Use of recombinant fibrillin-2 protein in the preparation of products for preventing or treating fibrillin-2 deficiency eye diseases

Abstract

The present invention discloses the use of recombinant fibrillin-2 protein in the preparation of products for preventing or treating fibrillin-2 deficiency eye diseases. The present invention is the first to construct a method for a mouse model of FBN2 gene-deficient eye diseases by intravitreal injection of an FBN2 gene interference vector AAV, and is the first to discover that intravitreal injection of FBN2 recombinant protein has a therapeutic effect on FBN2 gene-deficient eye diseases. The research of the present invention further proves a series of pathological and physiological changes caused by FBN2 gene deficiency in the development of ocular tissue retinopathy and myopia, and that intravitreal injection of exogenous FBN2 recombinant protein can make up for the problem of endogenous FBN2 deficiency in the FBN2 gene-deficient eye disease model, thereby achieving the effect of treating the disease, providing a new method for one-step targeted gene intervention research and indicating the research direction.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to use of recombinant fibrillin-2 protein in preparing products for preventing or treating fibrillin-2 deficiency eye diseases. Background Art

[0002] Fibrillin-2 (FBN2) gene mutation has been shown to be an important cause of early-onset macular degeneration (EOMD) and age-related macular degeneration (AMD). In 2014, FBN2 gene mutation was found to be autosomal dominant in AMD families. In 2016, AMD patients with FBN2 gene mutation were found to have clinical manifestations of drusen. More and more people are suffering from AMD, and in severe cases, they will lose their vision. FBN2 protein is involved in the formation of microfibrils and is essential for the formation and remodeling of ECM. It is involved in the formation of elastic fibers in connective tissue throughout the body. AMD patients with FBN2 gene mutation have reduced FBN2 protein expression or loss of function in their eyes, which changes its permeability, elastin fiber packaging, and flexibility, which is an important cause of retinopathy. However, the mechanism of action of FBN2 gene defect in the occurrence and development of retinopathy is still unclear.

[0003] Adeno-associated virus (AAV) has the advantages of a wide host range, multiple serotypes, high titer, high safety, high accuracy, stable expression, and low immunogenicity. It has been widely used in basic medical research and clinical trials and is regarded as one of the most promising research gene therapy vectors.

[0004] Protein targeted intervention is widely used in the treatment of ophthalmic diseases, such as antibodies (vascular endothelial growth factor, VEGF), etc., but there are no reports on the research of targeted drugs for FBN2 gene defective retinopathy and myopia. Therefore, this application provides a new strategy for the clinical diagnosis and treatment of FBN2 gene defective eye diseases. Summary of the invention

[0005] In view of the defects in the prior art, the main purpose of the present invention is to provide a use of recombinant fibrillin-2 protein in the preparation of products for preventing or treating fibrillin-2 deficiency eye diseases.

[0006] This application uses AAV as a vector to construct RNAi for gene-defective eye diseases, and injects recombinant proteins for targeted intervention therapy, providing a new method for targeted treatment of retinopathy. This application study found that the gene-defective retinal disease model that can be constructed by RNAi using AAV as a vector is mainly manifested by the appearance of drusen-like exudates in the fundus, rough reflective areas in retinal coherence optical tomography, and thinning of the outer granular layer of the retina, which are all clinical manifestations of retinopathy. After targeted intervention therapy with injection of recombinant protein, fundus exudation decreased, and the outer granular layer of the retina thickened, which played a certain therapeutic role in retinopathy.

[0007] In one aspect, the present invention provides a use of a recombinant fibrillin-2 protein in the preparation of a product for preventing or treating a fibrillin-2-deficient eye disease.

[0008] Furthermore, the fibrillin-2-deficient eye disease is selected from at least one of hereditary retinopathy, axial myopia and congenital macular degeneration.

[0009] Furthermore, the product is a medicine, a reagent or a vaccine.

[0010] In one embodiment, the present invention provides the use of recombinant fibrillin-2 protein for regulating electroretinogram waveform amplitude for non-therapeutic purposes;

[0011] Preferably, the regulating the electroretinogram waveform amplitude is to restore the electroretinogram waveform amplitude to normal;

[0012] Preferably, the electroretinogram waveform amplitude is selected from at least one of the dark-adapted rod cell response waveform amplitude, the dark-adapted maximum mixed response waveform amplitude, the dark-adapted oscillatory potential response waveform amplitude and the light-adapted cone cell response waveform amplitude.

[0013] In one embodiment, the present invention provides the use of recombinant fibrillin-2 protein for reducing retinal exudation and fundus deposits for non-therapeutic purposes.

[0014] In one embodiment, the present invention provides the use of recombinant fibrillin-2 protein for increasing the thickness of the retinal outer nuclear layer for non-therapeutic purposes.

[0015] In one embodiment, the recombinant fibrillin-2 protein is used for non-therapeutic purposes to reduce the axial length of the eye.

[0016] The present invention provides the use of recombinant fibrillin-2 protein for increasing the expression of fibrillin-2 for non-therapeutic purposes;

[0017] Preferably, the expression of fibrillin-2 is selected from the expression of fibrillin-2 gene or the expression of fibrillin-2 protein;

[0018] Preferably, the expression of the fibrillin-2 gene is the expression of the fibrillin-2 gene mRNA.

[0019] On the other hand, the present invention provides a product for preventing or treating fibrillin-2 deficiency eye disease, wherein the product contains recombinant fibrillin-2 protein as the only active ingredient, or is a composition containing recombinant fibrillin-2 protein.

[0020] Furthermore, the product is a medicine, and the composition also includes pharmaceutical excipients;

[0021] Preferably, the pharmaceutical excipient is selected from at least one of a diluent, an excipient, a flavoring agent and a sweetener;

[0022] Preferably, the drug preparation is at least one of a suspension, an emulsion, a solution, a syrup or an injection;

[0023] Preferably, the drug is administered by drip or injection;

[0024] Preferably, the drug is administered by intravitreal injection.

[0025] Furthermore, the content of recombinant fibrillin-2 protein in the product is 0.01~5.0 μg / μL; preferably, the content of recombinant fibrillin-2 protein in the product is 0.1~1.0 μg / μL; preferably, the content of recombinant fibrillin-2 protein in the product is 0.2~0.5 μg / μL.

[0026] The beneficial effects of the present invention are as follows:

[0027] 1. The present invention has demonstrated in the experiments of constructing an FBN2 gene defective eye disease model and injecting FBN2 protein that exogenous FBN2 recombinant protein can improve retinopathy and myopic axial length growth caused by FBN2 gene defect.

[0028] 2. The present invention can construct an FBN2 gene-deficient eye disease mouse model by injecting the FBN2 gene interference vector AAV, which is mainly manifested by yellow-white drusen-like deposits in the fundus, thinning of the ONL, axial lengthening, decreased ERG waveform amplitude showing an extinguished type, and decreased mRNA and protein expression of FBN2.

[0029] 3. The present invention can effectively treat FBN2 gene defective retinopathy by injecting exogenous FBN2 recombinant protein, reduce the generation of yellow-white drusen-like deposits in the fundus, increase ONL thickness, restore ERG waveform amplitude, and increase FBN2 mRNA and protein expression.

