CN111437398A

CN111437398A - Transthyretin entering eye and its application in preparing drops

Info

Publication number: CN111437398A
Application number: CN202010506950.7A
Authority: CN
Inventors: 辛瑜
Original assignee: Tongyan Shanghai Medical Equipment Co ltd
Current assignee: Yizhou Shanghai Biomedical Co ltd
Priority date: 2019-12-17
Filing date: 2020-06-05
Publication date: 2020-07-24
Anticipated expiration: 2040-06-05
Also published as: CN111437398B; CN110960687A

Abstract

The invention provides an application of transthyretin as a carrier for protein and/or polypeptide drugs to enter eyes through an ocular barrier, wherein the transthyretin is a protein consisting of amino acids shown in SEQ ID NO. 1 or mutation or modification thereof. The invention also provides application of the transthyretin and/or the fusion protein of the transthyretin and a medicament in preparing drops and the drops, wherein the medicament is a protein medicament and/or a polypeptide medicament. The transthyretin has better biocompatibility and safety in human bodies, and can effectively convey exogenous proteins and/or polypeptides into eyes to achieve the effect of treating eye diseases. By using the eye drop mode, when treating eye diseases such as DR, AMD, ROP and the like, transthyretin can cross a corneal barrier and enter a vitreous body and an eyeground, so that the eyeball retina leakage is obviously inhibited, the number of retinal new vessels is obviously reduced, and the pathological phenomenon of the eye diseases is effectively relieved.

Description

Transthyretin entering eye and its application in preparing drops

Technical Field

The invention relates to the technical field of medicines, in particular to application of transthyretin as a carrier for protein and/or polypeptide medicines entering eyes through an ocular barrier, application of the transthyretin and/or fusion protein consisting of the transthyretin and the protein and/or polypeptide medicines in preparation of drops and the drops.

Background

Diabetic Retinopathy (DR), abbreviated as "sugar network", is a clinical manifestation of Diabetic microangiopathy and one of the most serious complications of diabetes, and has become one of the major blinding eye diseases at present. Mainly under the microenvironment of long-term high sugar and hypoxia of eyes, the clinical symptoms of microvascular obstruction, microangioma, hemorrhage, vein dilatation, macular edema, neovascularization, massive vitreous hemorrhage, intraocular fibrosis, retinal detachment and the like gradually occur.

Age-related macular degeneration (AMD), is an aging change in the structure of the macular region. Mainly characterized in that the phagocytic digestion capacity of retinal pigment epithelial cells to the disc membrane of the extracellular segment is reduced, and as a result, the residual bodies of the disc membrane which are not completely digested are retained in the primary pulp of basal cells and are discharged out of the cells to be deposited on the Bruch membrane to form drusen. This change is more pronounced due to the structural and functional particularities of the macula. Drusen are also found in the elderly with normal vision, but as a result of the secondary pathological changes, macular degeneration occurs. Or cause rupture of Bruch membrane, choroidal capillaries enter under RPE (retinal pigment epithelium) and retinal nerve epithelium through the ruptured Bruch membrane to form choroidal neovascularization, which is subretinal neovascularization. Due to structural abnormality of the wall of the new blood vessel, the leakage and bleeding of the blood vessel are caused, and a series of secondary pathological changes are caused. Age-related macular degeneration mostly occurs above 45 years old, and the prevalence rate of age-related macular degeneration increases, which is currently an important disease causing blindness in the elderly.

Retinopathy of prematurity (ROP) refers to a premature infant who is under 36 weeks gestation, low birth weight, long-term oxygen intake, whose non-vascularized retina develops fibrohemangioma hyperplasia, contraction, and further causes tractional retinal detachment and blindness. Formerly known as Terry syndrome or retrolental fibroplasia, the latter only reflects late manifestations of the disease. The pregnancy period is shorter or the weight of the organism is lower, and the incidence rate can reach 60 to 80 percent. The etiology of the disease is that after the premature infant is born, the premature infant excessively absorbs oxygen in a high-oxygen environment in an incubator, the premature infant is in a relatively anoxic state after being removed from the incubator, and the retina which is not completely vascularized generates vasoconstriction and vascular proliferation on oxygen.

For the treatment of ocular diseases, it is desirable to deliver drugs effectively into the eye, particularly ocular diseases such as diabetic retinopathy, age-related macular degeneration, and retinopathy of prematurity; however, effective delivery of drugs to the eye is challenging due to the greater barrier presented to the eye. Accordingly, recently, ocular drug delivery systems have received a wide range of attention.

Depending on the ocular anatomy, ocular barriers include primarily the tear barrier, the corneal/conjunctival barrier, and the blood-ocular barrier; the multiple barriers can effectively block the invasion of exogenous chemical and biological molecules while protecting eyes, but also bring great obstruction to the transmission of drugs. Currently, the common administration modes of the eye mainly comprise conventional eye drops, subconjunctival injection and scleral administration, and intravitreal injection administration. Wherein, the contact time of the conventional eye drops and the ocular surface is short, and due to the structure and the characteristics of the conventional eye drops, some exogenous drugs and proteins are difficult to effectively enter eyes; subconjunctival and intravitreal injections often cause some trauma. The methods of retina laser treatment and vitrectomy combined with silicone oil filling have large damage and unsatisfactory vision recovery. The most common current treatment for DR, AMD and ROP is intravitreal injection of anti-vegf antibodies, but this approach often involves some trauma and multiple administrations require long intervals.

At present, some patent documents design functional small peptides capable of effectively breaking through various ocular barriers to reach eyes, such as patents of zhengying, xu et al (a new class of small peptides inhibiting new vessels and applications thereof, CN2013100527142), patents of suli et al (a class of small peptides inhibiting new vessels and applications thereof, CN201310058978.9), patents of yangjiu et al (a small molecule polypeptide for preventing and/or treating inflammatory responses and applications thereof).

The above studies have limitations, intraocular injection causes certain trauma, and the delivery efficiency of functional small peptide is low, and the half-life is short, so there is a need to find a drug and a drug administration method which are safer, more stable, effective and have no or less harm to eyes.

Disclosure of Invention

The invention provides an application of transthyretin (TTR) as a carrier of protein and/or polypeptide drugs entering eyes through an ocular barrier, an application of the transthyretin and/or a fusion protein composed of the transthyretin and the protein and/or the polypeptide drugs in the preparation of drops and the drops, aiming at overcoming the defect that the prior art has no administration method and drugs which can safely, stably and effectively deliver the drugs into the eyes without or with little harm to the eyes so as to treat the eye diseases such as Diabetic Retinopathy (DR), age-related macular degeneration (AMD), retinopathy of prematurity (ROP) and the like due to the existence of the ocular barrier.

Through a large number of experiments, the inventor unexpectedly discovers that transthyretin can effectively cross a corneal barrier to enter a vitreous body and an eyeground by using drops instead of a conventional injection mode in the field, and can also effectively convey exogenous protein and/or polypeptide, so that appropriate protein and/or polypeptide medicines can be selected according to the pathological characteristics of eye diseases and conveyed into eyes through the transthyretin, thereby achieving the effect of treating the eye diseases, and the invention has important application prospect in the field of medicines as a substitute of injection medicines.

Through a large number of experiments, the inventor also unexpectedly discovers that the transthyretin can obviously inhibit the eyeball retina from leaking, obviously reduce the number of retinal new vessels and effectively relieve eye diseases such as DR, AMD, ROP and the like after the transthyretin crosses a corneal barrier and enters the vitreous body and the fundus oculi. That is, transthyretin can treat or alleviate ocular diseases such as DR, AMD, ROP, etc. without fusion with protein and/or polypeptide drugs, and can reach sufficient therapeutic concentration and therapeutic time (long half-life).

Furthermore, those skilled in the art know only that hyaluronic acid has a moistening effect and do not know that hyaluronic acid can increase the transthyretin permeation amount, and the present inventors have further found that the use of hyaluronic acid can increase the transthyretin permeation amount in the vitreous cavity and fundus when preparing drops such as eye drops, thereby effectively increasing the transthyretin concentration.

In order to solve the above technical problems, the present invention provides, in a first aspect, a use of transthyretin as a carrier for proteinaceous and/or polypeptide drugs to enter the eye through the ocular barrier, said transthyretin being represented by (a), (b) or (c):

(a) a protein consisting of amino acids shown in SEQ ID NO. 1;

(b) a protein which is shown by a sequence with one or more amino acids substituted, deleted or added on the amino acid sequence in (a) and has the function of inhibiting angiogenesis and is derived from (a);

(c) a protein represented by a sequence modified in a hydrophilic or hydrophobic manner in the amino acid sequence in (a) or (b).

Preferably, in (b), the protein derived from (a) is substituted by 22 amino acids in the amino acid sequence in (a).

Preferably, in (b), the protein derived from (a) is obtained by 25 amino acid substitutions in the amino acid sequence in (a).

Preferably, in (b), the protein derived from (a) is a substitution of T, I, N, H, R, A, D, T, S, E, V, I, K, I, A, H, E, P102, R104, T123, K126 and/or E127 of the amino acid sequence in (a). more preferably, in (b), the protein derived from (a) is a substitution of T3, T5, I26, N27, H31, R34, A36, D39, T40, S50, E61, E63, V65, I68, K70, I, H90, P102, R104, T123, K126 and E127 of the amino acid sequence in (a). more preferably, in (b), the protein derived from (a) is a substitution of T3, T5, T26, N27, H31, R34, A36, D39, T40, S50, E61, E63, V65, I68, K70, I, H90, P102, R104, T123, K126 and E127. more preferably, T90, P102, E11, T3, T11, K70, E31, R31, S70, E68, E70, E61, E40, E68, E70, E.

Preferably, in (b), the protein derived from (a) is a protein obtained by deletion of 5 amino acids in the amino acid sequence in (a). More preferably, in the step (b), the protein derived from the protein of the step (a) is deleted at position 123-127 of the amino acid sequence of the protein of the step (a).

Preferably, in the step (b), the amino acid sequence of the protein derived from the step (a) is shown as SEQ ID NO. 9, SEQ ID NO. 10 or SEQ ID NO. 11.

Preferably, in the (c), the hydrophilic modification or the hydrophobic modification is performed on cysteine at position 10 on the amino acid sequence in (a). Preferably, the hydrophobic modification is a modification with a long chain hydrophobic segment such as n-dodecane. In a preferred embodiment of the present invention, the protein is a protein represented by a sequence in which cysteine at position 10 of the amino acid sequence in (a) is linked to n-dodecane through maleimide. In the present invention, the 10 th cysteine of the amino acid sequence in (a) may be linked to a fluorescent label such as 5-aminofluorescein to trace the modified protein.

Preferably, the transthyretin is expressed by fusion with the protein and/or polypeptide drug; the fusion is preferably to fuse the protein and/or polypeptide drug to the N-terminal or C-terminal of the transthyretin.

The microbial cells may be E.coli including, but not limited to, E.coli B L21, E.coli B L21 (DE3), E.coli JM109, E.coli DH5 α, E.coli K12^TMHigh Capacity endo toxin Removal SpinColums, ThermoFisher) and then through a 0.22 μm pore size filter membrane to remove residual bacteria.

Preferably, the protein and/or polypeptide drug comprises, but is not limited to lysozyme, albumin and/or EGFR antibody, and the molecular weight is not more than 45kDa, wherein the lysozyme is egg white lysozyme with GenBank accession number AA L69327.1.

