-3 with two pairs of disulfide bonds through dilution is about 10%, and the renaturation rate of the rClu-sample rAJ-6-1 prepared by the application through dilution is more than or equal to 50%, so that the renaturation rate is remarkably improved; rClu with more than two pairs of disulfide bonds is easy to generate mismatch through dilution and renaturation, and has extremely low biological activity; if the G25 column renaturation is adopted, the yield is lower. It should be noted that: wherein, the reduction treatment is to add a large amount of mercaptoethanol to completely open disulfide bonds of the protein sample, and the non-reduction treatment is to add no mercaptoethanol.

S24, performing molecular sieve chromatography after liquid exchange;

and (3) the renaturated solution obtained in the step (S4) is subjected to ammonium persulfate precipitation or ultrafiltration concentration, then is replaced by a phosphate buffer system (pH 7.4) and is subjected to molecular sieve column chromatography, and a molecular sieve gel filler adopts GE Superdex S200 or Bogurone Chromda200PG to obtain a sample rAJ-6-1.

The purity of the sample rAJ-6-1 is more than 95 percent through analysis, and the total purification yield is 10.2 percent; samples rAJ-6-2 (SEQ ID NO: 25) and AJ-6-3 (SEQ ID NO: 26) had similar results after purification by the above procedure. It should be noted that: the yields of the final samples purified by the above method after expression of rClu-rAJ-3 containing two pairs of disulfide bonds and rClu (SEQ ID NO: 1) -rAJ-1 containing five pairs of disulfide bonds are respectively 0.55% and 0.25%, the purities are respectively 61.6% and 62.4% far lower than that of the application and are mixtures, cannot be made into medicines, and the electrophoresis results are shown in FIG. 2.

The eukaryotic expression protein can be directly purified from recombinant apolipoprotein J host cell (e.g. CHO) culture solution without renaturation, and the eukaryotic expression apolipoprotein J mixture with nonuniform molecular weight can be obtained through the ion exchange and the molecular sieve column chromatography purification.

EXAMPLE 3 Effect of disulfide bond position on renaturation results

Recombinant plasmids were constructed with SEQ ID NOS.8, 12, 16, 20 and 24, respectively, and diluted and renatured after expression in accordance with the method of example 2, and the results are shown in Table 2.

TABLE 2 influence of disulfide bond position on renaturation ratio

In general, the spatial structure of proteins can affect the biological activity and immunogenicity, while five pairs of disulfide bonds formed at the above sites are necessary for natural rClu to maintain the spatial structure of proteins to exert their biological activity; in the application, eight of ten cysteines in the amino acid sequence are mutated into glycine to form rClu with a pair of disulfide bonds, so that the rClu still has good biological activity; meanwhile, the position of the disulfide bond can obviously influence the renaturation rate of dilution renaturation, and the renaturation rate of rClu of SEQ ID NO. 24 is the highest when the cysteine is at the 107 th position and the 263 th position, so that the effect is the best.

Example 4 in vitro Activity assay

Inflammatory response in human body can lead to the increase of MMP9 secretion and can generate destructive effect on local tissue structure; MMP9 is also one of the major matrix degrading enzymes on the corneal surface, and an increase in the density and activity of MMP-9 is observed in tears of patients with ocular surface diseases such as dry eye, and is considered as a causative agent of disruption of ocular surface barrier by desiccation stress in mice and humans. And apolipoprotein J is a natural inhibitor of MMP9, and can inhibit MMP9 enzyme activity, so that the apolipoprotein J is one of natural measures for dealing with inflammatory response in human body.

Enzyme chromatography (Zymography) is adopted to measure MMP9 enzyme activity by using an MMP9 test kit (Wuhansai biosciences Co., ltd.) to reflect the inhibition effect of rClu, human serum is taken as a reference, the human serum added with rAJ-6-3 and rAJ-1 is electrophoresed in SDS-Page gel containing gelatin, then the gel is incubated with an enzyme reaction buffer system, the MMP9 is activated to degrade the gelatin on the electrophoresis gel, and then gel staining and decoloring are carried out. The bands with MMP9 activity form transparent areas due to degradation of gelatin, while gelatin distributed at other positions without degradation forms dark background, so that transparent bands can be scanned to convert MMP9 enzyme activity, and the results are shown in Table 3 and FIG. 3.

Specific experimental methods are the same as those of optimizing gelatin zymography for detecting spontaneous hypertension rat plasma MMP-2 and MMP-9 activities (Li Bingwei, etc., 2013, basic medicine and clinic, pages 845-848.