[0030] 4. The present invention is the first to construct an animal model of FBN2 gene deficiency by intravitreal injection of FBN2 gene interference vector AAV, and is the first to find that intravitreal injection of FBN2 recombinant protein has a therapeutic effect on FBN2 gene deficiency eye diseases. The present invention's research further proves that FBN2 gene deficiency causes a series of pathological and physiological changes in the occurrence and development of retinopathy and myopia, and that intravitreal injection of exogenous FBN2 recombinant protein can make up for the endogenous deficiency of FBN2 in FBN2 gene deficiency eye diseases, thereby achieving the effect of treating the disease, providing a new method for the next step of targeted gene intervention research and indicating the research direction. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Results of scanning confocal ophthalmoscopy and optical coherence tomography after different treatments;

[0032] Figure 2 Results of Rod-b, Max-a, OPs total, and Cone-b waveform amplitudes after different processing;

[0033] Figure 3 Results of axial length after different treatments; a: apparent axial length diagram; b: axial length statistical diagram;

[0034] Figure 4 The results are for the mRNA expression of FBN2 gene in the retina after different treatments;

[0035] Figure 5 The results of ELISA test for FBN2 protein expression in the retina after different treatments;

[0036] Figure 6 The western blot results of FBN2 protein expression in the retina after different treatments;

[0037] In the above figure, * indicates that the difference between the normal control group and the negative control group under the same conditions is significant at the α=0.05 level; & indicates that the difference between the negative control group and the AAV group under the same conditions is significant at the α=0.05 level; # indicates that the difference between the AAV group and the AAV group under the same conditions is significant at the α=0.05 level. DETAILED DESCRIPTION

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0039] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. The purity of the FBN-2 recombinant protein used in the following examples is > 97%.

[0040] Example 1

[0041] The experiment used 8-week-old C57BL / 6J mice, SPF grade, and body weight of 21-23g. The mice were screened for eye health before enrollment to exclude corneal disease, cataracts, and retinal fundus diseases through scanning laser ophthalmoscope (SLO), optical coherence tomography (OCT), and electroretinogram (ERG).

[0042] Construction of FBN2 adenovirus-related interference vector: First, synthesize single-stranded DNA oligonucleotides, then generate double-stranded DNA oligonucleotides, and then directly connect them to the RNA interference vector after restriction digestion through the restriction sites contained at both ends (BamHI recognition sequence: 5' G^GATCC 3', EcoRI recognition sequence: 5' G^AATTC 3'); the correct clone is successfully constructed. Co-transfect AAV-293 cells, package the virus, collect the stock solution, concentrate and purify to obtain a concentrated solution. Extract the high-purity, endotoxin-free AAV vector, use HG transgene reagent to co-transfect the constructed viral vector and plasmid into AAV-293 cells, add Enhancing buffer 10-12 hours after transfection, and then replace fresh culture medium 8 hours later. After 48 hours of culture medium, the cells and supernatant including the virus are collected after the cells fall off, and then concentrated and purified to obtain AAV virus with a titer of 1.21E+12 VG / mL.

[0043] The amino acid sequence of FBN-2 recombinant protein is: TPGRYECNCPPDFQLNPTGVGCVDNRVGNCYLKFGPRGDGLSCNTEIGVGVSRSSCCCSLGKAWGNPCETCPPVNSTEYYTLCPGGEGFRPNPITIILEDIDECQELPGLCQ GGNCINTFGSFQCECPQGYYLSEDTRICEDIDECFAHPGVCGPGTCYNTLGNYTCICPPEYMQVNGGHNCMDMRKSFCYRSYNGTTCENELPFNVTKRMCCCTYNVGKAWNKPCEPCPTPGTADFKTIC.

[0044] The FBN-2 recombinant protein was efficiently produced by the host-vector system of E. coli. The buffer for diluting the FBN-2 recombinant protein included 20mM Tris, 150mM NaCL, 1mM EDTA, 1mM dithiothreitol DTT, 0.01% sodium lauryl sarcosine, 5% trehalose and proclin 300. The original concentration of the FBN-2 recombinant protein was 200μg / μL.

[0045] Sixty-three 8-week-old C57BL / 6J mice were randomly divided into 7 groups: normal control group, negative control group, AAV group, FBN2 ultra-low concentration group (0.3μg / eye), FBN2 low concentration group (0.75μg / eye), FBN2 medium concentration group (1.5μg / eye), and FBN2 high concentration group (3.0μg / eye) (n=9 / group). The normal control group was fed normally without any treatment; after enrollment, the normal group was fed normally, and the negative control group was injected with 3μL negative virus stock solution (AAV inactivated virus stock solution) into the vitreous cavity of both eyes; the AAV group, FBN2 ultra-low concentration group, FBN2 low concentration group, FBN2 medium concentration group, and FBN2 high concentration group were injected with 3μL AAV virus into the vitreous cavity of both eyes to establish the FBN2 gene defective eye disease model. After 2 weeks, the FBN2 ultra-low concentration group, FBN2 low concentration group, FBN2 medium concentration group, and FBN2 high concentration group were injected into the vitreous cavity of both eyes with a concentration of 0.1μg / μL, 0.25μg / μL, 0.50μg / μL, and 1μg / μL, with a dose of 3μL of FBN2 recombinant protein. The FBN2 ultra-low concentration group, FBN2 low concentration group, FBN2 medium concentration group, and FBN2 high concentration group were injected with 0.3μg / eye, 0.75μg / eye, 1.5μg / eye, and 3μg / eye of FBN2 recombinant protein, respectively, once a week for 3 consecutive times. Among them, the specific operation of the vitreous cavity injection includes: one day in advance, ofloxacin eye drops (3 times / d) were used to reduce eye inflammation in the negative control group, AAV group, and FBN2 injection group. Intraperitoneal injection of 10g•L -1 Sodium pentobarbital solution (50 mg kg -1 ) for anesthesia, ensure normal breathing, and drop mydriatic drugs to dilate the pupil. Place the anesthetized mouse under a microscope, disinfect the area around the eye with povidone-iodine, rinse off the surface povidone-iodine with 0.9% saline 1 minute later, then dry it with a dry sterile cotton swab, and use oxybuprocaine hydrochloride eye drops for ocular surface anesthesia. Clamp the bulbar conjunctiva with microscope toothed forceps, use a microsyringe (10L), insert the needle behind the corneoscleral limbus, clamp the needle hole for 30 seconds, release the forceps and drop the Debisu eye drops, and continue to drop Debisu (3 times) 1 day after surgery.

[0046] SPSS21.0 statistical software was used to analyze the experimental results data, and normal distribution and homogeneity of variance were tested. All experiments involved were multi-group experiments, and variance analysis with independent sample t test was used. Measurement data were expressed as x ± S, and variance analysis was used for differences between groups. Levene's test was used for homogeneity of variance of each indicator in each group. P < 0.05 was considered to be statistically significant.

[0047] Example 2

[0048] After each injection of FBN2 recombinant protein, SLO was used to detect fundus changes.

[0049] The specific operation of scanning confocal ophthalmoscopy for SLO examination includes: before the test, intraperitoneal injection of 10g·L -1 Sodium pentobarbital solution (50 mg kg -1 ) for anesthesia, dilate the pupil, and apply ofloxacin eye ointment to the cornea until the mouse is completely anesthetized. Align the mouse's eyes to the scanning laser ophthalmoscope lens, adjust the mouse's position, and take the computer monitor to capture a complete eye map as the standard. Take at least 3 pictures for each group and save the images. All operations are performed by the same researcher. The test results are shown in the figure. Figure 1 shown.

[0050] Depend on Figure 1 The results showed that after the injection of negative virus, the negative control group showed no significant changes in the retinal fundus map compared with the normal control group; after the injection of AAV virus, retinal exudation and yellow-white drusen-like deposits appeared in the AAV group and each FBN2 injection group. After one injection of FBN2 protein, there were no significant changes in the exudation and deposits of the FBN2 ultra-low concentration group, FBN2 low concentration group, FBN2 medium concentration group, and FBN2 high concentration group. After two injections of FBN2 protein, the retinal exudation and deposits in the FBN2 low concentration group were significantly reduced, the exudation and deposits in the FBN2 ultra-low concentration group and the medium concentration group were slightly reduced, and there was no significant change in the FBN2 high concentration group. After three injections of FBN2 protein, the retinal exudation and deposits in the FBN2 low concentration group were significantly reduced, the exudation and deposits in the FBN2 ultra-low concentration group and the medium concentration group were significantly reduced, and the exudation and deposits in the FBN2 high concentration group were slightly reduced and the changes were not obvious.