Preferably, the proteinaceous and/or polypeptide agents include those for the treatment of diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity.

Preferably, the nucleotide sequence for coding the transthyretin is shown as SEQ ID NO. 2.

Preferably, the transthyretin is expressed by using a recombinant expression vector, and a promoter in a skeleton plasmid of the recombinant expression vector is a rhamnothalic inducible promoter, preferably a rhaPBAD promoter.

Preferably, the transthyretin is expressed by using a recombinant expression vector, the skeleton vector of the recombinant expression vector can be pET-21a or a vector with homology of 25% or more, and the sequence of the vector with homology of 25% or more is preferably shown as SEQ ID NO. 8.

Preferably, the nucleotide sequence of the recombinant plasmid for expressing the transthyretin is shown as SEQ ID NO. 3.

Preferably, the transthyretin is expressed in microbial cells (in the form of transformants) and further purified, and the microbial cells may be E.coli including, but not limited to, E.coli B L21, E.coli B L21 (DE3), E.coli JM109, E.coli DH5 α, E.coli K12^TMHigh Capacity endo toxin Removal Spin Columns, ThermoFisher) and the residual bacteria were removed by 0.22 μm pore size filtration.

Preferably, the transthyretin is expressed by culturing a transformant containing the transthyretin gene until the OD600 of the obtained bacterial cell reaches 1.5 to 2.0 (e.g., 1.6, 1.7, 1.8 or 1.9).

Preferably, the expression of the transthyretin is induced by using an expression-inducing agent, the expression-inducing agent is 0.1-2% by mass, such as 0.2%, 0.3%, 0.4%, 0.5%, 0.7%, 0.8%, 1.2% or 1.6%, and the expression-inducing time is preferably 8-20h, such as 10h, 12h, 14h, 16h, 17h, 18h or 19 h; the expression inducing agent is preferably rhamnose or IPTG.

In a preferred embodiment of the invention, the application is to apply the fusion protein obtained by fusing and expressing the transthyretin and the protein and/or polypeptide drugs to eyes by means of eye dropping (for example, preparing into drops such as eye drops), wherein the dropping times are 1-3 times per day, and the dropping amount is 0.6-0.8nmol protein/eye. The drops may be administered 2 times daily, 1 drop at a time, for 3 months. The drops may be administered 2 times daily, 1 drop at a time, for 5 days. The drops may be administered 2 times daily, 1 drop at a time, for 3 weeks.

In order to solve the technical problems, the second aspect of the invention provides an application of transthyretin and/or a fusion protein of transthyretin and a medicament in preparing drops, wherein the medicament is a protein medicament and/or a polypeptide medicament, and the transthyretin is represented by (a), (b) or (c):

(a) a protein consisting of amino acids shown in SEQ ID NO. 1;

Preferably, the content of the fusion protein is more than or equal to 4 mu mol/L, preferably 10-15 mu mol/L.

Preferably, the transthyretin is present in an amount of ≥ 4 μmol/L, preferably 5-30 μmol/L, more preferably 10-20 μmol/L, e.g. 15, 20, 25 μmol/L.

Preferably, the drops further contain physiological saline.

Preferably, the drop also contains hyaluronic acid, and the content of the hyaluronic acid in percentage by mass and volume is less than or equal to 6%, preferably 1-4%, and more preferably 2%.

Preferably, the drops are preferably eye drops.

Preferably, the drops are preferably drops for the treatment of diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity.

Preferably, the number of drops per day is 1-3, each drop preferably being 0.6-0.8nmol protein per eye.

Preferably, said drops are administered 2 times daily, 1 drop at a time, for 3 months; and/or, said drops are administered 2 times daily, 1 drop at a time, for 5 days; and/or, the drops are administered 2 times daily, 1 drop at a time, for 3 weeks.

Preferably, the transthyretin is expressed by using a recombinant expression vector, the skeleton vector of the recombinant expression vector is pET-21a or a vector having homology of 25% or more, and the sequence of the vector having homology of 25% or more is preferably shown as SEQ ID NO. 8.

Preferably, the sequence of the fusion protein is shown as SEQ ID NO. 6 or SEQ ID NO. 7.

Preferably, the transthyretin is expressed by fusion with the protein and/or polypeptide drugs, the fusion is preferably to fuse the protein and/or polypeptide drugs at the N end or the C end of the transthyretin, the fusion of the transthyretin and the protein and/or polypeptide drugs is preferably to express in microbial cells, and the microbial cells can be escherichia coli, and the escherichia coli comprises but is not limited to E.coli B L21, E.coli B L21 (DE3), E.coli JM109, E.coli DH5 α and E.coli K12.

Preferably, the protein and/or polypeptide drugs comprise, but are not limited to lysozyme, albumin and/or EGFR antibody, the molecular weight of which is not more than 45kDa, the lysozyme is egg white lysozyme, the GenBank accession number of which is AA L69327.1, and the albumin is egg white albumin.

In order to solve the above technical problems, the third aspect of the present invention provides a drop (for example, in the form of eye drops) comprising transthyretin, and/or a fusion protein of transthyretin and a drug; the medicine is protein and/or polypeptide medicine, and the transthyretin is shown as (a), (b) or (c):

(a) a protein consisting of amino acids shown in SEQ ID NO. 1;

Preferably, when the drops contain a fusion protein of transthyretin and a protein and/or polypeptide drug, the content of the fusion protein is more than or equal to 4 mu mol/L, preferably 10-15 mu mol/L.

Preferably, when the drops contain transthyretin, the amount of transthyretin is ≥ 4 μmol/L, preferably 5-30 μmol/L, more preferably 10-20 μmol/L, e.g. 15, 20, 25 μmol/L.

Preferably, the drops further contain physiological saline.

In a preferred embodiment of the invention, said drops (for example in the form of eye drops) further comprise physiological saline and 1-6% hyaluronic acid.

In a preferred embodiment of the invention, said drops (for example in the form of eye drops) further comprise physiological saline and 1-4% hyaluronic acid.

In a preferred embodiment of the invention, said drops (for example in the form of eye drops) further comprise physiological saline and 2% hyaluronic acid.

Preferably, the drops are preferably eye drops.

In a preferred embodiment of the invention, the amount of transthyretin in said drops, e.g. eye drops, is 5-20 μmol/L, 2 times a day, 1 drop each time, for 3 months.

In a preferred embodiment of the invention, the amount of transthyretin in said drops, e.g. eye drops, is 10-15 μmol/L, 2 times a day.

In a preferred embodiment of the invention, the amount of transthyretin in said drops, e.g. eye drops, is 10 μmol/L, 2 times a day.

In a preferred embodiment of the invention, the amount of transthyretin in said drops, e.g. eye drops, is 5-20 μmol/L, 2 times a day, 1 drop each time, for 5 days.

In a preferred embodiment of the invention, the amount of transthyretin in said drops, e.g. eye drops, is 5-20 μmol/L, 2 times a day, 1 drop each time, for 3 weeks.

In the present invention, the listed eye drops and the amount of transthyretin in the drops are only suggested. It will be understood by those skilled in the art that dosages and amounts which are suitably adjusted for administration to a suitable subject in accordance with the present invention are also intended to be within the scope of the present invention.

The Transthyretin is found to enter cells mediated by a high density lipoprotein receptor SRB1 (L receptors, K.A., et al, Transthyretin upper protein in planar cells regulated by the high-density lipoprotein receptor, scanner receiver class B member1.mol Cell Endocrinol,2018.474: p.89-96). structurally (three-dimensional structure is shown in FIG. 1), the tetramer surface of Transthyretin has significant hydrophilic domains (SD) and hydrophobic domains (light color), the core hydrophobic domain of Transthyretin can carry strong hydrophobic Transthyretin molecules across various cells (FIG. 2). The amino acid sequences of Transthyretin of different species are highly conserved and the amino acid sequence of human Transthyretin is similar to that of mouse Transthyretin (3695/35. the Transthyretin is derived from mouse (3695).

During the process of preparing drops, the inventor finds that the recombinant expression level of the transthyretin in the escherichia coli is extremely low, and most of the transthyretin is in an insoluble inclusion body form. The common methods for improving the protein expression level include expression vector selection, fermentation condition optimization (temperature, pH value, time, inducer concentration and the like) or molecular chaperone co-expression assistance and the like, but the inventors of the present invention have tried that none of the known methods can effectively improve the expression level of transthyretin in escherichia coli. Through a large number of experiments, the inventor finds that a high-efficiency production plasmid of a mature fragment of transthyretin (NCBI Reference Sequence: NP-000362.1) can be constructed based on a pET-21a plasmid, after a promoter (such as a T7 promoter) on the plasmid is replaced by a rhamnoides inducible (such as a rhaPBAD promoter), or after codon optimization is carried out on a nucleotide Sequence of the pET-21a plasmid (the nucleic acid Sequence is shown as SEQ ID NO: 2), or after Sequence reconstruction is carried out on the pET-21a plasmid, or an OD value when the protein is expressed, the using amount of an agent for inducing protein expression and the time for inducing expression are optimized, so that the expression amount of the finally obtained transthyretin is obviously improved. Therefore, the present invention further provides a gene expressing transthyretin, a recombinant expression vector and a transformant comprising the same, a recombinant plasmid for expressing transthyretin, an expression method of transthyretin, and the like. When the gene for expressing the transthyretin, the recombinant plasmid for expressing the transthyretin or the expression method of the transthyretin is used for expression, the expression amount of the transthyretin is obviously improved. Specifically, the method comprises the following steps:

the fourth aspect of the invention also provides a gene for expressing transthyretin, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

The fifth aspect of the present invention also provides a recombinant expression vector containing the gene according to the fourth aspect of the present invention.

Preferably, the promoter in the skeleton vector of the recombinant expression vector is a rhamnoides inducible promoter; the rhamnothalic inducible promoter is preferably the rhaPBAD promoter.

Preferably, the skeleton vector of the recombinant expression vector is pET-21a or a vector having 25% or more homology with pET-21a, and the sequence of the vector having 25% or more homology with pET-21a is preferably shown as SEQ ID NO. 8.

More preferably, the nucleotide sequence of the recombinant expression vector is shown as SEQ ID NO. 3.

The sixth aspect of the invention also provides a recombinant plasmid, the sequence of which is shown as SEQ ID NO. 8.

The seventh aspect of the present invention also provides a recombinant plasmid for expressing transthyretin, the recombinant plasmid comprising a backbone plasmid and an expression fragment of transthyretin,

wherein the promoter in the skeleton plasmid is a rhamnoides inducible promoter; and/or the skeleton vector of the recombinant expression vector is pET-21a or a vector with homology of 25 percent or more with the pET-21a, and the sequence of the vector with homology of 25 percent or more with the pET-21a is preferably shown as SEQ ID NO. 8.

Preferably, the nucleotide sequence for coding the transthyretin expression fragment is shown as SEQ ID NO. 2.

Preferably, the rhamnong inducible promoter is a rhaPBAD promoter.

More preferably, the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO. 3.

The eighth aspect of the present invention also provides a transformant comprising the gene according to the fourth aspect of the present invention, or the recombinant expression vector according to the fifth aspect of the present invention, or the recombinant plasmid according to the sixth or seventh aspect of the present invention.