TABLE 3 Effect of different samples on MMP-9 Gray peak area

As can be seen from the above table, the inhibition of MMP9 enzyme activity by sample rAJ-6-3 prepared by the application is at least equivalent to sample rAJ-1, and the biological activity is good or even better.

EXAMPLE 5 in vivo Activity assay Mono-scopolamine induces dry eye

Female C57/BL6Shijh mice of 6 weeks of age were selected and subjected to low humidity combined subcutaneous injection of scopolamine (2 times/day, 0.75mg/mL,0.3 mL/piece/time) for 5 consecutive days to induce dry eye modeling for simulation; dry eye with reduced tear secretion is first modeled as D ₁ .25 eyes are selected according to the average value of the staining scores of the corneal fluorescein sodium and are randomly divided into five groups, namely a model group, a dosing group and a dosing group, PB buffer, rAJ-6-1 sample (20. Mu.g/ml), rAJ-1 sample (20. Mu.g/ml), cyclosporine eye drops (II) -Shenyang Xingzhi eye drops (Inc. of Shenyang Xingzhi eye drops (Shentian pharmaceutical (China) Inc.) eye drops (sodium hyaluronate eye drops) were treated by eye drops (4 times per day, 1-2. Mu.L per eye), all animals were treated while continuing to receive the molding treatment, as previously described.

After eyes were instilled with 2ul of 0.5% sodium fluorescein solution and washed out with normal saline after blinking, the staining of sodium fluorescein on the cornea was observed by slit lamp photographing, six animal eyes were stained and averaged for each group, before treatment (0 d) and 5d, 10d, 15d after dosing treatment, respectively, and the results are shown in table 4. The rClu samples are prepared by diluting rAJ-6-1 and rAJ-1 with PB buffer, and related experiments and detection methods are related to the prior art and are not described herein.

TABLE 5 influence of different samples on the scoring of sodium fluorescein staining

Apolipoprotein J is one of the protein components in human tears, and has protective effects on ocular surface tissues. When tear secretion is reduced, it is difficult to take on the original protective effect, resulting in dry eye. As shown in the table, the recombinant rClu (rAJ-6-1) containing 1 pair of disulfide bonds prepared by the application can be used as eye drops to supplement natural apolipoprotein J of ocular surface tissues, the fluorescence staining score drop amplitude is larger than rAJ-1 containing 5 pairs of disulfide bonds, and is equivalent to that of the commercial cyclosporine eye drops, and the recombinant rClu (rAJ-6-1) containing 1 pair of disulfide bonds has a certain inhibition effect on the increase of the staining score of mouse cornea fluorescein sodium induced by scopolamine combined with a low-humidity environment, so that the dry eye caused by tear secretion reduction can be improved and treated.

EXAMPLE 6 in vivo Activity assay Di-benzalkonium chloride induces Dry eye

Taking 20 female C57/BL6 mice of 6 weeks old as tested mice, adapting to the environment in an IVC system for 3 days, and carrying out local eye dropping on 0.075% benzalkonium chloride (in 1% PBS), 5 uL/eye, 2 times a day, and continuously 7 days to simulate xerophthalmia caused by cornea and conjunctiva injury; after the 7 th day of continuous modeling is finished, slit lamp photographing and fluorescent staining evaluation are carried out on all the tested mice, and 15 mice which are successfully modeled are screened to enter a later experiment.

The 15 mice that were successfully modeled were randomly divided into three groups and dosing was initiated on day 8, with groups 1, 2, and 3 using PB buffer, sodium hyaluronate eye drops, rAJ-6-1 samples (60 μg/ml, prepared by dilution with PB buffer), respectively. Daily 9:00, 17:00 continuation Point 0.075% BAC, 9 per day: 30, 12:30, 15:30, 18:30 each eye was instilled with the test article 4 times, 2. Mu.l of the test article was instilled with each eye, and the administration was continued for 7 days until the end of day 14, and the administration was stopped.

All mice tested were tested for aqueous tear secretion by phenol red cotton yarn method at the same time period before no molding on day 1, at day 7 and after completion of day 14, and the results are shown in fig. 4; after the eye surface detection is finished on the 14 th day of the experiment, the experiment mice are euthanized, then the whole eyes of the right eyes are dissected and removed, paraffin sections are prepared, and HE staining and PAS staining are carried out, and the results are shown in fig. 5 and 6, wherein the HE staining is the prior art.