[0051] Example 3

[0052] After each injection of FBN2 recombinant protein, ERG was used to detect retinal function. The specific operation of ERG included: ERG detection was performed in an absolutely dark environment. Before the test, the mice were placed in a dark environment for more than 8 hours for dark adaptation. Before the test, 10 g•L -1 Sodium pentobarbital solution (50 mg kg -1) for anesthesia, and dilate the pupil. Use oxybuprocaine hydrochloride eye drops for ocular surface anesthesia, and apply ofloxacin eye ointment to the cornea until the mouse is completely anesthetized. Place the animal on the operating table, and use the RETI port system to fix the gold ring corneal electrode (diameter 3.00mm) on the cornea of ​​both eyes of the mouse. Insert the needle-shaped reference electrode into the thick muscles on both sides of the left and right corners of the mouse's mouth, and slowly insert the needle-shaped ground electrode into the subcutaneous muscle of the right upper limb of the mouse. After the ground wire on the detection screen is stable, start recording the EEG Rod-ERG, Max-ERG, Ops total, and after 10 minutes of light adaptation, record the Cone-ERG and Flick-ERG waveforms, as well as the changes in each amplitude. Take 3 values ​​for each waveform and analyze them. The results are shown in the figure. Figure 2 shown.

[0053] (1) Dark-adapted rod photoreceptor response (Rod-b)

[0054] At the time of enrollment, the Rod-b wave values ​​of the normal control group, negative control group, AAV group, ultra-low FBN2 concentration group, low FBN2 concentration group, medium FBN2 concentration group, and high FBN2 concentration group were (140.40±18.79 μV, 138.41±17.42 μV, 141.35±15.80 μV, 140.22±18.33 μV, 140.97±17.58 μV, 139.14±15.36 μV, 141.35±18.03 μV), respectively.

[0055] After negative AAV injection, the amplitude of Rod-b waveform in negative control group did not change significantly at each time point compared with normal control (after negative AAV injection: 138.91±18.93 μV vs. 139.78±16.66 μV; FBN2 injection once: 142.91±17.44 μV vs. 140.97±16.09 μV, FBN2 injection twice: 141.16±15.09 μV vs. 141.29±19.72 μV; FBN2 injection three times: 136.91±17.73 μV vs. 145.38±18.31, P>0.05). After AAV injection, the amplitude of Rod-b waveform in each group became gentler and extinguished compared with negative control group (AAV group vs. negative control group: 27.18±4.21 vs. 138.91±18.93 μV, P>0.05). μV; FBN2 ultra-low concentration group vs. negative control group: 28.32±3.78 μV vs. 138.91±18.93 μV; FBN2 low concentration group vs. negative control group: 22.26±4.02 μV vs. 138.91±18.93 μV, FBN2 medium concentration group vs. negative control group: 23.45±4.27μV vs. 138.91±18.93 μV, FBN2 high concentration group vs. negative control group: 24.11±3.78 μV vs. 138.91±18.93μV, P<0.05).

[0056] After one injection of FBN2 protein, the Rod-b waveform amplitude of each group did not change significantly compared with the AAV group (FBN2 ultra-low concentration group vs. AAV group: 26.44±3.67 μV vs. 26.28±4.33 μV; FBN2 low concentration group vs. AAV group: 32.95±5.67 μV vs. 26.28±4.33 μV; FBN2 medium concentration group vs. AAV group: 30.38±4.29 μV vs. 26.28±4.33 μV; FBN2 high concentration group vs. AAV group: 27.55±1.79 μV vs. 26.28±4.33 μV, P>0.05). Compared with the AAV group, the Rod-b waveform amplitude of each group became gentle and showed an extinguishing type (AAV group vs. negative control group: 26.28±4.33μV vs. 142.91±17.44μV vs. μV; FBN2 ultra-low concentration group vs. negative control group: 26.44±3.67 μV vs. 142.91±17.44 μV; FBN2 low concentration group vs. negative control group: 32.95±5.67 μV vs. 142.91±17.44 μV; FBN2 medium concentration group vs. negative control group: 30.38±4.29 μV vs. 142.91±17.44 μV; FBN2 high concentration group vs. negative control group: 27.55±1.79 μV vs. 142.91±17.44 μV, P<0.05); the trends of each group compared with the normal control group were consistent with those compared with the negative control group.

[0057] After two injections of FBN2 protein, the waveform amplitude of the FBN2 low concentration group was increased compared with the AAV group (76.33±7.46 μV vs. 25.96±4.59 μV, P<0.05). There was no significant change in the Rod-b waveform amplitude of the FBN2 ultra-low concentration group, FBN2 medium concentration group, and FBN2 high concentration group compared with the AAV group (FBN2 ultra-low concentration group vs. AAV group: 28.97±5.00 μV vs. 25.96±4.59 μV; FBN2 medium concentration group vs. AAV group: 39.79±6.17 μV vs. 25.96±4.59 μV; FBN2 high concentration group vs. AAV group: 30.40±4.42 μV vs. 25.96±4.59 μV, P>0.05). Compared with the negative control group, the Rod-b waveform amplitude of each group was significantly lower (AAV group vs. negative control group: 25.96±4.59 μV vs. 141.16±15.09 μV; FBN2 ultra-low concentration group vs. negative control group: 28.97±5.00 μV vs. 141.16±15.09 μV; FBN2 low concentration group vs. negative control group: 76.33±7.46 μV vs. 141.16±15.09 μV; FBN2 medium concentration group vs. negative control group: 39.79±6.17 μV vs. 141.16±15.09 μV; FBN2 high concentration group vs. negative control group: 30.40±4.42 μV vs. 141.16±15.09 μV, P<0.05). The trends of each group compared with the normal control group were consistent with those compared with the negative control group.

[0058] After three injections of FBN2 protein, the Rod-b waveform amplitude of the FBN2 low-concentration group and the FBN2 medium-concentration group was significantly increased compared with the AAV group (FBN2 low-concentration group vs. AAV group: 118.33±12.42 μV vs. 26.25±3.88 μV; FBN2 medium-concentration group vs. AAV group: 77.00±8.51 μV vs. 26.25±3.88 μV, P<0.05). The Rod-b waveform amplitude of the FBN2 ultra-low-concentration group and the FBN2 high-concentration group was not significantly changed compared with the AAV group (FBN2 ultra-low-concentration group vs. AAV group: 31.25±4.79 μV vs. 26.25±3.88 μV; FBN2 high-concentration group vs. AAV group: 58.88±5.32 μV vs. 26.25±3.88 μV, P>0.05). Compared with the negative control group, the Rod-b waveform amplitude of the low FBN2 concentration group had no significant change (118.33±12.42 vs. 136.91±17.73 μV, P>0.05). The Rod-b waveform amplitude of the AAV group, ultra-low FBN2 concentration group, medium FBN2 concentration group, and high FBN2 concentration group were significantly lower (AAV group vs. negative control group: 26.25±3.88 μV vs. 136.91±17.73 μV; ultra-low FBN2 concentration group vs. negative control group: 31.25±4.79 μV vs. 136.91±17.73 μV; medium FBN2 concentration group vs. negative control group: 77.00±8.51 μV vs. 136.91±17.73 μV; high FBN2 concentration group vs. negative control group: 58.88±5.32 μV vs. 136.91±17.73 μV). μV, P>0.05); the trends of each group compared with the normal control group were consistent with those of the negative control group.