Preferably, the gene according to the fourth aspect of the present invention, the recombinant expression vector according to the fifth aspect of the present invention or the recombinant plasmid according to the sixth or seventh aspect of the present invention is introduced into a host, which is escherichia coli, preferably escherichia coli e.coli B L21 (DE3), e.coli TG1, e.coli JM109, e.coli DH5 α or e.coli K12.

The ninth aspect of the invention also provides a preparation method of transthyretin, which comprises the following steps:

(1) obtaining a transformant according to the eighth aspect of the present invention;

(2) screening the transformants, expressing and purifying the protein.

Preferably, the expression is carried out by culturing the transformant until the OD600 of the obtained bacterial cells reaches 1.5 to 2.0 (e.g., 1.6, 1.7, 1.8 or 1.9).

Preferably, the expression is induced by using an expression-inducing agent, the expression-inducing agent has a mass volume percentage of 0.1-2%, such as 0.2%, 0.3%, 0.4%, 0.5%, 0.7%, 0.8%, 1.2% or 1.6%, and the expression-inducing time is preferably 8-20h, such as 10h, 12h, 14h, 16h, 17h, 18h or 19 h; the expression inducing agent is preferably rhamnose or IPTG.

The tenth aspect of the present invention also provides an expression method of transthyretin, which comprises the steps of obtaining a transformant containing a gene of transthyretin, and screening, expressing, and purifying the transthyretin.

Based on the above, the inventors have also found through a large number of experiments that, after transthyretin enters the vitreous body and the fundus oculi across the corneal barrier, transthyretin can significantly inhibit the ocular retinal leakage, significantly reduce the retinal neovascularization number, and effectively alleviate eye diseases such as DR, AMD, ROP, etc. Therefore, the invention further provides the application of the transthyretin in preparing the medicines for treating eye diseases such as diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity and the like.

The eleventh aspect of the invention also provides the use of transthyretin in the manufacture of a medicament for the treatment of age-related macular degeneration, retinopathy of prematurity and/or diabetic retinopathy.

Preferably, the transthyretin is present in a non-injectable form, preferably as a wipe, preferably as a paste (e.g. an eye ointment) or as a gel (e.g. an ophthalmic gel), or as drops, for example as eye drops.

Preferably, the transthyretin is expressed using a gene according to the fourth aspect of the invention, a recombinant expression vector according to the fifth aspect of the invention, or a recombinant plasmid according to the sixth or seventh aspect of the invention, a transformant according to the eighth aspect of the invention.

More preferably, the content of transthyretin in the eye drops is more than or equal to 4 mu mol/L, preferably 5-30 mu mol/L, more preferably 10-20 mu mol/L, such as 15, 20 and 25 mu mol/L.

More preferably, the eye drops further contain physiological saline.

More preferably, the eye drops further contain hyaluronic acid, and the content of the hyaluronic acid in percentage by mass and volume is less than or equal to 6%, preferably 1-4%, and more preferably 2%.

Even more preferably, the eye drops are administered 2 times daily, 1 drop at a time, for 3 months; and/or, the eye drops are administered 2 times daily, 1 drop at a time, for 5 days; and/or, the eye drops are administered 2 times daily, 1 drop at a time, for 3 weeks.

The invention also provides the use of transthyretin and/or drops according to the third aspect of the invention for the treatment of an ocular disorder. The ocular disease is preferably diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity. The transthyretin is preferably as described above.

The present invention also provides a method of treating ocular diseases such as diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity and the like which comprises administering transthyretin as described above and/or drops according to the third aspect of the invention.

The present invention also provides a method of delivering a foreign protein and/or polypeptide into the eye through an ocular barrier using the transthyretin described above.

In a preferred embodiment of the present invention, the preparation method of the fusion protein comprises:(1) connecting the transthyretin and the coding gene of the drug protein and/or the polypeptide to a pET 21a (+) plasmid to obtain a recombinant plasmid; (2) transforming the recombinant plasmid constructed in the step (1) into a host cell for expression; (3) using TB culture medium, fermenting at 30-40 deg.C and OD_600nmInducing with 0.1-0.5mM IPTG for 8-16h when reaching 1.5-2.0; (4) purifying with nickel column affinity adsorption to obtain expressed target protein, and purifying with endotoxin adsorption column (Pierce)^TMHigh Capacity endo toxin removal Spin Columns, ThermoFisher) and the residual bacteria were removed by 0.22 μm pore size filtration.

In a preferred embodiment of the present invention, the transthyretin is expressed and purified by transforming the plasmid pETx-rhaPBAD-ttr (the whole plasmid has the nucleotide sequence shown in SEQ ID NO: 3) into E.coli B L21 (DE3) cells, culturing the recombinant E.coli B L21 (DE3) in L B culture medium, preparing seed solution, inoculating the seed solution into 5L TB culture medium at 37 deg.C and stirring paddle rotation speed of 150rpm until OD is reached₆₀₀1.5-2.0; adding 0.4-2% rhamnose for inducing for 16-20 hr. Endotoxin adsorption column (Pierce) was used^TMHigh Capacity endo toxin removal Spin Columns, ThermoFisher) and the residual bacteria were removed by 0.22 μm pore size filtration.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The abbreviations for the amino acids in the present invention are those conventional in the art unless otherwise specified, and the amino acids corresponding to the specific abbreviations are shown in Table 1-1.

TABLE 1-1

Codons corresponding to the amino acids are also conventional in the art, and the correspondence between specific amino acids and codons is shown in tables 1-2.

Tables 1 to 2

The positive progress effects of the invention are as follows: transthyretin has better biocompatibility and safety in human bodies, can pass through eye barriers per se, can effectively convey exogenous proteins and/or polypeptides into eyes to achieve the effect of treating eye diseases, and has important application prospect in the field of medicines as a substitute of injection medicines. In the invention, by using drops for eye dropping instead of direct injection, when the transthyretin is used for treating eye diseases such as DR, AMD, ROP and the like, the transthyretin can cross a corneal barrier and enter a vitreous body and an eyeground, the eyeball retina leakage is obviously inhibited, the number of retinal neovascularization is obviously reduced, and the pathological phenomena of the eye diseases such as DR, AMD, ROP and the like are effectively relieved.

Drawings

FIG. 1 shows the three-dimensional structure (PDB ID: 1ICT) of transthyretin (TTR).

Figure 2 shows that the TTR core hydrophobic domain carries a strongly hydrophobic thyroxine molecule across various cells.

FIG. 3 shows that human-derived TTR has > 95% similarity to SD rat, C57B L/6 mouse, rabbit-derived TTR amino acid sequences.

FIG. 4 is a diagram of pET 21a (+) -His-tag-TTR-X plasmid. The front end of the gene sequence is fused and expressed with a His-tag sequence and is connected into a plasmid through Nde I and EcoR I restriction enzyme cutting sites; "X" represents a protein fused to TTR.

Fig. 5 is an electropherogram of E.coli B L21 (DE3) expressing human transthyretin, fusion protein purified product and green fluorescent protein, egg white lysozyme and egg white albumin standard.

FIG. 6 shows TTR content in cornea, vitreous body and fundus samples (retina, choroid) of C57B L/6 mice and SD rats after dropping TTR.

FIG. 7 is a western-blot image of a target protein in the vitreous cavity of a human transthyretin and a fusion protein thereof after being dropped into the eye of rats and rabbits for two weeks, wherein the left eye is a dropping eye and the right eye is a control eye.

FIG. 8 shows retinal leakage and retinal neovascularization counts after STZ induction in TTR eye drops in DR SD rats.

Figure 9 shows that TTR eye drops prevented the molding process of ROP.

Figure 10 shows that TTR eye drops inhibited the pathological process of ROP.

Figure 11 shows that TTR eye drops inhibited the pathological process in the AMD model.

FIG. 12 shows the process of chemically modifying human TTR.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

A first part: preparation of transthyretin (TTR) and fusion protein thereof

Example 1: preparation of transthyretin

The method comprises the following steps:

(1) construction of recombinant plasmid pET 21a (+) -His-tag-TTR: His-tag-TTR (shown in SEQ ID NO: 4) with a nucleotide sequence (shown in SEQ ID NO:1, pET 21a purchased from ATCC) was synthesized, and the synthesized nucleotide sequence was ligated to pET 21a (+) with Nde I and EcoR I enzymes, and the sequence was verified to be successful.

(2) And (2) expression and purification of recombinant TTR, namely transforming the pET 21a (+) -His-tag-TTR plasmid constructed in the step (1) into E.coli B L21 (DE3) cells, culturing the obtained recombinant E.coli B L21 (DE3) in a L B culture medium to prepare a seed solution, inoculating the seed solution into a 5L TB culture medium at the temperature of 37 ℃ and the rotating speed of a stirring paddle of 150rpm until the seed solution is cultured to OD₆₀₀1.5-2.0; induction was carried out for 8-16h with the addition of 0.1-0.5mM IPTG (Table 1). And (3) performing high-pressure homogenization to break bacteria, and performing affinity adsorption on the supernatant through a nickel column to obtain the TTR. The obtained protein was passed through an endotoxin adsorption column (Pierce)^TMHigh Capacity endo toxin removal Spin Columns, ThermoFisher) and the residual bacteria were removed by 0.22 μm pore size filtration. TTR protein production was measured and the results are shown in Table 1. At OD₆₀₀When the concentration is 1.5-1.8, inducing with 0.3-0.5 mM IPTG for 12-14 h, and the protein yield is not less than 17mg/g wet thallus.

TABLE 1 expression of TTR under different Induction conditions

Example 2:

transthyretin-green fluorescent protein fusion protein (TTR-GFP) was prepared as follows:

(1) construction of recombinant plasmid pET 21a (+) -His-tag-TTR-GFP (the following recombinant plasmid pET 21a (+) -His-tag-TTR-X plasmid map is shown in FIG. 4): His-tag-TTR-GFP sequence shown as SEQ ID NO. 5 is synthesized, and is connected with pET 21a (+) by Nde I and EcoR I enzymes, and the construction is successful after sequencing verification.

(2) Expression of TTR-GFP fusion protein recombinant plasmid constructed in step (1) was transformed into E.coli B L21 (DE3) cells at an inoculum size of 5%, inoculated into 5L TB medium at 37 ℃,the rotation speed of the stirring paddle is 150rpm, and OD is cultured_600nmTo 1.5-2.0; adding 0.1-0.5mM IPTG to induce for 8-16 h; and (3) performing high-pressure homogenization to break bacteria, and performing affinity adsorption on the supernatant through a nickel column to prepare the TTR-GFP fusion protein. The obtained protein was passed through an endotoxin adsorption column (Pierce)^TMHigh CapacityEndotoxin Removal Spin Columns, ThermoFisher) and passed through a 0.22 μm pore size filter to remove residual bacteria. The production of TTR-GFP protein was measured and the results are shown in Table 2. At OD₆₀₀When the concentration is 1.5-1.9, inducing with 0.3-0.5 mM IPTG for 12h, and the protein yield is not less than 10mg/g wet thallus.

TABLE 2 expression of TTR-GFP under different Induction conditions

Example 3:

transthyretin-hen egg white lysozyme fusion protein (TTR-L ysozyme) was prepared as follows:

(1) the recombinant plasmid pET 21a (+) -His-tag-TTR-L ysozyme is constructed by synthesizing the His-tag-TTR-L ysozyme sequence shown in SEQ ID NO:6, connecting the His-tag-TTR-L ysozyme sequence with pET 21a (+) by Nde I and EcoR I enzymes, and verifying the sequence and successfully constructing the recombinant plasmid.