As can be seen from fig. 4, compared with before molding, the tear secretion of the mice is obviously reduced after the benzalkonium chloride is spotted for 7 days, which indicates that the benzalkonium chloride is spotted for significantly reducing the tear secretion, and the success of the construction of the benzalkonium chloride eye-induced dry eye model of the mice is verified. After eye drop treatment for 7 days, the tear secretion of the mice in the group 2 and the group 3 is obviously increased compared with that before the administration, and is obviously increased compared with the group 1, which shows that the rAJ-6-1 sample and the sodium hyaluronate eye drops prepared by the application can promote the tear secretion of the mice to be increased, but the phosphate buffer solution can not change the tear secretion condition of the mice.

As can be seen from fig. 5, the thickness of the cornea epithelium in the groups 1 and 2 is thinned, the number of the epithelial cell layers is 2-3, but the number of the cornea epithelium in the group 3 is still 5-6, and the thickness is obviously higher than that of the cornea epithelium in the groups 1 and 2, which indicates that the recombinant apolipoprotein J prepared by the application can obviously promote the recovery of the damaged mouse cornea epithelium to treat xerophthalmia caused by the recovery by combining with tear film mucin.

The conjunctival goblet cells mainly secrete mucin MUC5Ac and the like, become important components of the ocular tear film, keep a certain lubrication degree between the eyelid and the eyeball, reduce the movable friction force between the eyelid conjunctiva and the eyeball surface and maintain the moist microenvironment of the conjunctiva and the cornea epithelium. As can be seen from fig. 6, the number of goblet cells in the model for 7 days (D7) was significantly reduced compared to the control group, and the number of goblet cells in the groups 1, 2, and 2 was also down-regulated compared to the control group after 7 days (D14) of treatment, but the difference was not large compared to the number of goblet cells in the model for 7 days; the difference in goblet cell number of group 3 compared to the control group was not statistically significant, but significantly increased compared to 7 days of modeling (D7); whereas on treatment day 7 (D14) the goblet cell numbers were increased for groups 2, 3 compared to group 1, with the most significant increase in group 3, these results indicate that recombinant apolipoprotein J prepared according to the application significantly promoted conjunctival goblet cell production.

EXAMPLE 7 corneal epithelial injury repair experiment

7.1 Experimental methods

The number of the tested mice is 25, fluorescent staining is carried out first day before molding, slit lamp observation is carried out, 20 mice with normal cornea and negative fluorescent staining are selected, and the mice are randomly divided into five groups B1, B2, B3, B4 and B5; mice were anesthetized, placed in lateral position, and the eyeballs were exposed. Disinfecting around eyes with iodophor. The tobicarbamide eye drops are used for carrying out mydriasis. The 0.5% procaine hydrochloride eye drops are used for carrying out surface anesthesia by eye dropping. The central cornea was marked with a 2.5mm trephine under a surgical microscope, and the corneal epithelium in the mark was scraped with a corneal epithelium scraper to avoid damaging the corneal stroma, and molding was performed.

The mice are administrated for two days after molding, the test product is dripped into each eye for 4 times every day, 9 am, 12 am, 3 pm and 6 am are administrated every day, five groups B1, B2, B3, B4 and B5 are dripped into each eye respectively, the test product rAJ-1, rAJ-6-1, rAJ-3, sodium hyaluronate eye drops and PB buffer solution are dripped into each eye respectively, and the concentration of each group rAJ is 45 mug/ml.

7.2 detection and results

7.2.1 sodium fluorescein staining

After scraping 0h, 12h, 24h, 36h, 2 μl of 0.1% sodium fluorescein solution was used to drop eyes, and after blinking was rinsed with normal saline; the staining condition of corneal sodium fluorescein was observed by slit lamp photographing, the wound surface area was quantitatively determined by ImageJ software according to fluorescein staining images, the epithelial wound healing rate was calculated as the wound closure percentage at different time points, and the result is shown in fig. 7.