[0059] (2) Dark-adapted maximum mixed response (Max-a)

[0060] At the time of enrollment, the Rod-b wave values ​​of the normal control group, negative control group, AAV group, ultra-low FBN2 concentration group, low FBN2 concentration group, medium FBN2 concentration group, and high FBN2 concentration group were (-121.97±18.67 μV, -123.85±16.48 μV, -117.26±17.83 μV, -119.77±18.63 μV, -115.7±17.88 μV, -121.97±15.46 μV, -117.26±15.33 μV), respectively.

[0061] After negative AAV injection, the Max-a wave of the negative control group did not change significantly at each time point of the experiment compared with the normal control group (after negative AAV injection: -116.07±18.77 μV vs. -116.45±17.44 μV; FBN2 injection once: -117.05±15.83 μV vs. -117.45±17.33 μV; FBN2 injection twice: -118.29±17.42 μV vs. -116.45±18.68 μV; FBN2 injection three times: -116.05±16.71 μV vs. -119.5±17.33 μV, P>0.05). After AAV injection, the amplitude of the Max-a waveform of each group and the negative control group became gentle and showed an extinguishing type (AAV group vs. negative control group: -18.90±2.84 μV Compared with -116.07±18.77 μV; FBN2 ultra-low concentration group compared with negative control group: -17.19±2.67 μV vs. -116.07±18.77 μV; FBN2 low concentration group compared with negative control group: -15.65±2.35 μV vs. -116.07±18.77 μV; FBN2 medium concentration group compared with negative control group: -17.90±2.79 μV vs. -116.07±18.77 μV; FBN2 high concentration group compared with negative control group: -15.07±3.07 μV vs. -116.07±18.77 μV, P<0.05); the comparison trend of each group with the normal control group was consistent with that with the negative control group.

[0062] After one injection of FBN2 protein, the amplitude of electroretinogram waveform in the ultra-low concentration FBN2 group, low concentration FBN2 group, medium concentration FBN2 group, and high concentration FBN2 group was not significantly changed compared with the AAV group (ultra-low concentration FBN2 group vs. AAV group: -19.49±2.33 μV vs. -18.40±3.33 μV; low concentration FBN2 group vs. AAV group: -25.65±5.22 μV vs. -18.40±3.33 μV; medium concentration FBN2 group vs. AAV group: -22.53±4.67 μV vs. -18.40±3.33 μV; high concentration FBN2 group vs. AAV group: -19.90±3.67 μV vs. -18.40±3.33 μV, P>0.05); compared with the negative control group, the amplitude of MAX-a waveform in each group became gentle and showed an extinguishing type (AAV group vs. negative control group: -18.40±3.33μV vs. -117.05±15.83 μV; FBN2 ultra-low concentration group vs. negative control group: -19.49±2.33 μV vs. -117.05±15.83 μV; FBN2 low concentration group vs. negative control group: -25.65±5.22 μV vs. -117.05±15.83 μV; FBN2 medium concentration group vs. negative control group: -22.53±4.67 μV vs. -117.05±15.83 μV; FBN2 high concentration group vs. negative control group: -19.90±3.67 μV vs. -117.05±15.83, P<0.05) The comparison trends of each group with the normal control group were consistent with those with the negative control group.

[0063] After two injections of FBN2 protein, the Max-a waveform amplitude of the FBN2 low-concentration group and the FBN2 medium-concentration group was slightly increased compared with the AAV group (FBN2 low-concentration group vs. AAV group: -67.95±5.67 μV vs. -19.82±3.85 μV; FBN2 medium-concentration group vs. AAV group: -27.24±6.67 μV vs. -19.82±3.85 μV, P<0.05), and the Max-a waveform amplitude of the FBN2 ultra-low-concentration group and the FBN2 high-concentration group was not significantly changed compared with the AAV group (FBN2 ultra-low-concentration group vs. AAV group: -23.94±6.67 μV vs. -19.82±3.85 μV; FBN2 high-concentration group vs. AAV group: -24.54±5.44 μV vs. -19.82±3.85 μV, P<0.05). μV, P>0.05); compared with the negative control group, the amplitude of Max-a waveform in AAV group, FBN2 ultra-low concentration group, FBN2 low concentration group, FBN2 medium concentration group, and FBN2 high concentration group was lower (AAV group vs. negative control group: -19.82±3.85 μV vs. -118.29±17.42 μV; FBN2 ultra-low concentration group vs. negative control group: -23.94±6.67 μV vs. -118.29±17.42 μV; FBN2 low concentration group vs. negative control group: -67.95±5.67 μV vs. -118.29±17.42 μV; FBN2 medium concentration group vs. negative control group: -27.24±6.67 μV vs. -118.29±17.42 μV; FBN2 high concentration group vs. negative control group: -24.54±5.44 μV Compared with -118.29±17.42 μV, P<0.05); the trend of each group compared with the normal control group was consistent with that of the negative control group.

[0064] After 3 injections of FBN2 protein, the amplitude of Max-a waveform increased in the FBN2 low concentration group and FBN2 medium concentration group compared with the AAV group (FBN2 low concentration group vs. AAV group: -99.44±9.42 vs. -19.17±3.67 μV, FBN2 medium concentration group vs. AAV group: -65.27±8.67 μV vs. -19.17±3.67 μV, P<0.05). There was no significant change in the amplitude of Max-a waveform in the FBN2 ultra-low concentration group and FBN2 high concentration group compared with the AAV group (FBN2 ultra-low concentration group vs. AAV group: -24.72±4.33 μV vs. -19.17±3.67 μV; FBN2 high concentration group vs. AAV group: -30.57±5.88 μV vs. -19.17±3.67 μV, P>0.05). There was no significant difference in the Max-a waveform amplitude between the low-concentration FBN2 group and the medium-concentration FBN2 group and the negative control group (the low-concentration FBN2 group vs. the negative control group: -99.44±9.42 μV vs. -116.05±16.71 μV; the medium-concentration FBN2 group vs. the negative control group: -65.27±8.67 μV vs. -116.05±16.71 μV; P>0.05). The Max-a waveform amplitude of the AAV group, the ultra-low-concentration FBN2 group, and the high-concentration FBN2 group was significantly lower than that of the negative control group (the AAV group vs. the negative control group: -19.17±3.67 μV vs. -116.05±16.71 μV; the ultra-low-concentration FBN2 group vs. the negative control group: -24.72±4.33 μV vs. -116.05±16.71 μV). μV; FBN2 high concentration group vs. negative control group: -30.57±5.88 μV vs. -116.05±16.71 μV,); The trends of each group compared with the normal control group were consistent with those of the negative control group.

[0065] (3) Dark-adapted oscillatory potential response (OPs total)

[0066] At the time of enrollment, the OPs-total wave values ​​of the normal control group, negative control group, AAV group, ultra-low FBN2 concentration group, low FBN2 concentration group, medium FBN2 concentration group, and high FBN2 concentration group were (234.95±19.33μV, 226.28±20.21μV, 230.61±19.47μV, 231.45±20.22μV, 232.11±18.67μV, 226.58±21.03μV, 233.62±19.45 μV), respectively.