(2) Expression of TTR-L ysozyme fusion protein pET 21a (+) -His-tag-TTR-L ysozyme constructed in the step (1) is transformed into E.coli B L21 (DE3) cells, 5% of the inoculum size is inoculated into 5L TB culture medium at 37 ℃, the rotating speed of a stirring paddle is 150rpm, and culture OD is cultured_600nm1.5-2.0, adding 0.1-0.5mM IPTG to induce for 8-16h, breaking bacteria by high pressure homogenization, subjecting the supernatant to affinity adsorption with nickel column to obtain TTR-L ysozyme fusion protein, and subjecting the obtained protein to endotoxin adsorption column (Pierce)^TMHigh Capacity endo toxin Removal Spin Columns, ThermoFisher) and residual bacteria were removed through a 0.22 μm pore size filter, TTR-L ysozyme protein production was examined and the results are shown in Table 3.

TABLE 3 expression of TTR-L ysozyme under different induction conditions

Example 4:

transthyretin-egg white albumin (TTR-Ovalbumin) was prepared as follows:

(1) construction of recombinant plasmid pET 21a (+) -His-tag-TTR-Ovalbumin: His-tag-TTR-Ovalbumin sequence shown in SEQ ID NO. 7 is synthesized, and is connected with pET 21a (+) by Nde I and EcoR I enzyme, and the construction is successful after sequencing verification.

(2) The expression of the TTR-Ovalbumin fusion protein is realized by transforming the pET 21a (+) -His-tag-TTR-Ovalbumin plasmid constructed in the step (1) into E.coli B L21 (DE3) cells, inoculating the E.coli B L (DE3) cells into a 5L TB culture medium at the temperature of 37 ℃, the rotating speed of a stirring paddle is 150rpm, and culturing OD (origin-to-destination) is realized_600nmTo 1.5-2.0; adding 0.1-0.5mM IPTG to induce for 8-16 h; and (3) performing high-pressure homogenization to break bacteria, and performing affinity adsorption on the supernatant through a nickel column to prepare the TTR-Ovalbumin fusion protein. The obtained protein was passed through an endotoxin adsorption column (Pierce)^TMHigh Capacity endo toxin Removal Spin Columns, ThermoFisher) and the residual bacteria were removed by 0.22 μm pore size filtration. The production of TTR-Ovalbumin protein was measured, and the results are shown in Table 4.

TABLE 4 expression of TTR-Ovalbumin under different Induction conditions

The electrophorograms of E.coli B L21 (DE3) expressed transthyretin, the purified fusion protein product and the standards of green fluorescent protein, egg white lysozyme and egg white albumin are shown in FIG. 5.

Example 5 recombinant production of human transthyretin

(1) Construction of the recombinant plasmid pETx-rhaPBAD-ttr: the pET-21a plasmid (purchased from ATCC China center for culture Collection) is reconstructed (the difference of the sequence of the reconstructed plasmid and the original pET-21a plasmid is about 75 percent, the specific sequence is shown as SEQ ID NO: 8), rhaPBAD promoter (Rhamngtang inducible) is used for replacing T7 promoter, meanwhile, the rhaPBAD promoter is connected with human TTR optimized nucleic acid sequence (shown as SEQ ID NO: 2), and the whole nucleic acid sequence of the obtained plasmid is shown as SEQ ID NO: 3. The construction was successful by sequencing verification (the sequencing company is Nanjing Kingsrei Biotechnology Co., Ltd.).

(2) The expression and purification of the recombinant humanized TTR are carried out by transforming pETx-rhaPBAD-TTR plasmid constructed in the step (1) into E.coli B L21 (DE3) cells, culturing the obtained recombinant E.coli B L21 (DE3) in L B culture medium, preparing seed liquid, inoculating into 5L TB culture medium with 5% inoculation amount, culturing at 37 ℃ and 150rpm of stirring paddle until OD₆₀₀1.5-2.0; adding 0.4-2% rhamnose to induce for 16-20h (Table 5). Crushing thallus with high pressure homogenizer, and passing the supernatant through nickel (Ni)⁺) And (4) performing column chromatography to obtain the humanized TTR. Endotoxin adsorption column (Pierce) was used^TMHigh Capacity endo toxin removal Spin Columns, ThermoFisher) and the residual bacteria were removed by 0.22 μm pore size filtration. The obtained production amounts of human TTR protein are shown in Table 5. At OD₆₀₀When the concentration is 1.5-2.0%, 0.4-2% (mass volume percentage) rhamnose is used for inducing for 16-20h, and the obtained protein yield is more than or equal to 50mg/g wet thallus.

TABLE 5 recombinant expression of TTR of human origin

(3) Expression and purification of recombinant rat-derived TTR and mouse-derived TTR as described above, only the human-derived TTR-optimized nucleic acid sequence was replaced with the corresponding murine nucleic acid sequence, and the remaining steps were identical. Wherein, the amino acid sequence of rat TTR is shown as SEQ ID NO. 9, and the amino acid sequence of mouse TTR is shown as SEQ ID NO. 10.

(4) The protein product of the humanized mature TTR with the C-terminal-TNPKE lost is obtained by purification according to the recombinant expression step, the product is named as humanized TTR-C L, and the amino acid sequence is shown as SEQ ID NO. 11.

(5) Chemical modification of human TTR: designing and synthesizing a chemical modification group which is a hydrophobic modification segment (Ex 490nm, Em 520nm) formed by connecting maleimide, dodecane and 5-aminofluorescein, and then carrying out targeted chemical modification on the chemical modification group and the only cysteine (C) residue in the human TTR which is subjected to recombinant expression and purification. At 4 ℃, the ratio of TTR of human origin to the chemical modifying group is 1: 5 mol ratio was slowly shaken (the reaction process is shown in FIG. 12). After the reaction is terminated, removing residual chemical modification reagent by ultrafiltration, concentrating TTR, detecting a sample by a fluorescence spectrometer, exciting at 490nm wavelength, wherein the emission wavelength is 520nm, which indicates that the only cysteine residue of TTR is successfully subjected to targeted modification and is named as humanized TTR-Modified.

Example 6 alignment before and after optimization of human TTR nucleic acid sequences

The T7 promoter on the reconstructed pET-21a plasmid in the embodiment 5 is replaced by a rhaPBAD promoter (rhantan inducible), and is connected with human TTR with an unoptimized nucleic acid sequence to construct and obtain a recombinant plasmid pETx-rhaPBAD-TTR (unoptimized), namely the difference between the obtained recombinant plasmid and the recombinant plasmid pETx-rhaPBAD-TTR in the embodiment is only whether TTR is optimized or not. The recombinant human-derived TTR was expressed and purified by the same method as in section (2) of example 5, and the results are shown in Table 6 below. As can be seen from the table, TTR proteins are expressed, and when OD600 is 1.6-2.0, 1.2-2% rhamnose is used for inducing for 17-20 h, the obtained protein yield is not less than 17mg/g wet thallus, and the protein expression amount is high.

TABLE 6 recombinant expression of human TTR (pETx-rhaPBAD-TTR (not optimized))

A second part: TTR itself and fusion with protein can pass through corneal barrier

Example 7 eye drop form of humanized TTR into the vitreous chamber and fundus over the corneal Barrier

(1) The human TTR obtained in example 5 was prepared as 10 μmol/L (containing physiological saline), and C57B L/6 mice (purchased from the research center for experimental animals in the upper sea, 8 weeks old) and SD rats (purchased from the research center for experimental animals in the upper sea, 8 weeks old) were each subjected to eye drop (-30 μ l), sacrificed after waiting 3h, and the cornea, vitreous body and fundus sample (retina, choroid) were removed, respectively, and proteins were extracted, respectively.

(2) The human TTR obtained in example 5 was prepared as 5-20. mu. mol/L (containing physiological saline or 1-10% low molecular weight hyaluronic acid), and C57B L/6 mice (8 weeks old) and SD rats (8 weeks old) were respectively dropped with eye, and after 3-72 hours, the vitreum and fundus sample samples were sacrificed to extract proteins, and the content of human TTR in the vitreous and fundus samples of the C57B L/6 mice and SD rats was determined by E L ISA, which showed that the effect of the increase in TTR concentration on the concentration of the vitreous and fundus sample human TTR was not large when the TTR content in the eye drops was 10-15. mu. mol/L, the vitreous and fundus sample human TTR content reached the maximum peak after 3 hours of eye dropping, the increase in TTR concentration did not affect the concentration of the vitreous and fundus sample human TTR, and the hyaluronic acid sample human TTR concentration reached the maximum peak after 3 hours of eye dropping, and the hyaluronic acid content in the eye drops was close to the peak of TTR in the vitreous and fundus sample human TTR, and fundus sample, and mouse were found to show that the effective half-life of the TTR in the eye drops is similar to that the TTR in the eye drops 60, and rat (the effective peak of the TTR in the glass sample) was found in the eye drops, the effective time of the mice).

TABLE 7-1 humanized TTR crosses the corneal barrier of C57B L/6 mice and SD rats into the fundus

(3) The human TTR and human TTR-Modified obtained in example 5 were prepared in the form of 10. mu. mol/L (containing physiological saline or 2% low molecular weight hyaluronic acid), and C57B L/6 mice (8 weeks old) and SD rats (8 weeks old) were dropped into the eye, and after 3 to 72 hours, the vitreous body and fundus sample (retina, choroid) were sacrificed to extract protein, and the content of human TTR was measured in the vitreous body and fundus sample of C57B L/6 mice and SD rats by E L ISA using rabbit anti-His-tag antibody as the primary antibody and donkey anti-rabbit antibody as the secondary antibody, and the result showed that the human TTR-Modified was Modified with a long hydrophobic segment to enter the fundus significantly more efficiently than the unmodified human TTR (Table 7-2).

TABLE 7-2

Example 8:

the purified TTR protein (concentration of 4. mu. mol/L) obtained in example 1 after removal of endotoxin and bacteria was treated with a protein sample eye in the form of eye drops in healthy SD rats (rats norgeicus) (6 weeks old) and in New Zealand big ear rabbits (Orycola cuniculus) (2 months old,. about.2.5 kg), the protein sample eye was dropped in the left eye, and a normal saline solution was dropped in the right eye as a blank control, the number of times of dropping was 1 to 3 times per day, and the amount of dropping was 0.4 to 0.8nmol each time, and two weeks later, the eye was harvested and separated to obtain a vitreous body sample, and it was preliminarily verified by the western blot method (FIG. 7A) that TTR could enter the vitreous cavity from the ocular surface, the result of SD using Anti-tag antibody as an Anti-SD antibody showed that the signal intensity of exogenous TTR in the glass of SD rats was 28.7 times that in the right eye, the signal intensity of exogenous TTR in the left eye of the New Zealand big ear rabbits was 35.6.84 times of the eye, and the content of exogenous TTR in the rat was measured by the western blot method using ISA 0.6 to show that the content of exogenous TTR in the right eye, and the content of the rat in the rabbit eye was measured by the rabbit eye, and the content of exogenous TTR in the rabbit ISA 0.8 in the rabbit eye, and the rabbit content of the rabbit eye, and.