As can be seen from fig. 7, after 12 hours of eye drop treatment with different drugs, the five groups of mice had cornea epithelium wound healing rates of 22.88%, 30.17%, 25.91%, 26.68%, 18.34%, and the groups B1-B4 had no significant difference (P > 0.05) in cornea epithelium wound healing rates, with the group B2 mice having the highest cornea epithelium wound healing rate and the group B5 mice having the lowest cornea epithelium wound healing rate, respectively; after 24h treatment, the corneal epithelial wound healing rates of the five groups of mice were 81.26%, 87.91%, 81.89%, 83.53%, 69.47%, respectively, with the lowest corneal epithelial wound healing rate for group B5 mice, with significant differences (P < 0.05) compared to the other groups of mice; after 36h treatment, the corneal epithelial wounds of the five mice were substantially completely healed, the corneal surfaces of the mice of groups B1-B4 were smooth, and the mice of group B5 had rough corneal surfaces and positive for sodium fluorescein staining. The results show that after treatment, the B1-B4 test sample has a certain repairing effect on the mice with damaged corneal epithelium, wherein the B2 test sample has the best treatment effect.

7.2.2HE staining and immunofluorescent staining

After the eye surface detection is finished on the 4 th day of the experiment, the mice are euthanized, then the whole eyeballs and accessory tissues of the left eye and the right eye of each group of mice are taken out to prepare frozen sections, HE staining and immunofluorescence staining are respectively carried out, and the results are shown in fig. 8 and 9.

The immunofluorescence staining method comprises the following steps: 1) Taking out the slide from the refrigerator at-80 ℃ and airing, and placing the slide in cold acetone for fixing for 10min; 2) Washing with 1 XPBS three times for 5min each; 3) Dripping 0.2% TritonX-100 onto slide tissue, and penetrating into membrane for 20min; 4) Washing with 1 XPBS three times for 5min each; 5) 2% BSA was added dropwise to the slide tissue and blocked for 1h at room temperature; 6) Diluting the corresponding primary antibody (Ki 67, E-cadherin, PMN, F4/80) with 1% BSA, dripping the primary antibody after the blocking is finished, and placing the primary antibody in a wet box for incubation at 4 ℃ overnight; 7) Washing with 1 XPBS three times for 10min each; 8) Diluting the corresponding secondary antibody by using 1% BSA, dripping the secondary antibody, and placing the secondary antibody in a wet box for incubation for 1h at room temperature in a dark place; 9) Washing with 1 XPBS three times for 10min each; 10 Counterstaining the plaques with DAPI.

As shown in fig. 8, compared with the control group, the cornea epithelium of the mice in group B2 is complete, the thickness is similar to that of the control group, the matrix fibers are orderly arranged, and no inflammatory cell infiltration exists; the epithelia of the mice in the groups B1, B3 and B4 are similar in thickness, the thickness is thinner than that of the mice in the control group, a small amount of inflammatory cells infiltrate into the cornea stroma of the mice in the groups B1 and B3, and a large amount of inflammatory cells infiltrate into the cornea stroma of the mice in the group B4; the cornea epithelium of the mice in the B5 group is only of a single-layer structure, the cornea epithelium is not completely healed, and inflammatory cells infiltrate into cornea stroma.

As can be seen from fig. 9, after eye drop treatment, the five groups of mouse cornea epithelium have Ki67 and E-cadherin positive expression with different degrees, and the initial study of the use of a certain protein medicine in the groups B4 and B5 for treating the repair of mouse cornea epithelium injury has the least number of Ki67 positive cells, and the E-cadherin expression is reduced compared with the control group; the E-cadherin expression of the cornea epithelium of the mice in the B1 and B3 groups is similar, the number of Ki67 positive cells is increased compared with the control group, but the number of Ki67 positive cells is not significantly different from the number of Ki67 positive cells in the control group; the number of corneal epithelial Ki67 positive cells was significantly increased in group B2 mice compared to the control group (P < 0.005) and E-cadherein expression was consistent compared to the control group. The results show that the test products B1, B2 and B3 have the effect of promoting the proliferation of the corneal epithelial cells of the mice, and the medicine effect is that B2 is more than B1 and equal to B3.

After eye drop treatment, PMN and F4/80 positive expression occurs in the cornea stroma of five groups of mice to different degrees, and the number of PMN and F4/80 positive cells in the cornea stroma of five groups of mice is increased compared with that of a control group, wherein a small number of PMN and F4/80 positive cells exist in the cornea stroma of groups B1 and B3 of mice; the number of PMN and F4/80 positive cells in the cornea stroma of the mice in the B2 group is minimal and has no significant difference (P > 0.05) compared with the control group; a large number of PMN and F4/80 positive cells exist in cornea stroma of the mice in the groups B4 and B5, which shows that the test products B1, B2 and B3 have the functions of promoting proliferation of cornea epithelial cells, adhering cells and inhibiting inflammation on the mice with cornea epithelial damage, and the drug effect is that B2 is more than B1 and approximately equal to B3. In conclusion, the recombinant apolipoprotein J prepared by the application can be used as eye drops to supplement natural apolipoprotein J of ocular surface tissues, and simultaneously regulate apoptosis and/or cell proliferation signal channels through the surface receptors of the ocular conjunctival epithelial cells, promote the proliferation of the ocular conjunctival epithelial cells, inhibit inflammatory reaction, and further improve and treat the condition that the ocular conjunctiva is damaged caused by mechanical injury such as cataract surgery and the like.