[0067] After negative AAV injection, there was no significant change in the OPs-total wave at each time point of the experiment between the negative control group and the normal control group (after negative AAV injection: 229.28±19.67 μV vs. 238.56±20.44 μV; FBN2 injection once: 226.7±18.37 μV vs. 250.95±21.33 μV; FBN2 injection twice: 229.28±21.33 μV vs. 222.95±18.67 μV; FBN2 injection three times: 272.8±21.34 μV vs. 234.95±22.67 μV, P>0.05). After AAV injection, the amplitude of OPs-total waveform in each group became flatter and extinguished compared with the negative control group (AAV group vs. negative control group: 19.56±2.97 μV vs. 229.28±19.67 μV; FBN2 ultra-low concentration group vs. negative control group: 19.96±3.03μV vs. 229.28±19.67 μV; FBN2 low concentration group vs. negative control group: 22.75±3.57 μV vs. 229.28±19.67 μV; FBN2 medium concentration group vs. negative control group: 21.56±4.05 μV vs. 229.28±19.67 μV; FBN2 high concentration group vs. negative control group: 26.75±3.33 μV vs. 229.28±19.67 μV, P<0.05). The trends of each group compared with the normal control group were consistent with those of the negative control group.

[0068] After one injection of FBN2 protein, there was no significant change in the OPs-total waveform amplitude in the ultra-low FBN2 concentration group, low FBN2 concentration group, medium FBN2 concentration group, and high FBN2 concentration group compared with the AAV group (ultra-low FBN2 concentration group vs. AAV group: 25.48±3.44 μV vs. 22.23±3.90 μV; low FBN2 concentration group vs. AAV group: 29.73±4.03 μV vs. 22.23±3.90 μV; medium FBN2 concentration group vs. AAV group: 26.37±3.67 μV vs. 22.23±3.90 μV; high FBN2 concentration group vs. AAV group: 25.39±4.85 μV vs. 22.23±3.90 μV, P>0.05). Compared with the negative control group, the amplitude of OPs-total waveform in each group was gentle and showed an extinguishing type (AAV group vs. negative control group: 22.23±3.90 μV vs. 226.7±18.37 μV; FBN2 ultra-low concentration group vs. negative control group: 25.48±3.44 μV vs. 226.7±18.37 μV; FBN2 low concentration group vs. negative control group: 29.73±4.03 μV vs. 226.7±18.37 μV; FBN2 medium concentration group vs. negative control group: 26.37±3.67 μV vs. 226.7±18.37 μV; FBN2 high concentration group vs. negative control group: 25.39±4.85 μV vs. 226.7±18.37 μV, P<0.05). The trends of each group compared with the normal control group were consistent with those compared with the negative control group.

[0069] After two injections of FBN2 protein, the OPs-total waveform amplitude in the FBN2 low concentration group was larger than that in the AAV group (107.03±18.03 μV vs. 24.51±8.67 μV, P<0.05), while the OPs-total waveform amplitude in the FBN2 ultra-low-medium concentration group, FBN2 medium concentration group, and FBN2 high concentration group had no significant changes (FBN2 ultra-low concentration group vs. AAV group 35.03±9.55 μV vs. 24.51±8.67 μV; FBN2 medium concentration group vs. AAV group 44.91±14.53 μV vs. 24.51±8.67 μV; FBN2 high concentration group vs. AAV group 30.18±5.44 μV vs. 24.51±8.67 μV, P>0.05). Compared with the negative control group, the OPs-total waveform amplitude of each group became significantly flatter (AAV group vs. negative control group: 24.51±8.67 μV vs. 229.28±21.33 μV; FBN2 ultra-low concentration group vs. negative control group: 35.03±9.55 μV vs. 229.28±21.33 μV; FBN2 low concentration group vs. negative control group: 107.03±18.03 μV vs. 229.28±21.33 μV; FBN2 medium concentration group vs. negative control group: 44.91±14.53 μV vs. 229.28±21.33 μV; FBN2 high concentration group vs. negative control group: 30.18±5.44 μV vs. 229.28±21.33 μV, P<0.05). The trends of each group compared with the normal control group were consistent with those of the negative control group.

[0070] After three injections of FBN2 protein, the OPs total waveform amplitude in the FBN2 low concentration group was significantly increased compared with the AAV group (133.56±18.77 μV vs. 25.08±8.76 μV, P<0.05). There was no significant change in the OPs-total waveform amplitude in the FBN2 ultra-low concentration group, FBN2 medium concentration group, and FBN2 high concentration group compared with the AAV group (FBN2 ultra-low concentration group vs. AAV group: 37.83±6.97 μV vs. 25.08±8.76 μV; FBN2 medium concentration group vs. AAV group: 70.75±23.33 μV vs. 25.08±8.76 μV; FBN2 high concentration group vs. AAV group: 58.31±9.24 μV vs. 25.08±8.76 μV, P>0.05). Compared with the negative control group, the FBN2 low concentration group had no change in the OPs total waveform amplitude (133.56±18.77 μV vs. 272.8±21.34 μV, P>0.05). Compared with the negative control group, the OPs-total waveform amplitude was significantly flatter in the AAV group, the ultra-low FBN2 concentration group, the medium FBN2 concentration group, and the high FBN2 concentration group (AAV group vs. negative control group: 25.08±8.76 μV vs. 272.8±21.34 μV; the ultra-low FBN2 concentration group vs. negative control group: 37.83±6.97 μV vs. 272.8±21.34 μV; the medium FBN2 concentration group vs. negative control group: 70.75±23.33 μV vs. 272.8±21.34 μV; the high FBN2 concentration group vs. negative control group: 58.31±9.24 μV Compared with 272.8±21.34 μV, P<0.05); the trend of each group compared with the normal control group was consistent with that of the negative control group.

[0071] (4) Light-adapted cone cell response (Cone- b)

[0072] At the time of enrollment, the Cone-b wave values ​​of the normal control group, negative control group, AAV group, ultra-low FBN2 concentration group, low FBN2 concentration group, medium FBN2 concentration group, and high FBN2 concentration group were (72.33±8.03 μV, 72.61±9.64 μV, 69.88±9.59 μV, 72.46±10.33 μV, 74.53±10.67 μV, 72.61±11.44 μV, 69.88±9.82 μV), respectively.

[0073] After negative AAV injection, there was no significant change in the OPs-total wave at each time point of the experiment between the negative control group and the normal control group (after negative AAV injection: 69.56±11.33 μV vs. 71.21±10.17 μV; FBN2 injection once: 68.77±9.33 μV vs. 75.21±9.47 μV; FBN2 injection twice: 70.23±6.63 μV vs. 77.22±8.93 μV; FBN2 injection three times: 71.03±8.44 μV vs. 77.31±9.03 μV, P>0.05). After AAV injection, compared with the negative control group, the amplitude of Cone-b waveform in each group became gentle and showed an extinguishing type (AAV group vs. negative control group: 3.89±2.82 μV vs. 69.56±11.33 μV, FBN2 ultra-low concentration group vs. negative control group: 4.77±2.63 μV vs. 69.56±11.33 μV, FBN2 low concentration group vs. negative control group: 3.43±3.44 μV vs. 69.56±11.33 μV, FBN2 medium concentration group vs. negative control group: 3.68±2.51 μV vs. 69.56±11.33 μV, FBN2 high concentration group vs. negative control group: 3.47±1.67 μV vs. 69.56±11.33 μV, P<0.05). The trends of each group compared with the normal control group were consistent with those compared with the negative control group.

[0074] After one injection of FBN2 protein, there was no significant change in the Cone-b waveform amplitude between each protein injection group and the AAV group (FBN2 ultra-low concentration group vs. AAV group: 3.93±2.56 μV vs. 3.03±2.02 μV, FBN2 low concentration group vs. AAV group: 6.21±2.67 μV vs. 3.03±2.02 μV, FBN2 medium concentration group vs. AAV group: 4.44±3.33 μV vs. 3.03±2.02 μV, FBN2 high concentration group vs. AAV group: 3.99±1.95 μV vs. 3.03±2.02 μV, P>0.05). Compared with the negative control group, the amplitude of Cone-b waveform in each group became gentler and showed an extinguishing type (AAV group vs. negative control group: 3.03±2.02 μV vs. 68.77±9.33 μV, FBN2 ultra-low concentration group vs. negative control group: 3.93±2.56 μV vs. 68.77±9.33 μV, FBN2 low concentration group vs. negative control group: 6.21±2.67 μV vs. 68.77±9.33μV, FBN2 medium concentration group vs. negative control group: 4.44±3.33 μV vs. 68.77±9.33 μV, FBN2 high concentration group vs. negative control group: 3.99±1.95 μV vs. 68.77±9.33 μV, P<0.05). The trend of each group compared with the normal control group was consistent with that of the negative control group.