In addition, in FIG. 3, it is shown that the sequence similarity of human/rat/rabbit transthyretin reaches nearly 95% after homology alignment, and it can be seen that the bulk TTR positive signal in FIG. 7A is the intraocular homologous protein signal.

TABLE 8 Effect of TTR reaching the eyes of SD rats in different treatment regimes

TABLE 9 Effect of TTR in reaching eyes of New Zealand big ear rabbits with different treatment modes

Example 9:

the purified TTR-GFP protein (concentration 4. mu. mol/L) after endotoxin and bacteria removal of example 2 was treated by eye-dropping with healthy SD rats (rats norgeicus (6 weeks old) and New Zealand big ear rabbits (Oryctolaguscululus) (2 months old,. about.2.5 kg) with the left eye being a protein sample eye and the right eye being a blank control with physiological saline added thereto for 1 to 3 times per day at an amount of 0.4 to 0.8nmol each two weeks before the eyeball was extracted and separated to obtain a vitreous body sample, and the results of initial verification by the western-blot method (FIG. 7B) that TTR-GFP was able to enter the vitreous cavity from the ocular surface, the results of SD western-blot with Anti-GFP antibody as an SD showed that the signal intensity of exogenous GFP in the rat vitreous body was 62.3 times that of the right eye, the results of signal intensity of the Anti-GFP antibody in the left eye of the New Zealand big ear rabbit were 47.6 times the signal intensity of the Anti-GFP in the rat vitreous body, and the results of the Anti-GFP in vivo detection of the rabbit were carried out by the western blot method with Anti-GFP, and the Anti-GFP content of the rabbit, and the rat, the rabbit, the results of the antibody of the rabbit were carried out the antibody, the assay by the western blot method, the rabbit.

TABLE 10 Effect of TTR-GFP in different treatment modalities on reaching the eyes of SD rats

TABLE 11 Effect of TTR-GFP in reaching eyes of New Zealand big ear rabbits with different treatment modalities

Example 10:

the purified TTR-L ysozyme protein (concentration 4. mu. mol/L) prepared in example 3 after removal of endotoxin and bacteria was treated by eye dropping with healthy SD rats (rats norgeicus) (6 weeks old) and New Zealand big ear rabbits (Oryctolagus cuniculus) (2 months old, 2.5kg), the left eye was a protein sample eye, the right eye was a control eye to which physiological saline was added as a blank, the number of times of daily dropping was 1-3, the amount of each dropping was 0.4-0.8nmol, two weeks later, a vitreous body was extracted and isolated, and it was preliminarily verified by the western blot method (FIG. 7C) that TTR-L ysozyme could enter the vitreous cavity from the eye SD, the result of which the rabbit eye was treated with the antibody of Antarcti-567 ysozyme antibody was found to have a higher signal intensity in vivo as the rabbit eye L, the rabbit eye 356. 23. the rabbit eye was treated with the antibody of Western blot, and the rabbit eye was treated with the rabbit eye containing no exogenous antibody of the rabbit eye 23. the rabbit eye, the rabbit eye containing no exogenous rabbit antibody of the rabbit eye, the rabbit eye 23. the rabbit eye, the rabbit eye containing the rabbit eye 23. the rabbit eye, the rabbit eye No. 12. the rabbit eye was treated with the rabbit eye to which was treated with eye sample eye to which was treated with eye added with eye sample eye, the rabbit eye sample, the rabbit eye to which was treated with eye to which was added with the rabbit eye sample, the rabbit eye containing exogenous signal intensity of the rabbit eye, the rabbit eye to which was added with the rabbit eye to which was added.

TABLE 12 Effect of TTR-L ysozyme reaching the eyes of SD rats in different treatment regimes

TABLE 13 Effect of TTR-L ysozyme on reaching eyes of New Zealand big ear rabbits by different treatment methods

Example 11:

after removing endotoxin and bacteria in example 4, the prepared TTR-ovabumin protein (concentration of 4 μmol/L) was purified, a healthy SD rat (rats norgeicus) (6 weeks old) and new zealand big ear rabbit (orictolagus) (2 months old,. about.2.5 kg) were treated by eye dropping, a protein sample eye was dropped in the left eye, a normal saline solution was dropped in the right eye as a blank control, the number of times of dropping per day was 1 to 3 times, and the amount of dropping per time was 0.4 to 0.8 nmol.two weeks later, an eyeball was extracted and a vitreous body was obtained by separation, it was preliminarily verified by the western blot method (fig. 7D) that TTR-ovabumin was able from the surface of the eye into the vitreous cavity, the result of using an antibody of ansd-ovabumin an antibody as an antibody showed that the signal intensity of the SD rat with the foreign source ovabumin the left eye was 25.3 times that the rat with the foreign source of the foreign antibody of the right eye was 25.3 times of the right eye, the rat with the rabbit in the eye having the strong foreign antibody of the rabbit, and the signal intensity of the rat with the rabbit in the rabbit eye being measured by the rabbit eye of the rabbit with the rabbit eye having the foreign antibody content of the rabbit in the rabbit, the.

TABLE 14 Effect of TTR-Ovalbumin on reaching SD rat eyes under different treatment modes

TABLE 15 Effect of TTR-Ovalbumin on New Zealand big ear eyes with different treatments

And a third part: TTR for treating eye diseases such as diabetic retinopathy

Example 12 eye drop treatment of DR SD rats with human TTR

(1) SD rats of 8 weeks old, the weight of 200-250g, fasting for 12-18h, intraperitoneal injection of 2% STZ (60mg/kg) for 48h and 72h, tail-cutting and blood collection, blood glucose test paper detection is higher than 16.7mM, modeling is successful, the STZ rats are divided into 2 groups, one group is STZ without any treatment (5), the other group is human TTR eye drop (25) group prepared in example 5 (TTR (5-20 mu mol/L) (normal saline + 2% hyaluronic acid) is added to the left eye for 2 times per day), the right eye is added with normal saline + 2% hyaluronic acid, in addition, 1 group of normal rats are used as a control (5), after the rats are continuously raised for 3 months, the retinas are respectively stripped for EB staining, Trypsin enzymolysis is used for observing the density of new blood vessels, the result shows that after the diabetic rats are raised for 3 months, the STZ induces the retinal blood vessels to leak, the number of the new blood vessels is obviously improved, the phenomenon of retinal blood vessels is obviously dropped, the eyeball leakage phenomenon is obviously inhibited, and the clinical effect of the drop of the new blood vessels is shown in a 358% of the normal control (TTR, namely, the clinical effect of the TTR is obtained, and the clinical effect is showed, wherein the clinical effect of the TTR (L).

TABLE 16 treatment of STZ induced diabetic SD rat DR pathological conditions with humanized TTR

Example 13 treatment of ROP SD rats with eye drops of human TTR

One week old suckling mice (SD rats) were born and kept in a hyperbaric oxygen chamber, controls were kept in a normal environment (normal group, 6-8) and 1 time per day (30. mu.l each time) (6 ROP/TTR (model-making) group) were dropped to one group of suckling mice using 5-20. mu. mol/L of TTR (physiological saline + 2% hyaluronic acid) prepared in example 5. mu.l each time (ROP/TTR (model-making) group) in the hyperbaric chamber, 1 time per day (30. mu.l each time (ROP group, 24) was dropped to one group of suckling mice using physiological saline + 2% hyaluronic acid prepared in example 5 and 5-20. mu. mol/L each time per day was dropped to one group of suckling mice in the hyperbaric chamber for 5 days, 1 time per day (ROP/TTR (model-forming) group) was dropped to one group of suckling mice using physiological saline + 2% hyaluronic acid per day, and the mice in the ROP group were kept in a normal environment for 5 days and then the mice were sacrificed by using the remaining physiological saline + 2% hyaluronic acid as controls.

The results are shown in FIG. 9, which shows that the retinal EB staining pattern of the normal control group was normal during the molding process; the ROP group had significant areas of no perfusion and neovascularization; the ROP/TTR group which is molded and simultaneously dropped with TTR does not form obvious non-perfusion area and malformed new blood vessels; this indicates that the composition has a protective effect on normal blood vessels and an inhibitory effect on new blood vessels in the hypoxic state (FIG. 9).

TTR (10. mu. mol/L) was applied as eye drops to molded ROP suckling mice for 5 days, comparing ROP and ROP/TTR groups, a large number of leakage areas appeared after the malformed neovasculature in the ROP group was engorged with the retina at the later stage of the experiment (5 days), and TTR eye drops were able to reverse this trend (FIG. 10).

The molded ROP rats were dropped with different concentrations of TTR (5-20 μmol/L) for 5 days later in the experiment (5 days) where the malformed neovasculature in the ROP control group was engorged with retinal areas of profuse leakage and TTR drops reversed this trend, with the best 10 μmol/L TTR drop (table 17).

TABLE 17 pathological conditions of rat suckling rat ROP induced by hyperbaric oxygen chamber treated by humanized TTR

Example 14 eye drop treatment of human TTR for AMD C57B L/6 mice

A9-week-old C57/B L6 mouse was photocoagulated with krypton laser (647nm) at a power of 360mW, a diameter of 50um, a time of 0.05s, and 8 photocoagulation points per eye to induce choroidal neovascularization and gradually migrate toward retinal hyperplasia.A C57 mouse was subjected to TTR eye-dropping, 5-20. mu. mol/L of TTR (physiological saline + 2% hyaluronic acid) prepared in example 5 was added twice to the right eye daily, 30. mu. L each time, and a control (14 eye-dropped groups, 6 non-eye-dropped groups, and 2 non-laser-dropped groups) was added to the left eye, and after 2 weeks of eye-dropping, the animal was sacrificed, retinal Evans Blue was peeled off and retinal vascular leakage was observed, and neovascular density was observed by Trypsin enzymolysis.

The results are shown in FIG. 11, which shows that the EB staining pattern of the normal control group retinas is normal, that the AMD group has significant retinal leakage and neovascularization, and that the retinal leakage and neovascularization of the AMD/TTR group are significantly alleviated by 10 μmol/L TTR eye drops (FIG. 11).

AMD mice, which had been molded, were instilled with different concentrations of TTR (5-20. mu. mol/L) for two weeks, AMD control group showed more extravasation area and significantly increased number of new blood vessels, and TTR instillation reversed this trend, with the best effect of 10. mu. mol/L TTR instillation (Table 18).

TABLE 18 treatment of AMD pathological conditions in mice by human TTR induced by laser retinal photocoagulation C57B L/6

As can be seen from the above examples, the pathological states of DR, AMD and ROP can be effectively treated in the animal models of DR, AMD and ROP by the way of TTR dropping into the eye.