EXAMPLE 8 STZ-induced diabetic rat Dry eye

Male SD rats (weight 240-320 g/serving) of about 7-9 weeks old are taken and subjected to national science and technology limited SPF animal house adaptation environment for 3 days, and the standard of feeding environmental conditions is referred to national standard GB 14995-2010 of the people's republic of China.

Healthy rats were selected to be fasted without water for 16 hours, the skin at the injection site was disinfected, and a single bolus of STZ solution was injected intraperitoneally at 60mg/kg body weight. After the STZ was molded, 5% glucose was drunk overnight, blood was taken from the tail tip 1 week after STZ administration to measure blood glucose, and rats showing random blood glucose higher than 16.7mmol/L twice a week and symptoms of polydipsia, polyphagia, polyuria and emaciation were identified as diabetic models.

Part of the diabetic SD rat model over time may be complicated by diabetic dry eye complications; after molding for 4 weeks, the body weight, blood sugar value and tear secretion were measured, and the tear secretion and tear film rupture time were significantly shortened compared with those before molding, indicating that the molding was successful. Model mice were divided into 3 groups, i.e., experiment 1 group, 2 group, 3 group, each of which was 5, wherein experiment 1 group, 2 group, 3 group were continuously dosed with 0.1mol/L sodium citrate-citric acid (ph=4.5) buffer, commercially available sodium hyaluronate eye drops, rAJ-6-1 as therapeutic drug eye drops (concentration 60 μg/ml), each group was 5 μl/eye/each group, four times per day 09:00, 12:00, 15:00, 18:00 eye drops, and normal animals were used as control groups for 14 days. Blood glucose, tear film break time, and index of extent of corneal epithelial loss were measured on days 0 and 7 from the start of administration, and the results are shown in Table 6.

Wherein citrate buffer: precisely weighing 2.1g of citric acid, adding 100mL of ultrapure water, and preparing solution A; precisely weighing 2.94g of sodium citrate, adding 100mL of ultrapure water to prepare solution B, mixing A, B solution according to a ratio of 1:1.32 (V: V) to prepare 0.1mol/L sodium citrate-citric acid buffer solution (pH=4.5); STZ solution: precisely weighing 0.2g of STZ powder, placing into a dry sterile centrifuge tube, strictly avoiding light, dissolving in precooled sodium citrate-citrate buffer solution to prepare 11mg/ml solution, filtering and sterilizing by a microporous filter membrane of 0.22 mu m, requiring 5min to complete preparation, adding ice after preparation, and finishing using within 10 min. STZ and sodium citrate-citric acid buffer solution are stored at low temperature and are prepared for use.

The tear film rupture time detection method comprises the following steps: rats were induced for anaesthesia with 3-4% isoflurane concentration, 1-2% isoflurane concentration maintained for anaesthesia, gas flow: 0.6L/min. After the measurement of the lacrimal secretion, 10. Mu.L of 0.1% sodium fluorescein solution was aspirated by a pipette, and the solution was dropped into conjunctival sac, and the eye was opened and ruptured by hand 3 times, and observed under cobalt blue light of a slit lamp microscope. The stopwatch is timed, the time from the last instant to the occurrence of the 1 st black spot of the cornea is recorded, repeated for 3 times, and an average value is obtained. The time for which no black spot occurred was calculated as 60 s.

The cornea epithelial loss degree index detection method comprises the following steps: rats were induced for anaesthesia with 3-4% isoflurane concentration, 1-2% isoflurane concentration maintained for anaesthesia, gas flow: 0.6L/min. 2 mu L of 1% sodium fluorescein solution is sucked by a pipette, dripped into conjunctival sac, manually and transiently washed for 1min, the conjunctival sac is washed by pure water for 5 times, 1mL each time, redundant dye in the conjunctival sac is sucked out, the conjunctival sac is placed under a fluorescent body view mirror for photographing, and fluorescence intensity analysis is carried out on photographed pictures by using fluorescence intensity analysis software (Image-Pro Plus 6.0).