[0075] After two injections of FBN2 protein, there was no significant change in the Cone-b waveform amplitude in each protein injection group compared with the AAV group (FBN2 ultra-low concentration group vs. AAV group: 7.67±3.22 μV vs. 3.67±2.97 μV; FBN2 low concentration group vs. AAV group: 21.18±8.48 μV vs. 3.67±2.97 μV; FBN2 medium concentration group vs. AAV group: 10.86±4.03 μV vs. 3.67±2.97 μV; FBN2 high concentration group vs. AAV group: 8.05±3.33 μV vs. 3.67±2.97 μV, P>0.05). Compared with the negative control group, the amplitude of Cone-b waveform in each group was significantly flatter (AAV group vs. negative control group: 3.67±2.97 μV vs. 70.23±6.63 μV; FBN2 ultra-low concentration group vs. negative control group: 7.67±3.22 μV vs. 70.23±6.63 μV; FBN2 low concentration group vs. negative control group: 21.18±8.48 μV vs. 70.23±6.63μV; FBN2 medium concentration group vs. negative control group: 10.86±4.03 μV vs. 70.23±6.63 μV; FBN2 high concentration group vs. negative control group: 8.05±3.33 μV vs. 70.23±6.63 μV, P<0.05). The trends of each group compared with the normal control group were consistent with those compared with the negative control group.

[0076] After three injections of FBN2 protein, compared with the AAV group, the Cone-b waveform amplitude of the FBN2 low concentration group was significantly increased, 25.61±7.67 μV vs. 3.17±2.11 μV, P<0.05), while the Cone-b waveform amplitude of the FBN2 ultra-low concentration group, FBN2 medium concentration group, and FBN2 high concentration group had no significant changes (FBN2 ultra-low concentration group vs. AAV group: 8.55±5.42 μV vs. 3.17±2.11 μV, FBN2 medium concentration group vs. AAV group: 18.57±8.03 μV vs. 3.17±2.11 μV, FBN2 high concentration group vs. AAV group: 10.66±4.43 μV vs. 3.17±2.11 μV, P>0.05). Compared with the negative control group, the amplitude of Cone-b waveform in each group was significantly flatter, but the therapeutic effect of the ultra-low concentration FBN2 group was the most obvious (AAV group vs. negative control group: 3.17±2.11 μV vs. 71.03±8.44 μV; ultra-low concentration FBN2 group vs. negative control group: 8.55±5.42 μV vs. 71.03±8.44 μV; low concentration FBN2 group vs. negative control group: 25.61±7.67 μV vs. 71.03±8.44 μV; medium concentration FBN2 group vs. negative control group: 18.57±8.03 μV vs. 71.03±8.44 μV; high concentration FBN2 group vs. negative control group: 10.66±4.43 μV vs. 71.03±8.44 μV, P<0.05). The trends of each group compared with the normal control group were the same as those of the negative control group.

[0077] Example 4

[0078] After each injection of FBN2 recombinant protein, the axial length of the eye was measured using OCT.

[0079] The specific operation of optical coherence tomography (OCT) includes: before the test, 10 g·L -1 Sodium pentobarbital solution (50 mg kg -1 ) for anesthesia, dilate the pupil, and apply ofloxacin eye ointment to the cornea until the mouse is completely anesthetized. Put on a corneal contact lens with a diameter of 3mm and a refractive power of -25D for the mouse, align the mouse's eye with the scanning lens, adjust the mouse's position and the lens distance, and take the computer screen to take the accurate position of the tomographic image as the standard. Take at least 3 pictures for each group and save the images, take pictures and analyze the images, and measure the axial length of the eye. The results are as follows Figure 3As shown. After sh-AAV / AAV injection, the axial length of the negative control group was (3.29±0.09 mm), and the axial length of the AAV group and the FBN2 protein injection group was (3.31±0.11 mm, 3.31±0.12 mm), with no statistical significance (P>0.05). The recombinant protein group was injected with FBN2 recombinant protein three times, with an interval of 9 days each time; at the same time, the AAV group was injected with an equal amount of Ringer's injection buffer. After injection, the axial length of the AAV group was significantly greater than that of the negative control group and the normal control group (AAV group vs. negative control group: 3.68±0.10 mm vs. 3.40±0.03 mm; AAV group vs. negative control group: 3.68±0.10 mm vs. 3.40±0.09 mm, P<0.05). The axial length of the FBN2 low-concentration protein injection group was significantly lower than that of the AAV group (3.35±0.03 mm vs. 3.68±0.10 mm, P<0.05). There was no statistically significant difference between the axial length of the FBN2 low-concentration protein injection group and the negative control group and the normal control group (P>0.05).

[0080] Example 5

[0081] Six retinal tissues were selected from each group during the experiment. The extraction of total RNA from retinal tissues and the RT-PCR reaction were performed according to the following method.

[0082] 1. Extraction of total RNA

[0083] (1) Tissue grinding: Take out the frozen retinal tissue, transfer it to liquid nitrogen, grind it with a glass rod, add 200μl Trizol and grind it for 2 min. Repeat the grinding. When there is no visible retinal tissue in the EP tube, add 500μl Trizol and mix it by pipetting (Note: the sample volume cannot exceed 10% of the volume of Trizol reagent).

[0084] (2) Separation: Let stand for 5 minutes. After the nucleoprotein complex is completely separated, add 150 μl of chloroform (trichloromethane), shake vigorously several times, let stand for 2-3 minutes (15-30°C), and then centrifuge in a pre-cooled high-speed centrifuge at 4°C for 20 minutes at 14500g. After centrifugation, RNA is present in the upper liquid.

[0085] (3) RNA precipitation: transfer the upper transparent liquid to a 1.5 mL EP tube, add 0.3 mL of isopropanol, mix by pipetting, let stand at room temperature, and then centrifuge in a pre-cooled high-speed centrifuge at 4°C, 14,500 g for 20 min;

[0086] (4) RNA washing: remove the supernatant, add 1 ml of 75% ethanol prepared by 0.25 ml of DEPC water and 0.75 ml of anhydrous ethanol, and centrifuge in a precooled high-speed centrifuge at 4 °C, 14500 g for 20 min;

[0087] (5) RNA re-dissolution: remove the supernatant and centrifuge in a pre-cooled high-speed centrifuge at 14,500 g for 20 min at 4°C. Remove excess liquid with a pipette. After drying for 3 min, add 30 μl of DEPC water to dissolve the RNA precipitate and mix by pipetting.

[0088] (6) The OD260 / OD280 value of the RNA sample is meaningful when it is between 1.8 and 2.0. The RNA purity and concentration are measured using a K5600 ultra-micro spectrophotometer. Take 1 μL of RNA sample and divide it into smaller pieces to reduce the risk of repeated freezing and thawing. Then perform reverse transcription and divide the remaining RNA sample into smaller pieces and store them at -80°C to avoid RNA degradation.