Example 15 eye drop treatment of DR SD rats with human-derived TTR/rat-derived TTR

SD rats of 8 weeks old, with a weight of 200 & lt- & gt 250g, fasting for 12-18h, intraperitoneal injection of 2% STZ (60mg/kg) after 48h and 72h, tail-clipped blood collection, blood glucose dipstick detection of more than 16.7mM, molding success, the STZ rats were divided into 4 groups, one group was the human TTR eye drop (5) group prepared in example 5 (2 human TTR (10. mu. mol/L) (physiological saline + 2% hyaluronic acid) per day for the left eye, the right eye was the physiological saline + 2% hyaluronic acid), 1 group was the rat TTR eye drop (5) prepared in example 5, the left eye was the human TTR eye drop (10. mu. mol/L) (physiological saline + 2% hyaluronic acid) per day for the rat, the right eye was the human TTR-C L eye drop (5) group prepared in example 5, the human TTR-C eye drop (5) group prepared in example 5 was the human TTR eye drop (10. mu. mol/L) (physiological saline + 2% hyaluronic acid), the human TTR-C eye drop (5) group prepared in example 5) per day, the human eye drop (2 times of ModifR-C drop) group prepared in the left eye drop) for the left eye, the human eye drop (2 times of the human eye drop) group, the normal blood glucose drop) group, the rat eye drop-C drop (normal blood glucose drop) was observed as a clinical observation results, the rat eye drop-induced by injection, the rat eye drop-induced by the clinical observation, the rat-induced by the normal blood glucose drop-induced by the rat-induced by the normal blood glucose drop (normal blood glucose drop-induced by the rat eye drop-induced by the rat eye drop (2-induced by the rat) after the normal blood glucose-induced by the normal blood glucose drop-induced by the normal blood glucose-induced by the normal.

TABLE 19 treatment of STZ induced diabetic SD rat DR pathological conditions with human-derived TTR/rat-derived TTR

Example 16 eye drop treatment of ROP SD rats with human-derived TTR/rat-derived TTR

After breeding in a hyperbaric oxygen chamber for 5 days after birth, all the suckling rats (SD rats) are transferred from the hyperbaric oxygen chamber to a normal environment for breeding, and the molded ROP suckling rats are subjected to TTR (10 mu mol/L) eye dropping for 5 days.

TABLE 20 treatment of pathological conditions of hyperbaric oxygen chamber induced SD rat suckling rat ROP by humanized/rat TTR

Example 17 eye drop treatment of AMD C57B L/6 mice with human-derived/mouse-derived TTR

A9-week-old C57/B L mouse is subjected to photocoagulation on retina by krypton laser (647nm), has power of 360mW, diameter of 50um and time of 0.05s, has 8 photocoagulation points per eye, induces choroidal neovascularization and gradually migrates toward retina hyperplasia.A C57 mouse is subjected to TTR eye dropping, 10 mu mol/L of human TTR or mouse TTR (physiological saline + 2% hyaluronic acid) is added twice per day to the right eye, 30 mu L of each time, physiological saline + 2% hyaluronic acid is added to the left eye as a control (5 human TTR eye drops, 5 human TTR-C L eye drops, 5 human TTR-Modified eye drops, 5 mouse TTR eye drops, 5 non-eye drops), a non-laser photocoagulation mouse (non-eye drops) is used as a normal control group (5 eye drops), after 2 weeks, animals are sacrificed, retinal is stripped off, and is subjected to staining, the condition of blood vessels is observed, the retinal leakage of human origin is remarkably reduced, and the clinical signs of human retina leakage and the TTR-retinal leakage is remarkably reduced by TTR-derived from the TTR eye drop table.

TABLE 21 treatment of AMD pathological conditions in mice with human/mouse TTR induced by laser retinal photocoagulation C57B L/6

Comparative example 1 recombinant expression of humanized TTR Using different plasmids

(1) Construction of recombinant plasmid pET-28a-ttr, pQE-30-ttr, pQE-60-ttr: the mature TTR-optimized DNA sequence (shown as SEQ ID NO: 2) was ligated into pET-28a plasmid (pET-28a, pQE-30, pQE-60 purchased from ATCC) via Nde I and Hind III restriction sites to construct pET-28a-TTR plasmid; the DNA sequence optimized by mature TTR (shown as SEQID NO: 2) is connected and connected into pQE-30 plasmid through two enzyme cutting sites of BamH I and Hind III to construct pQE-30-TTR plasmid; the mature TTR optimized DNA sequence (shown as SEQ ID NO: 2) is connected into pQE-60 plasmid through two enzyme cutting sites of EcoR I and Hind III to construct pQE-60-TTR plasmid. The construction was successful by sequencing verification (Nanjing Kingsrei Biotech Co., Ltd.).

(2) Expressing and purifying the recombinant human TTR, namely converting pET-28a-TTR, pQE-30-TTR and pQE-60-TTR plasmids constructed in the step (1) and pETx-rhaPBAD-TTR constructed in the example 5 into E.coli B L21 (DE3) cells, culturing the obtained recombinant E.coli B L21 (DE3) in L B culture medium to prepare seed solution, inoculating the seed solution into 5L TB culture medium by 5 percent of inoculation amount at the temperature of 37 ℃, the rotating speed of a stirring paddle of 150rpm, culturing until OD600 is 1.5-2.0, adding 0.2mM IPTG into the first three culture media to induce 18h, adding 1.6 percent rhamnose into the last culture medium to induce 18h, crushing the thalli by a high-pressure homogenizer, and preparing the human TTR by Ni + from supernatant to prepare the human TTR through endotoxin adsorption column chromatography (Pierce)^TMHigh CapacityEndotoxin Removal Spin Columns, ThermoFisher) and passed through a 0.22 μm pore size filter to remove residual bacteria. The yields of the obtained human TTR protein are shown in Table 22.

TABLE 22 recombinant expression of human TTR using different plasmids

From the above results, it was found that no soluble expression was observed when expression was carried out using pET-28a, pQE-30, pQE-60 or the like.

Comparative example 2

With respect to Table 7 in example 7, the concentration of human TTR in the vitreous chamber was only 0.10. + -. 0.00. mu. mol/L when TTR was added dropwise at a concentration of 1. mu. mol/L.

Comparative example 3:

the present embodiment is the same as example 9, except that the GFP protein not fused to TTR (Genbank accession No. QAA95705.1) was expressed according to the method of example 2, and the eye drop test was performed on SD rats and New Zealand big ear rabbits using the same procedure as in example 9 using the GFP protein not fused to TTR, and it was revealed that GFP did not enter the vitreous body of SD rats and New Zealand big ear rabbits (tables 23 and 24).

TABLE 23 Effect of GFP content 0.6nmol added dropwise each time and reaching SD rat eyes after different times

Wherein "-" indicates no detection.

TABLE 24 Effect of GFP content 0.6nmol added dropwise each time and reaching the eyes of New Zealand big ears after different times

Fruit

Wherein "-" indicates no detection.

Comparative example 4:

the present embodiment is the same as example 10 except that L ysozyme protein not fused to TTR (Genbank accession No.: AA L69327.1) was expressed according to the method of example 3, and the same procedures as in example 10 were carried out to test SD rats and New Zealand big ear rabbits for eye drop of unfused L ysozyme protein, and it was revealed that L ysozyme did not enter the vitreous body of SD rats and New Zealand big ear rabbits (tables 25, 26).

TABLE 25 Effect of L ysozyme content 0.6nmol per drop, L ysozyme reaching SD rat eyes after different times of drop

TABLE 26 Effect of L ysozyme on reaching the eyes of New Zealand big ears under different treatment modes after L ysozyme content of 0.6nmol is added to each drop for different times

Comparative example 5:

the present embodiment is the same as example 11 except that the method of example 4 was used to express the ovabulin protein (UniProt accession No. P01012) which was not fused to TTR, and the results of the eye drop test of the unfused ovabulin protein in SD rats and new zealand big ear rabbits by the same procedure as in example 11 showed that ovabulin did not enter the vitreous body of SD rats and new zealand big ear rabbits (tables 27 and 28).

TABLE 27 Effect of Ovalbumin content of 0.6nmol added dropwise each time and reaching SD rat eyes after different times of daily dropwise addition

TABLE 28 Effect of Ovalbumin content of 0.6nmol added dropwise each time and reaching the eye of New Zealand big ear after different times of daily addition

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> Tongyan (Shanghai) medical Instrument Co., Ltd

<120> use of transthyretin in the eye and in the preparation of drops

<130>P20013185C

<150>201911302937.3

<151>2019-12-17

<160>11

<170>PatentIn version 3.5

<210>1

<211>127

<212>PRT

<213>Homo sapiens

<400>1

Gly Pro Thr Gly Thr Gly Glu Ser Lys Cys Pro Leu Met Val Lys Val

15 10 15

Leu Asp Ala Val Arg Gly Ser Pro Ala Ile Asn Val Ala Val His Val

20 25 30

Phe Arg Lys Ala Ala Asp Asp Thr Trp Glu Pro Phe Ala Ser Gly Lys

35 40 45

Thr Ser Glu Ser Gly Glu Leu His Gly Leu Thr Thr Glu Glu Glu Phe

50 55 60

Val Glu Gly Ile Tyr Lys Val Glu Ile Asp Thr Lys Ser Tyr Trp Lys

65 70 75 80

Ala Leu Gly Ile Ser Pro Phe His Glu His Ala Glu Val Val Phe Thr

85 90 95

Ala Asn Asp Ser Gly Pro Arg Arg Tyr Thr Ile Ala Ala Leu Leu Ser

100 105 110

Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val Thr Asn Pro Lys Glu

115 120 125

<210>2

<211>381

<212>DNA

<213>Artificial Sequence

<220>

<223> TTR mature fragment optimized nucleic acid sequence

<400>2

ggtccgaccg gtaccggtga atctaaatgc ccgctgatgg ttaaagttct ggacgctgtt 60

cgtggttctc cggctatcaa cgttgctgtt cacgttttcc gtaaagctgc tgacgacacc 120

tgggaaccgt tcgcttctgg taaaacctct gaatctggtg aactgcacgg tctgaccacc 180

gaagaagaat tcgttgaagg tatctacaaa gttgaaatcg acaccaaatc ttactggaaa 240

gctctgggta tctctccgtt ccacgaacac gctgaagttg ttttcaccgc taacgactct 300

ggtccgcgtc gttacaccat cgctgctctg ctgtctccgt actcttactc taccaccgct 360

gttgttacca acccgaaaga a 381

<210>3

<211>4693

<212>DNA

<213>Artificial Sequence

<220>

<223> plasmid Whole nucleic acid sequence

<400>3

aattcttaag aaggagatat acatatgaga ggatcgcatc atcatcatca tcatggatcc 60

ggtccgaccg gtaccggtga atctaaatgc ccgctgatgg ttaaagttct ggacgctgtt 120

cgtggttctc cggctatcaa cgttgctgtt cacgttttcc gtaaagctgc tgacgacacc 180

tgggaaccgt tcgcttctgg taaaacctct gaatctggtg aactgcacgg tctgaccacc 240

gaagaagaat tcgttgaagg tatctacaaa gttgaaatcg acaccaaatc ttactggaaa 300

gctctgggta tctctccgtt ccacgaacac gctgaagttg ttttcaccgc taacgactct 360

ggtccgcgtc gttacaccat cgctgctctg ctgtctccgt actcttactc taccaccgct 420

gttgttacca acccgaaaga atgaaagctt ctgttttggc ggatgagaga agattttcag 480

cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt tgcctggcgg 540

cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac gccgtagcgc 600

cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat caaataaaac 660

gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc 720

tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag 780

ggtggcgggc aggacgcccg ccataaactg ccaggcatca aattaagcag aaggccatcc 840

tgacggatgg cctttttgcg tttctacaaa ctcttttgtt tatttttcta aatacattca 900

aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 960

aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 1020

cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 1080

ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 1140

cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 1200

ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 1260

gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 1320

gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 1380

acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 1440

cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 1500

acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 1560

ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 1620

ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 1680

gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 1740

atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 1800

ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 1860

attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 1920

ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 1980

aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 2040

aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 2100

ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg 2160

tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 2220

ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 2280

cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 2340

agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 2400

gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 2460

ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 2520

tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 2580

tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 2640

cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 2700

tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 2760

gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 2820

atatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac 2880

tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga 2940

cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc 3000

cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg 3060

gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc 3120

cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt 3180

aagggcggtt ttttcctgtt tggtcacttg atgcctccgt gtaaggggga atttctgttc 3240

atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat 3300

gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg 3360

gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt 3420

ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct 3480

gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct 3540

caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca 3600

ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg 3660

atcatgcgca cccgtggcca ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg 3720

gagatggcgg acgcgatgga tatgttctgc caagggttgg tttgcgcatt cacagttctc 3780

cgcaagaattgattggctcc aattcttgga gtggtgaatc cgttagcgag gtgccgccgg 3840

cttccattca ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga 3900

caaggtatag ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg 3960

cataaatcgc cgtgacgatc agcggtccag tgatcgaagt taggctggta agagccgcga 4020

gcgatccttg aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca 4080

acgcgggcat cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc 4140

ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa 4200

tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg 4260

cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc 4320

ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa 4380

agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga 4440

ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc cttatgcgac tcctgcatta 4500

ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat 4560

gcatcgatca ccacaattca gcaaattgtg aacatcatca cgttcatctt tccctggttg 4620

ccaatggccc attttcctgt cagtaacgag aaggtcgcga attcaggcgc tttttagact 4680

ggtcgtaatg aac 4693

<210>4

<211>420

<212>DNA

<213>Artificial Sequence

<220>

<223>His-tag-TTR

<400>4

atgagaggat cgcatcatca tcatcatcat ggatccggtc cgaccggtac cggtgaatct 60

aaatgcccgc tgatggttaa agttctggac gctgttcgtg gttctccggc tatcaacgtt 120

gctgttcacg ttttccgtaa agctgctgac gacacctggg aaccgttcgc ttctggtaaa 180

acctctgaat ctggtgaact gcacggtctg accaccgaag aagaattcgt tgaaggtatc 240

tacaaagttg aaatcgacac caaatcttac tggaaagctc tgggtatctc tccgttccac 300

gaacacgctg aagttgtttt caccgctaac gactctggtc cgcgtcgtta caccatcgct 360

gctctgctgt ctccgtactc ttactctacc accgctgttg ttaccaaccc gaaagaatga 420

<210>5

<211>1137

<212>DNA

<213>Artificial Sequence

<220>

<223>His-tag-TTR-GFP

<400>5

atgcgtggtt ctcaccacca ccaccaccac ggttctggtc cgaccggtac cggtgaatct 60

aaatgcccgc tgatggttaa agttctggac gctgttcgtg gttctccggc tatcaacgtt 120

gctgttcacg ttttccgtaa agctgctgac gacacctggg aaccgttcgc ttctggtaaa 180

acctctgaat ctggtgaact gcacggtctg accaccgaag aagaatttgt tgaaggtatc 240

tacaaagttg aaatcgacac caaatcttac tggaaagctc tgggtatctc tccgttccac 300

gaacacgctg aagttgtttt caccgctaac gactctggtc cgcgtcgtta caccatcgct 360

gctctgctgt ctccgtactc ttactctacc accgctgttg ttaccaaccc gaaagaaggt 420

tctatgtcta aaggtgaaga actgttcacc ggtgttgttc cgatcctggt tgaactggac 480

ggtgacgtta acggtcacaa attctctgtt tctggtgaag gtgaaggtga cgctacctac 540

ggtaaactga ccctgaaatt catctgcacc accggtaaac tgccggttcc gtggccgacc 600

ctggttacca ccttcaccta cggtgttcag tgcttctctc gttacccgga ccacatgaaa 660

cagcacgact tcttcaaatc tgctatgccg gaaggttacg ttcaggaacg taccatcttc 720

ttcaaagacg acggtaacta caaaacccgt gctgaagtta aattcgaagg tgacaccctg 780

gttaaccgta tcgaactgaa aggtatcgac ttcaaagaag acggtaacat cctgggtcac 840

aaactggaat acaactacaa ctctcacaac gtttacatca tggctgacaa acagaaaaac 900

ggtatcaaag ttaacttcaa aatccgtcac aacatcgaag acggttctgt tcagctggct 960

gaccactacc agcagaacac cccgatcggt gacggtccgg ttctgctgcc ggacaaccac 1020

tacctgtcta cccagtctgc tctgtctaaa gacccgaacg aaaaacgtga ccacatggtt 1080

ctgctggaat ttgttaccgc tgctggtatc acccacggta tggacgaact gtacaaa 1137

<210>6

<211>864

<212>DNA

<213>Artificial Sequence

<220>

<223>His-tag-TTR-Lysozyme

<400>6

atgcgtggtt ctcaccacca ccaccaccac ggttctggtc cgaccggtac cggtgaatct 60

aaatgcccgc tgatggttaa agttctggac gctgttcgtg gttctccggc tatcaacgtt 120

gctgttcacg ttttccgtaa agctgctgac gacacctggg aaccgttcgc ttctggtaaa 180

acctctgaat ctggtgaact gcacggtctg accaccgaag aagaatttgt tgaaggtatc 240

tacaaagttg aaatcgacac caaatcttac tggaaagctc tgggtatctc tccgttccac 300

gaacacgctg aagttgtttt caccgctaac gactctggtc cgcgtcgtta caccatcgct 360

gctctgctgt ctccgtactc ttactctacc accgctgttg ttaccaaccc gaaagaaggt 420

tctatgcgtt ctctgctgat cctggttctg tgcttcctgc cgctggctgc tctgggtaaa 480

gttttcggtc gttgcgaact ggctgctgct atgaaacgtc acggtctgga caactaccgt 540

ggttactctc tgggtaactg ggtttgcgct gctaaattcg aatctaactt caacacccag 600

gctaccaacc gtaacaccga cggttctacc gactacggta tcctgcagat caactctcgt 660

tggtggtgca acgacggtcg taccccgggt tctcgtaacc tgtgcaacat cccgtgctct 720

gctctgctgt cttctgacat caccgcttct gttaactgcg ctaaaaaaat cgtttctgac 780

ggtaacggta tgaacgcttg ggttgcttgg cgtaaccgtt gcaaaggtac cgacgttcag 840

gcttggatcc gtggttgccg tctg 864

<210>7

<211>1581

<212>DNA

<213>Artificial Sequence

<220>

<223>His-tag-TTR-Ovalbumin

<400>7

atgcgtggtt ctcaccacca ccaccaccac ggttctggtc cgaccggtac cggtgaatct 60

aaatgcccgc tgatggttaa agttctggac gctgttcgtg gttctccggc tatcaacgtt 120

gctgttcacg ttttccgtaa agctgctgac gacacctggg aaccgttcgc ttctggtaaa 180

acctctgaat ctggtgaact gcacggtctg accaccgaag aagaatttgt tgaaggtatc 240

tacaaagttg aaatcgacac caaatcttac tggaaagctc tgggtatctc tccgttccac 300

gaacacgctg aagttgtttt caccgctaac gactctggtc cgcgtcgtta caccatcgct 360

gctctgctgt ctccgtactc ttactctacc accgctgttg ttaccaaccc gaaagaaggt 420

tctatgggtt ctatcggtgc tgcttctatg gaattttgct tcgacgtttt caaagaactg 480

aaagttcacc acgctaacga aaacatcttc tactgcccga tcgctatcat gtctgctctg 540

gctatggttt acctgggtgc taaagactct acccgtaccc agatcaacaa agttgttcgt 600

ttcgacaaac tgccgggttt cggtgactct atcgaagctc agtgcggtac ctctgttaac 660

gttcactctt ctctgcgtga catcctgaac cagatcacca aaccgaacga cgtttactct 720

ttctctctgg cttctcgtct gtacgctgaa gaacgttacc cgatcctgcc ggaatacctg 780

cagtgcgtta aagaactgta ccgtggtggt ctggaaccga tcaacttcca gaccgctgct 840

gaccaggctc gtgaactgat caactcttgg gttgaatctc agaccaacgg tatcatccgt 900

aacgttctgc agccgtcttc tgttgactct cagaccgcta tggttctggt taacgctatc 960

gttttcaaag gtctgtggga aaaagctttc aaagacgaag acacccaggc tatgccgttc 1020

cgtgttaccg aacaggaatc taaaccggtt cagatgatgt accagatcgg tctgttccgt 1080

gttgcttcta tggcttctga aaaaatgaaa atcctggaac tgccgttcgc ttctggtacc 1140

atgtctatgc tggttctgct gccggacgaa gtttctggtc tggaacagct ggaatctatc 1200

atcaacttcg aaaaactgac cgaatggacc tcttctaacg ttatggaaga acgtaaaatc 1260

aaagtttacc tgccgcgtat gaaaatggaa gaaaaataca acctgacctc tgttctgatg 1320

gctatgggta tcaccgacgt tttctcttct tctgctaacc tgtctggtat ctcttctgct 1380

gaatctctga aaatctctca ggctgttcac gctgctcacg ctgaaatcaa cgaagctggt 1440

cgtgaagttg ttggttctgc tgaagctggt gttgacgctg cttctgtttc tgaagaattt 1500

cgtgctgacc acccgttcct gttctgcatc aaacacatcg ctaccaacgc tgttctgttc 1560

ttcggtcgtt gcgtttctcc g 1581

<210>8

<211>4311

<212>DNA

<213>Artificial Sequence

<220>

<223> sequence of pET-21a plasmid after reconstruction

<400>8

aattcttaag aaggagatat acatatggga tcccatcatc atcatcatca ttgactgcag 60

ccaagcttct gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa 120

cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct 180

gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc 240

catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg 300

ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc 360

gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc 420

ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt 480

tctacaaact cttttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 540

aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 600

tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 660

aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 720

aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 780

tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc 840

aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 900

tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 960

ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 1020

taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 1080

agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 1140

caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 1200

tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 1260

gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 1320

cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 1380

caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 1440

ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 1500

aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 1560

gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 1620

atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 1680

tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 1740

gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 1800

actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 1860

gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 1920

agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 1980

ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 2040

aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 2100

cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 2160

gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 2220

cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 2280

cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 2340

gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt 2400

attttctcct tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac 2460

aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg 2520

gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 2580

ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 2640

ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg 2700

tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga 2760

agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg 2820

gtcacttgat gcctccgtgt aagggggaat ttctgttcat gggggtaatg ataccgatga 2880

aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac 2940

gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg 3000

gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc 3060

ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt 3120

acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc 3180

agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac 3240

cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg 3300

acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata 3360

tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa 3420

ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc 3480

ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac 3540

aatccatgcc aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag 3600

cggtccagtg atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg 3660

atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc 3720

ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa 3780

gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg 3840

tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc 3900

aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca 3960

gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc 4020

gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg 4080

catcggtcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt 4140

gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc atcgatcacc acaattcagc 4200