Table 6 weight and blood glucose statistics for mice of each group

TABLE 7 tear film rupture time and fluorescein staining results statistics for rats of each group

Modeling first diary was M1, and experimental first diary was D1.

As is clear from tables 6 to 7, the recombinant apolipoprotein J of the present application has no therapeutic effect on diabetes itself, but has an obvious therapeutic effect on the complication caused by diabetes, dry eye.

EXAMPLE 9 therapeutic action on glaucoma

Taking 55 male SD rats, taking 50 rats after quarantine is qualified, performing general anesthesia by using isoflurane, administering the procaine hydrochloride eye drops to experimental eyes (right eyes) for surface anesthesia, spreading eyelids, shearing bulbar conjunctiva along upper corner scleral margin, passively separating fascia, exposing episcleral veins, and cauterizing two episcleral veins beside rectus muscle and one episcleral vein on temporal side of the rats. Sign of successful cauterization: the blood vessels of the near-angle scleral edge are angry, and the blood flow of the far-angle scleral edge is disappeared without bleeding. After the operation, the conjunctiva is intermittently sutured by a 10-0 non-absorbable suture line, the levofloxacin eye drops are administered after suturing, the rat is put back into a cage after waking up, the levofloxacin eye drops are used for eye dropping after the operation for 2 times per day, and the drug is continuously used for one week.

On day 3 after molding, selecting 25 relatively uniform animals according to intraocular pressure and weight, randomly dividing the animals into an experiment 1 group, an experiment 2 group and an experiment 3 group, wherein each group comprises 5 animals; after the grouping, the right eye drops were carried out four times per day for 09:00, 12:00, 15:00, 18:00 eyes with 0.1mol/L of sodium citrate-citric acid (pH=4.5) buffer, commercial sodium hyaluronate eye drops, (concentration of 60. Mu.g/ml) rAJ-6-1 as therapeutic agent, and the administration was continued for 4 weeks. The intraocular pressure was measured with a rebound tonometer in a awake state of rats 1 time per week before, after 30 minutes after molding, before grouping, and after administration, and the average value was obtained 3 times, and the results are shown in table 8.

Table 8 statistics of ocular pressure data for different groups

As can be seen from table 8, the recombinant apolipoprotein J of the present application has a remarkable therapeutic effect on glaucoma.

Although the present application is disclosed above, the present application is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the application, and the scope of the application should be assessed accordingly to that of the appended claims.

Claims

1. Use of recombinant apolipoprotein J in the preparation of a medicament for the treatment of an ophthalmic disease, characterized in that said recombinant apolipoprotein J comprises an amino acid primary sequence encoding an apolipoprotein J, said amino acid primary sequence having two cysteines therein and a tertiary structure formed by the same having a pair of disulfide bonds.

2. The use according to claim 1, wherein at least 95% of the primary amino acid sequence is identical to SEQ ID No. 1.

3. The use according to claim 2, wherein the cysteine is located at amino acid positions 107 and 263, or at amino acid positions 99 and 273, or at amino acid positions 94 and 280, or at amino acid positions 91 and 283, or at amino acid positions 80 and 291 of SEQ ID NO. 1.

4. The use according to claim 1, wherein the recombinant apolipoprotein J further comprises a signal peptide sequence linked to the N-terminus of SEQ ID No. 1, at least 95% of the signal peptide sequence being identical to SEQ ID No. 2 or SEQ ID No. 3.

5. The use according to any one of claims 1 to 4, wherein the medicament is in the form of an eye drop or an eye ointment.

6. The use according to claim 5, wherein the ophthalmic disease is any of dry eye, glaucoma, keratoconjunctival damage, meibomian gland dysfunction.

7. The use according to claim 6, wherein the ophthalmic diseases are open angle glaucoma and dry fundus maculopathy.

8. A pharmaceutical formulation comprising recombinant apolipoprotein J comprising an amino acid primary sequence encoding apolipoprotein J having two cysteines in the amino acid primary sequence such that the spatial structure thereof forms a pair of disulfide bonds.

9. The pharmaceutical preparation according to claim 8, wherein the pharmaceutical preparation is an eye drop or an eye ointment, and the eye drop is an artificial tear or an anti-inflammatory agent.