[0089] 2. First-strand cDNA synthesis

[0090] (1) Add 1 μL Random 6mers, 2 μL RNase Free dH2O, dNTP Mixture, 1 μL Oligo dT Primer and 5 μL RNA sample in sequence, mix well and centrifuge;

[0091] (2) Incubate for 5 min in a 65°C metal thermostatic pot;

[0092] (3) Add 1 μL PrimeScript II RTase, 0.5 μL RNase Inhabitor, 4 μL 5× PrimeScript II Buffer, and 4.5 μL RNase Free dH2O in sequence, mix well, and centrifuge;

[0093] (4) Incubate for 10 min (30°C), then transfer to a 42°C water bath for 60 min;

[0094] (5) Place the sample in a 95°C metal thermostat to inactivate the reverse transcriptase (5 min).

[0095] 3. Design and synthesis of primers

[0096] The primer sequences were synthesized by Shanghai Bioengineering, and β-actin was used as the internal reference. The primer sequences are shown in Table 1.

[0097] Table 1 Sequence list of primers for retinal molecules

[0098]

[0099] 4. Real-time fluorescence quantitative PCR reaction

[0100] RT-PCR was used to detect the relative expression of FBN2 and β-actin mRNA in the retina of each group of mice. The FBN2 gene was set up with three replicate wells, using a 96-well plate with a total volume of 20 μL, including: 8 μL cDNA template diluted 20 times, 1 μL upstream and downstream primers, 10 μL LightCycler®480 SYBR Green I Master, sealing, and light centrifugation. The reaction conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 10 s, annealing at 54°C for 30 s, extension at 72°C for 30 s, a total of 45 cycles, and finally cooling at 40°C for 10 s. 2 －△△CT Method, β-actin was used as internal reference. －△△CT Methods The fold changes of gene expression levels in the FBN2 antibody group and PBS group relative to the normal control group were calculated, and the relative expression levels of FBN2 and β-actin mRNA in the retina of each group of mice were quantitatively analyzed. Figure 4 shown.

[0101] Depend on Figure 4 The results showed that after negative AAV injection, the negative control group [(3.08±0.39)×10 -3 ] and the normal control group [(3.16±0.45)×10 -3 ], the expression of FBN2 gene mRNA had no significant change (P>0.05). After AAV injection, compared with the negative control group [(3.16±0.45)×10 -3 ] compared with AAV group [(1.36±0.24)×10 -3 ], FBN2 ultra-low concentration group [(1.35±0.26)×10 -3 ], FBN2 low concentration group [(1.45±0.30)×10 -3 ], FBN2 medium concentration group [(1.54±0.41)×10 -3 ], FBN2 high concentration group [(1.55±0.04)×10 -3 ], the mRNA expression of FBN2 gene was significantly decreased (P<0.05).

[0102] After FBN2 protein was injected three times, the FBN2 low concentration group [(3.00±0.37)×10 -3 ] The mRNA expression of FBN2 gene was higher than that of AAV group [(1.48±0.60)×10 -3] was significantly increased (P<0.05); the ultra-low concentration of FBN2 group [(1.69±0.71)×10 -3 ], FBN2 medium concentration group [(2.01±0.22)×10 -3 ], FBN2 high concentration group [(1.51±0.37)×10 -3 ] compared with the AAV group [(1.48±0.60)×10-3], there was no significant change in the mRNA expression of FBN2 gene ( P>0.05).

[0103] FBN2 low concentration group [(3.00±0.37)×10 -3 ] The mRNA expression of FBN2 gene was significantly higher than that of negative control group [(3.22±0.30)×10 -3 ] had no significant change (P>0.05); AAV group [(1.48±0.60)×10 -3 ], FBN2 ultra-low concentration group [(1.69±0.71)×10 -3 ], FBN2 medium concentration group [(2.01±0.22)×10 -3 ], FBN2 high concentration group [(1.51±0.37)×10 -3 ] Compared with the negative control group [(3.22±0.30)×10-3], the mRNA expression of FBN2 gene was significantly decreased (P<0.05); the trend of each group compared with the normal control group was consistent with that compared with the negative control group.

[0104] Example 6

[0105] After the experiment, an overdose of 10 g·L -1 The mice were killed with sodium pentobarbital solution, the eyeballs were removed with curved forceps and rinsed in physiological saline, then placed under a Zeiss microscope, cut along the corneoscleral margin, and then the retina was gently separated with an iris restorer and stored in a sterile, enzyme-free 1.5ml EP tube, quick-frozen in liquid nitrogen, and stored at -80°C for later use. During the experiment, 6 retinal tissues were selected from each group and transferred to liquid nitrogen. Tissue lysis solution was added to the tissue, placed on ice, and ground with a glass rod, then the crushed homogenate was aspirated, and the supernatant was aspirated, and then ultrasonically crushed, and then the homogenate was centrifuged according to the above centrifugation conditions, and the supernatant was aspirated and stored at -80°C. The sample concentration was detected by the BCA method, and the ELISA test and analysis of FBN2 protein expression were performed according to the instructions. The specific steps are as follows:

[0106] (1) Equilibrate the reagent kit at room temperature for 60 minutes;

[0107] (2) Add 50 μL of standard to each standard well as required;

[0108] (3) Add 50 μL of test sample to each sample well and 50 μL of diluent to each blank well;

[0109] (4) Add 100 μL of HRP-labeled detection antibody to each well, seal the wells, and keep the incubation at 37°C for 60 min.

[0110] (5) Prepare washing solution: dilute to 1×;

[0111] (6) Discard the liquid and pat dry. Add 350 μL of washing solution to each well and pat dry after 1 min. Repeat 5 times.

[0112] (7) Add 50 μL of substrate (A, B) to each well and incubate at 37°C for 15 min (protect from light);

[0113] (8) Add 50 μL of stop solution to each well;

[0114] (9) Measure the OD value of each well at a wavelength of 450 nm;

[0115] (10) Calculate the relative content of the target protein in the retina based on the standard concentration gradient and OD value. Figure 5 shown.

[0116] Depend on Figure 5The results showed that after negative AAV injection, the protein expression in the negative control group (7.11±0.98μg / ml) was not significantly different from that in the normal control group (7.13±1.13 μg / ml) (P>0.05). After AAV injection, the protein expression in the AAV group (4.02±0.92 μg / ml), FBN2 ultra-low concentration group (4.21±0.96 μg / ml), FBN2 low concentration group (4.23±0.89 μg / ml), FBN2 medium concentration group (4.30±0.39μg / ml), and FBN2 high concentration group (4.00±0.70 μg / ml) was significantly decreased compared with the negative control group (7.11±0.98μg / ml) (P<0.05), and the trend of each group compared with the normal control group was consistent with that of the negative control group. After 3 injections of FBN2 protein, the protein expression in the FBN2 low concentration group (7.51±0.87 μg / ml) was significantly higher than that in the AAV group (4.21±0.79 μg / ml) (P<0.05). There was no significant change in the protein expression in the FBN2 ultra-low concentration group (4.58±0.80 μg / ml), FBN2 medium concentration group (6.12±0.77 μg / ml), and FBN2 high concentration group (5.01±0.36 μg / ml) compared with the AAV group (4.21±0.79 μg / ml) (P>0.05). The protein expression in AAV group (4.21±0.79 μg / ml), FBN2 ultra-low concentration group (4.58±0.80 μg / ml), FBN2 medium concentration group (6.12±0.77 μg / ml), and FBN2 high concentration group (5.01±0.36 μg / ml) was significantly decreased compared with the negative control group (7.70±0.94 μg / ml) (P<0.05); the comparison trend between each group and the normal control group was consistent with that between each group and the negative control group.

[0117] Example 7

[0118] To prepare protein samples, each group of samples was thoroughly mixed with SDS-PAGE protein loading buffer at a volume ratio of 5:1. Three samples were taken from each group and mixed in equal proportions. They were then boiled at 100°C for 6 min. After the samples were cooled to room temperature, they were placed in a -80°C refrigerator for later use.