aaattgtgaa catcatcacg ttcatctttc cctggttgcc aatggcccat tttcctgtca 4260

gtaacgagaa ggtcgcgaat tcaggcgctt tttagactgg tcgtaatgaa c 4311

<210>9

<211>127

<212>PRT

<213>Rattus norvegicus

<400>9

Gly Pro Gly Gly Ala Gly Glu Ser Lys Cys Pro Leu Met Val Lys Val

1 5 10 15

Leu Asp Ala Val Arg Gly Ser Pro Ala Val Asp Val Ala Val Lys Val

20 25 30

Phe Lys Lys Thr Ala Asp Gly Ser Trp Glu Pro Phe Ala Ser Gly Lys

35 40 45

Thr Ala Glu Ser Gly Glu Leu His Gly Leu Thr Thr Asp Glu Lys Phe

50 55 60

Thr Glu Gly Val Tyr Arg Val Glu Leu Asp Thr Lys Ser Tyr Trp Lys

65 70 75 80

Ala Leu Gly Ile Ser Pro Phe His Glu Tyr Ala Glu Val Val Phe Thr

85 90 95

Ala Asn Asp Ser Gly His Arg His Tyr Thr Ile Ala Ala Leu Leu Ser

100 105 110

Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val Ser Asn Pro Gln Asn

115 120 125

<210>10

<211>127

<212>PRT

<213>Mus musculus

<400>10

Gly Pro Ala Gly Ala Gly Glu Ser Lys Cys Pro Leu Met Val Lys Val

1 5 10 15

Leu Asp Ala Val Arg Gly Ser Pro Ala Val Asp Val Ala Val Lys Val

20 25 30

Phe Lys Lys Thr Ser Glu Gly Ser Trp Glu Pro Phe Ala Ser Gly Lys

35 40 45

Thr Ala Glu Ser Gly Glu Leu His Gly Leu Thr Thr Asp Glu Lys Phe

50 5560

Val Glu Gly Val Tyr Arg Val Glu Leu Asp Thr Lys Ser Tyr Trp Lys

65 70 75 80

Thr Leu Gly Ile Ser Pro Phe His Glu Phe Ala Asp Val Val Phe Thr

85 90 95

Ala Asn Asp Ser Gly His Arg His Tyr Thr Ile Ala Ala Leu Leu Ser

100 105 110

Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val Ser Asn Pro Gln Asn

115 120 125

<210>11

<211>122

<212>PRT

<213>Artificial Sequence

<220>

<223> human TTR-C L

<400>11

Gly Pro Thr Gly Thr Gly Glu Ser Lys Cys Pro Leu Met Val Lys Val

1 5 10 15

Leu Asp Ala Val Arg Gly Ser Pro Ala Ile Asn Val Ala Val His Val

20 25 30

Phe Arg Lys Ala Ala Asp Asp Thr Trp Glu Pro Phe Ala Ser Gly Lys

35 40 45

Thr Ser Glu Ser Gly Glu Leu His Gly Leu Thr Thr Glu Glu Glu Phe

50 5560

Val Glu Gly Ile Tyr Lys Val Glu Ile Asp Thr Lys Ser Tyr Trp Lys

65 70 75 80

Ala Leu Gly Ile Ser Pro Phe His Glu His Ala Glu Val Val Phe Thr

85 90 95

Ala Asn Asp Ser Gly Pro Arg Arg Tyr Thr Ile Ala Ala Leu Leu Ser

100 105 110

Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val

115 120

Claims

1. The use of transthyretin as a carrier for proteinaceous and/or polypeptide drugs to enter the eye through the ocular barrier, said transthyretin being as defined in (a), (b) or (c):

(a) a protein consisting of amino acids shown in SEQ ID NO. 1;

2. The use of claim 1, wherein in (b), the protein derived from (a) is substituted with 22 amino acids, 25 amino acids, or 5 amino acids in the amino acid sequence of (a);

and/or, in (c), the hydrophilic modification or hydrophobic modification is performed on the cysteine at position 10 on the amino acid sequence in (a);

preferably:

in (b), the protein derived from (a) is a substitution of T, I, N, H, R, A, D, T, S, E, V, I, K, I, A, H, E, P102, R104, T123, K126 and/or E127 of the amino acid sequence in (a), or a deletion at position 123 and 127 of the amino acid sequence in (a), preferably a substitution of T3, T5, I26, N27, H31, R34, A36, D39, T40, S50, E61, E63, V65, I68, K70, I, H90, P102, R104, T123, K126 and E127 of the amino acid sequence in (a), or a substitution of T3, T5, I26, N27, H31, R34, A36, D37, T38, K126, E70, E60, K70, E60, E68, K70, E60, E68, E60, E68, E38, E68, E70, E60, E68, E127, E60, E;

and/or, in (c), the protein is modified with a long-chain hydrophobic segment, such as n-dodecane, at the cysteine at position 10 of the amino acid sequence in (a);

more preferably:

in the step (b), the amino acid sequence of the protein derived from the step (a) is shown as SEQ ID NO. 9, SEQ ID NO. 10 or SEQ ID NO. 11;

and/or, in the (c), the protein is a protein shown in a sequence which is connected with n-dodecane modification on the 10 th cysteine on the amino acid sequence in the (a) through maleimide.

3. The use according to claim 1 or 2, wherein the transthyretin is expressed as a fusion with the proteinaceous and/or polypeptide drug; the fusion is preferably to fuse the protein and/or polypeptide drug at the N terminal or C terminal of the transthyretin; the transthyretin is preferably expressed in microbial cells and purified after being fused with the protein and/or polypeptide drug; the purification is preferably to remove endotoxin by an endotoxin adsorption column and then remove residual thalli by a filter membrane with the aperture of 0.22 mu m;

and/or the protein and/or polypeptide drugs comprise lysozyme, albumin and/or EGFR antibodies, and the molecular weight of the lysozyme is not more than 45kDa, the lysozyme is preferably egg white lysozyme, and the GenBank accession number of the lysozyme is AA L69327.1, and the albumin is preferably egg white albumin;

and/or, the protein and/or polypeptide drugs comprise protein and/or polypeptide drugs for treating diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity.

4. The use of any one of claims 1 to 3, wherein the nucleotide sequence encoding said transthyretin is as set forth in SEQ ID NO. 2;

and/or the transthyretin is expressed by using a recombinant expression vector, and a promoter in a skeleton plasmid of the recombinant expression vector is a rhamnothaliana inducible promoter, preferably a rhaPBAD promoter;

and/or, the transthyretin is expressed by using a recombinant expression vector, a skeleton vector of the recombinant expression vector is pET-21a or a vector having homology of 25% or more with the pET-21a, and the sequence of the vector having homology of 25% or more with the pET-21a is preferably shown as SEQ ID NO. 8;

and/or the nucleotide sequence of the recombinant plasmid for expressing the transthyretin is shown as SEQ ID NO. 3;

and/or the transthyretin is expressed in microbial cells, and is preferably purified, wherein the microbial cells are preferably escherichia coli, and the escherichia coli preferably comprises E.coli B L21, E.coli B L21 (DE3), E.coli JM109, E.coli DH5 α and E.coli K12, and the purification is preferably to remove endotoxin through an endotoxin adsorption column and remove residual thallus through a 0.22 mu m pore size filter membrane;

and/or, when expressing the transthyretin, by culturing a transformant containing the transthyretin gene until the OD600 of the obtained bacterial cell reaches 1.5-2.0, for example, 1.6, 1.7, 1.8 or 1.9;

and/or, when the transthyretin is expressed, inducing expression by using an expression-inducing agent, wherein the expression-inducing agent is 0.1-2% by mass, such as 0.2%, 0.3%, 0.4%, 0.5%, 0.7%, 0.8%, 1.2% or 1.6% by mass, and the expression-inducing time is preferably 8-20h, such as 10h, 12h, 14h, 16h, 17h, 18h or 19 h; the expression inducing agent is preferably rhamnose or IPTG.

5. The application of transthyretin and/or a fusion protein composed of the transthyretin and a medicament in preparing drops, wherein the medicament is a protein medicament and/or a polypeptide medicament, and the transthyretin is shown as (a), (b) or (c):

(a) a protein consisting of amino acids shown in SEQ ID NO. 1;

6. The use of claim 5, wherein in (b), the protein derived from (a) is substituted with 22 amino acids, 25 amino acids, or 5 amino acids in the amino acid sequence of (a);

preferably:

more preferably:

7. The use according to claim 5 or 6, wherein the content of the fusion protein is 4 μmol/L, preferably 10-15 μmol/L;

and/or the transthyretin is present in an amount of 4. mu. mol/L or more, preferably 5-30. mu. mol/L, more preferably 10-20. mu. mol/L, e.g., 15, 20, 25. mu. mol/L;

and/or, the drops also contain physiological saline;

and/or the drops also contain hyaluronic acid, wherein the content of the hyaluronic acid is less than or equal to 6 percent, preferably 1-4 percent, and more preferably 2 percent;

and/or, the drops are preferably eye drops;

and/or, the drops are preferably drops for the treatment of diabetic retinopathy, age-related macular degeneration and/or retinopathy of prematurity;

and/or, the drops are administered in 1-3 drops per day, preferably in an amount of 0.6-0.8nmol protein per eye;

and/or, said drops are administered 2 times daily, 1 drop at a time, for 3 months; and/or, said drops are administered 2 times daily, 1 drop at a time, for 5 days; and/or, said drops are administered 2 times daily, 1 drop at a time, for 3 weeks;

and/or, the nucleotide sequence for coding the transthyretin is shown as SEQ ID NO. 2;

and/or, when the transthyretin is expressed, inducing expression by using an expression-inducing agent, wherein the expression-inducing agent is 0.1-2% by mass, such as 0.2%, 0.3%, 0.4%, 0.5%, 0.7%, 0.8%, 1.2% or 1.6% by mass, and the expression-inducing time is preferably 8-20h, such as 10h, 12h, 14h, 16h, 17h, 18h or 19 h; the expression inducing agent is preferably rhamnose or IPTG;

and/or the sequence of the fusion protein is shown as SEQ ID NO. 6 or SEQ ID NO. 7;

and/or, the transthyretin is expressed by fusion with the protein and/or polypeptide drug; the fusion is preferably to fuse the protein and/or polypeptide drug at the N terminal or C terminal of the transthyretin; the transthyretin is preferably expressed in microbial cells and purified after being fused with the protein and/or polypeptide drug; the purification is preferably to remove endotoxin by an endotoxin adsorption column and then remove residual thalli by a filter membrane with the aperture of 0.22 mu m;

8. Drops, characterized in that they contain transthyretin and/or a fusion protein of transthyretin and a drug; the medicine is protein and/or polypeptide medicine, and the transthyretin is shown as (a), (b) or (c):

(a) a protein consisting of amino acids shown in SEQ ID NO. 1;

9. The drop of claim 8, wherein in (b), the protein derived from (a) is a 22 amino acid substitution, a 25 amino acid substitution, or a 5 amino acid deletion in the amino acid sequence of (a);

preferably:

more preferably:

10. Drops according to claim 8 or 9, wherein when said drops contain a transthyretin-drug fusion protein, said fusion protein is present in an amount of at least 4 μmol/L, preferably 10-15 μmol/L;

and/or, when said drops contain transthyretin, said transthyretin is present in an amount of ≥ 4 μmol/L, preferably 5-30 μmol/L, more preferably 10-20 μmol/L, e.g. 15, 20, 25 μmol/L;

and/or, the drops also contain physiological saline;

and/or, the drops are preferably eye drops;