[0119] 1. Glue preparation

[0120] (1) Brushing glass plate: use liquid detergent + water, rinse with distilled water, and air dry / blow dry;

[0121] (2) Fix with a splint, add 4-5 ml of anhydrous ethanol to test for leaks, and pour out if it is not full after 5 minutes;

[0122] (3) Prepare separation gel: ① Acrylamide (30%); ② 10% APS: Prepare and use immediately: 0.1 g ammonium persulfate; ③ Tns-HCI-SDS: Shake well before use, SDS crystallizes; ④ APS TEMED: Operate on ice; ⑤ Prepare magnesium acrylamide: APS TEMED is placed back in the 4°C refrigerator;

[0123] (4) After infusion, add anhydrous ethanol to level the liquid surface. Do not use water.

[0124] (5) Let stand for 45 minutes (can be longer but not shorter);

[0125] (6) Prepare 5% stacking glue, pour out the ethanol, tilt it, dry it with filter paper, and add stacking glue on the glass plate.

[0126] At the top, insert the blue comb as needed;

[0127] (7) Let it stand for 45 minutes, then pull the comb vertically upwards and slowly pull it out. Add the Mark and sample in the middle in turn. Place the two short plates inside out in the electrophoresis tank, add an appropriate amount of electrophoresis liquid between the two plates, and measure it on absorbent paper. If there is no leakage, continue adding and stop when it is close to the short plate.

[0128] 2. Electrophoresis and membrane transfer:

[0129] (1) Pour the diluted electrophoresis solution into the electrophoresis tank (with both ends facing inwards and upwards) and place it on a piece of paper to test for leaks;

[0130] (2) If there is no leakage, put the box in and fill it with electrophoresis solution, carefully pull out the comb, and pay attention to the normal tissue;

[0131] (3) Take out the sample and centrifuge it at 12000r / min, place it on ice, and pre-stain molecular proteins, etc.

[0132] (4) Load 5ul of Marker, extract the upper layer of the protein sample, and do not need to extract the bottom sediment. ① When loading the sample, the inclined surface of the sampler is against the rear glass plate and the sample is loaded slowly. ② Before changing the sample, rinse the sampler with distilled water several times;

[0133] (5) Fill the current tank. Note that the sampler can be used to prevent the sample from being washed out. The liquid is added outside the tank through the iron wire.

[0134] (6) 80V for 30mh and then switch to 100V for 120min;

[0135] (7) Pre-cool the 1× transfer buffer in a -80°C refrigerator for 20 minutes before transfer.

[0136] (8) Cut the filter paper PVDF membrane, pour the transfer solution into the tray to soak the filter paper, soak the PVDF in methanol for 30 minutes, and then rinse it with triple distilled water;

[0137] (9) After electrophoresis, remove the glass plate and cut off the upper layer of gel with a cutting board. Remove the lower gel and place it in a tray, paying attention to the front and back and left and right, and recover the electrophoresis solution.

[0138] (10) Blackboard → single layer sponge → three layers of filter paper → glue → PVDF membrane, three layers of filter low - sponge - transparent plate layer by layer;

[0139] (11) Put the plates together and fasten them tightly into the tank, black against black and red against red, put them into an ice basin, and fill it with transfer solution;

[0140] (12) After distinguishing the positive and negative poles, cover them tightly, 100V for 120min, ice at 4℃; 200mA / 100min;

[0141] (13) TBST: 10×TBST + 900 ml water + 500 ml Tween suspension, shake for 2 h;

[0142] (14) After transfer, cut off the upper left corner and remove it. Place the left and right corners in a sealing bag, pour in 10 ml of blocking solution, and shake for 1 hour. After the blocking is complete, wash with TBST for 3×5 minutes and then incubate with antibody eggs for detection;

[0143] (15) Recover the transfer solution, clean the basin, splint sponge, and sponge, first soak them in tap water and then in distilled water, take them out and place them on a tray to dry, 3 / 4 TBST dilution multiple see the instructions, diluent: 1 / 4 blocking solution;

[0144] (16) During the blocking process, primary antibodies are added to make pockets;

[0145] (17) The pockets are marked, the blocking solution is poured out, the strips are cut, and they are placed into the corresponding pockets according to the name, sealed, and the primary antibody is added. ① All strips are marked, ② The bubbles are removed and sealed, and ③ The pockets are used for the corresponding antibodies;

[0146] (18) After each strip is fixed, place it on a shaker for 0.5 h to 11 h (longer is better than shorter) and then store it at 4°C overnight.

[0147] 3. Secondary antibody development

[0148] (1) Place the membrane in a shaker for 45 minutes, but not more than 2 hours;

[0149] (2) TBST: 4x8ml wash;

[0150] (3) Make a secondary antibody pocket: prepare secondary antibody diluent, 1 / 4 blocking solution, 3 / 4 TBST;

[0151] (4) Place the strip in a pocket, add secondary antibody, remove bubbles, and shake for 1 hour;

[0152] (5) Prepare AB solution and recover secondary antibody;

[0153] (6) Wash the strips with TBST, 4 × 8 min; wash with TBS, 2 × 5 min (to prevent TST from interfering with development);

[0154] (7) Drop AB on a clean plate, contact the strip face-to-face, react for 2 minutes, and then expose. Save the strip, wash the developed strip twice with TBST, 10 minutes, 20 minutes for an unlimited time, wash again with TBST, and then seal with TBS; the test results are as follows: Figure 6 shown.

[0155] Table 2

[0156]

[0157] The FBN2 protein injection group was the FBN2 low-concentration group with the best therapeutic effect. Figure 6 The results showed that after negative AAV injection, the expression of FBN2 gene protein in the retina of the negative control group was not significantly changed compared with the normal control group (P>0.05); after AAV injection, the expression of FBN2 gene protein in the AAV group and the FBN2 injection group decreased (P<0.05). After FBN2 protein was injected three times, the expression of FBN2 gene protein in the retina of the FBN2 injection group was slightly lower than that of the normal control group (P>0.05); the expression of FBN2 gene protein in the retina was significantly increased compared with the AAV group (P<0.05).

[0158] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field. The above description is only the embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. Use of recombinant fibrillin-2 protein in the preparation of products that reduce retinal exudation and fundus deposits, regulate electroretinogram waveform amplitude, increase retinal outer nuclear layer thickness or reduce axial length; The product is a composition containing recombinant fibrillin-2 protein as the sole active ingredient or a recombinant fibrillin-2 protein; The content of recombinant fibrillin-2 protein in the product is 0.2-0.5 μg / μL; The product is a drug; the drug preparation is an injection; the drug is administered by intravitreal injection; The product increases the expression of fibrillin-2; the expression of fibrillin-2 is the expression of the fibrillin-2 gene; the expression of the fibrillin-2 gene is the expression of the fibrillin-2 gene mRNA; The regulating the electroretinogram waveform amplitude is to restore the electroretinogram waveform amplitude to normal; the electroretinogram waveform amplitude is selected from at least one of the dark-adapted rod cell response waveform amplitude, the dark-adapted maximum mixed response waveform amplitude, the dark-adapted oscillatory potential response waveform amplitude and the light-adapted cone cell response waveform amplitude; The amino acid sequence of FBN-2 recombinant protein is: TPGRYECNCPPDFQLNPTGVGCVDNRVGNCYLKFGPRGDGLSCNTEIGVGVSRSSCCCSLGKAWGNPCETCPPVNSTEYYTLCPGGEGFRPNPITIILEDIDECQELPGLCQ GGNCINTFGSFQCECPQGYYLSEDTRICEDIDECFAHPGVCGPGTCYNTLGNYTCICPPEYMQVNGGHNCMDMRKSFCYRSYNGTTCENELPFNVTKRMCCCTYNVGKAWNKPCEPCPTPGTADFKTIC.

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